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A section of DNA; the sequence of the plate-like units (nucleotides) in the center carries information. (Photo credit: Wikipedia)
DNA (Photo credit: Allen Gathman)
Triplex DNA
1. A Third Strand for DNA
The DNA double helix can under certain conditions accommodate a third strand in its major groove. Researchers in the UK have now presented a complete set of
four variant nucleotides that makes it possible to use this phenomenon in gene regulation and mutagenesis.
Natural DNA only forms a triplex if the targeted strand is rich in purines – guanine (G) and adenine (A) – which in addition to the bonds of the Watson-Crick base pairing can form two further hydrogen bonds, and
the ‘third strand’ oligonucleotide has the matching sequence of pyrimidines – cytosine (C) and thymine (T).
Any Cs or Ts in the target strand of the duplex will only bind very weakly, as they contribute just one hydrogen bond. Moreover, the recognition of G requires the C in the probe strand to be protonated, so triplex formation will only work at low pH.
To overcome all these problems, the groups of Tom Brown and Keith Fox at the University of Southampton have developed modified building blocks, and have now completed a set of
four new nucleotides, each of which will bind to one DNA nucleotide from the major groove of the double helix.1
They tested the binding of a 19-mer of these designer nucleotides to a double helix target sequence in comparison with the corresponding triplex-forming oligonucleotide made from natural DNA bases. Using fluorescence-monitored thermal melting and DNase I footprinting, the researchers showed that their construct forms stable triplex even at neutral pH. Tests with mutated versions of the target sequence showed that
three of the novel nucleotides are highly selective for their target base pair,
while the ‘S’ nucleotide, designed to bind to T, also tolerates C.
In principle, triplex formation has already been demonstrated as a way of inducing mutations in cell cultures and animal experiments.2
Since the pioneering work of Felsenfeld, Davies, & Rich (1), double-stranded polynucleotides containing
purines in one strand
and pydmidines in the other strand [such as poly(A)/poly(U), poly(dA)/poly(dT), or poly(dAG)/poly(dCT)]
have been known to be able to undergo a stoichiometric transition forming a triple-stranded structure containing one polypurine and two polypyrimidine strands. Early on, it was assumed that the third
strand was located in the major groove and associated with the duplex via non-Watson-Crick interactions now known as Hoogsteen pairing.
Triple helices consisting of one pyrimidine and two purine strands were also proposed. However, notwithstanding the fact that single-base triads in tRNAs tructures were well-documented, triple-helical DNA escaped wide attention before the mid-1980s.
The considerable modern interest in DNA triplexes arose due to two partially independent developments.
First, homopurine-homopyrimidine stretches in supercoiled plasmids were found
to adopt an unusual DNA structure, called
H-DNA which includes a triplex as the major structural element.
Secondly, several groups demonstrated that homopyrimidine and some
purine-rich oligonucleotidescan form stable and sequence-specific complexes with
corresponding homopurine-homopyrimidine sites on duplex DNA. These
complexes were shown to be triplex structures rather than D-loops, where the
oligonucleotide invades the double helix and displaces one strand.
A characteristic feature of all these triplexes is that the two chemically homologous strands (both pyrimidine or both purine) are antiparallel. These findings led explosive growth in triplex studies.
One can easily imagine numerous “geometrical” ways to form a triplex, and those that have been studied experimentally. The canonical intermolecular triplex consists of either
three independent oligonucleotide chains or
of a long DNA duplex carrying homopurine-homopyrimidine insert
and the corresponding oligonucleotide.
Triplex formation strongly depends on the oligonucleotide(s) concentration. A single DNA chain may also fold into a triplex connected by two loops. To comply with the sequence and
polarity requirements for triplex formation, such a DNA strand must have a peculiar sequence: It contains
a mirror repeat (homopyrimidine for YR*Y triplexes and homopurine for YR*R triplexes)
flanked by a sequence complementary to one half of this repeat.
Such DNA sequences fold into triplex configuration much more readily than do the corresponding intermolecular triplexes, because all triplex forming segments are brought together within the same molecule.
It has become clear recently, however, that both sequence requirements and chain polarity rules for triplex formation can be met by DNA target sequences built
of clusters of purines and pyrimidines. The third strand consists of
adjacent homopurine and homopyrimidine blocks forming Hoogsteen hydrogen bonds with purines
on alternate strands of the target duplex, and this strand switch preserves the proper chain polarity.
These structures, called alternate-strand triplexes, have been
experimentally observed as both intra- and intermolecular triplexes. These results increase the number of potential targets for triplex formation in natural DNAs somewhat by
adding sequences composed of purine and pyrimidine clusters, although
arbitrary sequences are still not targetable because strand switching is energetically unfavorable.
REFERENCES
Lyamichev VI, Mirkin SM, Frank-Kamenetskii MD. J. Biomol. Stract. Dyn. 1986; 3:667-69. http://JbiomolStractDyn.com/Lyamichev_VI/ Mirkin SM, Lyamichev VI, Drushlyak KN, Dobrynin VN0 Filippov SA, Frank-Kamenetskii MD. Nature 1987; 330:495-97. http://Nature.com/ Demidov V, Frank-Kamenetskii MD, Egholm M, Buchardt O, Nielsen PE. Nucleic Acids Res. 1993; 21:2103-7. http://NucleicAcidsResearch.com/ Mirkin SMo Frank-Kamenetskii MD. Anna. Rev. Biophys. Biomol. Struct. 1994; 23:541-76. http://AnnRevBiophysBiomolecStructure.com/ Hoogsteen K. Acta Crystallogr. 1963; 16:907-16 http://ActaCrystallogr.com/ Malkov VA, Voloshin ON, Veselkov AG, Rostapshov VM, Jansen I, et al. Nucleic Acids Res. 1993; 21:105-11. http://NucleicAcidsResearch.com/ Malkov VA, Voloshin ON, Soyfer VN, Frank-Kamenetskii MD. Nucleic Acids Res. 1993; 21:585-91 Chemy DY, Belotserkovskii BP, Frank-Kamenetskii MD, Egholm M, Buchardt O, et al. Proc. Natl. Acad. Sci. USA 1993; 90:1667-70 http://PNAS.org/
Triplex forming oligonucleotides (TFOs) bind in the major groove of duplex DNA with a high specificity and affinity. Because of these characteristics, TFOs have been proposed as homing devices for genetic manipulation in vivo.
These investigators review work demonstrating the ability of TFOs and related molecules to
alter gene expression and
mediate gene modification in mammalian cells.
TFOs can mediate targeted gene knock out in mice,
providing a foundation for potential application of these molecules in human gene therapy.
4. Novagon DNA
John Allen Berger, founder of Novagon DNA and The Triplex Genetic Code Over the past 12+ years, Novagon DNA has amassed a vast array of empirical findings which challenge the “validity” of the “central dogma theory”, especially the current five nucleotide Watson-Crick DNA and RNA genetic codes. DNA = A1T1G1C1, RNA =A2U1G2C2. We propose that our new Novagon DNA 6 nucleotide Triplex Genetic Code has more validity than the existing 5 nucleotide (A1T1U1G1C1) Watson-Crick genetic codes. Our goal is to conduct a “world class” validation study to replicate and extend our findings.
Triplex DNA Structures
Maxim D. Frank-Kamenetskii, Sergei M. Mirkin
A DNA triplex is formed when pyrimidine or purine bases occupyt he major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. Intermolecular triplexes are formed
between triplex forming oligonucleotides (TFO) and target sequences on duplex DNAI.ntramolecular triplexes are the major elements of H-DNA usnusual DNA structures, which are formed in homopurine-homopyrimidine regions of supercoiled DNAs. TFOs are promising gene-drugs, which can be used in an anti-gene strategy, that attempt to modulate gene activity in vivo. Numerous chemical modifications of TFO are known. In peptide nucleic acid (PNA), the sugarphosphate backbone is replaced with a protein-like backbone. PNAs form P-loops while interacting with duplex DNA forming triplex with one of DNA strands leaving the other strand displaced. Very unusual recombination or parallel triplexes, or R-DNA have been assumed to form under RecA protein in the course of homologous recombination.
Perspectives and Summary
Since the pioneering work of Felsenfeld, Davies, & Rich (1), double-stranded polynucleotides containing purines in one strand and pydmidines in the other strand [such as poly(A)/poly(U), poly(dA)/poly(dT), or poly(dAG)/poly(dCT)] have been known to be able to undergo a stoichiometric transition forming a triple-stranded structure containing one polypurine and two polypyrimidine strands (2-4). Early on, it was assumedth at the third strand was located in the major groove and associated with the duplex via non-Watson-Crick interactions now known as Hoogsteenp airing. Triple helices consisting of one pyrimidine and two purine strands were also proposed( 5, 6). However notwithstanding the fact that single-base triads in tRNAs tructures were well-documented (reviewed in 7), triple-helical DNA escaped wide attention before the mid-1980s.
The considerable modem interest in DNA triplexes arose due to two partially independent developments. First, homopurine-homopyrimidine stretches in supercoiled plasmids were found to adopt an unusual DNA structure, called H-DNA which includes a triplex as the major structural element (8, 9). Secondly, several groups demonstrated that homopyrimidine and some purine-rich oligonucleotides can form stable and sequence-specific complexes with corresponding homopurine-homopyrimidine sites on duplex DNA(1 0-12). These complexes were shown to be triplex structures rather than D-loops, where the oligonucleotide invades the double helix and displaces one strand. A characteristic feature of all these triplexes is that the two chemically homologous strands (both pyrimidine or both purine) are antiparallel. These findings led explosive growth in triplex studies.
During the study of intermolecular triplexes, it became clear that triplex-forming oligonucleotides (TFOs) might be universal drugs that exhibit sequence-specific recognition of duplex DNA.This is an exciting possibility because, in contrast to other DNA-binding drugs, the recognition principle of TFOs is very simple: Hoogsteen pairing rules between a purine strand of the DNA duplex and the TFO bases. However this mode of recognition is limited in that homopurinehomopyrimidine sites are preferentially recognized. Though significant efforts have been directed toward overcoming this limitation, the problem is still unsolved in general. Nevertheless, the high specificity of TFO-DNA recognition has led to the development of an “antigene” strategy, the goal of which is to modulate gene activity in vivo using TFOs (reviewed in 13).
Although numerous obstacles must be overcome to reach the goal, none are likely to be fatal for the strategy. Even if DNA TFOs proved to be unsuitable as gene-drugs, there are already many synthetic analogs that also exhibit triplex-type recognition. Among them are oligonucleotides with non-natural bases capable of binding the duplex more strongly than can natural TFOs.
Another promising modification replaces the sugar-phosphate backbone of ordinary TFO with an uncharged peptidelike backbone, called a peptide nucleic acid (PNA) (reviewed in 14). Homopyrimidine PNAs form remarkably strong and sequence-specific complexes with the DNA duplex via an unusual strand displacement reaction: Two PNA molecules form a triplex with one of the DNA strands, leaving the other DNA strand displaced (a “P-loop”) (15, 16). The ease and sequence specificity with which duplex DNA and TFOs formed triplexes seemed to support the idea (17) that the homology search preceding homologous recombination might occur via a triplex between a single DNA strand and the DNA duplex without recourse to strand separation in the duplex.
However, these proposed recombination triplexes are dramatically different from the orthodox triplexes observed experimentally. First, the recombination triplexes must be formed for arbitrary sequences and, second, the two identical strands in this triplex are parallel rather than antiparallel. Some data supported the existence of a special class of recombination triplexes, at least within the complex among duplex DNA, RecA protein, and single-stranded DNA (reviewed in Ref. 18), called R-DNA. A stereochemical model of R-DNA was published (19). However the structure of the recombinationi ntermediate is far from being understood, and some recent data strongly favor the traditional model of homology search via local strand separation of the duplex and D-loop formation mediated by RecA protein. Intramolecular triplexes (H-DNA) are formed in vitro under superhelical stress in homopurine-homopyrimidinem irror repeats. The average negative supercoiling in the cell is not sufficient to induce H-DNA formation in most cases.
Annu.Rev.Biochem 1995. 64:65-95
Doubling down: four-stranded, ‘quadruple helix’ DNA discovered
Published January 21, 2013
Quadruplex DNA strands are seen at left, while fluorescent stains at right reveal their presence in human cell nuclei and chromosomes. (Jean-Paul Rodriguez and Giulia Biffi)
60 years after scientists first described the “double helix” shape of human DNA, the chemical code of life, scientists have discovered the first quadruple helix — and it may help them prevent the runaway cell proliferation at the root of cancer.
“It’s been sixty years since its structure was solved but work like this shows us that the story of DNA continues to twist and turn,” said Julie Sharp, senior science information manager at Cancer Research UK.
‘The story of DNA continues to twist and turn.’
– Julie Sharp, senior science information manager at Cancer Research UK
The research, published Monday in the science journal Nature Chemistry, shows clearly a four-stranded DNA structure that the scientists dubbed a “G-quadruplex.” The name comes from the building block guanine, one of the chemical bases that form DNA, along with adenine, cytosine, and thymine (usually abbreviated to their first letter).
By targeting these DNA oddities with synthetic molecules that trap and contain them — preventing cells from replicating their DNA and consequently blocking cell division — it may be possible to halt the spread of cancer, the researchers said.
“We are seeing links between trapping the quadruplexes with molecules and the ability to stop cells dividing, which is hugely exciting,” said professor Shankar Balasubramanian from the University of Cambridge’s Department of Chemistry and Cambridge Research Institute, whose group produced the research.
“We’ve come a long way in 10 years, from simple ideas to really seeing some substance in the existence and tractability of targeting these funny structures,” he told the BBC.
“I’m hoping now that the pharmaceutical companies will bring this on to their radar and we can perhaps take a more serious look at whether quadruplexes are indeed therapeutically viable targets.”
The ability to target specific sequences of DNA through oligonucleotide-based triple-helix formation provides a powerful tool for genetic manipulation. Under experimental conditions, triplex DNA can inhibit DNA transcription and replication, generate site-specific mutations, cleave DNA, and induce homologous recombination. This review describes the binding requirements for triplex formation, surveys recent advancements in the chemistry and biology of triple helices, and considers several potential applications of triplex DNA for use in genetic therapy.
A Gold Nanoparticle Based Approach for Screening Triplex DNA Binders
Min Su Han, Abigail K. R. Lytton-Jean, and Chad A. Mirkin*
The publisher’s final edited version of this article is available at J Am Chem Soc
Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change does not occur in the presence of duplex DNA binders and therefore provides a significantly better screening process for triplex DNA binding molecules compared to standard methods.
Regulating gene expression by controlling nucleic acid transcription is a potential strategy for the treatment of genetic-based diseases. A promising approach involves the use of triplex forming oligonucleotides (TFOs).1 Triple helix nucleic acids, or triplex structures, are formed through sequence specific Hoogsteen, or reverse Hoogsteen, hydrogen bond formation between a single-stranded TFO and purine bases in the major groove of a target duplex.2 Because TFOs can achieve sequence-specific recognition of genomic DNA, they can, in principle, be used to modulate gene expression by interfering with transcription factors that bind to DNA. However, at present, only purine-rich sequences can be targeted and the resultant triplex structure is less stable than the analogous duplex. This inherent instability has prompted research efforts to develop molecules that selectively bind to such triplex structures to stabilize the TFO-duplex complex. Potentially, triplex specific binding molecules could be used in conjunction with TFOs to achieve control of gene expression.3 Molecules identified as triplex binders include benzoindoloquinoline, benzopyridoquinoxaline, naphthyquinoline, acridine, and anthraquinone derivatives.4 In the past, typical screening processes for identifying triplex binders have included competitive dialysis, mass spectroscopy, electrophoresis and UV/Vis melting experiments, most of which are not applicable to high-throughput screening processes.5 However, with the development of combinatorial libraries which can produce large numbers of potential drug candidates, high-throughput screening strategies have become a necessary part of drug development.6
Systems Integrated Biomedical Research
Cutting a SWATH through Personalized Medicine
The Institute for Systems Biology (ISB) signed a multi-year agreement with AB Sciex to collaborate on the development of methods and technology in proteomics mass spectrometry with the goal of redefining biomarker research and complement genomics through quantitative proteomics analysis. The aim is to help advance the development of a new approach to medical care.
Led by ISB president and co-founder Leroy Hood, M.D., Ph.D., ISB’s research is being accelerated by SWATH™ Acquisition, a data-independent acquisition (DIA) mass spectrometry workflow that reportedly can quantify virtually all detectable peptides and proteins in a sample from a single analysis. ISB will be using the AB Sciex TripleTOF® 5600+ System and an Eksigent ekspert™ nano-LC 400 System as the instrument platforms on which to conduct the protein identification and quantitation. The TripleTOF 5600+ System can reportedly provide the high speed necessary for SWATH Acquisition. ISB also plans to use SelexION™ technology, a recent advancement in differential ion mobility, in the future to advance its research.
“SWATH is a game-changing technique that essentially acts as a protein microarray and is the most reproducible way to generate comprehensive quantitation of the entire proteome,” says Dr. Hood, “It generates a digital record of the entire proteome that can be mined retrospectively for years to come.”
ISB shall support the development of SWATH libraries similar to its SRMAtlas project for the human proteome, pioneered by Rob Moritz, Ph.D., and his collaborators, and the proteomes of other clinically relevant organisms. “With complete proteome-wide libraries, ISB provides the basis to support comprehensive SWATH analysis,” said Dr. Moritz, who is ISB’s proteomics research director.
ISB aims to make the SWATH libraries available to the global scientific community to accelerate the use of SWATH for other biological research. ISB will develop new SWATH technologies and tools to enable the community to adopt comprehensive quantitative proteome analysis.
“Having the proteomics data standardized across laboratories and across samples really enables us to quantitate entire proteomes at a level that hasn’t been done before,” said Dr. Moritz. “We aim to define markers that can predict whether a patient will respond to a certain treatment or not, and applying SWATH will play a big part in taking our advancements to another level. Not only can we now complement the breadth of genomics, but we will have the much-needed libraries and software development going forward to make data-sharing quite easier and standardized.”
AB Sciex forged this alliance with ISB through the AB Sciex Academic Partnership Program to help broaden the availability of new technologies to researchers delving into OMICS research around the world.
“What ISB does with SWATH will set a new benchmark in proteomics research,” said Rainer Blair, president of AB Sciex. “Our collaboration with ISB will help drive SWATH into the mainstream of analytical science and make comprehensive, reproducible and simplified omics data more accessible to biologists around the world.”
SWATH Acquisition was first made available to the worldwide scientific community back in April through a collaboration between AB Sciex and ETH Zurich.
Genetics and Biophysics for Large Volumes of Data
Rresearchers used an interdisciplinary approach combining genetics and biophysics. “It is the first analysis to combine all known protein structures and genomes with folding rates as a physical parameter,” says Dr. Gräter.
The analysis of 92,000 proteins and 989 genomes can only be tackled with computational methods. The group of Gustavo Caetano-Anolles, head of the Evolutionary Bioinformatics Laboratory at Urbana-Champaign, had originally classified most structurally known proteins from the Protein Database (PDB) according to age. For this study, Minglei Wang in his laboratory identified protein sequences in the genomes, which had the same folding structure as the known proteins. He then applied an algorithm to compare them to each other on a time scale. In this way, it is possible to determine which proteins became part of which organism and when. After that, Cedric Debes, a member of Dr. Gräter’s group, applied a mathematical model to predict the folding rate of proteins. The individual folding steps differ in speed and can take from nanoseconds to minutes. No microscope or laser would be able to capture these different time scales for so many proteins. A computer simulation calculating all folding structures in all proteins would take centuries to run on a mainframe computer. This is why the researchers worked with a less data-intensive method. They calculated the folding speed of the single proteins using structures that have been previously determined in experiments: A protein always folds at the same points. If these points are far apart from each other, it takes longer to fold than if they lie close to each other. With the so-called Size-Modified Contact Order (SMCO), it is possible to predict how fast these points will meet and thus how fast the protein will fold, regardless of its length.
“Our results show that in the beginning there were proteins which could not fold very well,” Dr. Gräter summarizes. “Over time, nature improved protein folding so that eventually, more complex structures such as the many specialized proteins of humans were able to develop.”
Researchers develop Compilation of Protein Interaction Data
Posted on January 2, 2013 by grathbone
Researchers have created a platform detailing all atomic data on protein structures and protein interactions for eight organisms. Applying a singular homology-based modelling procedure they have brought together the information previously stored in diverse databases. Interactome3D has been compiled by scientists Roberto Mosca, Arnaud Ceol and Patrick Aloy as an open-access, free web platform as part of their work at the Institute for Research in Biomedicine.
For the first time the platform offers anonymous access to molecular details of protein interaction and 3D models. It means that researchers can easily find the atomic level detail that is fundamental to new discoveries in biology and pharmaceuticals.
Information on more than 12,000 protein interactions for eight model organisms – the plant Arabidopsis thaliana, the worm Caenorhabditis elegans, the fly Drosophila melanogaster, the bacteria Escherichia coli and Helicobacter pylori, the brewer’s yeast Saccharomyces cerevisiae, the mouse Mus musculus, and Homo sapiens – is included. These eight models are the most relevant for biomedical and genetic research.
Patrick Aloy, ICREA researcher at IRB Barcelona, said: “We have designed Interactome3D for molecular and cellular biologists. It is a well organised non-technical interface that presents the results in a simple manner.
With only a few clicks of the mouse, you can get the information you are looking for and you don’t have to be a bioinformatician to navigate around the platform, to look things up or to interpret the results.”
The platform is the result of more than four years of lab experience and collaboration, and the information it contains will be updated every six months, with up to 16,000 protein interaction details expected to be available soon.
Dual coding in alternative reading frames correlates with intrinsic protein disorder
Erika Kovacs, Peter Tompa, Karoly Liliom, and Lajos Kalmar1
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary
Edited* by Ada Yonath, Weizmann Institue, Rehovot, Israel, and approved January 29, 2010 (received for review July 14, 2009)
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 67 human genes with alternative splice variants comprising a dualcoding 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. In the case of þ1 frameshifts, the average level of disorder in the two frames is similarly high (47.2% in the ancestral frame, 58.2% in the derived frame, with the average level of disorder in human proteins being approximately 30%), whereas in the case of −1 frameshifts, there is a significant tendency to become more disordered upon shifting the frame (16.7% in the ancestral frame, 56.3% in the derived frame).
The regions encoded by the derived frame are mostly disordered (disorder percentage >50%) in 39 out of 62 cases, which strongly suggests that structural disorder enables these protein products to exist and function without the need of a highly evolved 3D fold.
The potential advantages are also demonstrated by the appearance of novel functions and the high incidence of transcripts escaping nonsense-mediated decay. By discussing several examples, we demonstrate that dual coding may be an effective mechanism for the evolutionary appearance of novel intrinsically disordered regions with new functions.
Alternative splicing ∣ nonsense-mediated decay ∣ unstructured protein
The process of alternative splicing (AS), in which different combinations of exons are joined together in mRNA maturation, enables several protein isoforms to be encoded by a single gene (1, 2). It is estimated that more than 75% of mammalian genes are alternatively spliced (1, 3) and in about 50% of all AS events the reading frame is altered (4), i.e., a certain stretch of DNA has the potential to be translated in different reading frames. The use of such alternative reading frames (ARFs), however, is often suppressed by a premature termination codon (PTC) that results in nonsense-mediated decay (NMD) of the mRNA product (5, 6). In mammals, a stop codon followed by an exon–exon junction more than 50–55 nucleotides downstream is recognized as a PTC (7) that regulates gene expression and/or acts as a surveillance mechanism against potentially harmful protein products.
A major concern with dual-coding in ARFs is that it gives rise to two intertwined polypeptide sequences which are highly unlikely to both result in two properly folded functional proteins. Thus, dual-coding has long been thought to be prevalent only in viruses and prokaryotes that are under pressure to maintain a compact genome (8, 9). Only relatively recently, results on functional pairs of proteins derived from ARFs (10–16) and bioinformatic studies of conserved overlapping open reading frames (ORFs) (16–19) have pointed to the likely importance of the use of ARFs in eukaryotes.
An enigmatic issue largely overlooked thus far is the protein structural impact of this phenomenon. Because folding of a polypeptide chain to a unique 3D state is a highly evolved feature www.pnas.org/cgi/doi/10.1073/pnas.0907841107 PNAS Early Edition ∣ 1 of 6
A series of novel 20-modiifed nucleoside 50-triphosphates was synthesized. The amino, imidazole, and carboxylate functionalities were attached to the 5-position of pyrimidine base of these molecules through alkynyl and alkyl spacers, respectively. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.
Recently, much attention has been focused on the development of functionalized nucleotides suitable for in vitro selection with the hope of increasing the potential of nucleic acids for binding and catalysis. For RNA in vitro selections modifications should be at the nucleotide level so that they can be incorporated simply and efficiently using RNA polymerase without problematic side reactions associated with synthetic posttranscriptional modification.
English: The structure of DNA showing with detail showing the structure of the four bases, adenine, cytosine, guanine and thymine, and the location of the major and minor groove. (Photo credit: Wikipedia)
English: A model of a DNA tetrahedron. Each edge of the tetrahedron is a 20bp DNA duplex, and each vertex is a three-arm junction. In this model each basepair is represented by five pseudo-atoms, representing the two sugars, the two phosphates, and the major groove. The scale bar is 1 nm. (Photo credit: Wikipedia)
From left to right, the structures of A-, B- and Z-DNA. The structure a DNA molecule depends on its environment. In aqueous enviromnents, including the majority of DNA in a cell, B-DNA is the most common structure. The A-DNA structure is dominates in dehydrated samples and is similar to the double-stranded RNA and DNA/RNA hybrids. Z-DNA is a rarer structure found in DNA bound to certain proteins. (Photo credit: Wikipedia)
Article 1.1 Advances in the Understanding of the Human Genome The Initiation and Growth of Molecular Biology and Genomics- Part I
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 http://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:
the progress made by X-ray crystallographers in studying organic macromolecules;
the growing evidence supplied by geneticists that it was DNA, not protein, in chromosomes that was responsible for heredity;
Erwin Chargaff’s experimental finding that there are equal numbers of A and T bases and of G and C bases in DNA;
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.
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/25664997http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851785 http://www.pnas.org/content/107/12/5429.full.pdf
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:
broken ends of chromosomes will fuse, 2-by-2, and
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.
The conclusion was that cells sense the presence in their nuclei of ruptured ends of chromosomes
then activate a mechanism that will bring together and then unite these ends
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
both accidental breaks and
programmed breaks may be frequent.
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
duplications,
deficiencies,
inversions,
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.
Source: 1983 Nobel Lecture. Barbara McClintock. THE SIGNIFICANCE OF RESPONSES OF THE GENOME TO CHALLENGE.
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.
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.
English: The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle. Produced at WikiPathways. (Photo credit: Wikipedia)
Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
Reporter& Curator: Larry Bernstein, MD, FCAP
Unlocking the diversity of genomic expression within tumorigenesis and “tailoring” of therapeutic options
1. Reshaping the DNA landscape between diseases and within diseases by the linking of DNA to treatments
In the NEW York Times of 9/24,2012 Gina Kolata reports on four types of breast cancer and the reshaping of breast cancer DNA treatment based on the findings of the genetically distinct types, which each have common “cluster” features that are driving many cancers. The discoveries were published online in the journal Nature on Sunday (9/23). The study is considered the first comprehensive genetic analysis of breast cancer and called a roadmap to future breast cancer treatments. I consider that if this is a landmark study in cancer genomics leading to personalized drug management of patients, it is also a fitting of the treatment to measurable “combinatorial feature sets” that tie into population biodiversity with respect to known conditions. The researchers caution that it will take years to establish transformative treatments, and this is clearly because in the genetic types, there are subsets that have a bearing on treatment “tailoring”. In addition, there is growing evidence that the Watson-Crick model of the gene is itself being modified by an expansion of the alphabet used to construct the DNA library, which itself will open opportunities to explain some of what has been considered junk DNA, and which may carry essential information with respect to metabolic pathways and pathway regulation. The breast cancer study is tied to the “Cancer Genome Atlas” Project, already reported. It is expected that this work will tie into building maps of genetic changes in common cancers, such as, breast, colon, and lung. What is not explicit I presume is a closely related concept, that the translational challenge is closely related to the suppression of key proteomic processes tied into manipulating the metabolome.
2. Fiddling with an expanded genetic alphabet – greater flexibility in design of treatment (pharmaneogenesis?)
Diagram of DNA polymerase extending a DNA strand and proof-reading. (Photo credit: Wikipedia)
A clear indication of this emerging remodeling of the genetic alphabet is a new
study led by scientists at The Scripps Research Institute appeared in the
June 3, 2012 issue of Nature Chemical Biology that indicates the genetic code as
we know it may be expanded to include synthetic and unnatural sequence pairing (Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought, Genome). They infer that the genetic instructions for living organisms
that is composed of four bases (C, G, A and T)— is open to unnatural letters. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications. The implications of the application of this would further expand the translation of portions of DNA to new transciptional proteins that are heretofore unknown, but have metabolic relavence and therapeutic potential. The existence of such pairing in nature has been studied in Eukariotes for at least a decade, and may have a role in biodiversity. The investigators show how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. This could as well be translated into human diversity, and human diseases.
The Romesberg laboratory collaborated on the new study and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). It had been clear that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication, but that is not the case because other bonds can be in play.
The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion that resembles a ‘mispair.’ In test after test, the NaM-5SICS pair was efficiently replicable even though it appeared that the DNA polymerase didn’t recognize it. Their structural data showed that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium.
The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could be used to extend the DNA alphabet and that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been developed allowing for life based on other genetic systems.
3. Studies that consider a DNA triplet model that includes one or more NATURAL nucleosides and looks closely allied to the formation of the disulfide bond and oxidation reduction reaction.
This independent work is being conducted based on a similar concep. John Berger, founder of Triplex DNA has commented on this. He emphasizes Sulfur as the most important element for understanding evolution of metabolic pathways in the human transcriptome. It is a combination of sulfur 34 and sulphur 32 ATMU. S34 is element 16 + flourine, while S32 is element 16 + phosphorous. The cysteine-cystine bond is the bridge and controller between inorganic chemistry (flourine) and organic chemistry (phosphorous). He uses a dual spelling, using sulfphur to combine the two referring to the master catalyst of oxidation-reduction reactions. Various isotopic alleles (please note the duality principle which is natures most important pattern). Sulfphur is Methionine, S adenosylmethionine, cysteine, cystine, taurine, gluthionine, acetyl Coenzyme A, Biotin, Linoic acid, H2S, H2SO4, HSO3-, cytochromes, thioredoxin, ferredoxins, purple sulfphur anerobic bacteria prokaroytes, hydrocarbons, green sulfphur bacteria, garlic, penicillin and many antibiotics; hundreds of CSN drugs for parasites and fungi antagonists. These are but a few names which come to mind. It is at the heart of the Krebs cycle of oxidative phosphorylation, i.e. ATP. It is also a second pathway to purine metabolism and nucleic acids. It literally is the key enzymes between RNA and DNA, ie, SH thiol bond oxidized to SS (dna) cysteine through thioredoxins, ferredoxins, and nitrogenase. The immune system is founded upon sulfphur compounds and processes. Photosynthesis Fe4S4 to Fe2S3 absorbs the entire electromagnetic spectrum which is filtered by the Allen belt some 75 miles above earth. Look up chromatium vinosum or allochromatium species. There is reasonable evidence it is the first symbiotic species of sulfphur anerobic bacteria (Fe4S4) with high potential mvolts which drives photosynthesis while making glucose with H2S.
He envisions a sulfphur control map to automate human metabolism with exact timing sequences, at specific three dimensional coordinates on Bravais crystalline lattices. He proposes adding the inosine-xanthosine family to the current 5 nucleotide genetic code. Finally, he adds, the expanded genetic code is populated with “synthetic nucleosides and nucleotides” with all kinds of customized functional side groups, which often reshape nature’s allosteric and physiochemical properties. The inosine family is nature’s natural evolutionary partner with the adenosine and guanosine families in purine synthesis de novo, salvage, and catabolic degradation. Inosine has three major enzymes (IMPDH1,2&3 for purine ring closure, HPGRT for purine salvage, and xanthine oxidase and xanthine dehydrogenase.
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)
3. Nutritional regulation of gene expression, an essential role of sulfur, and metabolic control
Finally, the research carried out for decades by Yves Ingenbleek and the late Vernon Young warrants mention. According to their work, sulfur is again tagged as essential for health. Sulfur (S) is the seventh most abundant element measurable in human tissues and its provision is mainly insured by the intake of methionine (Met) found in plant and animal proteins. Met is endowed with unique functional properties as it controls the ribosomal initiation of protein syntheses, governs a myriad of major metabolic and catalytic activities and may be subjected to reversible redox processes contributing to safeguard protein integrity.
Consuming diets with inadequate amounts of methionine (Met) are characterized by overt or subclinical protein malnutrition, and it has serious morbid consequences. The result is reduction in size of their lean body mass (LBM), best identified by the serial measurement of plasma transthyretin (TTR), which is seen with unachieved replenishment (chronic malnutrition, strict veganism) or excessive losses (trauma, burns, inflammatory diseases). This status is accompanied by a rise in homocysteine, and a concomitant fall in methionine. The ratio of S to N is quite invariant, but dependent on source. The S:N ratio is typical 1:20 for plant sources and 1:14.5 for animal protein sources. The key enzyme involved with the control of Met in man is the enzyme cystathionine-b-synthase, which declines with inadequate dietary provision of S, and the loss is not compensated by cobalamine for CH3- transfer.
As a result of the disordered metabolic state from inadequate sulfur intake (the S:N ratio is lower in plants than in animals), the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. They both stimulate comparable remethylation reactions from homocysteine (Hcy), indicating that Met homeostasis benefits from high metabolic priority. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing reducing molecules implicated in the regulation of the 3 desulfuration pathways
4. The effect on accretion of LBM of protein malnutrition and/or the inflammatory state: in closer focus
Hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly and liver production of TTR integrates the dietary and stressful components of any disease spectrum. Thus we have a depletion of visceral transport proteins made by the liver and fat-free weight loss secondary to protein catabolism. This is most accurately reflected by TTR, which is a rapid turnover protein, but it is involved in transport and is essential for thyroid function (thyroxine-binding prealbumin) and tied to retinol-binding protein. Furthermore, protein accretion is dependent on a sulfonation reaction with 2 ATP. Consequently, Kwashiorkor is associated with thyroid goiter, as the pituitary-thyroid axis is a major sulfonation target. With this in mind, it is not surprising why TTR is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequaled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyper-homocysteinemic states.
Individuals submitted to N-restricted regimens are basically able to maintain N homeostasis until very late in the starvation processes. But the N balance study only provides an overall estimate of N gains and losses but fails to identify the tissue sites and specific interorgan fluxes involved. Using vastly improved methods the LBM has been measured in its components. The LBM of the reference man contains 98% of total body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass). The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g).
TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline with an accelerated decrease after 65 years. Sulfur Methylation (SM) undergoes a 15% reduction in size per decade, an involutive process. The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation or in critically ill patients whose muscle mass undergoes proteolysis.
5. The results of the events described are:
Declining generation of hydrogen sulfide (H2S) from enzymatic sources and in the non-enzymatic reduction of elemental S to H2S.
The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions.
Elemental S operates as co-factor of several (apo)enzymes critically involved in the control of oxidative processes.
Combination of protein and sulfur dietary deficiencies constitute a novel clinical entity threatening plant-eating population groups. They have a defective production of Cys, GSH and H2S reductants, explaining persistence of an oxidative burden.
6. The clinical entity increases the risk of developing:
cardiovascular diseases (CVD) and
stroke
in plant-eating populations regardless of Framingham criteria and vitamin-B status.
Met molecules supplied by dietary proteins are submitted to transmethylation processes resulting in the release of Hcy which:
either undergoes Hcy — Met RM pathways or
is committed to transsulfuration decay.
Impairment of CβS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. The data show that combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.
Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.
7. The dysfunctional metabolism in transitional cell transformation
A third development is also important and possibly related. The transition a cell goes through in becoming cancerous tends to be driven by changes to the cell’s DNA. But that is not the whole story. Large-scale techniques to the study of metabolic processes going on in cancer cells is being carried out at Oxford, UK in collaboration with Japanese workers. This thread will extend our insight into the metabolome. Otto Warburg, the pioneer in respiration studies, pointed out in the early 1900s that most cancer cells get the energy they need predominantly through a high utilization of glucose with lower respiration (the metabolic process that breaks down glucose to release energy). It helps the cancer cells deal with the low oxygen levels that tend to be present in a tumor. The tissue reverts to a metabolic profile of anaerobiosis. Studies of the genetic basis of cancer and dysfunctional metabolism in cancer cells are complementary. Tomoyoshi Soga’s large lab in Japan has been at the forefront of developing the technology for metabolomics research over the past couple of decades (metabolomics being the ugly-sounding term used to describe research that studies all metabolic processes at once, like genomics is the study of the entire genome).
Their results have led to the idea that some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer. The collaborators have published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism. The researchers summarize that shows how accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell. It shifts the balance of metabolic processes and disrupts the cell in ways that could favor development of cancer. This is of particular interest because “fumarate” is the intermediate in the TCA cycle that is converted to malate.
Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. (Photo credit: Wikipedia)
The Keio group is able to label glucose or glutamine, basic biological sources of fuel for cells, and track the pathways cells use to burn up the fuel. As these studies proceed, they could profile the metabolites in a cohort of tumor samples and matched normal tissue. This would produce a dataset of the concentrations of hundreds of different metabolites in each group. Statistical approaches could suggest which metabolic pathways were abnormal. These would then be the subject of experiments targeting the pathways to confirm the relationship between changed metabolism and uncontrolled growth of the cancer cells.
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