Posts Tagged ‘DNA replication’

Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on Novel Targets and Therapies 2:35 PM

Reporter: Stephen J. Williams, PhD


Session VMS.ET04.01 – Novel Targets and Therapies

Targeting chromatin remodeling-associated genetic vulnerabilities in cancer: PBRM1 defects are synthetic lethal with PARP and ATR inhibitors

Presenter/AuthorsRoman Merial Chabanon, Daphné Morel, Léo Colmet-Daage, Thomas Eychenne, Nicolas Dorvault, Ilirjana Bajrami, Marlène Garrido, Suzanna Hopkins, Cornelia Meisenberg, Andrew Lamb, Theo Roumeliotis, Samuel Jouny, Clémence Astier, Asha Konde, Geneviève Almouzni, Jyoti Choudhary, Jean-Charles Soria, Jessica Downs, Christopher J. Lord, Sophie Postel-Vinay. Gustave Roussy, Villejuif, France, The Francis Crick Institute, London, United Kingdom, Institute of Cancer Research, London, United Kingdom, Sage Bionetworks, Seattle, WA, Institute of Cancer Research, London, United Kingdom, Institute of Cancer Research, London, United Kingdom, Institut Curie, Paris, France, Université Paris-Sud/Université Paris-Saclay, Le Kremlin-Bicêtre, France, Gustave Roussy Cancer Campus and U981 INSERM, ATIP-Avenir group, Villejuif, FranceDisclosures R.M. Chabanon: None. D. Morel: None. L. Colmet-Daage: None. T. Eychenne: None. N. Dorvault: None. I. Bajrami: None. M. Garrido: None. S. Hopkins: ; Fishawack Group of Companies. C. Meisenberg: None. A. Lamb: None. T. Roumeliotis: None. S. Jouny: None. C. Astier: None. A. Konde: None. G. Almouzni: None. J. Choudhary: None. J. Soria: ; Medimmune/AstraZeneca. ; Astex. ; Gritstone. ; Clovis. ; GSK. ; GamaMabs. ; Lilly. ; MSD. ; Mission Therapeutics. ; Merus. ; Pfizer. ; PharmaMar. ; Pierre Fabre. ; Roche/Genentech. ; Sanofi. ; Servier. ; Symphogen. ; Takeda. J. Downs: None. C.J. Lord: ; AstraZeneca. ; Merck KGaA. ; Artios. ; Tango. ; Sun Pharma. ; GLG. ; Vertex. ; Ono Pharma. ; Third Rock Ventures. S. Postel-Vinay: ; Merck KGaA. ; Principal investigator of clinical trials for Gustave Roussy.; Boehringer Ingelheim. ; Principal investigator of clinical trials for Gustave Roussy.; Roche. ; Principal investigator of clinical trials for Gustave Roussy. Benefited from reimbursement for attending symposia.; AstraZeneca. ; Principal investigator of clinical trials for Gustave Roussy.; Clovis. ; Principal investigator of clinical trials for Gustave Roussy.; Bristol-Myers Squibb. ; Principal investigator of clinical trials for Gustave Roussy.; Agios. ; Principal investigator of clinical trials for Gustave Roussy.; GSK.AbstractAim: Polybromo-1 (PBRM1), a specific subunit of the pBAF chromatin remodeling complex, is frequently inactivated in cancer. For example, 40% of clear cell Renal Cell Carcinoma (ccRCC) and 15% of cholangiocarcinoma present deleterious PBRM1 mutations. There is currently no precision medicine-based therapeutic approach that targets PBRM1 defects. To identify novel, targeted therapeutic strategies for PBRM1-defective cancers, we carried out high-throughput functional genomics and drug screenings followed by in vitro and in vivo validation studies.
Methods: High-throughput siRNA-drug sensitization and drug sensitivity screens evaluating > 150 cancer-relevant small molecules in dose-response were performed in Pbrm1 siRNA-transfected mouse embryonic stem cells (mES) and isogenic PBRM1-KO or -WT HAP1 cells, respectively. After identification of PBRM1-selective small molecules, revalidation was carried out in a series of in-house-generated isogenic models of PBRM1 deficiency – including 786-O (ccRCC), A498 (ccRCC), U2OS (osteosarcoma) and H1299 (non-small cell lung cancer) human cancer cell lines – and non-isogenic ccRCC models, using multiple clinical compounds. Mechanistic dissection was performed using immunofluorescence, RT-qPCR, western blotting, DNA fiber assay, transcriptomics, proteomics and DRIP-sequencing to evaluate markers of DNA damage response (DDR), replication stress and cell-autonomous innate immune signaling. Preclinical data were integrated with TCGA tumor data.
Results: Parallel high-throughput drug screens independently identified PARP inhibitors (PARPi) as being synthetic lethal with PBRM1 defects – a cell type-independent effect which was exacerbated by ATR inhibitors (ATRi) and which we revalidated in vitro in isogenic and non-isogenic systems and in vivo in a xenograft model. PBRM1 defects were associated with increased replication fork stress (higher γH2AX and RPA foci levels, decreased replication fork speed and increased ATM checkpoint activation), R-loop accumulation and enhanced genomic instability in vitro; these effects were exacerbated upon PARPi exposure. In patient tumor samples, we also found that PBRM1-mutant cancers possessed a higher mutational load. Finally, we found that ATRi selectively activated the cGAS/STING cytosolic DNA sensing pathway in PBRM1-deficient cells, resulting in increased expression of type I interferon genes.
Conclusion: PBRM1-defective cancer cells present increased replication fork stress, R-loop formation, genome instability and are selectively sensitive to PARPi and ATRi through a synthetic lethal mechanism that is cell type-independent. Our data provide the pre-clinical rationale for assessing PARPi as a monotherapy or in combination with ATRi or immune-modulating agents in molecularly-selected patients with PBRM1-defective cancers.

1057 – Targeting MTHFD2 using first-in-class inhibitors kills haematological and solid cancer through thymineless-induced replication stress

Presenter/AuthorsThomas Helleday. University of Sheffield, Sheffield, United KingdomDisclosures T. Helleday: None.AbstractSummary
Thymidine synthesis pathways are upregulated pathways in cancer. Since the 1940s, targeting nucleotide and folate metabolism to induce thymineless death has remained first-line anti-cancer treatment. Recent discoveries that showing cancer cells have rewired networks and exploit unique enzymes for proliferation, have renewed interest in metabolic pathways. The cancer-specific expression of MTHFD2 has gained wide-spread attention and here we describe an emerging role for MTHFD2 in the DNA damage response (DDR). The folate metabolism enzyme MTHFD2 is one of the most consistently overexpressed metabolic enzymes in cancer and an emerging anticancer target. We show a novel role for MTHFD2 being essential for DNA replication and genomic stability in cancer cells. We describe first-in-class nanomolar MTHFD2 inhibitors (MTHFD2i), with protein co-crystal structures demonstrating binding in the active site of MTHFD2 and engaging with the target in cells and tumours. We show MTHFD2i reduce replication fork speed and induce replication stress, followed by S phase arrest, apoptosis and killing of a range of haematological and solid cancer cells in vitro and in vivo, with a therapeutic window spanning up to four orders of magnitude compared to non-transformed cells. Mechanistically, MTHFD2i prevent thymidine production leading to mis-incorporation of uracil into DNA and replication stress. As MTHFD2 expression is cancer specific there is a potential of MTHFD2i to synergize with other treatments. Here, we show MTHFD2i synergize with dUTPase inhibitors as well as other DDR inhibitors and demonstrate the mechanism of action. These results demonstrate a new link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically.
MTHFD2, one-carbon metabolism, folate metabolism, DNA replication, replication stress, synthetic lethal, thymineless death, small-molecule inhibitor, DNA damage response



1060 – Genetic and pharmacologic inhibition of Skp2, an E3 ubiquitin ligase and RB1-target, has antitumor activity in RB1-deficient human and mouse small cell lung cancer (SCLC)

Hongling ZhaoVineeth SukrithanNiloy IqbalCari NicholasYingjiao XueJoseph LockerJuntao ZouLiang ZhuEdward L. Schwartz. Albert Einstein College of Medicine, Bronx, NY, Albert Einstein College of Medicine, Bronx, NY, Albert Einstein College of Medicine, Bronx, NY, University of Pittsburgh Medical Center, Pittsburgh, PA, Albert Einstein College of Medicine, Bronx, NY
 H. Zhao: None. V. Sukrithan: None. N. Iqbal: None. C. Nicholas: None. Y. Xue: None. J. Locker: None. J. Zou: None. L. Zhu: None. E.L. Schwartz: None.
The identification of driver mutations and their corresponding targeted drugs has led to significant improvements in the treatment of non-small cell lung cancer (NSCLC) and other solid tumors; however, similar advances have not been made in the treatment of small cell lung cancer (SCLC). Due to their aggressive growth, frequent metastases, and resistance to chemotherapy, the five-year overall survival of SCLC is less than 5%. While SCLC tumors can be sensitive to first-line therapy of cisplatin and etoposide, most patients relapse, often in less than 3 months after initial therapy. Dozens of drugs have been tested clinically in SCLC, including more than 40 agents that have failed in phase III trials.
The near uniform bi-allelic inactivation of the tumor suppressor gene RB1 is a defining feature of SCLC. RB1 is mutated in highly aggressive tumors, including SCLC, where its functional loss, along with that of TP53, is both required and sufficient for tumorigenesis. While it is known that RB1 mutant cells fail to arrest at G1/S in response to checkpoint signals, this information has not led to effective strategies to treat RB1-deficient tumors, and it has been challenging to develop targeted drugs for tumors that are driven by the loss of gene function.
Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/p53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, and we confirmed this was the mechanism of protection in the RP-Skp2 KO mice by using the knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind to and/or inhibit Skp2 induced apoptosis and inhibited SCLC cell growth. In a panel of SCLC cell lines, growth inhibition by a Skp2 inhibitor was not correlated with sensitivity/resistance to etoposide. Targeting Skp2 also had in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using the genetic and pharmacologic approaches, antitumor activity was seen in vivo in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. The identification and validation of an actionable target downstream of RB1 could have a broad impact on treatment of SCLC and other advanced tumors with mutant RB1, for which there are currently no targeted therapies available.

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New DNA replication mechanism

Curator: Larry H. Bernstein, MD, FCAP



Structural Study Points to New DNA Replication Mechanism


GEN  http://www.genengnews.com/gen-news-highlights/structural-study-points-to-new-dna-replication-mechanism/81252345/


This movie shows the helicase protein complex from all angles, and reveals how its shape changes back and forth between two forms. The research team hypothesizes that the rocking action of this conformational change could help split the DNA double helix and move the helicase along one strand so it can be copied by DNA polymerase.


These are two images showing the structure of the helicase protein complex from above. (a) A surface-rendered three-dimensional electron density map as obtained by cryo-EM. (b) A computer-generated ‘ribbon diagram’ of the atomic model built based on the density map. The helicase has three major components: the Mcm2-7 hexamer ring in green, which encircles the DNA strand; the Cdc45 protein in magenta; and the GINS 4-protein complex in marine blue. Cdc45 and GINS recruit and tether other replisome components to the helicase, including the DNA polymerases that copy each strand of the DNA. [Brookhaven National Laboratory]


A collaborative team of researchers from the U.S. Department of Energy’s Brookhaven National Laboratory, Stony Brook University, Rockefeller University, and the University of Texas have just released detailed structural images of DNA helicase from yeast and are proposing a novel mechanism for how the molecular machinery functions. The scientists believe their new data could provide valuable insight into ways that DNA replication can go askew.

“DNA replication is a major source of errors that can lead to cancer,” explained senior study author Huilin Li, Ph.D., a professor with a joint appointment at Brookhaven Lab and Stony Brook University. “The entire genome, all 46 chromosomes, gets replicated every few hours in dividing human cells, so studying the details of how this process works may help us understand how errors occur.”

The findings from this study were published recently in Nature Structural & Molecular Biology through an article entitled “Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation.”

This current study builds upon previous work that produced the first-ever images of the complete DNA-copying protein complex, called the replisome. That study provided a surprising revelation about the location of the DNA-copying enzymes, DNA polymerases. This new study focuses on the atomic-level details for the helicase portion of the protein complex—the part that encircles and splits the DNA double helix so the polymerases can synthesize two new daughter strands.

As they had done in their previous work, Dr. Li and his colleagues produced high-resolution images of the helicase using cryo-electron microscopy (cryo-EM). This technique holds an advantage over other EM methods in that proteins can be studied in solution, closely replicating intracellular conditions.

“You don’t have to produce crystals that would lock the proteins in one position,” Dr. Li remarked. It’s an important point because helicase is a molecular machine made of 11 associated proteins that must be flexible to work. “You have to be able to see how the molecule moves to understand its function,” Dr. Li said.

Once the images of the replication machinery were assembled, the investigators were able to map out the locations of the individual amino acids that make up the helicase complex in each conformation. Then, combining those maps with existing biochemical knowledge, they came up with a mechanism for how the helicase works.

“One part binds and releases energy from a molecule called ATP. It converts the chemical energy into a mechanical force that changes the shape of the helicase,” Dr. Li stated. The molecule subsequently ejects the drained ATP and the helicase complex reverts to its original shape so a new ATP molecule can come in and begin the process again.

“It looks and operates similar to an old-style pumpjack oil rig, with one part of the protein complex forming a stable platform, and another part rocking back and forth,” Dr. Li noted. The researchers postulate that the rocking motion would nudge the DNA strands apart and move the helicase along the double helix in a linear fashion.

This direct translocation mechanism appears to be quite different from the way helicases are thought to operate in more primitive organisms such as bacteria, where the entire complex is believed to rotate around the DNA. However, there is biochemical evidence to support the idea of linear motion, including the fact that the helicase can still function even when the ATP hydrolysis activity of some, but not all, of the components is knocked out by mutation.

“We acknowledge that this proposal may be controversial, and it is not really proven at this point, but the structure gives an indication of how this protein complex works, and we are trying to make sense of it,” Dr. Li stated.


Decades Old DNA Replication Models Called into Question

GEN  http://www.genengnews.com/gen-news-highlights/decades-old-dna-replication-models-called-into-question/81251929/?kwrd=Replisome

Decades Old DNA Replication Models Called into Question


A series of electron micrographs show the barrel-shaped helicase, which is the enzyme that separates the two DNA strands, along with other components of the replisome, including polymerase-epsilon (green).[Brookhaven National Laboratory]


Previously (left), the replisome’s two polymerases (green) were assumed to be below the helicase (tan), the enzyme that splits the DNA strands. The new images reveal one polymerase is located at the front of the helicase, causing one strand to loop backward as it is copied (right). [Brookhaven National Laboratory]


It may be time to update biology texts to reflect newly published data from a collaborative team of scientists at Rockefeller University, Stony Brook University, and the U.S. Department of Energy’s Brookhaven National Laboratory. Using cutting-edge electron microscopy (EM) techniques, the investigators gathered the first ever images of the fully assembled replisome, providing new insight into the molecular mechanisms of replication.

“Our finding goes against decades of textbook drawings of what people thought the replisome should look like,” remarked co-senior author Michael O’Donnell, Ph.D., professor and head of Rockefeller’s Laboratory of DNA Replication. “However, it’s a recurring theme in science that nature does not always turn out to work the way you thought it did.”

The researcher’s findings focused on the replisome found in eukaryotic organisms, a category that includes a broad swath of living things, including humans and other multicellular organisms. Over the past several decades, there has been an array of data describing the individual components comprising the complex nature of replisome. Yet, until now no pictures existed to show just how everything fit together.

“This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell,” explained co-senior author Huilin Li, Ph.D., biologist with joint appointments at Brookhaven Lab and Stony Brook University. “These new images show the fully assembled and fully activated ‘helicase’ protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two ‘polymerase’ enzymes that duplicate each strand to copy the genome.”

The image and implications from this study were described in a paper entitled “The architecture of a eukaryotic replisome,” published recently through Nature Structural & Molecular Biology.

Traditional models of DNA replication show the helicase enzyme moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands.

However, the images that the researchers collected of intact replisomes revealed that only one of the polymerases is located at the back of the helicase. The other is on the front side of the helicase, where the helicase first encounters the double-stranded helix. This means that while one of the two split DNA strands is acted on by the polymerase at the back end, the other has to thread itself back through or around the helicase to reach the front-side polymerase before having its new complementary strand assembled.

“DNA replication is one of the most fundamental processes of life, so it is every biochemist’s dream to see what a replisome looks like,” stated lead author Jingchuan Sun, EM biologist in Dr. Li’s laboratory. “Our lab has expertise and a decade of experience using electron microscopy to study DNA replication, which has prepared us well to tackle the highly mobile therefore very challenging replisome structure. Working together with the O’Donnell lab, which has done beautiful, functional studies on the yeast replisome, our two groups brought perfectly complementary expertise to this project.”

The positioning of one polymerase at the front of the helicase suggests that it may have an unforeseen function—the possibilities of which the collaborative group of scientists is continuing to study. Whatever the function the offset polymerase ends up having, Drs. Li and O’Donnell hope that it will not only provide them better insight into the replication machinery but that they may uncover useful information that can be exploited for disease intervention.

“Clearly, further studies will be required to understand the functional implications of the unexpected replisome architecture reported here,” the scientists concluded.


The architecture of a eukaryotic replisome

Jingchuan SunYi ShiRoxana E GeorgescuZuanning YuanBrian T ChaitHuilin Li Michael E O’Donnell

Nature Structural & Molecular Biology 2015; 22:976–982     http://dx.doi.org:/10.1038/nsmb.3113

At the eukaryotic DNA replication fork, it is widely believed that the Cdc45–Mcm2–7–GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ε is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α–primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ε, first threading through the Mcm2–7 ring and then making a U-turn at the bottom and reaching Pol εat the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.


Figure 4: Subunit proximities within CMGE determined by chemical cross-linking with mass spectrometry readout (CX-MS).close

Subunit proximities within CMGE determined by chemical cross-linking with mass spectrometry readout (CX-MS).

CMGE was cross-linked with a lysine-specific bifunctional cross-linker, then fragmented by proteolysis, and cross-linked peptides were identified by mass spectrometry. (a) Overview of cross-links observed within the region of Pol2 …


Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation

Zuanning YuanLin BaiJingchuan SunRoxana GeorgescuJun LiuMichael E O’Donnell & Huilin Li

Nature Structural & Molecular Biology 8 Feb 2016    http://dx.doi.org:/10.1038/nsmb.3170

The CMG helicase is composed of Cdc45, Mcm2–7 and GINS. Here we report the structure of theSaccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7–4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2–7, braced by Cdc45–GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2–7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.


Figure 1: Cryo-EM and overall structure of theS. cerevisiae CMG complex.

Cryo-EM and overall structure of the S. cerevisiae CMG complex.

(a) A typical motion-corrected raw image from ~8,000 images of frozen CMG particles recorded on a direct detector. (b) Six selected 2D averages representing the particles in different views. (c) 3D cryo-EM map of CMG, color-coded accord…



Figure 2: Structure and interactions of yeast GINS and Cdc45.

Structure and interactions of yeast GINS and Cdc45.

(a) The full-length GINS structure in top and side views. Domain A is shown in cartoon and domain B in surface. Top, schematic showing that all four subunits have a similar two-domain architecture, but domains A and B in Psf2 and Psf3 a…



Figure 3: Side-by-side comparison of conformer I and conformer II in the Mcm2–7 region of CMG helicase.

Side-by-side comparison of conformer I and conformer II in the Mcm2-7 region of CMG helicase.

(a,b) Comparison of the two conformations, shown in cartoon representation and viewed from the right side, from Cdc45 and GINS (which are both removed for clarity) with the CTD motor ring on top and the NTD ring at the bottom. The two b…



Figure 6: Pol2 footprint on the atomic model of CMG helicase.

Pol2 footprint on the atomic model of CMG helicase.

(a) The two-domain architecture of Pol2, the catalytic subunit of the Pol ε complex. The N-terminal half contains the polymerase and exonuclease activities. The C-terminal half is homologous to a B-family polymerase but lacks enzymatic…

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CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics – Part IIB

Curator: Larry H Bernstein, MD, FCAP

Part I: The Initiation and Growth of Molecular Biology and Genomics – Part I From Molecular Biology to Translational Medicine: How Far Have We Come, and Where Does It Lead Us?


Part II: CRACKING THE CODE OF HUMAN LIFE is divided into a three part series.

Part IIA. “CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way” reviews the Human Genome Project and the decade beyond.


Part IIB. “CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics” lays the manifold multivariate systems analytical tools that has moved the science forward to a groung that ensures clinical application.


Part IIC. “CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease “ will extend the discussion to advances in the management of patients as well as providing a roadmap for pharmaceutical drug targeting.


To be followed by:
Part III will conclude with Ubiquitin, it’s role in Signaling and Regulatory Control.

Part IIB. “CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics” is a continuation of a previous discussion on the role of genomics in discovery of therapeutic targets titled, Directions for Genomics in Personalized Medicinewhich focused on:

  • key drivers of cellular proliferation,
  • stepwise mutational changes coinciding with cancer progression, and
  • potential therapeutic targets for reversal of the process.

It is a direct extension of The Initiation and Growth of Molecular Biology and Genomics – Part I 

These articles review a web-like connectivity between inter-connected scientific discoveries, as significant findings have led to novel hypotheses and many expectations over the last 75 years. This largely post WWII revolution has driven our understanding of biological and medical processes at an exponential pace owing to successive discoveries of
  • chemical structure,
  • the basic building blocks of DNA  and proteins, of
  • nucleotide and protein-protein interactions,
  • protein folding,
  • allostericity,
  • genomic structure,
  • DNA replication,
  • nuclear polyribosome interaction, and
  • metabolic control.


In addition, the emergence of methods for

  • copying,
  • removal
  • insertion, and
  • improvements in structural analysis
  • developments in applied mathematics have transformed the research framework.

This last point,

  • developments in applied mathematics have transformed the research framework, is been developed in this very article

CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics – Part IIB

Computational Genomics

1. Three-Dimensional Folding and Functional Organization Principles of The Drosophila Genome

Sexton T, Yaffe E, Kenigeberg E, Bantignies F,…Cavalli G. Institute de Genetique Humaine, Montpelliere GenomiX, and Weissman Institute, France and Israel. Cell 2012; 148(3): 458-472.

Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation.

250px-DNA_labeled  DNA diagram showing base pairing      circular genome map

Here we present a high-resolution chromosomal contact map derived from

  • a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei.
  • the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks.
  • Chromosomal contacts are hierarchically organized between domains.
  • Global modeling of contact density and clustering of domains show that inactive
  • domains are condensed and confined to their chromosomal territories, whereas
  • active domains reach out of the territory to form remote intra- and interchromosomal contacts.

Moreover, we systematically identify

  • specific long-range intrachromosomal contacts between Polycomb-repressed domains.

Together, these observations

  • allow for quantitative prediction of the Drosophila chromosomal contact map,
  • laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system.


2A. Architecture Reveals Genome’s Secrets

Three-dimensional genome maps – Human chromosome

Genome sequencing projects have provided rich troves of information about

  • stretches of DNA that regulate gene expression, as well as
  • how different genetic sequences contribute to health and disease.

But these studies miss a key element of the genome—its spatial organization—which has long been recognized as an important regulator of gene expression.

  • Regulatory elements often lie thousands of base pairs away from their target genes, and recent technological advances are allowing scientists to begin examining
  • how distant chromosome locations interact inside a nucleus.
  • The creation and function of 3-D genome organization, some say, is the next frontier of genetics.

Mapping and sequencing may be completely separate processes. For example, it’s possible to determine the location of a gene—to “map” the gene—without sequencing it. Thus, a map may tell you nothing about the sequence of the genome, and a sequence may tell you nothing about the map.  But the landmarks on a map are DNA sequences, and mapping is the cousin of sequencing. A map of a sequence might look like this:
On this map, GCC is one landmark; CCCC is another. Here we find, the sequence is a landmark on a map. In general, particularly for humans and other species with large genomes,

  • creating a reasonably comprehensive genome map is quicker and cheaper than sequencing the entire genome.
  • mapping involves less information to collect and organize than sequencing does.

Completed in 2003, the Human Genome Project (HGP) was a 13-year project. The goals were:

  • identify all the approximately 20,000-25,000 genes in human DNA,
  • determine the sequences of the 3 billion chemical base pairs that make up human DNA,
  • store this information in databases,
  • improve tools for data analysis,
  • transfer related technologies to the private sector, and
  • address the ethical, legal, and social issues (ELSI) that may arise from the project.

Though the HGP is finished, analyses of the data will continue for many years. By licensing technologies to private companies and awarding grants for innovative research, the project catalyzed the multibillion-dollar U.S. biotechnology industry and fostered the development of new medical applications. When genes are expressed, their sequences are first converted into messenger RNA transcripts, which can be isolated in the form of complementary DNAs (cDNAs). A small portion of each cDNA sequence is all that is needed to develop unique gene markers, known as sequence tagged sites or STSs, which can be detected using the polymerase chain reaction (PCR). To construct a transcript map, cDNA sequences from a master catalog of human genes were distributed to mapping laboratories in North America, Europe, and Japan. These cDNAs were converted to STSs and their physical locations on chromosomes determined on one of two radiation hybrid (RH) panels or a yeast artificial chromosome (YAC) library containing human genomic DNA. This mapping data was integrated relative to the human genetic map and then cross-referenced to cytogenetic band maps of the chromosomes. (Further details are available in the accompanying article in the 25 October issue of SCIENCE).

Tremendous progress has been made in the mapping of human genes, a major milestone in the Human Genome Project. Apart from its utility in advancing our understanding of the genetic basis of disease, it  provides a framework and focus for accelerated sequencing efforts by highlighting key landmarks (gene-rich regions) of the chromosomes. The construction of this map has been possible through the cooperative efforts of an international consortium of scientists who provide equal, full and unrestricted access to the data for the advancement of biology and human health.

There are two types of maps: genetic linkage map and physical map. The genetic linkage map shows the arrangement of genes and genetic markers along the chromosomes as calculated by the frequency with which they are inherited together. The physical map is representation of the chromosomes, providing the physical distance between landmarks on the chromosome, ideally measured in nucleotide bases. Physical maps can be divided into three general types: chromosomal or cytogenetic maps, radiation hybrid (RH) maps, and sequence maps.
 ch10f3  radiation hybrid maps   ch10f2  subchromosomal mapping

2B. Genome-nuclear lamina interactions and gene regulation.

Kind J, van Steensel B. Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.
The nuclear lamina, a filamentous protein network that coats the inner nuclear membrane, has long been thought to interact with specific genomic loci and regulate their expression. Molecular mapping studies have now identified
  • large genomic domains that are in contact with the lamina.
Genes in these domains are typically repressed, and artificial tethering experiments indicate that
  • the lamina can actively contribute to this repression.
Furthermore, the lamina indirectly controls gene expression in the nuclear interior by sequestration of certain transcription factors.
Mol Cell. 2010; 38(4):603-13.          http://dx.doi.org/10.1016/j.molcel.2010.03.016
Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I,  …., van Steensel B.  Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.
To visualize three-dimensional organization of chromosomes within the nucleus, we generated high-resolution maps of genome-nuclear lamina interactions during subsequent differentiation of mouse embryonic stem cells via lineage-committed neural precursor cells into terminally differentiated astrocytes.  A basal chromosome architecture present in embryonic stem cells is cumulatively altered at hundreds of sites during lineage commitment and subsequent terminal differentiation. This remodeling involves both
  • individual transcription units and multigene regions and
  • affects many genes that determine cellular identity.
  •  genes that move away from the lamina are concomitantly activated;
  • others, remain inactive yet become unlocked for activation in a next differentiation step.

lamina-genome interactions are widely involved in the control of gene expression programs during lineage commitment and terminal differentiation.

 view the full text on ScienceDirect.
Graphical Summary
PDF 1.54 MB
Referred to by: The Silence of the LADs: Dynamic Genome-…
Authors:  Daan Peric-Hupkes, Wouter Meuleman, Ludo Pagie, Sophia W.M. Bruggeman, et al.
  • Various cell types share a core architecture of genome-nuclear lamina interactions
  • During differentiation, hundreds of genes change their lamina interactions
  • Changes in lamina interactions reflect cell identity
  • Release from the lamina may unlock some genes for activation

Fractal “globule”

About 10 years ago—just as the human genome project was completing its first draft sequence—Dekker pioneered a new technique, called chromosome conformation capture (C3) that allowed researchers to get a glimpse of how chromosomes are arranged relative to each other in the nucleus. The technique relies on the physical cross-linking of chromosomal regions that lie in close proximity to one another. The regions are then sequenced to identify which regions have been cross-linked. In 2009, using a high throughput version of this basic method, called Hi-C, Dekker and his collaborators discovered that the human genome appears to adopt a “fractal globule” conformation—

  • a manner of crumpling without knotting.


In the last 3 years, Jobe Dekker and others have advanced technology even further, allowing them to paint a more refined picture of how the genome folds—and how this influences gene expression and disease states.  Dekker’s 2009 findings were a breakthrough in modeling genome folding, but the resolution—about 1 million base pairs— was too crude to allow scientists to really understand how genes interacted with specific regulatory elements. The researchers report two striking findings.

First, the human genome is organized into two separate compartments, keeping

  • active genes separate and accessible
  • while sequestering unused DNA in a denser storage compartment.
  • Chromosomes snake in and out of the two compartments repeatedly
  • as their DNA alternates between active, gene-rich and inactive, gene-poor stretches.

Second, at a finer scale, the genome adopts an unusual organization known in mathematics as a “fractal.” The specific architecture the scientists found, called

  • a “fractal globule,” enables the cell to pack DNA incredibly tightly —

the information density in the nucleus is trillions of times higher than on a computer chip — while avoiding the knots and tangles that might interfere with the cell’s ability to read its own genome. Moreover, the DNA can easily Unfold and Refold during

  • gene activation,
  • gene repression, and
  • cell replication.

Dekker and his colleagues discovered, for example, that chromosomes can be divided into folding domains—megabase-long segments within which

  • genes and regulatory elements associate more often with one another than with other chromosome sections.

The DNA forms loops within the domains that bring a gene into close proximity with a specific regulatory element at a distant location along the chromosome. Another group, that of molecular biologist Bing Ren at the University of California, San Diego, published a similar finding in the same issue of Nature.  Dekker thinks the discovery of [folding] domains will be one of the most fundamental [genetics] discoveries of the last 10 years. The big questions now are

  • how these domains are formed, and
  • what determines which elements are looped into proximity.

“By breaking the genome into millions of pieces, we created a spatial map showing how close different parts are to one another,” says co-first author Nynke van Berkum, a postdoctoral researcher at UMass Medical School in Dekker‘s laboratory. “We made a fantastic three-dimensional jigsaw puzzle and then, with a computer, solved the puzzle.”

Lieberman-Aiden, van Berkum, Lander, and Dekker’s co-authors are Bryan R. Lajoie of UMMS; Louise Williams, Ido Amit, and Andreas Gnirke of the Broad Institute; Maxim Imakaev and Leonid A. Mirny of MIT; Tobias Ragoczy, Agnes Telling, and Mark Groudine of the Fred Hutchison, Cancer Research Center and the University of Washington; Peter J. Sabo, Michael O. Dorschner, Richard Sandstrom, M.A. Bender, and John Stamatoyannopoulos of the University of Washington; and Bradley Bernstein of the Broad Institute and Harvard Medical School.

2C. three-dimensional structure of the human genome

Lieberman-Aiden et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science, 2009; DOI: 10.1126/science.1181369.
Harvard University (2009, October 11). 3-D Structure Of Human Genome: Fractal Globule Architecture Packs Two Meters Of DNA Into Each Cell. ScienceDaily.   Retrieved February 2, 2013, from        http://www.sciencedaily.com/releases/2009/10/091008142957

Using a new technology called Hi-C and applying it to answer the thorny question of how each of our cells stows some three billion base pairs of DNA while maintaining access to functionally crucial segments. The paper comes from a team led by scientists at Harvard University, the Broad Institute of Harvard and MIT, University of Massachusetts Medical School, and the Massachusetts Institute of Technology. “We’ve long known that on a small scale, DNA is a double helix,” says co-first author Erez Lieberman-Aiden, a graduate student in the Harvard-MIT Division of Health Science and Technology and a researcher at Harvard’s School of Engineering and Applied Sciences and in the laboratory of Eric Lander at the Broad Institute. “But if the double helix didn’t fold further, the genome in each cell would be two meters long. Scientists have not really understood how the double helix folds to fit into the nucleus of a human cell, which is only about a hundredth of a millimeter in diameter. This new approach enabled us to probe exactly that question.”

The mapping technique that Aiden and his colleagues have come up with bridges a crucial gap in knowledge—between what goes on at the smallest levels of genetics (the double helix of DNA and the base pairs) and the largest levels (the way DNA is gathered up into the 23 chromosomes that contain much of the human genome). The intermediate level, on the order of thousands or millions of base pairs, has remained murky.  As the genome is so closely wound, base pairs in one end can be close to others at another end in ways that are not obvious merely by knowing the sequence of base pairs. Borrowing from work that was started in the 1990s, Aiden and others have been able to figure out which base pairs have wound up next  to one another. From there, they can begin to reconstruct the genome—in three dimensions.

4C profiles validate the Hi-C Genome wide map

Even as the multi-dimensional mapping techniques remain in their early stages, their importance in basic biological research is becoming ever more apparent. “The three-dimensional genome is a powerful thing to know,” Aiden says. “A central mystery of biology is the question of how different cells perform different functions—despite the fact that they share the same genome.” How does a liver cell, for example, “know” to perform its liver duties when it contains the same genome as a cell in the eye? As Aiden and others reconstruct the trail of letters into a three-dimensional entity, they have begun to see that “the way the genome is folded determines which genes were

2D. “Mr. President; The Genome is Fractal !”

Eric Lander (Science Adviser to the President and Director of Broad Institute) et al. delivered the message on Science Magazine cover (Oct. 9, 2009) and generated interest in this by the International HoloGenomics Society at a Sept meeting.

First, it may seem to be trivial to rectify the statement in “About cover” of Science Magazine by AAAS.

  • The statement “the Hilbert curve is a one-dimensional fractal trajectory” needs mathematical clarification.

The mathematical concept of a Hilbert space, named after David Hilbert, generalizes the notion of Euclidean space. It extends the methods of vector algebra and calculus from the two-dimensional Euclidean plane and three-dimensional space to spaces with any finite or infinite number of dimensions. A Hilbert space is an abstract vector space possessing the structure of an inner product that allows length and angle to be measured. Furthermore, Hilbert spaces must be complete, a property that stipulates the existence of enough limits in the space to allow the techniques of calculus to be used. A Hilbert curve (also known as a Hilbert space-filling curve) is a continuous fractal space-filling curve first described by the German mathematician David Hilbert in 1891,[1] as a variant of the space-filling curves discovered by Giuseppe Peano in 1890.[2] For multidimensional databases, Hilbert order has been proposed to be used instead of Z order because it has better locality-preserving behavior.

Representation as Lindenmayer system
The Hilbert Curve can be expressed by a rewrite system (L-system).

Alphabet : A, B

Constants : F + –                                                                                                                                      119px-Hilbert3d-step3                             120px-Hilbert512

Axiom : A

Production rules:

A → – B F + A F A + F B –

B → + A F – B F B – F A +

Here, F means “draw forward”, – means “turn left 90°”, and + means “turn right 90°” (see turtle graphics).


While the paper itself does not make this statement, the new Editorship of the AAAS Magazine might be even more advanced if the previous Editorship did not reject (without review) a Manuscript by 20+ Founders of (formerly) International PostGenetics Society in December, 2006.

Second, it may not be sufficiently clear for the reader that the reasonable requirement for the DNA polymerase to crawl along a “knot-free” (or “low knot”) structure does not need fractals. A “knot-free” structure could be spooled by an ordinary “knitting globule” (such that the DNA polymerase does not bump into a “knot” when duplicating the strand; just like someone knitting can go through the entire thread without encountering an annoying knot): Just to be “knot-free” you don’t need fractals. Note, however, that

  • the “strand” can be accessed only at its beginning – it is impossible to e.g. to pluck a segment from deep inside the “globulus”.

This is where certain fractals provide a major advantage – that could be the “Eureka” moment for many readers. For instance,

  • the mentioned Hilbert-curve is not only “knot free” –
  • but provides an easy access to “linearly remote” segments of the strand.

If the Hilbert curve starts from the lower right corner and ends at the lower left corner, for instance

  • the path shows the very easy access of what would be the mid-point
  • if the Hilbert-curve is measured by the Euclidean distance along the zig-zagged path.

Likewise, even the path from the beginning of the Hilbert-curve is about equally easy to access – easier than to reach from the origin a point that is about 2/3 down the path. The Hilbert-curve provides an easy access between two points within the “spooled thread”; from a point that is about 1/5 of the overall length to about 3/5 is also in a “close neighborhood”.

This may be the “Eureka-moment” for some readers, to realize that

  • the strand of “the Double Helix” requires quite a finess to fold into the densest possible globuli (the chromosomes) in a clever way
  • that various segments can be easily accessed. Moreover, in a way that distances between various segments are minimized.

This marvellous fractal structure is illustrated by the 3D rendering of the Hilbert-curve. Once you observe such fractal structure, you’ll never again think of a chromosome as a “brillo mess”, would you? It will dawn on you that the genome is orders of magnitudes more finessed than we ever thought so.

Those embarking at a somewhat complex review of some historical aspects of the power of fractals may wish to consult the ouvre of Mandelbrot (also, to celebrate his 85th birthday). For the more sophisticated readers, even the fairly simple Hilbert-curve (a representative of the Peano-class) becomes even more stunningly brilliant than just some “see through density”. Those who are familiar with the classic “Traveling Salesman Problem” know that “the shortest path along which every given n locations can be visited once, and only once” requires fairly sophisticated algorithms (and tremendous amount of computation if n>10 (or much more). Some readers will be amazed, therefore, that for n=9 the underlying Hilbert-curve helps to provide an empirical solution.

refer to pellionisz@junkdna.com

Briefly, the significance of the above realization, that the (recursive) Fractal Hilbert Curve is intimately connected to the (recursive) solution of TravelingSalesman Problem, a core-concept of Artificial Neural Networks can be summarized as below.

Accomplished physicist John Hopfield (already a member of the National Academy of Science) aroused great excitement in 1982 with his (recursive) design of artificial neural networks and learning algorithms which were able to find reasonable solutions to combinatorial problems such as the Traveling SalesmanProblem. (Book review Clark Jeffries, 1991, see also 2. J. Anderson, R. Rosenfeld, and A. Pellionisz (eds.), Neurocomputing 2: Directions for research, MIT Press, Cambridge, MA, 1990):

“Perceptions were modeled chiefly with neural connections in a “forward” direction: A -> B -* C — D. The analysis of networks with strong backward coupling proved intractable. All our interesting results arise as consequences of the strong back-coupling” (Hopfield, 1982).

The Principle of Recursive Genome Function surpassed obsolete axioms that blocked, for half a Century, entry of recursive algorithms to interpretation of the structure-and function of (Holo)Genome.  This breakthrough, by uniting the two largely separate fields of Neural Networks and Genome Informatics, is particularly important for

  • those who focused on Biological (actually occurring) Neural Networks (rather than abstract algorithms that may not, or because of their core-axioms, simply could not
  • represent neural networks under the governance of DNA information).

DNA base triplets

3A. The FractoGene Decade

from Inception in 2002 to Proofs of Concept and Impending Clinical Applications by 2012

  1. Junk DNA Revisited (SF Gate, 2002)
  2. The Future of Life, 50th Anniversary of DNA (Monterey, 2003)
  3. Mandelbrot and Pellionisz (Stanford, 2004)
  4. Morphogenesis, Physiology and Biophysics (Simons, Pellionisz 2005)
  5. PostGenetics; Genetics beyond Genes (Budapest, 2006)
  6. ENCODE-conclusion (Collins, 2007)

The Principle of Recursive Genome Function (paper, YouTube, 2008)

  1. Cold Spring Harbor presentation of FractoGene (Cold Spring Harbor, 2009)
  2. Mr. President, the Genome is Fractal! (2009)
  3. HolGenTech, Inc. Founded (2010)
  4. Pellionisz on the Board of Advisers in the USA and India (2011)
  5. ENCODE – final admission (2012)
  6. Recursive Genome Function is Clogged by Fractal Defects in Hilbert-Curve (2012)
  7. Geometric Unification of Neuroscience and Genomics (2012)
  8. US Patent Office issues FractoGene 8,280,641 to Pellionisz (2012)


When the human genome was first sequenced in June 2000, there were two pretty big surprises. The first was thathumans have only about 30,000-40,000 identifiable genes, not the 100,000 or more many researchers were expecting. The lower –and more humbling — number

  • means humans have just one-third more genes than a common species of worm.

The second stunner was

  • how much human genetic material — more than 90 percent — is made up of what scientists were calling “junk DNA.”

The term was coined to describe similar but not completely identical repetitive sequences of amino acids (the same substances that make genes), which appeared to have no function or purpose. The main theory at the time was that these apparently non-working sections of DNA were just evolutionary leftovers, much like our earlobes.

If biophysicist Andras Pellionisz is correct, genetic science may be on the verge of yielding its third — and by far biggest — surprise.

With a doctorate in physics, Pellionisz is the holder of Ph.D.’s in computer sciences and experimental biology from the prestigious Budapest Technical University and the Hungarian National Academy of Sciences. A biophysicist by training, the 59-year-old is a former research associate professor of physiology and biophysics at New York University, author of numerous papers in respected scientific journals and textbooks, a past winner of the prestigious Humboldt Prize for scientific research, a former consultant to NASA and holder of a patent on the world’s first artificial cerebellum, a technology that has already been integrated into research on advanced avionics systems. Because of his background, the Hungarian-born brain researcher might also become one of the first people to successfully launch a new company by using the Internet to gather momentum for a novel scientific idea.

The genes we know about today, Pellionisz says, can be thought of as something similar to machines that make bricks (proteins, in the case of genes), with certain junk-DNA sections providing a blueprint for the different ways those proteins are assembled. The notion that at least certain parts of junk DNA might have a purpose for example, many researchers now refer to with a far less derogatory term: introns.

In a provisional patent application filed July 31, Pellionisz claims to have unlocked a key to the hidden role junk DNA plays in growth — and in life itself. His patent application covers all attempts to count, measure and compare the fractal properties of introns for diagnostic and therapeutic purposes.

3B. The Hidden Fractal Language of Intron DNA

To fully understand Pellionisz’ idea, one must first know what a fractal is.

Fractals are a way that nature organizes matter. Fractal patterns can be found in anything that has a nonsmooth surface (unlike a billiard ball), such as coastal seashores, the branches of a tree or the contours of a neuron (a nerve cell in the brain). Some, but not all, fractals are self-similar and stop repeating their patterns at some stage; the branches of a tree, for example, can get only so small. Because they are geometric, meaning they have a shape, fractals can be described in mathematical terms. It’s similar to the way a circle can be described by using a number to represent its radius (the distance from its center to its outer edge). When that number is known, it’s possible to draw the circle it represents without ever having seen it before.

Although the math is much more complicated, the same is true of fractals. If one has the formula for a given fractal, it’s possible to use that formula

  • to construct, or reconstruct,
  • an image of whatever structure it represents,
  • no matter how complicated.

The mysteriously repetitive but not identical strands of genetic material are in reality building instructions organized in a special type

  • of pattern known as a fractal.  It’s this pattern of fractal instructions, he says, that
  • tells genes what they must do in order to form living tissue,
  • everything from the wings of a fly to the entire body of a full-grown human.

In a move sure to alienate some scientists, Pellionisz has chosen the unorthodox route of making his initial disclosures online on his own Web site. He picked that strategy, he says, because it is the fastest way he can document his claims and find scientific collaborators and investors. Most mainstream scientists usually blanch at such approaches, preferring more traditionally credible methods, such as publishing articles in peer-reviewed journals.

Basically, Pellionisz’ idea is that a fractal set of building instructions in the DNA plays a similar role in organizing life itself. Decode the way that language works, he says, and in theory it could be reverse engineered. Just as knowing the radius of a circle lets one create that circle, the more complicated fractal-based formula would allow us to understand how nature creates a heart or simpler structures, such as disease-fighting antibodies. At a minimum, we’d get a far better understanding of how nature gets that job done.

The complicated quality of the idea is helping encourage new collaborations across the boundaries that sometimes separate the increasingly intertwined disciplines of biology, mathematics and computer sciences.

Hal Plotkin, Special to SF Gate. Thursday, November 21, 2002.                          http://www.junkdna.com/Special to SF Gate/plotkin.htm (1 of 10)2012.12.13. 12:11:58/


3C. multifractal analysis

The human genome: a multifractal analysis. Moreno PA, Vélez PE, Martínez E, et al.

BMC Genomics 2011, 12:506. http://www.biomedcentral.com/1471-2164/12/506

Background: Several studies have shown that genomes can be studied via a multifractal formalism. Recently, we used a multifractal approach to study the genetic information content of the Caenorhabditis elegans genome. Here we investigate the possibility that the human genome shows a similar behavior to that observed in the nematode.
Results: We report here multifractality in the human genome sequence. This behavior correlates strongly on the

  • presence of Alu elements and
  • to a lesser extent on CpG islands and (G+C) content.

In contrast, no or low relationship was found for LINE, MIR, MER, LTRs elements and DNA regions poor in genetic information.

  • Gene function,
  • cluster of orthologous genes,
  • metabolic pathways, and
  • exons tended to increase their frequencies with ranges of multifractality and
  • large gene families were located in genomic regions with varied multifractality.

Additionally, a multifractal map and classification for human chromosomes are proposed.


we propose a descriptive non-linear model for the structure of the human genome,

This model reveals

  • a multifractal regionalization where many regions coexist that are far from equilibrium and
  • this non-linear organization has significant molecular and medical genetic implications for understanding the role of
  • Alu elements in genome stability and structure of the human genome.

Given the role of Alu sequences in

  • gene regulation,
  • genetic diseases,
  • human genetic diversity,
  • adaptation
  • and phylogenetic analyses,

these quantifications are especially useful.

MiIP: The Monomer Identification and Isolation Program

Bun C, Ziccardi W, Doering J and Putonti C.Evolutionary Bioinformatics 2012:8 293-300.    http://dx.goi.org/10.4137/EBO.S9248

Repetitive elements within genomic DNA are both functionally and evolutionarilly informative. Discovering these sequences ab initio is

  • computationally challenging, compounded by the fact that
  • sequence identity between repetitive elements can vary significantly.

Here we present a new application, the Monomer Identification and Isolation Program (MiIP), which provides functionality to both

  • search for a particular repeat as well as
  • discover repetitive elements within a larger genomic sequence.

To compare MiIP’s performance with other repeat detection tools, analysis was conducted for

  • synthetic sequences as well as
  • several a21-II clones and
  • HC21 BAC sequences.

The primary benefit of MiIP is the fact that it is a single tool capable of searching for both

  • known monomeric sequences as well as
  • discovering the occurrence of repeats ab initio, per the user’s required sensitivity of the search.

Methods for Examining Genomic and Proteomic Interactions

1. An Integrated Statistical Approach to Compare Transcriptomics Data Across Experiments: A Case Study on the Identification of Candidate Target Genes of the Transcription Factor PPARα

Ullah MO, Müller M and Hooiveld GJEJ. Bioinformatics and Biology Insights 2012:6 145–154.       http://dx.doi.org/10.4137/BBI.S9529

http://www.la- press.com/
http://bionformaticsandBiologyInsights.com/An_Integrated_Statistical_Approach_to_Compare_ transcriptomic_Data_Across_Experiments-A-Case_Study_on_the_Identification_ of_Candidate_Target_Genes_of_the Transcription_Factor_PPARα/
Corresponding author email: guido.hooiveld@wur.nl

An effective strategy to elucidate the signal transduction cascades activated by a transcription factor is to compare the transcriptional profiles of wild type and transcription factor knockout models. Many statistical tests have been proposed for analyzing gene expression data, but most

  • tests are based on pair-wise comparisons. Since the analysis of microarrays involves the testing of multiple hypotheses within one study, it is
  • generally accepted that one should control for false positives by the false discovery rate (FDR). However, it has been reported that
  • this may be an inappropriate metric for comparing data across different experiments.

Here we propose an approach that addresses the above mentioned problem by the simultaneous testing and integration of the three hypotheses (contrasts) using the cell means ANOVA model.

These three contrasts test for the effect of

  • a treatment in wild type,
  • gene knockout, and
  • globally over all experimental groups.

We illustrate our approach on microarray experiments that focused on the identification of candidate target genes and biological processes governed by the fatty acid sensing transcription factor PPARα in liver. Compared to the often applied FDR based across experiment comparison, our approach identified a conservative but less noisy set of candidate genes with same sensitivity and specificity. However, our method had the advantage of

  • properly adjusting for multiple testing while
  • integrating data from two experiments, and
  • was driven by biological inference.

We present a simple, yet efficient strategy to compare

  • differential expression of genes across experiments
  • while controlling for multiple hypothesis testing.

2. Managing biological complexity across orthologs with a visual knowledgebase of documented biomolecular interactions

Vincent VanBuren & Hailin Chen.   Scientific Reports 2, Article number: 1011  Received 02 October 2012 Accepted 04 December 2012 Published 20 December 2012

The complexity of biomolecular interactions and influences is a major obstacle to their comprehension and elucidation. Visualizing knowledge of biomolecular interactions increases comprehension and facilitates the development of new hypotheses. The rapidly changing landscape of high-content experimental results also presents a challenge for the maintenance of comprehensive knowledgebases. Distributing the responsibility for maintenance of a knowledgebase to a community of subject matter experts is an effective strategy for large, complex and rapidly changing knowledgebases.
Cognoscente serves these needs by

  • building visualizations for queries of biomolecular interactions on demand,
  • by managing the complexity of those visualizations, and
  • by crowdsourcing to promote the incorporation of current knowledge from the literature.

Imputing functional associations between biomolecules and imputing directionality of regulation for those predictions each

  • require a corpus of existing knowledge as a framework to build upon. Comprehension of the complexity of this corpus of knowledge
  • will be facilitated by effective visualizations of the corresponding biomolecular interaction networks.


was designed and implemented to serve these roles as

  • a knowledgebase and
  • as an effective visualization tool for systems biology research and education.

Cognoscente currently contains over 413,000 documented interactions, with coverage across multiple species.  Perl, HTML, GraphViz1, and a MySQL database were used in the development of Cognoscente. Cognoscente was motivated by the need to

  • update the knowledgebase of biomolecular interactions at the user level, and
  • flexibly visualize multi-molecule query results for heterogeneous interaction types across different orthologs.

Satisfying these needs provides a strong foundation for developing new hypotheses about regulatory and metabolic pathway topologies.  Several existing tools provide functions that are similar to Cognoscente, so we selected several popular alternatives to

  • assess how their feature sets compare with Cognoscente ( Table 1 ). All databases assessed had
  • easily traceable documentation for each interaction, and
  • included protein-protein interactions in the database.

Most databases, with the exception of BIND,

  • provide an open-access database that can be downloaded as a whole.

Most databases, with the exceptions of EcoCyc and HPRD, provide

  • support for multiple organisms.

Most databases support web services for interacting with the database contents programatically, whereas this is a planned feature for Cognoscente.

  • INT, STRING, IntAct, EcoCyc, DIP and Cognoscente provide built-in visualizations of query results,
  • which we consider among the most important features for facilitating comprehension of query results.
  • BIND supports visualizations via Cytoscape. Cognoscente is among a few other tools that support multiple organisms in the same query,
  • protein->DNA interactions, and
  • multi-molecule queries.

Cognoscente has planned support for small molecule interactants (i.e. pharmacological agents).  MINT, STRING, and IntAct provide a prediction (i.e. score) of functional associations, whereas
Cognoscente does not currently support this. Cognoscente provides support for multiple edge encodings to visualize different types of interactions in the same display,

  • a crowdsourcing web portal that allows users to submit interactions
  • that are then automatically incorporated in the knowledgebase, and displays orthologs as compound nodes to provide clues about potential
  • orthologous interactions.

The main strengths of Cognoscente are that

  1. it provides a combined feature set that is superior to any existing database,
  2. it provides a unique visualization feature for orthologous molecules, and relatively unique support for
  3. multiple edge encodings,
  4. crowdsourcing, and
  5. connectivity parameterization.

The current weaknesses of Cognoscente relative to these other tools are

  • that it does not fully support web service interactions with the database,
  • it does not fully support small molecule interactants, and
  • it does not score interactions to predict functional associations.

Web services and support for small molecule interactants are currently under development.

Other related articles on thie Open Access Online Sceintific Journal, include the following:

Big Data in Genomic Medicine                    lhb                          https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair S Saha                                                                                   https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-in-transcription-ubiquitination-and-dna-repair/

Computational Genomics Center: New Unification of Computational Technologies at Stanford A Lev-Ari    https://pharmaceuticalintelligence.com/2012/12/03/computational-genomics-center-new-unification-of-computational-technologies-at-stanford/

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1 (pharmaceuticalintelligence.com) A Lev-Ari https://pharmaceuticalintelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2 A Lev-Ari

Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3 A Lev-Ari https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-research-part-3/

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial” A Lev-Ari    https://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors S Saha

Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence A Lev-Ari


Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition sjwilliams


Directions for genomics in personalized medicine lhb https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis. Sjwilliams

Mitochondrial fission and fusion: potential therapeutic targets? Ritu saxena    https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

Mitochondrial mutation analysis might be “1-step” away ritu saxena  https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

mRNA interference with cancer expression lhb https://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

Expanding the Genetic Alphabet and linking the genome to the metabolome https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-metabolome/

Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression Analysis A Lev-Ari


Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis lhb https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/

Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology A Lev-Ari https://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-agricultural-biotechnology/

2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.  https://pharmaceuticalintelligence.com/2013_Genomics

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1 https://pharmaceuticalintelligence.com/Paradigm Shift in Human Genomics_/

English: DNA replication or DNA synthesis is t...

English: DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is paramount to all life as we know it. (Photo credit: Wikipedia)

Français : Deletion chromosomique

Français : Deletion chromosomique (Photo credit: Wikipedia)

A slight mutation in the matched nucleotides c...

A slight mutation in the matched nucleotides can lead to chromosomal aberrations and unintentional genetic rearrangement. (Photo credit: Wikipedia)

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From Molecular Biology to Translational Medicine: How Far Have We Come, and Where Does It Lead Us?

The Initiation and Growth of Molecular Biology and Genomics, Part I

Curator: Larry H Bernstein, MD, FCAP

Introduction and purpose

This material will cover the initiation phase of molecular biology, Part I; to be followed by the Human Genome Project, Part II; and concludes with Ubiquitin, it’s Role in Signaling and Regulatory Control, Part III.
This article is first a continuation of a previous discussion on the role of genomics in discovery of therapeutic targets titled Directions for genomics in personalized medicine https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

The previous article focused on key drivers of cellular proliferation, stepwise mutational changes coinciding with cancer progression, and potential therapeutic targets for reversal of the process. It also covers the race to delineation of the Human Genome, discovery methods and fundamental genomic patterns that are ancient in both animal and plant speciation.

This article reviews the web-like connections between early and later discoveries, as significant finding has led to novel hypotheses and many more findings over the last 75 years. This largely post WWII revolution has driven our understanding of biological and medical processes at an exponential pace owing to successive discoveries of chemical structure, the basic building blocks of DNA and proteins, of nucleotide and protein-protein interactions, protein folding, allostericity, genomic structure, DNA replication, nuclear polyribosome interaction, and metabolic control. In addition, the emergence of methods for copying, removal and insertion, and improvements in structural analysis as well as developments in applied mathematics have transformed the research framework.

In the Beginning

During the Second World War we had the discoveries of physics and the emergence out of the Manhattan Project of radioactive nuclear probes from E.O. Lawrence University of California Berkeley Laboratory. The use of radioactive isotopes led to the development of biochemistry and isolation of nucleotides, nucleosides, enzymes, and filling in of details of pathways for photosynthesis, for biosynthesis, and for catabolism.
Perhaps a good start of the journey is a student of Neils Bohr named Max Delbruck (September 4, 1906 – March 9, 1981), who won the Nobel prize for discovering that bacteria become resistant to viruses (phages) as a result of genetic mutations, founded a new discipline called Molecular Biology, lifting the experimental work in Physiology to a systematic experimentation in biology with the rigor of Physics using radiation and virus probes on selected cells. In 1937 he turned to research on the genetics of Drosophila melanogaster at Caltech, and two years later he coauthored a paper, “The growth of bacteriophage”, reporting that the viruses replicate in one step, not exponentially. In 1942, he and Salvador Luria of Indiana University demonstrated that bacterial resistance to virus infection is mediated by random mutation. This research, known as the Luria-Delbrück experiment, notably applied mathematics to make quantitative predictions, and earned them the 1969 Nobel Prize in Physiology or Medicine, shared with Alfred Hershey. His inferences on genes’ susceptibility to mutation was relied on by physicist Erwin Schrödinger in his 1944 book, What Is Life?, which conjectured genes were an “aperiodic crystal” storing code-script and influenced Francis Crick and James D. Watson in their 1953 identification of cellular DNA’s molecular structure as a double helix.

Watson-Crick Double Helix Model

A new understanding of heredity and hereditary disease was possible once it was determined that DNA consists of two chains twisted around each other, or double helixes, of alternating phosphate and sugar groups, and that the two chains are held together by hydrogen bonds between pairs of organic bases—adenine (A) with thymine (T), and guanine (G) with cytosine (C). Modern biotechnology also has its basis in the structural knowledge of DNA—in this case the scientist’s ability to modify the DNA of host cells that will then produce a desired product, for example, insulin.
The background for the work of the four scientists was formed by several scientific breakthroughs:

  1. the progress made by X-ray crystallographers in studying organic macromolecules;
  2. the growing evidence supplied by geneticists that it was DNA, not protein, in chromosomes that was responsible for heredity;
  3. Erwin Chargaff’s experimental finding that there are equal numbers of A and T bases and of G and C bases in DNA;
  4. and Linus Pauling’s discovery that the molecules of some proteins have helical shapes.

In 1962 James Watson (b. 1928), Francis Crick (1916–2004), and Maurice Wilkins (1916–2004) jointly received the Nobel Prize in physiology or medicine for their 1953 determination of the structure of deoxyribonucleic acid (DNA), performed with a knowledge of Chargaff’s ratios of the bases in DNA and having  access to the X-ray crystallography of Maurice Wilkins and Rosalind Franklin at King’s College London. Because the Nobel Prize can be awarded only to the living, Wilkins’s colleague Rosalind Franklin (1920–1958), who died of cancer at the age of 37, could not be honored.
Of the four DNA researchers, only Rosalind Franklin had any degrees in chemistry. Franklin completed her degree in 1941 in the middle of World War II and undertook graduate work at Cambridge with Ronald Norrish, a future Nobel Prize winner. She returning to Cambridge after a year of war service, presented her work and received the PhD in physical chemistry. Franklin then learned the  X-ray crystallography in Paris and rapidly became a respected authority in this field. Returning to returned to England to King’s College London in 1951, her charge was to upgrade the X-ray crystallographic laboratory there for work with DNA.

bt2304  Rosalind Franklin, crystallographer

Cold Spring Harbor Laboratory

I digress to the beginnings of the Cold Spring Harbor Laboratory. A significant part of the Laboratory’s life revolved around education with its three-week-long Phage Course, taught first in 1945 by Max Delbruck, the German-born, theoretical-physicist-turned-biologist. James D Watson first came to Cold Spring Harbor Laboratory with his thesis advisor, Salvador Luria, in the summer of 1948. Over its more than 25-year history, the Phage Course was the training ground for many notable scientists. The Laboratory’s annual scientific Symposium, has provided a unique highly interactive education about the exciting field of “molecular” biology. The 1953 symposium featured Watson coming from England to give the first public presentation of the DNA double helix. When he became the Laboratory’s director in 1968 he was determined to make the Laboratory an important center for advancing molecular biology, and he focused his energy on bringing large donations to the enterprise CSHNL. It became a magnate for future discovery at which James D. Watson became the  Director in 1968, and later the Chancellor. This contribution has as great an importance as his Nobel Prize discovery.

Biochemistry and Molecular Probes comes into View

Moreover, at the same time, the experience of Nathan Kaplan and Martin Kamen at Berkeley working with radioactive probes was the beginning of an establishment of Lawrence-Livermore Laboratories role in metabolic studies, as reported in the previous paper. A collaboration between Sid Collowick, NO Kaplan and Elizabeth Neufeld at the McCollum Pratt Institute led to the transferase reaction between the two main pyridine nucleotides.  Neufeld received a PhD a few years later from the University of California, Berkeley, under William Zev Hassid for research on nucleotides and complex carbohydrates, and did postdoctoral studies on non-protein sulfhydryl compounds in mitosis. Her later work at the NIAMDG on mucopolysaccharidoses. The Lysosomal Storage Diseases opened a new chapter on human genetic diseases when she found that the defects in Hurler and Hunter syndromes were due to decreased degradation of the mucopolysaccharides. When an assay became available for α-L-iduronidase in 1972, Neufeld was able to show that the corrective factor for Hurler syndrome that accelerates degradation of stored sulfated mucopolysaccharides was α-L-iduronidase.


The Hurler Corrective Factor. Purification and Some Properties (Barton, R. W., and Neufeld, E. F. (1971) J. Biol. Chem. 246, 7773–7779)
The Sanfilippo A Corrective Factor. Purification and Mode of Action (Kresse, H., and Neufeld, E. F. (1972) J. Biol. Chem. 247, 2164–2170)

I mention this for two reasons:
[1] We see a huge impetus for nucleic acids and nucleotides research growing in the 1950’s with a post WWII emergence of work on biological structure.
[2] At the same time, the importance of enzymes in cellular metabolic processes runs parallel to that of the genetic code.

In 1959 Arthur Kornberg was a recipient of the Nobel prize for Physiology or Medicine based on his discovery of “the mechanisms in the biological synthesis of deoxyribonucleic acid” (DNA polymerase) together with Dr. Severo Ochoa of New York University. In the next 20 years Stanford University Department of Biochemistry became a top rated graduate program in biochemistry. Today, the Pfeffer Lab is distinguished for research into how human cells put receptors in the right place through Rab GTPases that regulate all aspects of receptor trafficking. Steve Elledge (1984-1989) at Harvard University is one of  its graduates from the 1980s.

Transcription –RNA and the ribosome

In 2006, Roger Kornberg was awarded the Nobel Prize in Chemistry for identifying the role of RNA polymerase II and other proteins in transcribing DNA. He says that the process is something akin to a machine. “It has moving parts which function in synchrony, in appropriate sequence and in synchrony with one another”. The Kornbergs were the tenth family with closely-related Nobel laureates.  The 2009 Nobel Prize in Chemistry was awarded to Venki Ramakrishnan, Tom Steitz, and Ada Yonath for crystallographic studies of the ribosome. The atomic resolution structures of the ribosomal subunits provide an extraordinary context for understanding one of the most fundamental aspects of cellular function: protein synthesis. Research on protein synthesis began with studies of microsomes, and three papers were published on the atomic resolution structures of the 50S and 30S the atomic resolution of structures of ribosomal subnits in 2000. Perhaps the most remarkable and inexplicable feature of ribosome structure is that two-thirds of the mass is composed of large RNA molecules, the 5S, 16S, and 23S ribosomal RNAs, and the remaining third is distributed among ~50 relatively small and innocuous proteins. The first step on the road to solving the ribosome structure was determining the primary structure of the 16S and 23S RNAs in Harry Noller’s laboratory. The sequences were rapidly followed by secondary structure models for the folding of the two ribosomal RNAs, in collaboration with Carl Woese, bringing the ribosome structure into two dimensions. The RNA secondary structures are characterized by an elaborate series of helices and loops of unknown structure, but other than the insights offered by the structure of transfer RNA (tRNA), there was no way to think about folding these structures into three dimensions. The first three-dimensional images of the ribosome emerged from Jim Lake’s reconstructions from electron microscopy (EM) (Lake, 1976).

Ada Yonath reported the first crystals of the 50S ribosomal subunit in 1980, a crucial step that would require almost 20 years to bring to fruition (Yonath et al., 1980). Yonath’s group introduced the innovative use of ribosomes from extremophilic organisms. Peter Moore and Don Engelman applied neutron scattering techniques to determine the relative positions of ribosomal proteins in the 30S ribosomal subunit at the same time. Elegant chemical footprinting studies from the Noller laboratory provided a basis for intertwining the RNA among the ribosomal proteins, but there was still insufficient information to produce a high resolution structure, but Venki Ramakrishnan, in Peter Moore’s laboratory did it with deuterated ribosome reconstitutions. Then the Yale group was ramping up its work on the H. marismortui crystals of the 50S subunit. Peter Moore had recruited long-time colleague Tom Steitz to work on this problem and Steitz was about to complete the final event in the pentathlon of Crick’s dogma, having solved critical structures of DNA polymerases, the glutaminyl tRNA-tRNA synthetase complex, HIV reverse transcriptase, and T7 RNA polymerase. In 1999 Steitz, Ramakrishnan, and Yonath all presented electron density maps of subunits at approximately 5 Å resolution, and the Noller group presented 10 Å electron density maps of the Thermus 70S ribosome. Peter Moore aptly paraphrased Churchill, telling attendees that this was not the end, but the end of the beginning. Almost every nucleotide in the RNA is involved in multiple stabilizing interactions that form the monolithic tertiary structure at the heart of the ribosome.
Williamson J. The ribosome at atomic resolution. Cell 2009; 139:1041-1043.    http://dx.doi.org/10.1016/j.cell.2009.11.028/      http://www.sciencedirect.com/science/article/pii/S0092867409014536

This opened the door to new therapies.  For example, in 2010 it was reported that Numerous human genes display dual coding within alternatively spliced regions, which give rise to distinct protein products that include segments translated in more than one reading frame. To resolve the ensuing protein structural puzzle, we identified human genes with alternative splice variants comprising a dual coding region at least 75 nucleotides in length and analyzed the structural status of the protein segments they encode. The inspection of their amino acid composition and predictions by the IUPred and PONDR® VSL2 algorithms suggest a high propensity for structural disorder in dual-coding regions.
Kovacs E, Tompa P, liliom K, and Kalmar L. Dual coding in alternative reading frames correlates with intrinsic protein disorder. PNAS 2010.   http://www.jstor.org/stable/25664997   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851785

In 2012, it was shown that drug-bound ribosomes can synthesize a distinct subset of cellular polypeptides. The structure of a protein defines its ability to thread through the antibiotic-obstructed tunnel. Synthesis of certain polypeptides that initially bypass translational arrest can be stopped at later stages of elongation while translation of some proteins goes to completion. (Kannan K, Vasquez-Laslop N, and Mankin AS. Selective Protein Synthesis by Ribosomes with a Drug-Obstructed Exit Tunnel. Cell 2012; 151; 508-520.) http://dx.doi.org/10.1016/j.cell.2012.09.018     http://www.sciencedirect.com/science/article/pii/S0092867412011257

Mobility of genetic elements

Barbara McClintock received the Nobel Prize for Medicine for the discovery of the mobility of genetic elements, work that been done in that period. When transposons were demonstrated in bacteria, yeast and other organisms, Barbara rose to a stratospheric level in the general esteem of the scientific world, but she was uncomfortable about the honors. It was sufficient to have her work understood and acknowledged. Prof. Howard Green said of her, “There are scientists whose discoveries greatly transcend their personalities and their humanity. But those in the future who will know of Barbara only her discoveries will know only her shadow”.
“In Memoriam – Barbara McClintock”. Nobelprize.org. 5 Feb 2013   http://www.nobelprize.org/nobel_prizes/medicine/laureates/1983/mcclintock-article.html/

She introduced her Nobel Lecture in 1983 with the following observation: “An experiment conducted in the mid-nineteen forties prepared me to expect unusual responses of a genome to challenges for which the genome is unprepared to meet in an orderly, programmed manner. In most known instances of this kind, the types of response were not predictable in advance of initial observations of them. It was necessary to subject the genome repeatedly to the same challenge in order to observe and appreciate the nature of the changes it induces…a highly programmed sequence of events within the cell that serves to cushion the effects of the shock. Some sensing mechanism must be present in these instances to alert the cell to imminent danger, and to set in motion the orderly sequence of events that will mitigate this danger”. She goes on to consider “early studies that revealed programmed responses to threats that are initiated within the genome itself, as well as others similarly initiated, that lead to new and irreversible genomic modifications. These latter responses, now known to occur in many organisms, are significant for appreciating how a genome may reorganize itself when faced with a difficulty for which it is unprepared”.

An experiment with Zea conducted in the summer of 1944 alerted her to the mobility of specific components of genomes involved the entrance of a newly ruptured end of a chromosome into a telophase nucleus. This experiment commenced with the growing of approximately 450 plants in the summer of 1944, each of which had started its development with a zygote that had received from each parent a chromosome with a newly ruptured end of one of its arms. The design of the experiment required that each plant be self-pollinated to isolate from the self-pollinated progeny new mutants that were expected to appear, and confine them to locations within the ruptured arm of a chromosome. Each mutant was expected to reveal the phenotype produced by a minute homozygous deficiency. Their modes of origin could be projected from the known behavior of broken ends of chromosomes in successive mitoses. Forty kernels from each self-pollinated ear were sown in a seedling bench in the greenhouse during the winter of 1944-45.

Some seedling mutants of the type expected overshadowed by segregants exhibiting bizarre phenotypes. These were variegated for type and degree of expression of a gene. Those variegated expressions given by genes associated with chlorophyll development were startingly conspicuous. Within any one progeny chlorophyll intensities, and their pattern of distribution in the seedling leaves, were alike. Between progenies, however, both the type and the pattern differed widely.

The effect of X-rays on chromosomes

Initial studies of broken ends of chromosomes began in the summer of 1931. By 1931, means of studying the beads on a string hypothesis was provided by newly developed methods of examining the ten chromosomes of the maize complement in microsporocytes in meiosis. The ten bivalent chromosomes are elongated in comparison to their metaphase lengths. Each chromosome

  • is identifiable by its relative length,
  • by the location of its centromere, which is readily observed at the pachytene stage, and
  • by the individuality of the chromomeres strung along the length of each chromosome.

At that time maize provided the best material for locating known genes along a chromosome arm, and also for precisely determining the break points in chromosomes that had undergone various types of rearrangement, such as translocations, inversions, etc.
The recessive phenotypes in the examined plants arose from loss of a segment of a chromosome that carried the wild-type allele, and X-rays were responsible for inducing these deficiencies. A conclusion of basic significance could be drawn from these observations:

  1. broken ends of chromosomes will fuse, 2-by-2, and
  2. any broken end with any other broken end.

This principle has been amply proved in a series of experiments conducted over the years. In all such instances the break must sever both strands of the DNA double helix. This is a “double-strand break” in modern terminology. That two such broken ends entering a telophase nucleus will find each other and fuse, regardless of the initial distance that separates them, soon became apparent.

During the summer of 1931 she had seen plants in the maize field that showed variegation patterns resembling the one described for Nicotiana.  Dr. McClintock was interested in selecting the variegated plants to determine the presence of a ring chromosome in each, and in the summer of 1932 with Dr. Stadler’s generous cooperation from Missouri, she had the opportunity to examine such plants. Each plant had a ring chromosome, but It was the behavior of this ring that proved to be significant. It revealed several basic phenomena. The following was noted:

In the majority of mitoses

  • replication of the ring chromosome produced two chromatids completely free from each other
  • could separate without difficulty in the following anaphase.
  • sister strand exchanges do occur between replicated or replicating chromatids
  • the frequency of such events increases with increase in the size of the ring.
  • these exchanges produce a double-size ring with two centromeres.
  • Mechanical rupture occurs in each of the two chromatid bridges formed at anaphase by passage of the two centromeres on the double-size ring to opposite poles of the mitotic spindle.
  • The location of a break can be at any one position along any one bridge.
  • The broken ends entering a telophase nucleus then fuse.
  • The size and content of each newly constructed ring depend on the position of the rupture that had occurred in each bridge.
  1. The conclusion was that cells sense the presence in their nuclei of ruptured ends of chromosomes
  2. then activate a mechanism that will bring together and then unite these ends
  3. this will occur regardless of the initial distance in a telophase nucleus that separated the ruptured ends.

The ability of a cell to

  • sense these broken ends,
  • to direct them toward each other, and
  • then to unite them so that the union of the two DNA strands is correctly oriented,
  • is a particularly revealing example of the sensitivity of cells to all that is going on within them.

Evidence from gave unequivocal support for the conclusion that broken ends will find each other and fuse. The challenge is met by a programmed response. This may be necessary, as

  1. both accidental breaks and
  2. programmed breaks may be frequent.
  3. If not repaired, such breaks could lead to genomic deficiencies having serious consequences.

A cell capable of repairing a ruptured end of a chromosome must sense the presence of this end in its nucleus. This sensing

  • activates a mechanism that is required for replacing the ruptured end with a functional telomere.
  • that such a mechanism must exist was revealed by a mutant that arose in the stocks.
  • this mutant would not allow the repair mechanism to operate in the cells of the plant.

Entrance of a newly ruptured end of a chromosome into the zygote is followed by the chromatid type of breakage-fusion-bridge cycle throughout mitoses in the developing plant.
This suggested that the repair mechanism in the maize strains is repressed in cells producing

  • the male and female gametophytes and
  • also in the endosperm,
  • but is activated in the embryo.

The extent of trauma perceived by cells

  • whose nuclei receive a single newly ruptured end of a chromosome that the cell cannot repair,
  • and the speed with which this trauma is registered, was not appreciated until the winter of 1944-45.

By 1947 it was learned that the bizarre variegated phenotypes that segregated in many of the self-pollinated progenies grown on the seedling bench in the fall and winter of 1944-45, were due to the action of transposable elements. It seemed clear that

  • these elements must have been present in the genome,
  • and in a silent state previous to an event that activated one or another of them.

She concluded that some traumatic event was responsible for these activations. The unique event in the history of these plants relates to their origin. Both parents of the plants grown in 1944 had contributed a chromosome with a newly ruptured end to the zygote that gave rise to each of these plants.
Detection of silent elements is now made possible with the aid of DNA cloning method. Silent AC (Activator) elements, as well as modified derivatives of them, have already been detected in several strains of maize. When other transposable elements are cloned it will be possible to compare their structural and numerical differences among various strains of maize. In any one strain of maize the number of silent but potentially transposable elements, as well as other repetitious DNAs, may be observed to change, and most probably in response to challenges not yet recognized.
Telomeres are especially adapted to replicate free ends of chromosomes. When no telomere is present, attempts to replicate this uncapped end may be responsible for the apparent “fusions” of the replicated chromatids at the position of the previous break as well as for perpetuating the chromatid type of breakage-fusion-bridge cycle in successive mitoses.
In conclusion, a genome may react to conditions for which it is unprepared, but to which it responds in a totally unexpected manner. Among these is

  • the extraordinary response of the maize genome to entrance of a single ruptured end of a chromosome into a telophase nucleus.
  • It was this event that was responsible for activations of potentially transposable elements that are carried in a silent state in the maize genome.
  • The mobility of these activated elements allows them to enter different gene loci and to take over control of action of the gene wherever one may enter.

Because the broken end of a chromosome entering a telophase nucleus can initiate activations of a number of different potentially transposable elements,

  • the modifications these elements induce in the genome may be explored readily.

In addition to

modifying gene action, these elements can

  • restructure the genome at various levels,
  • from small changes involving a few nucleotides,
  • to gross modifications involving large segments of chromosomes, such as
  1. duplications,
  2. deficiencies,
  3. inversions,
  4. and other reorganizations.

In the future attention undoubtedly will be centered on the genome, and with greater appreciation of its significance as a highly sensitive organ of the cell,

  • monitoring genomic activities and correcting common errors,
  • sensing the unusual and unexpected events,
  • and responding to them,
  • often by restructuring the genome.

We know about the elements available for such restructuring. We know nothing, however, about

  • how the cell senses danger and instigates responses to it that often are truly remarkable.


In 2009 the Nobel Prize in Physiology or Medicine was awarded to Elizabeth Blackburn, Carol Greider and Jack Szoztak for the discovery of Telomerase. This recognition came less than a decade after the completion of the Human Genome Project previously discussed. Prof. Blackburn acknowledges a strong influence coming from the work of Barbara McClintock. The discovery is tied to the pond organism Tetrahymena thermophila, and studies of yeast cells. Blackburn was drawn to science after reading the biography of Marie Curie by her daughter, Irina, as a child. She recalls that her Master’s mentor while studying the metabolism of glutamine in the rat liver, thought that every experiment should have the beauty and simplicity of a Mozart sonata. She did her PhD at the distinguished Laboratory for Molecular Biology at Cambridge, the epicenter of molecular biology sequencing the regions of bacteriophage phiX 174, a single stranded DNA bacteriophage. Using Fred Sanger’s methods to piece together RNA sequences she showed the first sequence of a 48 nucleotide fragment to her mathematical-gifted Cambridge cousin, who pointed out repeats of DNA sequence patterns! She worked on the sequencing of the DNA at the terminal regions of  the short “minichromosomes” of the ciliated protozoan Tetrahymena thermophile at Yale in 1975. She continued her research begun at Yale at UCSF funded by the NIH based on an intriguing audiogram showing telomeric DNA in Tetrahymena.
I describe the work as follows:

  • Prof. Blackburn incorporated 32P isotope labelled deoxynucleoside residues into the rDNA molecules for DNA repair enzymatic reactions and found that
  • the end regions were selectively labeled by combinations of 32P isotope radiolabled nucleoside triphosphate, and by mid-year she had an audiogram of the depurination products.
  • The audiogram showed sequences of 4 cytosine residues flanked by either an adenosine or a guanosine residue.
  • In 1976 she had deduced a sequence consisting of a tandem array of CCCAA repeats, and subsequently separated the products on a denaturing gel electrophoresis that appeared as tiger stripes extending up the gel.
  • The size of each band was 6 bases more than the band below it.

Telomere must have a telomerase!

The discovery of the telomerase enzyme activity was done by the Prize co-awardee, Carol Greider. They were trying to decipher the structure right at the termini of telomeres of both cliliated protozoans and yeast plasmids. The view that in mammalian telomeres there is a long protruding G-rich strand does not take into account the clear evidence for the short C strand repeat oligonucleotides that she discovered. This was found for both the Tetrahymena rDNA minichromosome molecules and linear plasmids purified from yeast.
In contrast to nucleosomal regions of chromosomes, special regions of DNA, for example

  • promoters that must bind transcription initiation factors that control transcription, have proteins other than the histones on them.
  • The telomeric repeat tract turned out to be such a non-nucleosomal region.

They  found that by clipping up chromatin using an enzyme that cuts the linker between neighboring nucleosomes,

  • it cut up the bulk of the DNA into nucleosome-sized pieces
  • but left the telomeric DNA tract as a single protected chunk.

The resulting complex of the telomeric DNA tract plus its bound cargo of protective proteins behaved very differently, from nucleosomal chromatin, and concluded that it had no histones or nucleosomes.

Any evidence for a protein on the bulk of the rDNA molecule ends, such as their behavior in gel electrophoresis and the appearance of the rDNA molecules under the electron microscope, was conspicuously lacking. This was reassuring that there was no covalently attached protein at the very ends of this minichoromosome. Despite considerable work, she was unable to determine what protein(s) would co-purify with the telomeric repeat tract DNA of Tetrahymena. It was yeast genetics and approaches done by others that turned out to provide the next great leaps forward in understanding telomeric proteins. Carol Greider, her colleague, noticed the need to scale up the telomerase activity preparations and they used a very large glass column for preparative gel filtration chromatography.

Jack W Szostak at the Howard Hughes Medical Institue at Harvard shared in the 2009 Nobel Prize. He became interested in molecular biology taking a course on the frontiers of Molecular Biology and reading about the experiments of Meselson-Stahl barely a decade earlier, and learned how the genetic code had been unraveled. The fact that one could deduce, from measurements of the radioactivity in fractions from a centrifuge tube, the molecular details of DNA replication, transcription and translation was astonishing. A highlight of his time at McGill was the open-book, open-discussion final exam in this class, in which the questions required the intense collaboration of groups of students.

At Cornell, Ithaca, he collaborated with  John Stiles and they came up with a specific idea to chemically synthesize a DNA oligonucleotide of sufficient length that it would hybridize to a single sequence within the yeast genome, and then to use it as an mRNA and gene specific probe. At the time, there was only one short segment of the yeast genome for which the DNA sequence was known,

  • the region coding for the N-terminus of the iso-1 cytochrome c protein,

intensively studied by Fred Sherman
The Sherman lab, in a tour de force of genetics and protein chemistry, had isolated

  • double-frameshift mutants in which the N-terminal region of the protein was translated from out-of-frame codons.
  • Protein sequencing of the wild type and frame-shifted mutants allowed them to deduce 44 nucleotides of DNA sequence.

If they could prepare a synthetic oligonucleotide that was complementary to the coding sequence, they could use it to detect the cytochrome-c mRNA and gene. At the time, essentially all experiments on mRNA were done on total cellular mRNA. Ray Wu was already well known for determining the sequence of the sticky ends of phage lambda, the first ever DNA to be sequenced, and his lab was deeply involved in the study of enzymes that could be used to manipulate and sequence DNA more effectively, but would not take on a project from another laboratory. So John went to nearby Rochester to do postdoctoral work with Sherman, and he was able to transfer to Ray Wu’s laboratory. In order to carry out his work, Ray Wu sent him to Saran Narang’s lab in Ottawa, and he received training there under Keichi Itakura, who synthesized the Insulin gene. A few months later, he received several milligrams of our long sought 15-mer. In collaboration with John Stiles and Fred Sherman, who sent us RNA and DNA samples from appropriate yeast strains, they were able to use the labeled 15-mer as a probe to detect the cyc1 mRNA, and later the gene itself. He notes that one of the delights of the world of science is that it is filled with people of good will who are more than happy to assist a student or colleague by teaching a technique or discussing a problem. He remained in Ray’s lab after completion of the PhD upon the arrival of Rodney Rothstein from Sherman’s lab in Rochester, who introduced him to yeast genetics, and he was prepared for the next decade of work on yeast.

  • first in recombination studies, and
  • later in telomere studies and other aspects of yeast biology.

His studies of recombination in yeast were enabled by the discovery, in Gerry Fink’s lab at Cornell, of a way to introduce foreign DNA into yeast. These pioneering studies of yeast transformation showed that circular plasmid DNA molecules could on occasion become integrated into yeast chromosomal DNA by homologous recombination.

  • His studies of unequal sister chromatid exchange in rDNA locus resulted in his first publication in the field of recombination.

The idea that you could increase transformation frequency by cutting the input DNA was pleasingly counterintuitive and led us to continue our exploration of this phenomenon. He gained an appointment to the Sidney-Farber Cancer Institute due to the interest of Prof. Ruth Sager, who gathered together a great group of young investigators. In work spearheaded by his first graduate student, Terry Orr-Weaver, on

  • double-strand breaks in DNA
  • and their repair by recombination (and continuing interaction with Rod Rothstein),
  • they were attracted to what kinds of reactions occur at the DNA ends.

It was at a Gordon Conference that he was excited hearing a talk by Elizabeth Blackburn on her work on telomeres in Tetrahymena.

  • This led to a collaboration testing the ability of Tetrahymena telomers to function in yeast.
  • He performed the experiments himself, and experienced the thrill of being the first to know that our wild idea had worked.
  • It was clear from that point on that a door had been opened and that they were going to be able to learn a lot about telomere function from studies in yeast.
  • Within a short time he was able to clone bona fide yeast telomeres, and (in a continuation of the collaboration with Liz Blackburn’s lab)
  • they obtained the critical sequence information that led (them) to propose the existence of the key enzyme, telomerase.

A fanciful depiction evoking both telomere dynamics and telomere researchers, done by the artist Julie Newdoll in 2008, elicits the idea of a telomere as an ancient Sumarian temple-like hive, tended by a swarm of ancient Sumarian Bee-goddesses against a background of clay tablets inscribed with DNA sequencing gel-like bands.
Dr. Blackburn recalls owing much to Barbara McClintock for her scientific findings, but also, Barbara McClintock also gave her advice in a conversation with her in 1977, during which

  • she had unexpected findings with the rDNA end sequences.
  • Dr. McClintock urged her to trust in intuition about the scientific research results.

This advice was surprising then because intuitive thinking was not something that she accepted to be a valid aspect of being a biology researcher.
MLA style: “Elizabeth H. Blackburn – Biographical”. Nobelprize.org. 5 Feb 2013. http://www.nobelprize.org/nobel_prizes/medicine/laureates/2009/blackburn.html


In this Part I of a series of 3, I have described the

  • emergence of Molecular Biology and
  • closely allied work on the mechanism of Cell Replication and
  • the dependence of metabolic processes on proteins and enzymatic conversions through a surge of
  • post WWII research that gave birth to centers for basic science research in biology and medicine in both US and in England, which was preceded by work in prewar Germany. This is to be followed by further developments related to the Human Genome Project.
  • Transcription initiation (Photo credit: Wikipedia)
  • Schematic relationship between biochemistry, genetics, and molecular biology (Photo credit: Wikipedia)
  • Central dogma of molecular biology (Photo credit: Wikipedia)


Transcription initiation

Transcription initiation (Photo credit: Wikipedia)

Schematic relationship between biochemistry, g...

Schematic relationship between biochemistry, genetics, and molecular biology (Photo credit: Wikipedia)

Central dogma of molecular biology

Central dogma of molecular biology (Photo credit: Wikipedia)








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BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair S Saha

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Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence A Lev-Ari

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition sjwilliams

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How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis. SJ Williams

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Mitochondrial Damage and Repair under Oxidative Stress lhb

Mitochondria: More than just the “powerhouse of the cell” Ritu Saxena

Mitochondrial mutation analysis might be “1-step” away Ritu Saxena

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Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression Analysis A Lev-Ari

Gastric Cancer: Whole-genome reconstruction and mutational signatures A Lev-Ari

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Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell(1)

Reporter, Writer: Stephen J. Williams, Ph.D.

Most tumors exhibit a level of diversity, at the cellular, histologic, and even genetic level (2).  This genetic heterogeneity within a tumor has been a focus of recent research efforts to analyze the characteristics, expression patterns, and genetic differences between individual tumor cells.  This genetic diversity is usually manifested as single nucleotide variations (SNV) and copy number variations (CNV), both of which provide selection pressures in both cancer and evolution.

As cancer research and personalized medicine is focused on analyzing this tumor heterogeneity it has become pertinent view the tumor as a heterogeneous population of cells instead of as a homogenous mass.  In, fact, studies have suggested that cancer cell lines growing on plastic in culture, even though thought of as clonogenic, can actually display a varied degree of expression differences between neighboring cells growing on the same dish.  Indeed, cancer stem cells show an asynchronous cell division, for example a parent CD133-positive cell will divide into a CD133-positive and a CD133-negative cell(3). In addition, the discovery that circulating tumor cells (a rare population of circulating cells in the blood) can be prognostic of outcome in cancer such as inflammatory breast cancer(4), it is ever more important to develop methods to analyze single cell populations.

Harvard University researchers, Dr. Chenghang Zong, Sijia Lu, Alec Chapman and Sunney Xie developed a new amplification method utilizing multiple annealing and looping-based amplification cycles (MALBAC)(1).   A quasilinear preamplification process is used on pictograms of DNA genomic fragments (form 10 to 100 kb) isolated from a single cell.   This is performed to reduce the bias associated with nonlinear DNA amplification.  A series of random primers (which the authors termed MALBAC primers, constructed with a common sequence tags) are annealed at low temperature (0 °C). PCR rounds produce semiamplicons.  Further rounds of amplification, after a step of looping the amplicons, result in full amplicons with complementary ends.  When the two ends hybridize to form the looped DNA, this prevents use of this loop structure as a template, therefore leading to a close-to–linear amplification.    The process allows for a higher fidelity of DNA replication and the ability to amplify a whole genome.  The amplicons are then sequenced either by whole-genome sequencing methods using Sanger-sequencing to verify any single nucleotide polymorphisms.  This procedure of MALBAC-amplification resulted in coverage of 85-93% of the genome of a single cell.

As proof of principle, the authors used MALBAC to amplify the DNA of single SW480 cancer cells (picked from a clonally expanded population of a heterogeneous population (the bulk DNA).  Comparison of the MALBAC method versus the MDA method revealed copy number variations (CNV) between three individual cells, which had been picked from the clonally expanded pool. Their results were in agreement with karyotyping studies on the SW480 cell line.  Meticulous quality controls were performed to limit contamination, high false positive rates of SNV detection due to amplification bias, and false positives due to amplification or sequencing errors.

Interestingly, the authors found 35 unique single nucleotide variations which h had occurred from 20 cell divisions from a single SW480 cancer cell.  This resulted in an estimated 49 mutations which occurred in 20 generations, yielding a mutation rate of 2.5 nucleotides per generation.  In addition, the authors were able to map some of these mutations on various chromosomes and perform next-gen sequencing (deep sequencing) to verify the nucleotide mutations and found an unusually high purine-pyrimidine exchange rate.

In a subsequent paper, investigators from the same group at Harvard used this technology to sequence 99 sperm cells from a single individual to study genetic diversity created during meiotic recombination, a mechanism involved in evolution and development(5).


1.            Zong, C., Lu, S., Chapman, A. R., and Xie, X. S. (2012) Science 338, 1622-1626

2.            Cooke, S. L., Temple, J., Macarthur, S., Zahra, M. A., Tan, L. T., Crawford, R. A., Ng, C. K., Jimenez-Linan, M., Sala, E., and Brenton, J. D. (2011) British journal of cancer 104, 361-368

3.            Guo, R., Wu, Q., Liu, F., and Wang, Y. (2011) Oncology reports 25, 141-146

4.            Giuliano, M., Giordano, A., Jackson, S., Hess, K. R., De Giorgi, U., Mego, M., Handy, B. C., Ueno, N. T., Alvarez, R. H., De Laurentiis, M., De Placido, S., Valero, V., Hortobagyi, G. N., Reuben, J. M., and Cristofanilli, M. (2011) Breast cancer research : BCR 13, R67

5.            Lu, S., Zong, C., Fan, W., Yang, M., Li, J., Chapman, A. R., Zhu, P., Hu, X., Xu, L., Yan, L., Bai, F., Qiao, J., Tang, F., Li, R., and Xie, X. S. (2012) Science 338, 1627-1630

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Synthetic Biology

This collection aims to highlight PLOS ONE‘s role in the emerging interdisciplinary field of synthetic biology. The collection has its roots in PLOS ONE‘s very first issue, which included two publications from the field and since then, the number of synthetic biology articles published by the journal has grown steadily. As the field continues to develop, this collection will be updated to include new publications, thereby tracking the evolution of this dynamic research area.

Synthetic biology occurs at the intersection of a number of traditional disciplines, including biology, chemistry, and engineering. It aims to create biological systems that can be programmed to do useful things such as producing drugs and biofuel. The interdisciplinary nature of synthetic biology can make it difficult to publish in traditional journals. PLOS ONE‘s broad scope, however, allows for the publication of work crossing many traditional research boundaries, making it an ideal venue for many different types of synthetic biology publications. In addition, the journal’s focus on rigorous peer review without considering impact has made it possible to publish a body of articles that truly reflects the multifaceted nature of this research area.

One overarching theme of synthetic biology is standardization, which can only be achieved through concerted community effort. To this end, each article published in PLOS ONE can be the start of a lively conversation. The ability to comment on articles provides the community with a means to engage in a dialogue focused on specific articles, and the “Share this Article” feature allows readers to quickly send an article they find interesting to their entire networks, because all the content is openly accessible.

Articles in the Synthetic Biology Collection are presented in order of publication date and new articles will be added as they are published. PLOS ONE welcomes submissions in this field.

Collection Citation: Synthetic Biology (2012) PLOS Collections:http://www.ploscollections.org/syntheticbiology

Image Credit: Ivan Morozov (Virginia Bioinformatics Institute)



PLOS ONE Launches Synthetic Biology Collection

By Rachel Bernstein
Posted: August 15, 2012

Today PLOS ONE is happy to announce the launch of the Synthetic Biology Collection, including over 50 papers published in the last six years that illustrate the many facets of this dynamically evolving research area.

Synthetic biology is an innovative emerging field that exists at the intersection of many traditional disciplines, including biology, chemistry, and engineering, with aims to create biological systems that can be programmed to do useful things like produce drugs or biofuels, among other applications. Despite its potential, the heavily interdisciplinary nature of the research can make it difficult to publish in traditional discipline-specific journals.

However, PLOS ONE’s broad scope allows for the publication of work crossing many traditional research boundaries, making it an ideal venue for many different types of synthetic biology research. For example, the papers in the collection cover topics including DNA synthesis and assembly, standardized biological “parts” akin to interchangeable mechanical parts, protein engineering, and complex network and pathway analysis and modeling, as described in theCollection Overview written by collection editors Jean Peccoud of Virginia Tech and Mark Isalan of the Centre for Genomic Regulation.

The Collection has roots in PLOS ONE’s very first issue, which included two publications from the field. Since then, the number of synthetic biology articles published in the journal has grown steadily. The collection launched today highlights selected synthetic biology articles published in PLOS ONE since 2006, and it is intended to be a growing resource that will be updated regularly with new papers as the field continues to grow and develop.

Collection Citation: Synthetic Biology (2012) PLOS Collections:http://www.ploscollections.org/syntheticbiology

Image Credit: Ivan Morozov (Virginia Bioinformatics Institute)



The PLOS ONE Synthetic Biology Collection: Six Years and Counting

Jean Peccoud, Mark Isalan

Published 15 Aug 2012 | info:doi/10.1371/journal.pone.0043231

The PLOS ONE Synthetic Biology Collection: Six Years and Counting 

Jean Peccoud1,2*, Mark Isalan3

1 Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, United States of America, 2 Center for Systems Biology of Engineered Tissues, Institute for Critical Technologies and Applied Science, Virginia Tech, Blacksburg, Virginia, United States of America, 3 EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain


Since it was launched in 2006, PLOS ONE has published over fifty articles illustrating the many facets of the emerging field of synthetic biology. This article reviews these publications by organizing them into broad categories focused on DNA synthesis and assembly techniques, the development of libraries of biological parts, the use of synthetic biology in protein engineering applications, and the engineering of gene regulatory networks and metabolic pathways. Finally, we review articles that describe enabling technologies such as software and modeling, along with new instrumentation. In order to increase the visibility of this body of work, the papers have been assembled into the PLOS ONE Synthetic Biology Collection (www.ploscollections.org/synbio). Many of the innovative features of the PLOS ONE web site will help make this collection a resource that will support a lively dialogue between readers and authors of PLOS ONE synthetic biology papers. The content of the collection will be updated periodically by including relevant articles as they are published by the journal. Thus, we hope that this collection will continue to meet the publishing needs of the synthetic biology community.

Citation: Peccoud J, Isalan M (2012) The PLOS ONE Synthetic Biology Collection: Six Years and Counting. PLoS ONE 7(8): e43231. doi:10.1371/journal.pone.0043231

Editor: Wei Ning Chen, Nanyang Technological University, Singapore


Received: May 23, 2012; Accepted: July 16, 2012; Published: August 15, 2012

Copyright: © 2012 Peccoud, Isalan. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: JP is supported by National Science Foundation Awards 0850100 and 0963988 and by grants R01-GM078989 and R01-GM095955 from the National Institutes of Health. MI is funded by FP7 ERC 201249 ZINC-HUBS, Ministerio de Ciencia e Innovacion grant MICINN BFU2010-17953 and the MEC-EMBL agreement. The funders had no role in the preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: peccoud@vt.edu


Synthetic biology is an emerging transdisciplinary field at the intersection between many engineering and scientific disciplines such as biology, chemical engineering, chemistry, electrical engineering, or computer science. The scientific milestone that inspired the development of synthetic biology is often regarded as the description of two artificial gene networks in the same issue of Nature in 2000 [1][2]. However, the year 2004 marks the emergence of synthetic biology as a scientific community. This is the year of the first synthetic biology conference, the first iGEM competition –where students compete to build biological systems (http://igem.org/) ― and the creation of the synthetic biology page on Wikipedia. Two years later, the first issue of PLOS ONE included two synthetic biology articles [3][4], marking the beginning of a trend. Since then, PLOS ONE has published a large number of articles covering all aspects of the field. Synthetic biologists resolutely push the limits of their specialties in ways that few established journals have been able to appreciate. Since the result is often more “how to build something that works” rather than primary biological insight, the papers can be hard to place in classical journals. Many synthetic biology authors have benefited from the innovative PLOS ONE editorial policy to publish scientifically sound research, irrespective of its anticipated significance.

The purpose of this article is to introduce the PLOS ONE Synthetic Biology Collection (www.ploscollections.org/synbio/). The collection highlights selected synthetic biology articles published in PLOS ONE since 2006, putting them together in one place for easy perusal. The website is intended to be a growing resource that will be updated regularly.

We review the collection here by organizing it into some broad categories: DNA synthesis and assembly, Biological parts, Protein engineering, Networks and pathways, Synthetic life, Software and modeling, and Instruments. The classification is our own; since many synthetic biology papers cited in this review span more than one category, it was sometimes difficult to assign them to one category rather than another. Nonetheless, this structure should aid in navigating the 50+ papers currently included in the collection.

Summary of Papers Included in the Collection 

DNA Synthesis and Assembly

Synthetic biology projects often begin with the assembly of complicated, multi-component gene constructs. Therefore, both DNA assembly and cloning technologies are critical enablers of synthetic biology. Not surprisingly, many recent PLOS ONE papers propose methods to improve the efficiency of the fabrication step of synthetic biology projects. For example, Golden Gate Cloning [5] is a one-step DNA assembly protocol that can join at least nine distinct DNA fragments into one plasmid vector. The technique employs type IIs restriction enzymes that cut DNA at some distance from their cognate DNA-binding site, thus allowing flexibility and uniqueness in the compatible sticky ends that are generated. A related technique is GoldenBraid Assembly [6], that also uses type IIs restriction enzymes, but applies them iteratively to standardized DNA parts (see the ‘Biological parts’ section below). This allows the indefinite growth of reusable gene modules. Similarly, type IIs restriction enzymes have been used to make a hierarchical modular cloning system aimed at making eukaryotic multigene constructs [7].

‘One-pot’ assembly and cloning systems are being developed by many groups, and the ideal systems use as few standardized components as possible. Circular polymerase extension cloning (CPEC) fits into this category, using a single polymerase to assemble and clone multiple inserts with any vector, in a one-step in vitro reaction [8]. Alternatively, successive hybridization assembling (SHA) also employs a single reaction in vitro [9].

As well as cloning one desired multi-component construct, many projects require degenerate cloning or mutagenesis to make combinatorial libraries of gene variants. The OmniChange technique, which simultaneously saturates five independent codons, has therefore been developed to generate full-length gene libraries with 5 degenerate NNK-codons while avoiding PCR-amplification [10]. Large libraries of genetic sequences can be derived from oligonucleotides synthetized in a microarray, and later pooled in libraries from which more complex sequences can be derived [11]. By combining linear DNA amplification and PCR, DNA libraries with hundreds to thousands of members can be synthesized.

PCR methods themselves can have certain limitations, such as difficulties in amplifying GC-rich DNA targets. One study optimized polymerase chain assembly (PCA) and ligase chain reaction (LCR) methods for the construction of two GC-rich gene fragments implicated in tumorigenesis, IGF2R and BRAF [12]. They found that LCR was superior and benefited from the addition of DMSO and betaine.

The many synthesis and assembly methods presented in the collection can be combined to streamline the fabrication steps of synthetic biology projects, by producing collections of standardized biological parts. Standard parts are themselves a distinctive feature of synthetic biology, as reviewed below.

Biological Parts

The Registry of Standard Biological Parts (www.partsregistry.org), based on the original vision of Tom Knight, is providing a rich collection of components for synthetic biology projects. Several articles in the PLOS ONE collection reflect the importance of this resource. For example, a global analysis of the Registry clone collection [13] helped identify certain discrepancies between the sequences recorded in the database and the physical sequences of some clones in the collection. These results prompted a change in the quality control of the submissions to the Registry that has greatly improved the overall quality of the collection. Moreover, the analysis of parts usage patterns led to organizational guidelines that may help design and manage these new types of scientific resources. As most parts in the registry are for prokaryotes, a eukaryotic collection of 52 parts was developed and is available for distribution[14]. This includes multiple cloning sites (MCS), common protein tags, protein reporters and selection markers, amongst others. Furthermore, most of the parts were designed in a format to allow fusions that maintain the reading frame.

As well as standardized coding regions, synthetic biology projects require well-characterized promoters to achieve desired expression strengths. In one study, a single yeast promoter was mutated to make a fine-graded output range promoter library [15]. Transcription Activator-Like Orthogonal Repressors were then developed synthetically to control expression of these promoters in an orthogonal manner. Such orthogonality or ‘non-cross-reactivity’ is necessary for engineering larger synthetic gene circuits that do not interfere with the physiology of the biological chassis in which they operate. Mammalian synthetic promoters have also been developed by analyzing motifs found in highly active human promoters. Thus, by modulating the amount of sequences rich in GC and CpGs, custom designed promoters were obtained [16].

Finally, entirely de novo parts that are found nowhere in nature have been engineered to slot into biological systems. Using E. coli lacking conditionally essential genes, entirely new functional proteins were obtained from scaffolds of randomized 4-helix bundles, rescuing stalled growth [17]. Similarly, a synthetic ATP-binding protein, evolved entirely from non-natural sequences, was expressed in E. coli, altering the levels of intracellular ATP [18]. Protein engineering approaches are thus a potential source of many new parts, as well as forming a branch of synthetic biology in their own right.

Protein Engineering

Protein engineering can take many forms, from directed evolution methods to protein design. The PLOS ONE Synthetic Biology Collection includes a wide range of studies in this broad field.

Phage display is one of the classic tools of protein engineering, allowing combinatorial libraries of randomized proteins to be selected from the surface of bacteriophages. Phage display was used to generate a new class of binding proteins targeted to the pointed-end of actin [19]. These proteins, called synthetic antigen binders (sABs), were based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a new “reduced genetic code” phage display library.

An example of targeted protein design was the design of a dual reporter, Gemini [20]. Here, β-galactosidase (β-gal) α-fragment was fused to GFP, resulting in increased β-gal activity and some decrease in GFP sensitivity. GFP was also modified in a study where the ten proline residues of enhanced green fluorescent protein (EGFP) were replaced by (4R)- and (4S)-fluoroprolines (FPro) [21]. In this way, protein folding and stability could be tuned.

A promising advance in the field of engineering custom sequence-specific DNA-binding proteins is the use of Transcription Activator-Like (TAL) proteins. Modular TAL units specify A, C, G or T and can be concatenated to make long designer DNA-binding domains. Thus, Golden TAL Technology [22] has adapted Golden Gate Cloning [5] for engineering new TAL proteins. These were shown to function in human and plant cells and to target activation of both exogenous and endogenous genes, after fusion with a VP16 activation domain.

As well as single proteins, entire pathways can nowadays be engineered. Computational redesign was used to create new periplasmic binding proteins in plants, to act as biosensors in combination with a histidine kinase signaling cascade [23]. This resulted in transcription factor activation and ‘de-greening’ of plants in response to small-molecule stimuli. As can be seen from this example and the ones below, the move from single protein engineering to network engineering is one of the main driving forces in synthetic biology.

Networks and Pathways

One of the first, and now most-cited, synthetic biology papers in PLOS ONE was the study on fitness-induced attractor selection [3]. Here, a synthetic mutual inhibition gene network was built in E. coli, with two states, green (GFP) and red (RFP), that were mutually exclusive. By attaching a fitness pressure to one of the states (i.e. a gene required for growth in the absence of glutamine), the authors demonstrated that the cells switched stochastically into the fittest state, restoring growth. In other words, by changing to a glutamine-free medium, the red cells switched to green, even in the absence of formal signaling machinery. This work has important messages for potential new mechanisms in gene regulation, where underlying fitness pressures can ultimately determine how much a gene is expressed, simply according to need.

Other small bacterial networks have been built to include a heritable sequential memory switch, using the fim and hin inversion recombination systems [24], and an E. coli strain for use as a ‘chemical recording device’ [25]. In the latter, the authors created a synthetic chemically sensitive genetic toggle switch to activate appropriate fluorescent protein indicators (GFP, RFP) and along with a cell division inhibitor (minC). Moving to yeast, one example of network engineering was the reconstruction of a human p53-Mdm2 negative feedback module in S. cerevisiae [26]. In this example, many aspects of p53 regulation in mammals were maintained, such as Mdm2-dependent targeting of p53 for degradation, sumoylation at lysine 386 and further regulation of this process by p14ARF. In mammalian systems, a synthetic tetracycline regulator positive feedback loop was stably integrated and yielded a bimodal expression response because such cells can only be “OFF” or “ON” [27].

One unusual work in synthetic biology aimed to rewire and control cell shape in yeast, by changing the inputs into the α-factor pathway [28]. This pathway can give rise to multiple mating projections, upon prolonged activation. The authors tested genetic manipulations that ultimately gave rise to single or multiple projections, in the absence of the natural input, α-factor.

A group of papers in the collection explore ‘synthetic ecology’, where consortia of different cells interact to give patterns at a population level. For example, by engineering two strains of E. coli, one study was able to achieve synthetic biofilms with spatial self-organization [29]. The consortia achieved defined layered structures and had unexpected growth advantages. A second paper describes a systems composed of two quorum-sensing signal transduction circuits that allowed the authors to build a synthetic ecosystem where the population dynamics could be tuned by varying the environmental signals [30]. Third, quorum components were also used in a study which generated robust but unexpected oscillations in E. coli by building synthetic suicide circuits [31]. In fact, the quorum components proved to be unnecessary to achieve oscillations: there was a density-dependent plasmid amplification that gave rise to population-level negative feedback, ultimately resulting in the cycles. As in other areas of synthetic biology, the process of building systems often leads to surprises which can result in useful new engineering tools, or to a better understanding of the underlying biological processes [32].

Pathway engineering for the production of useful chemical or product synthesis is a major field within synthetic biology. For example, an engineered yeast that efficiently secretes penicillin was built by transplanting synthesis pathway components into a host that is more suited for pharmaceutical production [33]. Artemisinin derivatives are key components of malaria therapies and their synthesis is a high-profile goal of synthetic biology because extraction from slow-growing plants currently limits supply. Consequently, one study achieved high-level production of an artemisinin precursor in E. coli[34]. Another striking synthesis paper demonstrates a synthetic enzymatic pathway consisting of 13 enzymes for high-yield hydrogen production from starch and water [35]. Building such large systems is extremely challenging; as a result, these articles have received a lot of attention.

Synthetic Life

Synthetic life is among the most controversial of synthetic biology aims, and has received a lot of attention, even in the mainstream press. Public concerns of possible biological threats resulting from the misuse of these technologies prompted the development of new biosecurity policies [36].

One branch of this field is the de novo chemical synthesis and assembly of whole plasmids, viruses and genomes which are then transplanted into host cells. The pX1.0 plasmid is an example of a fully chemically-synthesized plasmid designed by calculating consensus sequences from 8 plasmids [37], while removing genes involved in antibiotic resistance and virulence. The plasmid not only replicated inE. coli, but could also self-transfer by conjugation into two other enterobacter species. A chemical synthesis approach was also used to construct whole genomes of bacteriophage G4 (around 10 kilobases in length), resulting in infectious viruses that could pass from one strain of E. coli to another[38].

One group has the ambitious long-term aim of building a synthetic chloroplast, and has begun by transplanting photosynthetic bacteria into eukaryotic cells to see whether they can achieve synthetic symbiosis [39]. Remarkably, the authors showed that some cyanobacteria were relatively harmless in zebrafish embryos, compared to E. coli. Furthermore, by engineering invasins into the cyanobacteria, they were able to invade and divide inside mammalian macrophages. Synthetic biology is only limited by our imagination, and one can speculate that entire free-living synthetic lifeforms could find their place in the collection in the not-too-distant future.

Software and Modeling

As the number of biological parts for synthetic biology increases, databases and design methods must evolve. For example, to help researchers search and retrieve biological parts, the Knowledgebase of Standard Biological Parts (SBPkb) is a Semantic Web resource for synthetic biology [40].

The collection also includes two articles presenting Computer Assisted Design software tools. Eugene is a human readable language to specify synthetic biological designs based on biological parts. It also provides a very expressive constraint system to drive the automatic creation of composite parts or devices from a collection of individual parts [41]. Alternatively, the Proto platform also provides a high-level biologically-oriented programming language [42]. Specifications are compiled from regulatory motifs, optimized, then converted into computational simulations for numerical verification.

Ultimately the design tools are only as good as the underlying mathematical models they rely on to make predictions of design behaviors. The collection includes a number of articles applying mathematical modeling approaches rooted in various engineering specialties to the design of synthetic genetic constructs.

Modeling gene networks is at the interface of systems and synthetic biology, and many PLOS ONE modeling papers aim to guide bioengineering projects. A recent example of adapting modeling for re-engineering properties into a system used a standardized synthetic yeast network from the In-vivo Reverse-engineering and Modeling Assessment (IRMA) [43]. Reverse engineering itself was used in a study which ultimately provided guidelines for chemotaxis pathway redesign [44]. Statecharts are used to describe dynamical systems, but have not been applied to gene networks. By doing so explicitly, one study was able to model network motifs and combine them in a complicated interlocked feed-forward loop network [45].

Two-component systems are common regulatory motifs in bacteria, and comprise a kinase that senses environmental signals together with a regulator that mediates the cell response. A recent study asked the question, “what happens if you add a third component that interacts with either of the other two?”[46]. Estimating the parameter space associated with a particular function is very valuable for guiding synthetic engineering approaches, as is determining whether a function is theoretically possible at all. For example, using a geometric argument, it was shown that, surprisingly, even monomer regulators can achieve bistability. This demonstrates the possibility of switch-like behavior in feedback autoloops without resorting to multimer regulators [47].

thumbnailFigure 1. Historical distribution of synthetic biology articles published by PLOS ONE.

This figure reports the number of articles in the collection published between 2006 and 2011. It shows a rapid growth of synthetic biology that reflects the growth of the journal and the increased familiarity of synthetic biologists with PLOS ONE.


By combining experiments and computation, one study was able to derive design algorithms for altering synonymous codons in proteins, resulting in drastic expression differences of the same protein sequence[48]. For example, with DNA polymerase and single chain antibodies, expression could be predictably tuned to obtain concentrations ranging from undetectable to 30% of cellular protein. Importantly, using partial least squares regression, the authors noticed that favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. This is an important discovery for building genetic constructs that express appropriately inside the target cells.

Computation is a key function of biological networks and several studies in the collection present schemes to achieve this. The first is implemented at the level of chemical reactions and describes functions such as an inverter, an incrementer, a decrementer, a copier, a comparator, a multiplier, an exponentiator, a raise-to-a-power operation, and a logarithm in base two [49]. A key simplification is that the scheme uses only two reaction rates (“fast” and “slow”). A second study models a synthetic gene network to perform frequency multiplication [50]. Both of these studies assume deterministic relationships between input and outputs. Recently, the deterministic assumption has been challenged by experimental and theoretical works analyzing the importance of noise in the dynamics of gene networks [51]. This trend is illustrated in the collection by an article demonstrating that reliable timing of decision-making processes (choosing between multistable states) can be accomplished for large enough population sizes, as long as cells are globally coupled by chemical means [52]. Modeling can often reveal subtle non-intuitive designs, and, as a means of guiding synthetic biology, is likely to become an even larger field in the future.

thumbnailFigure 2. Relationships between article-level metrics.

For articles published between 2006 and 2009, there is a positive correlation between the number of times an article is cited in the scientific literature and the number of times it is viewed (A). For articles published between 2010 and 2012, there is a positive relationship between the number of views and the number of citations in the Mendeley social network (B). Metrics, such as number of views and citations in social media, give readers and authors an estimate of the scientific impact of individual articles well before they receive citations in scientific literature.



Nowadays, new technology and machinery is an important driving force for both primary biological discovery and for synthetic biology. A neat example is provided by the use of inkjet printer technology to provide low-cost high-resolution tools; a bacterial piezoelectric inkjet printer was designed to print out different strains of bacteria or chemicals in small droplets onto a flat surface at high resolution [53]. Another group used an inkjet for continuous dosing of diffusible regulators to a gel culture of E. coli, allowing 2D spatiotemporal regulation [54]. Precise spatiotemporal control of cells can also be achieved with microfluidics, and a recent report grew dividing yeast cells in a remarkable planar array [55]. Transient pulses of gene expression could be triggered by briefly inducing the GAL1 or MET3 promoters, resulting in coherent induction of cell division across the cell cluster. Other novel culture systems presented in the collection include the development of a 3-D cell culture system using a designer peptide nanofiber scaffold that self-assembled [4]. The peptide could be linked to functional motifs for cell adhesion, differentiation, and bone marrow homing for use with mouse adult neural stem cells.

The Synthetic Biology Collection: A Dynamic Community Resource Top

It is remarkable that the collection includes several articles originating from engineers and computer scientists who traditionally publish their work in conference proceedings rather than the journals available to life-scientists. PLOS ONE’s indifference to subject matter made it possible to publish an unprecedented body of articles that reflects the multi-faceted nature of synthetic biology. No less remarkable is the observation that PLOS ONE published several articles originating from iGEM projects[13][41][56].

Since 2006, the number of synthetic biology articles published by the journal has been growing steadily (Figure 1). This evolution is consistent with the social trends in synthetic biology that have been mapped in an interesting bibliometric analysis included in the collection [57]. This is an indication that the synthetic biology community is becoming more aware of the services provided by the journal. Looking forward, the collection will make it easier to identify synthetic biology articles among the quickly growing volume of articles published by the journal each day. The content of the collection will be updated periodically as new synthetic biology articles are published by the journal.

Although Journal Impact Factors are a widely-discredited form of evaluating the quality of individual papers, all too often they are still used. Thus, it is imperative to find a better alternative. One of the most exciting features of the PLOS ONE web site is the Metrics tab, displaying article-based metrics that can be used to assess the impact of individual articles. These metrics naturally include traditional indicators, such as the number of citations. The two articles of the collection published in 2006 have been cited 70 and 84 times so far. Almost all the articles published in 2007 and 2008 have received more than 10 citations. The lag between the publication of an article and its citation by others is well known. Fortunately, the Metrics tab also includes more innovative indicators that give the authors and readers alike a real-time estimate of the ‘impact’ of an article. The number of times an article is viewed is an important indicator. Since PLOS ONE is an online journal, all readers view articles online in one way or another. As a result, we hypothesized that the number of times an article was viewed should be a good predictor of the number of citations it will receive. Using data reported in Table S1, we analyzed the relationship between views and citation numbers for articles included in the collection that were published between 2006 and 2009. Figure 2 shows that there is a positive correlation between the two metrics. That relationship does not hold when including more recent articles because of a difference in timing between viewing and citing activities. Articles typically receive a substantial number of views in the first few months after publication, but it takes a few years before they are cited. The 20 articles of the collection published in 2011 have recorded a lot of views, but have not had the time to be cited in the literature yet.

A non-conventional form of citations displayed in the Metrics tab is the number of times an article is bookmarked in social media. We have reported the Mendeley (www.mendeley.com) data in Table S1.Figure 2 shows that there is a positive relationship between the number of views and the number of times articles are bookmarked in this network, at least for the most recent articles of the collection. Older articles are under-represented in Mendeley because this network was not available at the time these articles were published. It will be interesting to see if citations of the collection articles in social media will be a better predictor of citations in the scientific literature than the number of views.

One overarching theme of synthetic biology is standardization [58][59], which can only be achieved through concerted efforts by members of the community. The field has therefore been deeply influenced by the development of resources such as the Registry of Standard Biological Parts (www.partsregistry.org ). More recently, the development of SBOL, the Open Language for Synthetic Biology (www.sbolstandard.org) illustrates the need to agree on data formats suitable to the development of software tool chains necessary to support experimental efforts. Each article published in PLOS ONE can be the start of a lively conversation. The journal web site provides authors and readers alike with a detailed vision of community connections. The “Share this article” feature allows readers to quickly send an article they find interesting to their networks. The comments tab of the articles provides the community with means to engage in a dialogue focused on specific articles [5][35][48][55]. This feature can also be used by authors to provide updated information about the work presented in the article [13].

When working at its best, science should be an active conversation that keeps refining ideas. We believe that PLOS ONE provides the ideal venue to achieve this, and we hope that the collection will inspire further progress in synthetic biology. Ultimately, we hope that having a clear repository in PLOS ONE should further increase its attractiveness as a home for publishing synthetic biology.

Table S1.

Article-level statistics for the Synthetic Biology Collection.


Author Contributions

Wrote the paper: JP MI.

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A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

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Published 14 May 2012 | info:doi/10.1371/journal.pone.0036982

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Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

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Two Component Systems: Physiological Effect of a Third Component

Baldiri Salvado, Ester Vilaprinyo, Hiren Karathia, Albert Sorribas, Rui Alves

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In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

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Chemical Synthesis of Bacteriophage G4

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OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons

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Microarray Generation of Thousand-Member Oligonucleotide Libraries

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A Modular Cloning System for Standardized Assembly of Multigene Constructs

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A Multi-Functional Synthetic Gene Network: A Frequency Multiplier, Oscillator and Switch

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Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium

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Programmable Ligand Detection System in Plants through a Synthetic Signal Transduction Pathway

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De Novo Designed Proteins from a Library of Artificial Sequences Function inEscherichia Coli and Enable Cell Growth

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Oscillations by Minimal Bacterial Suicide Circuits Reveal Hidden Facets of Host-Circuit Physiology

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DMSO and Betaine Greatly Improve Amplification of GC-Rich Constructs in De Novo Synthesis

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An Environment-Sensitive Synthetic Microbial Ecosystem

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Reverse Engineering of Bacterial Chemotaxis Pathway via Frequency Domain Analysis

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How to Turn a Genetic Circuit into a Synthetic Tunable Oscillator, or a Bistable Switch

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Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression

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A Man-Made ATP-Binding Protein Evolved Independent of Nature Causes Abnormal Growth in Bacterial Cells

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A Modified Consumer Inkjet for Spatiotemporal Control of Gene Expression

Daniel J. Cohen, Roberto C. Morfino, Michel M. Maharbiz

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Design Parameters to Control Synthetic Gene Expression in Escherichia coli

Mark Welch, Sridhar Govindarajan, Jon E. Ness, Alan Villalobos, Austin Gurney, Jeremy Minshull, Claes Gustafsson

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Synthetic Morphology Using Alternative Inputs

Hiromasa Tanaka, Tau-Mu Yi

Published 10 Sep 2009 | info:doi/10.1371/journal.pone.0006946

Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways

Jiayuan Quan, Jingdong Tian

Published 30 Jul 2009 | info:doi/10.1371/journal.pone.0006441

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet

Published 14 May 2009 | info:doi/10.1371/journal.pone.0005553

Monomeric Bistability and the Role of Autoloops in Gene Regulation

Stefanie Widder, Javier Macía, Ricard Solé

Published 30 Apr 2009 | info:doi/10.1371/journal.pone.0005399

Timing Cellular Decision Making Under Noise via Cell–Cell Communication

Aneta Koseska, Alexey Zaikin, Jürgen Kurths, Jordi García-Ojalvo

Published 13 Mar 2009 | info:doi/10.1371/journal.pone.0004872

High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli

Hiroko Tsuruta, Christopher J. Paddon, Diana Eng, Jacob R. Lenihan, Tizita Horning, Larry C. Anthony, Rika Regentin, Jay D. Keasling, Neil S. Renninger, Jack D. Newman

Published 16 Feb 2009 | info:doi/10.1371/journal.pone.0004489

Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory

Timothy S. Ham, Sung K. Lee, Jay D. Keasling, Adam P. Arkin

Published 30 Jul 2008 | info:doi/10.1371/journal.pone.0002815

Targeted Development of Registries of Biological Parts

Jean Peccoud, Megan F. Blauvelt, Yizhi Cai, Kristal L. Cooper, Oswald Crasta, Emily C. DeLalla, Clive Evans, Otto Folkerts, Blair M. Lyons, Shrinivasrao P. Mane, Rebecca Shelton, Matthew A. Sweede, Sally A. Waldon

Published 16 Jul 2008 | info:doi/10.1371/journal.pone.0002671

Bimodal and Hysteretic Expression in Mammalian Cells from a Synthetic Gene Circuit

Tobias May, Lee Eccleston, Sabrina Herrmann, Hansjörg Hauser, Jorge Goncalves, Dagmar Wirth

Published 04 Jun 2008 | info:doi/10.1371/journal.pone.0002372

Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

Thomas Steiner, Petra Hess, Jae Hyun Bae, Birgit Wiltschi, Luis Moroder, Nediljko Budisa

Published 27 Feb 2008 | info:doi/10.1371/journal.pone.0001680

Reconstitution of Mdm2-Dependent Post-Translational Modifications of p53 in Yeast

Barbara Di Ventura, Charlotta Funaya, Claude Antony, Michael Knop, Luis Serrano

Published 30 Jan 2008 | info:doi/10.1371/journal.pone.0001507

A Microfluidic Device for Temporally Controlled Gene Expression and Long-Term Fluorescent Imaging in Unperturbed Dividing Yeast Cells

Gilles Charvin, Frederick R. Cross, Eric D. Siggia

Published 23 Jan 2008 | info:doi/10.1371/journal.pone.0001468

Printing Multistrain Bacterial Patterns with a Piezoelectric Inkjet Printer

Jack Merrin, Stanislas Leibler, John S. Chuang

Published 25 Jul 2007 | info:doi/10.1371/journal.pone.0000663

High-Yield Hydrogen Production from Starch and Water by a Synthetic Enzymatic Pathway

Y.-H. Percival Zhang, Barbara R. Evans, Jonathan R. Mielenz, Robert C. Hopkins, Michael W.W. Adams

Published 23 May 2007 | info:doi/10.1371/journal.pone.0000456

Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

Fabrizio Gelain, Daniele Bottai, Angleo Vescovi, Shuguang Zhang

Published 27 Dec 2006 | info:doi/10.1371/journal.pone.0000119

Adaptive Response of a Gene Network to Environmental Changes by Fitness-Induced Attractor Selection

Akiko Kashiwagi, Itaru Urabe, Kunihiko Kaneko, Tetsuya Yomo

Published 20 Dec 2006 | info:doi/10.1371/journal.pone.0000049


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