Feeds:
Posts
Comments

Posts Tagged ‘reproductive research’


A Magnetically controlled Mechanical Propeller for Immotile Sperm

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Researchers from the Institute for Integrative Nanosciences, IFW Dresden, Germany and Material Systems for Nanoelectronics, Chemnitz University of Technology, Germany have developed something known as the spermbot, a remotely controlled sperm movement controlling robot that could help create babies of the future. It is a magnetically powered robotic “suit” that can strap itself to individual sperm and help guide it faster towards the egg. According to the inventors all the initial tests with the spermbot have delivered promising results.

The purpose of the spermbot is to solve one of the widely talked about causes of infertility in men which is poor motility of sperm. Low sperm motility, or otherwise healthy sperm that just can’t swim, can be a big factor in infertility. While the development of the spermbot is in its early stages, this is already being talked about as a promising alternative to existing popular techniques that are expensive and come with a high failure rate. These include methods like in-vitro fertilization and artificial insemination. Only 30 percent of the traditional “spray-and-pray” approach ends up with success, which warranted the need for an alternative procedure like the spermbot. According to the report, initial experiments show a marked increase in the probability of the spermbot-assisted sperm to reach its intended destination. The process of fertilization can be completed inside the body or in the lab, inside a petri-dish.

The spermbot is a coat of microscopic metal polymers shaped into a helix. It can attach itself to the tail of the spermatozoid, and then, using a hybrid micromotor, it can help propel the sperm faster towards the egg. The direction the sperm needs to take is controlled using a rotating magnetic field. In fact, even the motion of the sperm can be remote-controlled by simply adjusting this magnetic field. Once the spermbot propels the sperm towards the egg and the sperm manages to implant itself into the egg, the bionic part of the spermbot detaches itself from the tail.

While the initial experiments look promising, there is still some way to go before the spermbot technique is regularly used. To start off, scientists have very few sample size to correctly evaluate the results, and unless more comprehensive tests are carried out, it would not be possible to start using them on human subjects. Another major stumbling block is that there is currently no way to film the spermbot in action while it is moving inside the body. This also means that doctors would not be able to correctly direct it towards the egg. Another concern is the response of the body’s own immune system to the spermbot. The use of the spermbot could trigger a reaction from the body’s immune system, the results of which cannot be predicted without comprehensive clinical trials. The idea of the spermbot looks promising right now, but it is still too early to call it a replacement to the tried and tested methods like in-vitro fertilization and artificial insemination. In fact, it would take a few years for the procedure to be made available to patients if clinical trials are successfully completed.

References:

http://pubs.acs.org/doi/abs/10.1021/acs.nanolett.5b04221

http://www.acs.org/content/acs/en/pressroom/presspacs/2016/acs-presspac-january-13-2016/spermbots-could-help-women-trying-to-conceive-video.html

http://www.inquisitr.com/2711435/spermbot-robot-sperm-infertility-treatment/#utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+google%2FyDYq+%28The+Inquisitr+-+News%29

http://www.slate.com/articles/video/video/2016/01/spermbot_attached_to_sperm_and_delivers_it_quickly_to_an_egg_video.html

http://www.sciencemag.org/news/2016/01/video-motorized-spermbot-helps-sperm-reach-egg

Read Full Post »


English: This diagram shows the chromosomes of...

This diagram shows the chromosomes of Drosophila melanogaster approximately to scale. Chromosome sizes were based on basepair lengths given on the NCBI map viewer, and A. B. Carvalho, 2002. Curr. Op. Genet. & Devel. 12:664-668. Centimorgan distances were derived from selected loci listed in the NCBI website. (credit  Wikipedia)

Introduction

Generally speaking sexually reproducing species are composed of individuals of two complementary mating types or sexes.  An essential aspect of the developmental history of each individual is thus sex determination and differentiation. There exist two sex determination mechanisms, somatic and germline, that based on the chromosomal mechanism in the Drosophila melanogaster.  In the somatic sex determination mechanism, each individual assesses the ratio of X-chromosomes to autosomal chromosome sets), the X:A ratio provides the primary sex-determining signal   (reviewed by Cline and Meyer, 1996).  When X:A=1, female differentiation ensues (Bridges, 1925), along with the male-mode of X-chromosome dosage compensation.  The X:A ratio is calculated within each cell of the developing embryo, 2 hrs after fertilization. The X:A ratio determines the sex in Drosophila (Bridges, 1916, 1921, 1925) in a somatic-cell-autonomous manner that occurs early in embryonic development (Baker and Belote, 1983; Baker, 1989). Females possess two X-chromosomes, and males possess one X-chromosome and one Y-chromosome.   The Y-chromosome is required only for spermatogenesis (Lindsley and Tokuyasu 1980; Bridges 1986), and will not be considered further.  The number of X-chromosomes is counted through a mechanism involving positive-acting X-chromosome-encoded transcription factors, termed X-numerator elements (Cline, 1988), negative-acting autosome-encoded transcription factors or denominators, and signal transduction factors provided maternally.  Among the X-numerators are sisterless-a, sisterless-b (sis-b), sisterless-c, and runt (Schurpbach, 1985; Cline, 1986, 1988; Steinmann-Zwicky et al., 1989; Parkhurst et al., 1990; Ericson and Cline, 1991, 1993; Estes, 1995; Hoshijima et al., 1995; reviewed by Cline, 1993).

The best candidate for a denominator gene is the deadpan (dpn) locus.  Both daughterless (da) and extramacrochaete (emc) fulfill the role of maternally contributed transduction loci (Cline, 1976; Cronmiller et al., 1988).  Both in vitro biochemical evidence and in vivo genetic evidence support the idea that transcription factors of the basic-helix-loop-helix (bHLH) family are able to form homo- and hetero-dimers; thus the X:A ratio counting mechanism seems to involve the relative affinities and chromosome-dependent stoiciometries of the bHLH proteins SIS-B, DA, EMC, and DPN.  When X:A=1, sufficient SIS-B protein is synthesized so that it can effectively compete with the EMC and DPN proteins for binding to DA protein.  DA:SIS:B heterodimers then bind to so-called establishment promoter (Pe) elements of the SXL gene and activates its transcription, resulting in an early burst of SXL protein that sets splicing and dosage compensation in to female-specific modes.  When X:A=0.5, too little SIS-B is produced, and DA protein remains sequestered with EMC and DPN.  The Sxl Pe remains inactive, and splicing and dosage compensation enters male-specific modes. In response to X:A ratio=1, an embryo specific promoter of the gene called Sex-lethal (Sxl) is activated (Keyes et al., 1932).

Sxl protein that acts as a master gene for the somatic germline sex determination, has three somatic functions. First, Sxl protein carries out autoregulation at the level of pre-mRNA splicing.  Second, Sxl controls female-specific differentiation at the level of pre-RNA splicing and polyadenylation at least two genes that code for transcription factors that effect terminal differentiation. Third, Sxl protein negatively regulates X-chromosome dosage compensation.  It does so in two ways, by alternative RNA splicing of a normally male-specific gene, and by translation-level regulation of many X-chromosomal transcripts during embryogenesis. In the male, with Sxl in the off state, male differentiation occurs because tra is in the off state and therefore the differentiation-effector transcription factors are produced in alternative male-specific modes.  Dosage compensation is active, and the male X-chromosome is decorated by a minimum of four proteins and two RNA molecules that form a complex along the entire chromosome (reviewed by Cline and Meyer, 1996).  Transcription of the male X-chromosome is elevated two-fold, and it produces the same amount of RNA per template as found in females.

Germline pathway for sex determination and dosage compensation is different than the somatic sex determination mechanism.  (Figure 1) Figure 1: Sex determination of D. melanogaster (1998)The vast majority of somatic sex determination loci have no function in germline cells.  For example, none of the X-chromosome numerators is required for proper oogenesis (Granadino et al., 1989, 1992; Steinmann-Zwicky 1991), despite the fact that proper oogenesis requires that X:A =1 in the germline (Schupbach, 1982, 1985) nor are tra, tra-2, and dsxF required for oogenesis.  Sxl and snf have germline functions but the former is not a binary switch gene between oogenesis and spermatogenesis (Despande et al., 1996; Bopp et al., 1993, 1995; Hager et al., 1997). Systematic screens for female-sterile mutations have identified a large number of genes required for normal oogenesis (e.g. Gans et al., 1975; Mohler, 1977; Perrimon et al., 1986; Schupbach and Wieschaus, 19889, 1991).  Female-sterility can arise in diverse ways, but one interesting class of mutations is germline-dependent and causes an “ovarian tumor” phenotype.  “Ovarian tumor” mutations cause under-developed ovaries, in which egg chambers and ovarioles are filled with an excess of undifferentiated germ cells that have adopted male-like characteristics that include a prominent spherical nucleus, assembly of mitocondria around the nucleus, and mis-expression of male-specific marker genes (Oliver et al., 1988, 1990, 1993; Steinmann-Zwicky, 1988, 1992; Bopp et al., 1993; Pauli et al., Wei et al., 1994).  Among the “ovarian tumor” class of genes are ovo, ovarian tumor (otu), fused, and two genes with somatic phenotypes, namely snf and Sxl. Strong mutations at the ovo and otu loci result in ovaries totally devoid of germ cells (King and Killey, 1982; Busson et al., 1983; Oliver et al., 1987; Mevel-Ninio et al., 1989; Rodesh et al., 1995), Weaker mutations at both loci result in viable germline cells that have abnormal male-like splicing at the Sxl gene (Oliver et al, 1993). The overall conclusion is that oogenesis requires a chromosomally female germline is wild type for ovo, otu, Sxl, and snf.  If one of these genes is defective, either the germline will die or male-like differentiation and tumor formation ensure.

However, there are soma-germline interactions for a normal sex determination. (Figure 2) Figure 2: Somatic-Germline Interactions. (1998)Unlike the somatic regulatory hierarchy, which genetic mosaic experiments clearly showed functions in cell-autonomous fashion, sexual differentiation of the germline requires inductive signaling from somatic cells.  This was shown by use of pole cell transplantation, the method of making mosaics in which germline cells surgically transferred from donor embryos  (Schubach. 1985; Steinmann-Zwicky et al., 1989).  These experiments show that proper germline differentiation requires a combination of germline-autonomous chromosomal cues and proper signaling from the soma.  Evidence with tra and dsx mutant somatic hosts indicates these soma-germline interactions have detectable effects by larval stages (Steinmann-Zwicky., 1996).

The ovo gene is genetically complex.  At least three transcripts are produced from the ovo region (Mevel-Ninio et al, 1991, 1995, 1996; Garfinkel et al., 1992, 1994).  Two of these are germline-specific and correspond to the ovo function, while the third corresponds to the somatic-epidermal, non-sex-specific shavenbaby (svb) function.  (For a schematic of the gene map please refer to Figure3) 

 The ovo function is transcribed from two closely spaced germline-specific promoters, ovo a and ovob, give rise to 5-kb mRNAs (Mevel-Ninio et al., 1991, 1995; Garfinkel et al., 1992, 1994).   First identified  promoter was ovob  Garfinkel et al., (1994)  and the leader exon it forms is called Exon 1b, 1028-codon-long open reading frame that contains four Cys2-His2 fingers at the carboxy terminus; protein MW of 110.6 kD.  A second germline promoter, ovoa, was identified by Mevel-Ninio et al (1995), 1400 codons long, and predicts a 150.8-kD protein.  This Exon 1a contains an in-frame AUG upstream of the translation start in Exon 2 utilized by the OvoB open reading frame.  The OvoB mRNA isoforms is predominant during adult life, with the OvoA isoforms only appearing during Stage 14 of oogenesis (Mevel-Ninio et al., 1991, 1996; Garfinkel., 1994).  The ovo zinc finger domain binds to its own germline promoter regions, to the otu promoter region (Garfinkel et al., 1997; Lee, 1998; Lee and Garfinkel 1998).  This is consistent with ovo playing an important role in a sex determination hierarchy operating in germline cells that involves these other genes. The svb function is transcribed from an incompletely characterized somatic promoter that forms a 7.1 kb poly(A)+ mRNA (Garfinkel et al., 1994).  This transcript accumulates 9-12-hr post-fertilization, in the somatic tissues that later in embryogenesis form the cuticular structures affected by svb mutations.  Wieschaus et al. (1984) observed that ventral denticle belts and dorsal hairs are defective in svb mutations; hence the name, and svb mutations are polyphasic larval lethals. Exons and exon segments that are found in all mRNA forms coded by the region correspond to genomic DNA where so-called svb-ovo- mutations map (Mevel-Ninio et al., 1989; Garfinkel 1992).  Finally, somatic-specific exons, exon segments, and transcriptional regions correspond to region mutable to the svb- ovo- phenotype.  Since al known mRNA forms utilize the same splice junctions to join Exon3 to Exon4, all protein forms coded by the locus are believed to contain the same four zinc fingers at the carboxy terminus.   A wide variety of evidence points to ovo playing a critical role in germline sex determination.  High-level of ovo transcription in germline cells, as detected with Xgal staining of ovo promoter-lacZ constructs requires that they have a female karyotype (Oliver et al., 1994).  Chromosomally male germline cells have low levels of ovo transcription even if the soma is transformed towards female through the use of hs-traF cDNA minigenes.  Likewise, chromosomally female germline cells have high levels of ovo transcription even if the soma is anatomically male through the action of tra loss-of-function mutations.  This argues that high-level of ovo transcription is a germline X: A ratio-autonomous property, and stands in contrast to related experiments with otu.  In the case of otu, there is evidence that chromosomally male germline cells, which normally have no need of otu+ function at all, require otu- for proliferation when they are in a female host (Nagoshi et al., 1995). The D. melanogaster ovo gene is required for cell viability and differentiation of female germ cells, apparently playing a role in germline sex determination.  While female X: A ratio in germline cells is required for high levels of ovo germline promoters.  Therefore we undertook to identify trans-acting regulatory regions of the X-chromosome, with a particular interest in identifying candidate germline X-chromosome numerator elements. In this study, I screened  X-chromosome using 45 deficiency strains, I found that these trans-regulating regions were grouped into 12 loci based on overlapping cytology.  Five regions were trans-regulating activators, and seven were trans-regulating repressors; extrapolating to the entire genome, this result predicts nearly 85 loci.  A subset of the dozen X-chromosomal regions correlated with previously identified E(ovoD) and Su(ovoD) loci (Pauli et al., 1995).  

Materials and Methods

 

Fly Strains and Growth Flies were maintained on standard yeast/cornmeal medium and kept at 25oC and 18oC unless otherwise indicated.  Mutants are described in Lindsley and Zimm (1992).  The ovo3U21 and ovo4B8 were obtained from Brian Oliver of NIH;  OvoD1rS1 FM3 is from the Garfinkel lab collection.  The remaining stocks were obtained from the Bloomington Stock Center (see Table 2.1 for the list of stocks that had been used and Figure 2.1 for their location on the X Chromosome). 

Outcrosses Outcrosses were designed to create transgenic flies so that screening of the X chromosome for trans-regulators of ovo in the germline can be done.   Virgin female flies were collected 14 hour long windows at 18oC or 8 hour long windows at 25oC, during which newly emerged males remained immature.  Collected females were kept 3-5 days to make sure they are virgin before outcrossing them.  Heterozygous virgin females (5-7), carrying deficiency X-chromosomes balanced over first chromosome balancers were mated with males homozygous for either of two P-element transformation constructs of a lacZ reporter gene fused to the ovo promoter.  Both events were inserted on third chromosome.  They were grown at 25oC unless otherwise noted. The control class of F1 progeny has a complete X-chromosome pair, whereas the experimental class has one complete and one deficient X chromosome in its genome.  The [ovo::lacZ constructs] were designed by Oliver et al., (1994).  In this study two of their strains, ovo4B8 (pCOW+1.9) and ovo3U21 (pCOW-2.1) respectively, were used to determine the ovo promoter activity.

Outcrosses to Remove Duplications Several X-chromosome deficiencies in the Bloomington collection are carried in males, with compensatory duplications of X material on an autosome.  These had to be crossed to eliminate the duplications (Fig 2.4).  This was done as follows:  FM3/FM7a virgin flies were mated to Df/Y; Dp males.  Among the F1 progeny, half of the Df/(FM3 or FM7a) daughters will carry the unwanted duplication, and half will be free of the duplication.  In some cases, presence of the duplication could be determined from the females’ phenotypes.  In other cases, up to twenty individuals virgin Df(FM3 or FM7) F1 progeny were backcrossed to FM7a/Y males to establish stocks.  In the F2, absence of the duplication could be established by examining sons; in all cases, the Df is male-lethal unless “rescued” by the duplication.  Also FM3 is itself male lethal.  Thus, single-female stocks that produce only FM7a sons had the desired genotypes and were kept for experiments.

X-Gal Staining In this assay ovaries from two-day-old adults were dissected in Drosophila Ringer’s solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10mM TrisHCl, pH 6.8).  Then, these tissues were transferred to a microtiter plate and fixed in 1% gluteraldehyde, 50mM Na-cacodylyte acid solution for 15 minutes. After rinsing the tissues, three times for 5 minutes each staining buffer (7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 1.0 mM MgCl2, 0.15 mM NaCl), they were transferred to incubation buffer (staining buffer, 5 mM Fe2 (CN)3, 5 mM Fe3 (CN)2, 0.2% X-Gal) for an hour at 37oC.  Next, tissues were washed three times 5 minutes each in washing buffer, which is a 1 mM EDTA, added PBS (130 mM NaCl, 7 mM Na2HPO4*2H2O, 3 mM NaH2PO4*2H2O, pH 7.0) solution.  Finally, the tissues were dehydrated in ethanol solutions of increasing concentrations (50%, 75%, 95%) and mounted on a slide in Permount.  Preparate concentrations were examined under a compound microscope to make correlations between staining and gene activity. Although it was easy to determine positive and negative controls, but this assay wasn’t sensitive enough to see subtle differences due to effects of deleted regions on ovo promoters driving LacZ.

Histochemical Assay of LacZ Activity This method allowed us to make quantitative measurements of lacZ activity due to ovo promoter function in animals heterozygous for X-chromosome deletions.  Emerging F1 flies were collected and aged for two days before dissecting ovaries under a dissecting microscope.  For each soluble assay, 10 flies were dissected.  This is repeated at least seven assays (N, sample number) completed per stock for each construct.  Ovaries from ten dissected outcrossed flies were out into eppendorf tubes containing 100ml of Assay Buffer (50 mM K-phosphate, 1 mM MgCl2 at pH 7.8) and homogenized about 20 strokes.  For each dissected pair of ovaries 100 ml  of assay buffer was used and the volume was completed to appropriate amount.  After centrifuging for one minute, 20 ml of the supernatant was transferred into 980 ml of assay buffer (Simon and Lis, 1987; Ashburner, 1989) to make 2mM chlorophenol red-beta-D-galactopyranoside (CPRG).  Absorbance at 574 nm was measured at half hour time intervals starting from zero to two hours hydrolysis of CPRG by chlorophenol (red CPRG).  CPR has a molar extinction coefficient of 75,000 M-1 cm-1 (Boehringer-Manheim data sheet) and this is a very easily detected product of b-galactoside enzyme activity. Range finding experiments showed that 2mM of CPRG gives linear data for 2-3 hours often, color changes could be seen with the unaided eye. Two controls are shown in Figure 2.8 that validates CPRG for this work.  Ovaries from a non-transformed strain (y w RD) were used to prepare soluble extracts.  A near zero-absorbance at 574 nm was observed that did not appreciably change over several hours.  In contrast, ovarian extracts from the ovo promoter-lacZ transformant strain ovo3U21 and ovo4B8 (Oliver et al, 1994) showed a steep linear increase in A 574 during the same period.  The slopes of these lines were proportional to the amount of ovo3U21 and ovo4B8 extract added.

Bradford (1976) Assay For Protein This protein determination method is based on the binding of Coomasie Brilliant Blue G-250 to the protein.  Preparation of protein reagent was done according to Bradford (1976).  After 100 mg of Coomasie Brilliant Blue G-250 was dissolved in 50 ml 95% ethanol, and then 100 ml 85% (w/v) phosphoric acid was added.  The resulting solution was diluted to a final volume of 1 liter [final concentrations in the reagent were 0.01% (w/v) Coomasie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid].  20ml of prepared soluble extract from the dissected tissues were used.  This volume is diluted to 0.1ml with ddH2O, then 5ml of protein reagent was added to the test tube and contents were mixed.  The absorbance at 595nm was measured after 2 min and before 1 hr in 3 ml cuvettes against a reagent blank prepared from 0.1 ml of the appropriate buffer and 5 ml of protein reagent.  A standard curve using known quantities of bovine serum albumin (BSA) was constructed.  Soluble extract absorbances were plotted on the standard curve and protein amount interpolated.

Statistical Analysis Average specific activity is calculated as nanomoles of substrate used per hour per nanogram protein expressed (nmole CPRG liberated /ng / hr).  Sample number (N) always exceeded seven.  Mean specific activity and standard error of the mean (SEM) were calculated for each experimental and control class.  The F test was used to determine whether variances were equal, and therefore,, which type of student’s t-test calculation was appropriate.  A significant difference between experimental and control values was identified by a P < 0.05 for the t-test score.

RESULTS

In this study and ovo mechanism study, the X-chromosome was screened, using 56 different deficiency strains    Table 1: List of Stocks for X-chromosome Screening (1998)Table 2: Stocks Made in This Study for X-Chromosome Screening Table 1: Stocks for Negative Autoregulation of ovo (1998)  to identify transregulation of ovo Table 3: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo3U21Table 4: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo4B8 (Results)

The results are given in three sections: X chromosome deficiency screening, negative autoregulation of ovo exhibited by deficiencies removing ovo, and gene dose analysis using P element transformants carrying extra copies of ovo.

X Chromosome Screening The presence of polytene chromosomes in the salivary glands, which have distinctive, banding patterns allows the map positions of genes to be correlated with physical features of the chromosomes.  Breakpoint locations rearrangements, and the locations of cloned sequences can be easily established.  Each of the major chromosome arms is divided into 20 numbered segments, except chromosome 4, which is divided into 4 regions.  Each numbered region is then divided into six consecutive lettered regions, and each lettered region into numbered bands, for example 4E1. The precise relationship between physical length and the numbering scheme depends on local topography (Lefevre, 1976).  In the summary tables, each deficiency listed according to cytological positions. The map of the X chromosome, including the deficiencies used in this study is given in Materials and Methods (Fig 1). Figure 1: Sex determination of D. melanogaster (1998) In Drosophila melanogaster germ cells, ovo has a primary role in female sex specific cell viability, proliferation and differentiation.  Ovo responds to the number of X-chromosomes as assessed by high level expression (Oliver et al., 1994).  Thus, the ovo promoter may be dependent upon X germline numerator elements.  To identify possible trans-regulators of the ovo germline promoter (and, I hope, to identify germline numerators) I undertook deficiency screen for quantitative effects on ovo::lacZ reporter constructs.  Determination of trans-regulation effect by any of the deletion mutant, was based on two general rules.  If the excised part of the X chromosomes has any genes with the positive regulatory effects on ovo gene activity, then the levels of LacZ reporter gene function will be reduced in experimentals compared to control siblings.  If the experimental class results in the elevation of the LacZ activity by producing high levels of enzyme compared to controls, the elevated region having removed a repression locus. Significant effects were determined by statistical analysis, which using a student’s t-test P value is less than or equal to 0.05.  X-chromosome screening results are presented in Table 3.1 and 3.2.  The entire X-chromosome deficiency set was tested twice: once with a 3.3kb ovo promoter fragment driving LacZ (strain ovo3u21), and separately with a 3.1kb ovo promoter (ovo4B8).  Of  45 deficiencies that represent about 70% of the X-chromosome 17 deficiencies had significant effects in both ovo3U21 and ovo4B8 reporter activity, 1 deficiency had significant effects on only ovo3U21 and only 1 deficiency effect on ovo4B8.  Some of these deficiencies partly overlap, allowing the identification of 11 regions that apparently contain trans-acting modifiers of ovo promoter activity six are positive regulators and five are negative.

Region 1-4.  This region covers the eight overlapping deficiency lines, Df(1) BA1, Df(1)sc14, Df(1)64c18, Df(1)JC19, Df(1)dm75e19, Df(1)N8, Df(1)A113, DF(1)JC70.  For three of them, Df(1)A113, Df(1)JC70, and Df(1)BA1, the student’s t-test probabilities show a significant difference between control and experimental siblings.  The remaining strain has no significant trans-regulation effect on ovo gene activity.  Df(1)BA1 enhanced the ovo gene expression activity about 20% when either ovo3U21 or ovo4B8 is used.  It was suggested that a suppressor of ovoD (1F-2B+ locus) maps within 1E3-4 to 2B3-4 because of the dramatic gene dose effect of this region on the development of ovoD2/+ ovaries (Pauli et al, 1995).  In contrast, it was found that Df(1)A113 and Df(1)JC70 have repressing effects on ovo expression.  Df(1)A113 (3D6-E1; 4F5) removes several genes beside ovo, showed a very significant repression effect in outcrosses, about 82% and 47% (e/C), in ovo3U21 and ovo4B8 respectively.  That data obtained in Df/+ females has a particular quantitative significance, which implies that the missing loci have the complementary effect. It was shown that this region is contains a gene or genes resulting in genetic unbalance (Cline et al., 1987).  Also, Oliver et al., (1988) show that in deficiency lines, which they have used, strains removing both ovo and snf together are reducing viability of the progeny, that is, there is a synergistic interaction between ovo and snf.  

Region 5-8.  Twelve overlapping deletions have been tested in this region.  Two deletions Df(1)N73 (5C3-5;5E-8) and Df(1)Lz90b24 (8B-D) caused very significant repressing effects, implying the presence of trans-activating loci, one deletion Df(1)RA2 (7D10;8A4-5) resulted in heterozygous experimentals with significant elevation in LacZ compared to siblings, implying a trans-repressor locus.  It has been reposted that Df(1)RA2 strongly enhances ovoD  phenotypes due to the function of otu+ in germline sex determination (Pauli et al., 1993).  However, since out protein is cytoplasmic, it is unlikely that the Df(1)RA2 effect on ovo::lacZ promoter activity is due to changing dosage of otu.  It is also suggested that there is a synergistic interaction between ovo and lozenge, eye phenotype, which is deleted by Df(1)Lz90b24, and here the data showed a trans-activating effect due to this deletion.  The other deletions do not cause any significant effect on gene activity.

Region 9-10.  In this cytological position nine deficiency lines had been tested.  Since this region was very dense for putative trans-regulation repressors, it was group in a small region.  Among nine of the deficiencies were used six of them showed a repressor effect.  These effective regions were: Df91)vL15, Df(1)N110, Df(1)HC133, Df(1)vL11, Df(1)KA7, and Df(1)N71.  This region seems to have a very important effect on ovo, since in the 9Bto 10F interval there are various levels of repressor effect.  Two common overlapping regions were found; one was from 9C4 to 9D1-2, and the other was from 10A to10F6.  Other repressor effects from strongest to weakest was Df(1)vL11 (9C4;10A1-2), Df(1)HC133 (9B9-10;9E-F), Df(1)N110 (9B3-4;9D1-2), and Df(1)v-L15 (9B1-2;10A1-2), Df(1)KA7 (10A9;10F6-7) breakpoint was outside the first loci in the examined region.  Df(1)Ka7 and Df(1)vL15 show about 20% increase in the heterozygous siblings, the longest and the shortest breakpoints, respectively.  Three out of five repressing effect intervals, Df(1)v-L11 (9C4; 10A1-2), Df (1)HC133 (9B9-10; 9E-F), Df(1) N110 (9C4; 10A1-2) is the strongest of all in Df/+ and bearing the common region among the five strains, which is 9C4; 10A1-2.  

Region 11-13.     Eight deficiency lines were in this region, Df(1)JA26, Df(1)HF368, Df(1)N12, Df(1)C246, Df(1)g, Df(1) RK2, Df(1)RK4, and Df(1) sd 72b   .  It has been found that this region involves five overlapping deletions that gave rise to repressing effect on ovo gene expression.  According to common regions of the cytological positions, these overlapping deletions were grouped into three loci.  These three common regions, which are responsible from trans-regulation activity of ovo, reside on 11D0F; 12B-D, and 13F-B regions of the X-chromosome.  Df(1)N12 (11D12;11F1-2) and Df(1)C246 (11D-E; 12A1-2) were in the 11D-F loci, Df(1)g (12B;12E8) and Df(1)RK2 (12D2-E1; 13A2-5) were in the 12B0D region, and Df(1)sd72B (13F1-14B1) in the 13B-14B loci, all of which in this examined region showed a repressor activity. The strongest effect among the X-chromosome screening was located in 11D1-11F1-2 excised region of X-chromosome, this deletion corresponds to Df(1)N12 strain, which shows a significant effect as well as high gene activity repression, Around 140% to 240% E/C in Df/+ flies for both ovo::LacZ constructs.  In addition, it has been reported that reduced dose of the 11D-F region results in synergistic mutant phenotypes with a number of somatic sex determination genes (Belote et., 1985).  Furthermore, Flybase reports that this region seems to include locus involved in early sex determination examined by Scott and baker (1986). However, ambiguities in deficiency breakpoint assignments complicate interpretation.  For example, first loci, which includes Df(1)N12 and Df(1)C246 due to uncertainty at the distal end breakpoints of Df(1)C246 (12D-e; 12A1-2); the trans-acting repressor of ovo maybe located in 11E-F rather than 11D-F. Similarly, for the second loci in this region ambiguity at the distal breakpoint of Df(1)RK2 also cause a dilemma about the location of the trans-acting repressor, since the question was the common region between Df(1)g and Df(1)RK2 was whether in the 12D-E or in the 2E1-2E8 of X-chromosome. On the other hand, the last loci were determined by the only one deficiency strain.  In this case, the problem was whether determination of the loci was accurate enough, or whether another locus is involved in repressing of ovo reporter activity which Df(1)sd72b (13F114B1) may have a common region with.  This deficiency removes several lethal mutations, Myb, sd (scalloped), shi (shibiri), and exd (extradenticle).  Two genes previously cloned in the 13F cytological region are the Drosophila c-myb oncogene homolog (Katzen et al, 1985) and a G protein b-subunit (Yarfitz et al 1988).  It has been suggested that the sd+ gene might be associated with more than one product (perhaps a differential processing) or it might reflect differential tissue and/or temporal regulation (Campbell et al., 1991).

Region 14-20.   In this region eight deficiency strains, Df(1)4b18, Df(1)rD1, Df(1)B, Df(1)N19, Df(1)JA27, Df(1)HF396, DF(1)DCB1, and Df(1) A-209, were tested.  According to measured specific activities Df(1)4b18 (14B8; 14C1) and DF(1) B (15F9=16A6-7) showed significant activating effect on ovo promoter, activity of the former was weaker than that of latter.  Since there is no common region between these two putative trans-acting activators, interpretations of the results gave rise to two loci, 14B8-14C1 and 15F-16A1; 16A6-9. In addition, the Flybase report for Df(1) shows that 70 deletion that breaks within the second exon of the non A (no on or transient A) gene from Stanewsky et al (1993). As a result of X-chromosome screening, 45 deficiency strains were tested and found 17 regions were trans-regulating ovo promoter.  These regions were classified into 12 loci according to their overlapping common regions.  Among these, six, of which were showing trans-acting activator effect, and seven, of which were responsible for trans-acting repressor effect on ovo promoter.   Furthermore, one deficiency strain, Df(1)sc14, showed a significant trans-acting repressor effect in only ovo4B8 strain but not in ovo3U21 strain.  This maybe explained by position effect of P[ovo::LacZ] construct due to landing on P element transposase onto insertion site or by difference between the size of the ovo::LacZ constructs, e.g. ovo3U21 carries 200 bp longer than ovo4B8 at the N-terminal end that may cause a better translation product.  Consequently, among the X-chromosome screening data, it was found that two of the deficiency lines. Df(1)A113 and Df(1)JC70, which are removing ovo and snf along with the several genes due to deletions, and correspond to one loci acting as an repressor, were taking into more detailed investigations.  These results suggested a negative autoregulation mechanism in the ovo promoter.  Therefore, negative autoregulation of ovo was examined with three approaches: ovo point mutations, more defined deficiency strain, and downstream genes.

DISCUSSION

  The sex determination involves complex set of mechanisms.  The fly is chosen to be studied since Drosophila is inexpensive to rear, generates large numbers of progeny, and has nearly a century of accumulated data upon which to design experiments.  Mutational analysis of cell biological and developmental process is relatively simple, even if the resulting mutations are organism-lethal when homozygous.  This is decided advantage over mammalian genetics, in which lethal mutations often die in utero, which complicates the ability to examine and interpret mutant phenotypes. The Drosophila genome is one-twentieth the size of the mammalian genome, making insertional mutagenesis and positional cloning much less difficult.  Additionally, mammalian genetics lacks genetic tools such as balancers that make the maintenance of sterile and lethal-mutations nearly trouble free in Drosophila.  Nematodes have many of the same conveniences as Drosophila, with the added advantage of a highly stereotyped pattern of embryonic (and post-hatching) cell lineages.  The more-regulative character of Drosophila development induces complications lacking from worm genetics, with respect to cellular level analysis of mutant phenotypes.  Perhaps, the most compelling reason to take advantage of the specialized properties of Drosophila, is the extent to which prior studies have shown that genes, proteins, and developmental pathways and processes are conserved among metazoan groups.  We can, with high confidence, study sex determination in Drosophila with a reasonable confidence that what we learn can be extrapolated to other species, including man and his clinical diseases.

  The deletion mapping technique was used to identify the locations of genes that are required for ovo trans-regulation.  Each deficiency line removes several to many genes from the genome.  A sufficiently complete set of overlapping deletions can allow, potentially, every individual trans-acting gene to be localized. Seventeen deficiencies that have effects on the ovo germline promoters are shown in Table 4.1.  Twelve deficiencies showed repressor effects, and five deficiencies showed activator effects.  Deleted regions may affect any of several processes, such as numerator elements, cell viability and differentiation, dosage compensation, and response to inductive signals from soma.  Determination of which gene within a specific region is responsible for the effect on ovo requires more defined deletions or having null alleles for each gene. Estimation of the Number of Trans-Regulators.  Among the seventeen deficiencies in Table 4.1, overlapping common regions identify seven that function as trans-acting repressor loci, and five that function as trans-acting activator loci.  Thus, the entire euchromatic X-chromosome may have as many as ≈10 repressor genes and ≈7 activator genes for the ovo germline promoters.  If these results were extrapolated to the entire fly genome, ≈50 repressors and ≈35 activators of ovo transcription are predicted.  These are underestimates from the data, since any given deleted common region need not remove exactly one relevant gene. Is it reasonable for nearly 85 genes to be involved in regulating the ovo germline promoters?  Precedents from other developmental control systems suggest this is not an implausibly high number.

Regulation of the master sex determination gene Sxl is complex.  To establish somatic sex determination in the early embryo, nine genes are required to activate the Sxl early promoter.  These are sis-a, sis-b, sis-c, run, da, emc, gro, dpn, and her.  In biochemical terms, most are DNA-binding proteins.  In genetic terms, some are positive and are others are negative regulators.  Maintenance of Sxl expression involves positive autoregulation at the level of pre-mRNA alternative splicing.  At least five genes are known to play specific roles in this process: Sxl itself, snf, vir, her, and fl(2)d.  Function of Sxl in the germline is regulated in several ways.  Germline-specific transcriptional control of Sxl is still conjectural, but it is clear that the somatic functioning numerator elements play no role in the germline.  It is possible that ovo may play an important role in germline transcriptional control of Sxl (e.g., Lee. 1998); certainly it has an indirect role (e.g., Oliver et al., 1993).  Splicing-level autoregulation of Sxl is active in the female germline, and it involves the same genes that function in this process in somatic cells.  Once Sxl protein is produced in female germline cells, the otu protein plays an important role in this relocalization into the nucleus.  Thus, a minimum of sixteen genes is required for proper regulation of Sxl.

Establishment of the body plan in Drosophila is also under complex transcriptional control.  Maternally localized RNA and protein molecules establish the gross body axes: anterior-posterior and dorsal-ventral.  Hierarchically organized sets of zygotically activated genes are transcribed, and their protein products serve to refine the body axes into progressively finer-grained structures.  The metameric anterior-posterior body axis is specified by so-called gap genes, pair rule genes, and segment polarity genes, which create the segment-sized repeating units of the body.  Homeotic genes encoded by the Antennapedia Complex (ANT-C) and bithorax Complex (BX-C) then confer position-specific identities upon each segment. During the cellular blastoderm stage, gap genes and maternal coordinate genes regulated the activation of primary pair rule genes such as even-skipped (eve).  These are expressed in seven one-segment-wide stripes that alternate with on-segment-wide regions of non-expressing cells.  For example, the second stripe of eve expression is positively regulated by hunchback and bicoid, and negatively regulated by giant and Kruppel.  All four proteins directly bind to a 500-bp-long “eve-stripe 2 enhancer.”  Binding have giant and Kruppel is competitive with binding of hunchback  and bicoid, and vice versa.  Thus, spatially controlled concentrations of giant, Kruppel, bicoid, and hunchback proteins result in spatially restricted activation or repression of the eve stripe 2 enhancer.  The remaining six stripes of eve expression are similarly controlled by other DNA-binding proteins, which are acting another discrete stripe-specific enhancers. Ectopic expression of homeotic genes can have disastrous effects on development.  Thus, a special heterochromatin-like mechanism functions to ensure that ANT-C and BX-C genes are inactive in cells and tissues that do not require their expression.  Stable repression is mediated by the Polycomb class of proteins, which number over forty. Each of these examples illustrates that developmental control of individual gene transcription is mediated by both positive and negative effectors, and that sometimes the number of such upstream regulators numbers between one and several dozen.  Thus, our estimate of 85 regulators of the ovo germline promoters is not out of line with other developmentally regulated systems.

Evaluation of Candidate Loci Within Common Regions.   Based overlapping cytology, seventeen deficiencies that affected the ovo germline promoter fell into twelve common regions.  Each of these will be discussed in turn below. Of particular interest was the relationship each of our trans-acting may have with Su(ovoD) and E(ovoD) loci identified in a generic screen by Pauli et al. (1995).  In general, it is not straightforward to suggest identities between Su(ovoD) or E(ovoD) loci and our trans-acting repressor or activator loci because of the dissimilar means of assaying these gene-dose-sensitive interactions.  We use quantitative measures of LacZ reporter activity as a proxy for ovo transcription, while Pauli et al. (1995) use semi-quantitative measures of vitellogenesis.

Region 1 (polytene bands 1A1; 2A1-4):  The distal region of the X-chromosome showed a trans-regulating activator effect on the ovo promoters.  This region includes the acheate-scute complex (AS-C), home of the X-chromosome numerator element sis-b (Cline, 1988; Parkhurst and Ish-Horowicz, 1990), also known as scute-T4.  This numerator has no function in the female germline (Granadino et al., 1989).  Pauli et al., (1995), using other deficiency strains affecting this section of the X-chromosome, identified a strong Su(ovoD) locus in the polytene region 1E3-4; 2B3-4 that may correspond with our trans-activator.  Flybase indicates that this region contains over 100 genes, among them 23 unassigned open reading frames, 33 genes defined by apparent visible mutations, 53 lethal genes,, and two female sterile loci.

Region 2 (polytene bands 4C15-16; 4F15):  This region includes the ovo and snf loci, and was identified by Pauli et al., (1995) as a strong E(ovoD) due to the effects of these loci.  Further discussion is deferred to mechanism of ovo autoregulation, which deal with ovo negative regulation. Region 3 (polytene bands 5C3-5; 5E8):  This region has a trans-regulatory activation effect on the ovo germline promoters.  Deficiency for this region showed no interaction with ovoD in the vitellogenesis assay (Pauli et al., 1995).  Examination of Flybase records for this region reveals over twenty genes, and no strong candidates that may account for the interaction with the ovo promoters.

Region 4 (polytene bands 7D10; 8A4-5):  Results  showed that this region contains a transacting-repressor of ovo germline promoter activity.  This region reported by Pauli et al. (1995) to contain a strong E(ovoD) locus, which was identified as the ovarian tumor gene (Pauli et al., 1993, 1995).  It is virtually certain that the repressor-of-ovo is distinct from otu.  First, the otu protein is cytoplasmic and plays a role in egg chamber cytoskeletal function (Nagoshi et al., 1997).  Second, the ovo protein binds to the otu promoter in vitro (Garfinkel et al., 1997; Lee, 1998, Lee and Garfinkel 1998; Lu et al., 1998).  Third, under certain conditions, in vivo activity of the otu promoter is dependent upon ovo protein production (Hager and Cline, 1997; Lu et al., 1998).  Examination of Flybase reveals that this region contains fifty genes mutable to lethal, visible, or female-sterile phenotypes, but none appear to be a strong candidate for the repressor-of-ovo locus.

Region 5 (polytene bands 8B5-8; 8DE):  This region also has an apparent repressor of ovo germline promoter activity.  Deficiency for this region showed no interaction with ovoD mutations in the Pauli et al. (1995) vitellogenesis assay.  Examination of Flybase reveals that this region contains thirty genes mutable to lethal, visible, or female sterile phenotypes.  One gene stands out as a candidate for the repressor, namely, lozenge.  This is a complex locus that is mutable to female sterility (Green and Green, 1949, 1956), and it is named for a reduced-eye, smoothened-eye, mutant phenotypes.  Interestingly, certain ovo-mutant alleles are called “lozenge-like” in recognition of a similar eye defect (Oliver et al., 1987; Mevel-Ninio et al., 1989; Garfinkel et al., 1992).  The lz gene codes for a transcription factor (Dag et al., 1996). Region 6 (polytene bands 9C4; 9D1-2):  The cytological assignment of this region is based on the overlap of three deficiencies:  Df(1)N110, Df(1)H133, and Df(1)v L11.  Together, they mark a trans-acting repressor of ovo promoter activity.  According to  Pauli et al. (1995), only two of these three deficiencies behaved as if they exposed an E(ovoD) locus, while the third had no effect.  In combination with positive results from other deficiencies, Pauli et al. positioned the E(ovoD) locus at cytological region 9E-F.  Thus, it is again possible that the repressor-of-ovo we identified is distinct from a nearby E(ovoD) locus, and is among the half-dozen loci identified by Flybase as mapping into this interval.

Region 7 (polytene bands 10A6; 10F6-7):  This region contains a trans-acting repressor of ovo promoter activity.  According to Pauli et al. (1995), the defining deficiency had no significant interaction with ovoD alleles.  Examination of Flybase reveals that this region includes the somatic X-chromosome numerator element sis-a, which also has no function in germline development (Granadino et al., 1989, 1990, 1997).  Given the extent of this region, it is not  surprising that Flybase identifies 65 genes with diverse phenotypes and biochemical roles; however no strong candidate locus that may count for the repressor-of-ovo locus is apparent.

Region 8 (polytene bands11D1-2; 11F1-2):   This region contains perhaps the strongest trans-acting repressor of ovo promoter activity in the survey: deficiency heterozygous experimentals had 2-2.5 fold more lacZ specific activity in their ovaries that the balancer carrying controls.  According to Pauli et al (1995), one of the two deficiencies defining this common region showed a statistically weak enhancement of ovoDalleles, while the other had a significant Su(ovoD) phenotype.  Likewise, Belote et al. (1985) and Scott and Baker (1986) reported that the same deficiency later shown to have Su(ovoD) activity also interacted with loci in the somatic sex determination pathway.  It is an open question how these three results relate to one another.  Among sixteen genes that map into this region are two signal transduction loci: the Mek3 gene, a serine-threonine-specific protein kinase in the MAP kinase pathway, and a beta subunit of the heterotrimeric GTP-binding protein. A solitary female-sterile, fs(1) K4, also maps roughly into this region; it is germline-dependent, and yields fragile eggs, a phenotype occasionally seen in the eggs laid by ovoD3/+ females.

Region 9 (polytene bands 12D2-12E1; 12E8):  This region contains a trans-acting repressor of ovo promoter activity.  According to Pauli et al. (1995), neither deficiency defining this common region interacted with ovoDalleles.  This region contains the yolkless gene (DiMario et al., 1987), which has been cloned and codes for a member of 35 known genes, including a cluster of tRNA genes, the male-germline-specific Stellate genes, and several lethal and female-sterile genes.

Region 10 (polytene bands 13F1; 14B1):  This region contains a trans-acting repressor of ovo promoter activity.  Again, no significant interaction with ovoD allel4es was observed by Pauli et al. (1995).  Podry, Katzen and others have extensively mutagenized this region due to its containing shibiri (the Drosophila homolog of dynamin), c-myb, another Gb subunit, and the homeodomain protein extradenticle.  Their work revealed a total of twenty lethal genes, ten apparent visibles, and over a half-dozen unassigned open reading frames.

Region 11 (polytene bands 14B8; 14C1):  This region contains a trans-acting activator of ovo promoter activity.  According to Pauli et al., (1995), the defining deficiency had no significant interaction with ovoD alleles.  This region is surprisingly dense genetically, as it apparently contains over forty genes.  Several behavioral genes coding for neuronal functions map here, including nonA, paralytic, and easily shocked.  The nonA gene codes for an RNA-binding protein, and is mutable to a variety of phenotypes including recessive lethality, male-courtship-strong abnormalities, and defective vision.  The location of para (a sodium channel) is particularly intriguing since parats mutations fail to complement certain napts alleles, and nap genetically overlaps the dosage compensation function maleless.  Mutations in maleless are unique among the known dosage compensation loci in having a mutant phenotype in germline clones, and they are said to suppress the female-germline-lethality of ovo null mutations.  The easily shocked locus codes for ethanolmine kinase, and mutations at this locus also interact with mle.

Region 12 (polytene bands 15F9-16A1; 16A7):  This region contains a trans-acting activator of ovo promoter activity.  According to Pauli et al. (1995), the defining deficiency had no significant interaction with ovoDalleles.  Examination of Flybase reveals that this region contains at least a dozen female-sterile loci, a dozen lethal loci (including the Bar homeodomain protein gene). There is an ambiguity in compared mean of activities.  According to the negative autoregulation mechanism, there suppose to be a linear decrease pattern correlated to increase in copy of ovo.  However, the pattern of the gene dose was reaching plato, when three copies of ovo were present in the genome. Yet, this also shows that there is a protection mechanism that counts the number of ovo versus number of X chromosome exists.  Therefore, the sex determination mechanism turns off the extra ovo in the system immediately. 

Consequently, the system prohibits more wrong information to be processed according to its default setting where if the X:A ratio equals to one the outcome is going to be prepared as female, if not turn off the mechanism towards male-like, sterile mode, or death at the embryonic stage.  This discontinuity in the linear correlation may be due to position effect of P[w+ ovo+].  Future Directions and Concluding Remarks The results of this study suggest that the ovo germline promoters are regulated by a large set of upstream factors.  Nearly a dozen of these maps to the X-chromosome, some to region that are well characterized genetically.  Further deficiency mapping experiments, and assessment of the phenotypes of single-P insertion lines with female-sterile or perhaps lethal phenotypes, would be required to identify the relevant genes.  Some regions contain candidate loci that have been cloned (e.g. lozenge); in this example, either in vitro DNA-binding experiments using Lz protein and the ovo promoter region, or computational assessment of the likelihood that the ovo promoter contains binding sites for Lz can be done. Another potential upstream factor not assessed in these experiments is the ecdysone regulatory hierarchy.  The steroid ecdysone is the endocrine hormone that controls molting and metamorphosis in arthropods.  It is an allosteric effector for a heterodimeric receptor of the steroid-receptor superfamily.  The ovaries of adult females manufacture their own ecdysone, and the gene for the rate-limiting steroidogenic enzyme transcribed beginning in Stage 7-8 egg chambers.  This stage immediately precedes the onset of the highest level of ovo transcription (Mevel-Ninio et al., 1991; Garfinkel et al., 1994).  Mutations in the E74 and E75 genes, when made homozygous in germline clones, cause arrest of oogenesis at Stage 7-8, as if egg chambers are unable to respond to endogenous ecdysone and continue differentiation.  Both E74 and E75 code for transcription factors that are induced as immediate-early primary responses to added ecdysone both in-vivo and in tissue culture assays.  Thus, it is reasonable to suggest that one or both of these proteins will bind to the ovo germline promoter in an in vivo effect on expression of the ovo::lacZ reporter using the methods established in this dissertation.  

Acknowledgement:  This work had been comppleted in the laboratory of Dr. Mark Garfinkel at Illinois Institute of Technology.   Dr. Demet Sag initiated the project with her own  ideas, was fully supported by Turkish National Merit Fellowship, and  earned NATO Advanced Science institute  Grant on Genome Structure and Functional Genomics, Elba Island, Italy, accepted to work with Dr. Mevel Ninio, based on the proposal submitted by Demet Sag on Molecular Mechanism of  ovo, through EMBO long term scholarship in France.

BIBLIOGRAPHY
  1. Ashburner, M., Drosophila Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 317-318, 1989
  2. Baker, B.S., “Sex in flies: the splice of life,” Nature 340, pp.521-524, 1989.
  3.  Baker, B.S. and Belote, J.M., “Sex determination an dosage compensation in Drosophila melanogaster” Ann. Rev of Genetics 17, pp345-393, 1983.
  4. Baker B.S. and Ridge, K.A., “Sex and the single cell I. On the action of major loci affecting sex determination in Drosophila melanogaster,” Genetics 94, pp.383-423, 1980.
  5.  Bell, L.R. Horabin, J.I., Schedl, P., and Cline, T.W., “Sex-lethal, a Drosophila sex determination switch gene, exhibits sex specific RNA spilicing and sequence similarity to RNA binding proteins,” Cell 55, pp.1037-1046, 1988.
  6. Bell, L.R. Horabin, J.I., Schedl, P., and Cline, T.W., Positive autoregulation of Sex-lethal by alternative spilicing maintains the female determined state in Drosophila, Cell Vol 65, pp.229-239, 1991.
  7.  Belote, J.M., Handler, A.M., Wolfner, M.F., Livak, K.J., and Baker, B.S., Sex-specific regulation of yolk protein expression in Drosophila, Cell 40, pp.339-348, 1985.
  8.  Bohringer- Manheim Product Support, Chloroform Red-beta-D_Galactopyranoside sodium salt (CPRG) provided by ID#0223p, June 1986 dated information, fax received on 2/26/1996.
  9.  Bopp, D., Bell, L.R., Cline, T.W., Schedl, P. Developmental distribution of female specific Sex-Lethal proteins in Drosophila melanogaster, Genes and Development 5, pp.403-415,, 1991.
  10.  Bopp, D., Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila, Development 122, pp.971-982, 1996.
  11.  Bopp, D., Horabin, J.I., Lersch, R.A., Cline, T.W., Schedl, P., Expression of the Sex-lethal gene is controlled at multiple levels during Drosophila oogenesis, Development 118, pp.797-812, 1993.
  12.  Bradford, M. M., A Rapid and sensitive method for the Qantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Analytical Biochemistry 72, pp.248-254, 1976.
  13.  Bridges, C.B., Sex in relation to chromosomes, Am. Nat.59, pp.127-137, 1925.
  14.  Bridges, C.B., Triploid intersexes in Drosophila melanogaster, Science 54, pp.252-254, 1921.
  15.  Bridges, C.B., Non-disjunction as proof of the chromosome theory of heredity, Genetics 1, pp.1-52, 1916.
  16.  Burtis, K.C. and Baker, B.S., Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoded related sex-specific yolk protein gene enhancer, The EMBO J., Vol. 10, No. 9, pp.2557-2582, 1991.
  17.  Busson, D., Gans, M., Komitopoulou, and Masson, M., Genetic Analysis of three dominant female-sterile mutations located on the X-chromosome of Drosophila melanogaster, Genetics 105, pp.309-325, 1983.
  18.  Campbell, S.D., Duttoroy, A., Katzen, A.L., and Chovnick, A., Cloning and characterization of the scalloped region of Drosophila melanogaster, Genetics 127, pp.367-380, 1991.
  19.  Cline, T.W., The Drosophila sex determination signals: how do flies count two?, Ann. Rev. Genet 30, pp637-702, 1996.
  20.  Cline, T.W., Evidence that sisterless-a and sisterless-b are two of several discrete numerator elements of the X/A ratio sex-determination signal in Drosophila that switch Sxl between two alternative stable expression states, Genetics 119, pp.829-862, 1988.
  21.  Cline, T.W., Re-evaluation of the functional relationship in Drosophila between a small region on the X-chromosome (3E8-4F11) and the sex determination gene, Sex-lethal, Genetics 116, s:12, 1987.
  22.  Cline, T.W., A female specific lethal lesion in an X-linked positive regulator of the Drosophila sex determination gene, Sex-lethal, Genetics 113, pp.641-663, 1986.
  23.  Cline, T.W., Autoregulatory functioning of a Drosophila gene product that establishes and maintains the sexuality determined state, Genetics 107, pp.231-277, 1984.
  24.  Cline, T.W., Functioning of the gene daughterless and Sex-lethal in Drosophila germ cells, Genetics 107, s16-17, 1983.
  25.  Cline, T.W., A sex specific temperature-sensitive maternal effect of the daughterless mutation of Drosophila melanogaster, Genetics 84, pp.723-742, 1976.
  26.  Cronmiller, C., Schedl, P., Cline, T.W., Molecular characterization of daughterless, a Drosophila sex determination gene with multiple roles in development, Genes Dev. 2: 155-167, 1988.
  27.  Despande, G., Samuels., M.E., and Schedl, P.D. “Sex-lethal interacts with splicing in vitro and in vivo, Molecular and Cellular Biology, Vol. 16, No 8, pp. 5036-5047, 1996.
  28.  Despande, Stukey, J., and Schedl, P., scute (sis-b) function in Drosophila sex determination, Moll. Cell Biology 15, pp. 4430-4440, 1995.
  29.  DiMario, P.J., and Mahowald, A.P., Female sterile (1) yolkless: A recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within the developing oocytes, J. Cell Biology 105: 199-206, 1987.
  30.  Erickson, J.W. and Cline, T.W., A bZIP protein, sisterless-a, collaborates with bHLH transcription factors early in Drosophila development to determine sex, Genes and Dev. 7: 1688-1702, 1993.
  31.  Erickson, J.W. and Cline, T.W., Molecular nature of the Drosophila sex determination signal and its link to neuorogenesis, Science 251, pp. 1071-1074, 1991.
  32.  Estes, P.A., Keyes, L.N., and Schedl, P., Multiple response elements in the sex-lethal early promoter ensure its female-specific expression pattern, Mol Cell Biol 15, pp. 904-917, 1995.
  33.  Flickinger, T.W. and Salz, H.K., The Drosophila sex determination gene snf encodes a nuclear protein with sequence and functional similarity to the mammalian U1A snRNP, Gene and Development 8, pp. 914-925, 1994.
  34.  Flybase, The Drosophila genetic database, http://www.flybase.bio.indiana.edu, 1998.
  35.  Gans, M.C., Audit and Massson, M., Isolation and characterization of sex-linked female-sterile mutations in Drosophila melanogaster, Genetics 81, pp. 683-704, 1975.
  36.  Garfinkel, M.D., Lee, S., and Sigar, I., DNA-binding targets of the Drosophila melanogaster OVO protein, 38th Annual Drosophila Research Conference, Chicago, IL, 1997.
  37.  Garfinkel, M.D., Wang, J., Liang, Y., and Mahowald, A. P., Multiple products from the shavenbaby-ovo gene region of Drosophila melanogaster: relationship to genetic complexity, Molecular and Cell Biology, Vol. 14, No., 10, pp. 6809-6818, 1994.
  38.  Garfinkel, M.D., Lohe, A.H., and Mahowald, A.P., Molecular genetics of the Drosophila melanogaster ovo locus, a gene required for sex determination of germline cells, Genetics 130, pp. 791-803, 1992.
  39.  Gollin, S. M. and King, R. C., Studies on fs(1)1621, a mutation producing ovarian tumors in Drosophila melanogaster, Developmental Genetics 2, pp. 203-218, 1981.
  40.  Granadino, B., Compuzano, S., and Sanchez, L., The Drosophila melanogaster fl(2)d, a gene needed for Sex-lethal expression in Drosophila melanogaster, Genetics 130, pp. 597-612, 1990.
  41.  Granadino, B., Santamaria, P., and Sanchez, L., Sex-determination in the germ line of Drosophila melanogaster: activation of the gene Sex-lethal, Development 118, pp. 813-816, 1993.
  42.  Granadino, B., Juan, A. B. S. B, Santamaria, P., Sanchez, L., Distinct mechanisms of splicing regulation in vivo by the Drosoophila protein Sex-lethal, PNAS USA, 94, pp. 7343-7348, 1997.
  43.  Hager, J.H. and Cline, T.W., Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex-determination, Development 124, pp. 5033-5048, 1997.
  44.  Hilfiker, A., Amrein, H., H., Dobendorfer, A., Schneiter, R, and Nuthiger, R., The gene virilizer is required for female-specific splicing controlled by Sxl, master gene for development in Drosophila, Development 121, pp. 4017-4026, 1995.
  45.  Horabin, J.I., Bopp, D., Waterburry, J., and Schedl, P., Selection and maintenance of sexual identity in the Drosophila melanogaster, Genetics 141, pp. 1521-1565, 1995.
  46.  Horabin, J. I. And Schedl, P., Regulated spilicing of the Drosophila Sex-lethal male exon involves a blockage mechanism, Moll. Cell. Biol. 13, pp. 1408-1414, 1993.
  47.  Horabin, J.L., and Schedl, P., Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5’ splice site, Moll. Cell. Biol. 13: pp. 7734-7746, 1993.
  48.  Hoshijima, K., Kohyama, A., Watakabe, I., Inonue, K., Sakamato, H., and Shimura, Y., Transcriptiuonal regulation of the Sex-lethal gene by helix-loop-helix proteins, Nucleic Acids Res. 23, pp. 3441-3448, 1995.
  49.  Inonue, K., Hojhijima, K., Sakamato,, H., and Shimura, Y., Binding of the Drosophila Sex-lethal gene product to the alternative splice site of transformer primary transcript, Nature 344, pp. 461-463, 1990.
  50.  Keyes, L.N., Cline, T.W., and Schedl, P., The primary sex-determination signal of Drosophila acts at the level of transcription, Cell, Vol. 68, pp. 933-943, 1992.
  51.  Komitopoulou, K., Gans, M., Margaritis, L.H., Kafatos, F.C., and Masson, M., Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster with special attention to eggshell mutants, Genetics 105: 897-921, 1983.
  52.  Lee, S., DNA binding targets of the Drosophila melanogaster OVO protein, PhD Dissertation, Illinois Institute of Technology, Chicago, IL, USA, 1998.
  53.  Lee, S. and Garfinkel, M.D., DNA-binding targets of the Drosophila melanogaster OVO protein, Nucleic Acid. Res.
  54.  Linsley, D.L., and Zimm, G., The genome of the Drosophila melanogaster, Academic Press, San Diego, New York, 1980.
  55.  Lu, J., Andrews, J., Pauli, D., and Oliver, B., Drosophila OVO zinc finger protein regulates ovo and ovarian tumor target promoters, Dev. Genes. Evol., pp. 1-10, 1998.
  56.  Luccesi, J.C. and Manning, E., Gene dosage compaensation in Drosophila melanogaster, Adv. Genetics 24, pp. 371-429, 1987.
  57.  Madl, J.E., and Herman, R.K., Polyploids and sex determination in Caenornabtidis elegans, Genetics 93, pp. 393-402, 1979.
  58.  Mevel-Ninio, M., Mariol, M.C. and Gans, M., Mobilization of the gypsy and copia retrotransposans in Drosophila melanogaster induces reversion of the ovoD dominant female-sterile-mutations: molecular analysis of revertant alleles, EMBO J. 8, pp. 1549-1558, 1989.
  59.  Mevel-Ninio, M., Terracol, R., and Kafatos, F.C., The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development, EMBO J. 10, pp.2259-2266, 1991.
  60.  Mevel-Ninio, M., Guenal, I., and Limburg-Bouchen, B., Production of dominant female sterility in Drosophila melanogaster by insertion of the ovoD1 allele autosomes: use of transformed starins to generate germline mosaic, Mechanism of development 45, pp. 155-162, 1994.
  61.  Mevel-Ninio, M., Terracol, R., Salles, C., Vincent, A., and Payre, F., ovo, a Drosophila gene required for ovarian development, is specially expressed in the germline and shares most of its coding sequences with shavenbaby, a gene involved in embryo patterning, Mecahnism of Development 49, pp. 83-95, 1995.
  62.  Mevel-Ninio, M., Fouilloux, E., Genal, I.  and Vincent, A., The three point dominant female-staerile mutations of Drosophila ovo gene are point mutations that create new translation-initiatorAUG codons, Development 122, pp. 4131-4138, 1996.
  63.  Mohler, J.D., Developmental genetics of the Drosophila egg. I.: Identification of 50 sex-linked cistrons with maternal effects on embryonic development, genetics 85, pp. 259-272, 1977.
  64.  Nagai, K., Oubridge, C., Jessen, T. H., Li, J., and Evans, P.R., Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A, Nature 348, pp. 515-520, 1990.
  65.  Nagoshi, R.N., McKeown, M., Burtis, K.C., Belote, J.M., and Baker, B., The control of alternative spilicing at genes regulating differentiation in D. melanogaster, Cell, Vol. 53, pp.229-236, 1988.
  66.  Nagoshi, R.N., and Baker, B., Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex specific RNA splicing patterns, Genes and Development 4, pp. 89-97, 1990.
  67.  Nagoshi, R.N., Patton, J.S., Bae, E., and Geyer, P., The somatic sex determines the requirement for ovarian tumor gene activity in the proliferation of the Drosophila germline, development 121, pp.579-587, 1995.
  68.  Nothiger, R., and Steinmann-Zwicky, M., Meier-Gerschwiller, P., and Weber, T., Sex determination in the germline of Drosophila depends on genetic signals and inductive somatic factors, development 107, pp.505-518, 1989.
  69.  Oliver, B., Singer, J., Laget, V., Pennetta, G. and Pauli, D., Function of Drosophila melanogaster ovo– in germ line sex determination depend on X-Chromosome number, Development 120, pp.1-11, 1994.
  70.  Oliver, B., Kim, Y. and Baker, B., Sex-lethal, master and slave: a hierarchy of germ line sex determination in Drosophila, Development 119, pp. 897-908, 1993.
  71.  Oliver, B., Pauli, D., and Mahowald, A.P., Genetic evidence that the ovo locus is involved in Drosophila germ line sex determination, Genetics 125, pp. 535-550, 1990.
  72.  Oliver, B., Perrimon, N, an Mahowald, A.P., The ovo locus is required for sex-specific germ line maintenance in Drosophila, Genes and Development 1, pp. 913-923, 1987.
  73.   Oubridge, C., Ito, N., Evans, P.R., Teo, C.H., and Nagai, K., Crystal structure at the 1.92A resolution of the RNA binding domain of the U1A splicesomal protein completed with an RNA hairpin, Nature 372, pp.432-438, 1994.
  74.  Pauli, D., Oliver, B., and Mahowald, A.P., Identifications of regions interacting with ovo D mutations: potential new genes involved in germline sex determination in Drosophila melanogaster, Genetics 139, pp.713-732, 1995.
  75.  Pauli, D., Oliver, B., and Mahowal, A.P., The role of the ovarian tumor locus in Drosophila melanogaster germ line sex determination, Development 119, pp.123-134, 1993.
  76.  Pauli, D. and Mahowald, A.P., Germline sex determination in Drosophila melanogaster, Trends in Genetics, Vol. 6, No. 8, pp.259-264, 1990.
  77.  Parkhurst, S.M., Bopp, D., and Ish-Horowicz,  X:A ratio, the primary sex determining signal in Drosophila, is transduced by helix-loop-helix proteins, Cell, Vol. 63, pp.1179-1191, 1990.
  78.  Perrimon, N., Mohler, D., Engsttrom, L., and Mahowald, A.P., X-linked female-sterile loci in Drosophila melanogaster, Genetics 113, pp.695-712, 1986.
  79.  Perrimon, N., Engstrom, L., and Mahowald, A.P., The effects of zygotic lethal mutations on female-germ-line functions in Drosophila, Developmental Biology 105, pp. 404-414, 1984.
  80.  Perrimon, N., Clonal analysis of dominant female-sterile, germline-dependent mutations in Drosophila melanogaster, Genetics 108, pp.927-939, 1984.
  81.  Perrimon, N. and Gans, M., Clonalo analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female sterile mutation Fs(1)K1237, Developmental Biology 100, pp. 365-373, 1983.
  82.  Rodesch, C., Geyer, P.K., Patton, J.S., Bae, E., and Nagoshi, R.N., Developmental analysis of the ovarian tumor gene during Drosophila oogenesis, Genetics 141, pp.191-202, 1995.
  83.  Sag-Ozkol, D., Tekin, S., Garfinkel, M.D., Gene-dose sensitive trans-acting regulators of the Drosophila melanogaster germline promoter, 38th Annual Drosophila Research Conference, Chicago, IL, USA, 1997.
  84.  Sag-Ozkol, D., and Garfinkel, M.D., Negative autoregulation of Drosophila melanogaster female germline specific gene, ovo (in preparation).
  85.  Sag-Ozkol, D., and Garfinkel, M.D., X-chromosome screening of Drosophila melanogaster to find numerator elements of germline sex determination (in preparation).
  86.  Salz, H.K. and Flickinger, T.W., Both loss of function and gain-of-function mutations in snf define a role for snRNP proteins in regulating Sex-lethal pre-mRNA splicing in Drosophila development, Genetics 144, pp.95-108, 1996.
  87.  Salz, H.K., Maine, E.M., Keyes, L.N., Samuels, M.E., Cline, T.W., and Schedl, P., The Drosophila female-specific sex-determination gene, Sex-lethal has stage-, tissue-, and sex-specific RNAs suggesting multiple models of regulation, Genes and Development 3, pp.708-709, 1989.
  88.  Salz, H.K., Cline, T.W., and Schedl, P., Functional changes associated with structural alterations induced by mobilization of a p element inserted in the Sex-lethal gene of Drosophila, Genetics 117, pp.221-231, 1987.
  89.  Sanchez, L., Granadino, B., and Torres, M., Sex determination in Drosophila melanogaster, X-linked genes involved in the initial step of Sex-lethal activation, Developmental Genetics 15: 251-264, 1994.
  90.  Sass, G., Mohler, J.D., Walsh, R.C., Kalfayan, L.J. and Searles, L.L., Structure an the expression of hybrid dysgenesis-induced alleles of the ovarian-tumor (otu) gene in Drosophila melanogaster, Genetics 133, pp.253-263, 1993.
  91.  Sass, G., Comer, A.R. and Searles, L.L., The ovarian tumor protein isoforms of Drosophila melanogaster exhibit differences in function, expression, and localization, developmental Biology 167, pp.201-212, 1995.
  92.  Schedl, A, Ross, A., Lee, M., Engelkamp, D., Rashbass, van Heyningen, V., and Hastie, N., Influence of PAX6 gene dosage on development: over-expression causes sever eye abnormalities, Cell 86, pp.71-82, 1992.
  93.  Schupbach, T., and Wieschhaus, E., Female sterile mutations on the second chromosome of Drosophila melanogaster II mutations blocking oogenesis an altering egg morphology, Genetics 129, pp.1119-1136, 1991.
  94.  Shupbach, T., an Wieschaus, E., Female sterile mutations on the second chromosome of Drosophila melanogaster I. Maternal effect mutations, Genetics 121, pp.101-17, 1989.
  95.  Schupbach, T., Normal female germ cell differentiation requires the female X-chromosome to autosome ratio and expression of Sex-lethal in Drosophila melanogaster, Genetics 109, pp.529-548, 1985.
  96.  Simon, J.A. and Lis, J.T., A germline transformation analysis reveals flexibility in the organization of the heat-shock consensus elements, Nucleic Acids Research, Vol 15, No.7, 1987.
  97.  Staab, H., Heller, A., Steinmann-Zwicky, M., Somatic sex determining signals act on XX germ cells in Drosophila embryos, Development 122, pp.4065-4071, 1996.
  98.  Staab, H., and Steinmann-Zwicky, M., Female germ cells of Drosophila require zygotic ovo and out product for survival in larvae and pupae, Mech. Dev. 54, pp.205-210, 1995.
  99.  Stanewsky, R., Rendahl, K.G., Dill, M., and Saumweber, H., Genetic and molecular analysis of the X-chromosomal region 14B17-14C4 in Drosophila melanogaster: Loss of function in NONA, a nuclear protein common to many cell types, results in specific physiological and behavioral defects, Genetics 135, pp.419-442, 1993.
  100.   Steinman-Zwicky, M., Sex determination of the Drosophila germ line: tra and dsx control somatic inductive signals, Development 120, pp. 707-716, 1994.
  101.  Steinman-Zwicky, M., Sxl in the germline of Drosophila: A target for somatic late induction, Developmental Genetics 15, pp.265-274, 1994.
  102.  Steinman-Zwicky, M., Sex determination in Drosophila: sis-b, a major numerator element of the X:A ratio in the soma, does not contribute to the X:A ratio in germ line, Development 117, pp. 763-767, 1993.
  103.  Steinman-Zwicky, M., How do the germ cells choose their sex? Drosophila as a paradigm, Bioassays 14 (8), pp.513-518, 1992.
  104.  Steinman-Zwicky, M.,  Anrein, H. and Nothiger, R., Genetic control of sex determination in Drosophila, Advanced Genetics 27, pp.189-237, 1990.
  105.  Steinman-Zwicky, M.,  Schmid, H. and Nothiger, R., Cell-autonomous an inductive signals can determine the sex of the germ line of Drosophila by regulating the gene Sxl, Cell, Vol. 57, pp.157-166, 1989.
  106.  Steinman-Zwicky, M., Sex determination in Drosophila. The X-chromosomal gene liz is required for Sxl activity, The EMBO Journal 7, pp.3889-3898, 1988.
  107.  Steinman-Zwicky, M. and Nothiger, R., The small region on the X chromosome of Drosophila regulates a key gene that controls sex determination and dosage compensation, Cell, Vol. 42, pp.877-887, 1985.
  108.   Sosnowski, B. A., Belote, J. M. and McKeown, M., Sex specific alternative spilicing of RNA gene results from sequence-dependent splice site blockage, Cell, Vol. 3, pp.449-459, 1989.
  109.   Yarfitz, S., Provost, N. M., and Hurley, J. B., Cloning of Drosophila melanogaster guanine nucleotide regulatory protein subunit gene and characterization of its expression during development, PNAS USA 85, pp.7134-7138, 1988.
  110. Wieschaus, E., Audit, C., and Masson, M., A clonal analysis of the rules of somatic cells and germline during oogenesis in Drosophila, Developmental Biology 88, pp.92-103, 1981.
  111.  Wieschaus, E., Nusslein-Volhard, C., an Jurgen, G., Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Part III. Zygotic loci on the X-chromosome and fourth chromosome, Roux. Arch. Dev. Biol., 193, pp.296-307, 1984.

FIGURES and TABLES:

Figure 1: Sex determination of D. melanogaster (1998)

Figure 2: Somatic-Germline Interactions. (1998)

Figure 3: Molecular Structure of the ovo locus

Figure 4: In vivo Biochemical_genetic Assay for Regulators

Figure 5: ovo-LacZ Reporter Construction. (1998)

Figure 6 : Establishing Stocks From Duplication Carrying Lines.

Figure 7: Control Assay for b-galactosidase Assay. (1998).

Table 1: List of Stocks for X-chromosome Screening (1998)

Table 2: Stocks Made in This Study for X-Chromosome Screening

Table 3: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo3U21

Table 4: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo4B8 (Results)

Table 5: Deficiency Lines Affecting the ovo Gene Activity (X-chromosome screening result)

 

Previously Posted:  

ovo Female Germline Specific Drosophila melanogaster Gene has two auto-regulation mechanism: negative and positive

Blavatar

 

 

Read Full Post »

What is the Future for Genomics in Clinical Medicine?


What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Introduction

This is the last in a series of articles looking at the past and future of the genome revolution.  It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE.  But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down.  The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts.  What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways.  This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.  I illustrate this with some very new observations.

Gutenberg Redone

The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.

Topol_splash_image

In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.

We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.

For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.

The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.

I shall however, use some new information that gives real cause for joy.

Reprogramming Alters Cells’ Fate

Kathy Liszewski
Gordon Conference  Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”  They report two critical steps both of which are needed for exploring epigenetic reprogramming.  “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.  Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus st...

Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

English: Embryonic Stem Cells. (A) shows hESCs...

English: Embryonic Stem Cells. (A) shows hESCs. (B) shows neurons derived from hESCs. (Photo credit: Wikipedia)

Transforming growth factor beta (TGF-β) is a s...

Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)

Pioneer Transcription Factors

Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

ME Hubbi, K Shitiz, DM Gilkes, S Rey,….GL Semenza. Johns Hopkins University SOM
Sci. Signal 2013; 6(262) 10pgs. [DOI: 10.1126/scisignal.2003417]   http:dx.doi.org/10.1126/scisignal.2003417

http://SciSignal.com/A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication/

Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).

Identification of a Candidate Therapeutic Autophagy-inducing Peptide

Nature 2013;494(7436).    http://nature.com/Identification_of_a_candidate_therapeutic_autophagy-inducing_peptide/   http://www.ncbi.nlm.nih.gov/pubmed/23364696
http://www.readcube.com/articles/10.1038/nature11866

Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.

Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of

Histone H4 and Is Associated with Silent Chromatin

K Nishioka, JC Rice, K Sarma, H Erdjument-Bromage, …, D Reinberg.   Molecular Cell, Vol. 9, 1201–1213, June, 2002, Copyright 2002 by Cell Press   http://www.cell.com/molecular-cell/abstract/S1097-2765(02)00548-8

http://www.sciencedirect.com/science/article/pii/S1097276502005488           http://www.ncbi.nlm.nih.gov/pubmed/12086618
http://www.cienciavida.cl/publications/b46e8d324fa4aefa771c4d6ece4d2e27_PR-Set7_Is_a_Nucleosome-Specific.pdf

We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Next-Generation Sequencing vs. Microarrays

Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.

Gene Expression

Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.

Diagnostics

They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing.
Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.

Sequencing Hones In on Targets

Greg Crowther, Ph.D.

GEN Feb 2013

Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.

“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.

Summary

We have finished a breathtaking ride through the genomic universe in several sessions.  This has been a thorough review of genomic structure and function in cellular regulation.  The items that have been discussed and can be studied in detail include:

  1.  the classical model of the DNA structure
  2. the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
  3. the nature of the tight folding of the chromatin in the nucleus
  4. intramolecular bonds and short distance hydrophobic and hydrophilic interactions
  5. trace metals in molecular structure
  6. nuclear to membrane interactions
  7. the importance of the Human Genome Project followed by Encode
  8. the Fractal nature of chromosome structure
  9. the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
  10. Enzymatic components of gene regulation (ligase, kinases, phosphatases)
  11. Methods of computational analysis in genomics
  12. Methods of sequencing that have become more accurate and are dropping in cost
  13. Chromatin remodeling
  14. Triplex and quadruplex models not possible to construct at the time of Watson-Crick
  15. sequencing errors
  16. propagation of errors
  17. oxidative stress and its expected and unintended effects
  18. origins of cardiovascular disease
  19. starvation and effect on protein loss
  20. ribosomal damage and repair
  21. mitochondrial damage and repair
  22. miscoding and mutational changes
  23. personalized medicine
  24. Genomics to the clinics
  25. Pharmacotherapy horizons
  26. driver mutations
  27. induced pluripotential embryonic stem cell (iPSCs)
  28. The association of key targets with disease
  29. The real possibility of moving genomic information to the bedside
  30. Requirements for the next generation of electronic health record to enable item 29

Other Related articles on this Open Access Online Scientific Journal, include the following:

https://pharmaceuticalintelligence.com/2013/01/14/oogonial-stem-cells-purified-a-view-towards-the-future-of-reproductive-biology/   SSaha

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ RSaxena

https://pharmaceuticalintelligence.com/2012/08/22/a-possible-light-by-stem-cell-therapy-in-painful-dark-of-osteoarthritis-kartogenin-a-small-molecule-differentiates-stem-cells-to-chondrocyte-healthy-cartilage-cells/   ASarkar and RSaxena

https://pharmaceuticalintelligence.com/2012/08/07/human-embryonic-pluripotent-stem-cells-and-healing-post-myocardial-infarction/    LHB

https://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/  SJWilliams

https://pharmaceuticalintelligence.com/2013/01/09/gene-therapy-into-healthy-heart-muscle-reprogramming-scar-tissue-in-damaged-hearts/ ALev-Ari

https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/  SJWilliams

https://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/  TBarliya

Read Full Post »


Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations.  Author, Reporter: Stephen J. Williams, Ph.D.

The fall 2012 Meeting of the Mid-Atlantic Society of Toxicology (MASOT) focused on the challenges and solutions in developing proper Development and Reproductive Toxicology (DART) studies with regards to the newer classes of bio-therapeutics such as vaccines, antibody-based therapies, and viral-based therapies.  The full meeting and MASOT links can be found at http://www.masot.org.   The overall synopsis of the meeting talks agreed, that although the general aim and design of DART studies for biological are very similar to DART studies for small molecule therapeutics, it is more necessary to take into consideration the pharmacodynamics, pharmacokinetic differences between biologics and small molecules.   In addition it is imperative to use pharmacologically-relevant species, such as non-rodent (guinea pig and non-human primate). The meeting was highlighted by the keynote speaker, Dr. A. Wallace Hayes, renowned board-certified toxicologist, committee and expert panel member for National Academy of Sciences, NIEHS, EPA and Department of Defense, and editor of well-known textbooks including Principles and Methods of Toxicology.  Dr. Hayes discussed a timeline of milestones in the field of toxicology.

The following are the meeting talk abstracts as well as notes for each presenter.

What’s So Different About DART Assessment of Biologics? Christopher Bowman Ph.D., DABT (Pfizer, Inc.)

Abstract:  The aim of developmental and reproductive toxicity (DART) safety assessment of a biologic is no different from that of a small molecule. Both cases consist of evaluating the potential for maternal toxicity, pre- and postnatal development toxicity (including juvenile toxicity) and effects of fertility (reproduction).  The differences lie in the in the product attributes of a specific biologic, the pharmacological response, the potential for undesirable toxicities and how these product attributes influence and are influenced by the biology.  Thus the primary challenge for developing a DART strategy for a biologic are derived from the complexities of these biomolecules and how that dictates a case-by-case strategy for appropriately evaluating the potential for developmental and reproductive toxicity. Most protein biologics have very limited potential for off-target toxicities, but this is not necessarily the case for other modalities such as anti-sense oligonucleotides and antibody-drug-conjugates.  In these cases, off-target toxicities can be a major feature of the DART safety assessment.  The most noticeable difference in DART assessment of biologics is the need to conduct these studies in pharmacologically relevant species and how that can influence the overall nonclinical strategy (including DART).  This has led to increased use of non-human primates as a model system and led to optimizations of this model for this purpose and revisions to international guidelines.

Notes:   Dr. Bowman emphasized the need to understand the type of biological you are testing and to both devise DART studies based on this information, additional endpoint you may want, as well as carefully choosing the correct species most relevant to the biologic.  He highlighted general differences between small molecules versus a biologic with respect to their pharmacology.  These differences are summarized in the Table below:

  Small Molecule Biologic-based therapy
Species specificity Low High
Route of administration Usually oral Parental
ADME (PK, bio-distribution etc.) Wide distribution Low distribution

He noted that clinical trials for biologics rarely include reproductive toxicity so the preclinical DART study is of utmost importance.  He also emphasized that currently, the FDA requires two species for DART testing of small molecule therapies (usually one rodent and one non-rodent).  However this is not possible with many biologics as species is to be taken in consideration when designing a meaningful DART study.  Study designs can be like most DART studies but want to have a steady exposure during fetal organogenesis, use high doses (10 times the clinical dose) to achieve maximal pharmacology, confirm exposure to fetus and to F1 generation, and determine embryolethality.  Some biologics like interferon and insulin-growth factor receptor (IGFR) antagonists are fetal abortifactants. In fact Lucentis (Ranibizumab) and Macugen (Pegaptanib) were approved with no or little DART studies, however these drugs showed reproductive toxicity, resulting in warning concerning pregnancy on the label. Also important is the effect on the immune system and reproductive system of offspring, as well as the pharmacodynamics profile in the offspring.

Species Selection for Reproductive and Developmental Toxicity Testing of Biologics; Elise M. Lewis, Ph.D. (Charles River Preclinical Services)

Abstract:  Regulatory guidelines for developmental and reproductive toxicology studies require selection of “relevant” animal models as determined by kinetic, pharmacological, and preceding toxicological data.  Rats, mice, and rabbits are the preferred animal models for these studies based on historical experience and well-established procedures and study protocols.  However, due to species specificity and immunogenicity issues, developmental and reproductive toxicology testing for biologics is limited to a pharmacologically relevant animal model as described in the ICH s6 guideline.

Notes:  Dr. Lewis notes that DART studies in guinea pigs and hamsters represent a cost effective alternative to large animal models as well as the benefit of shorter duration and ability to assess mating behavior.  She also notes that reproductive toxicology of vaccines should be done in an animal model that can elicit an immune-response to the vaccine, especially to determine any maternal-fetal interaction.  For example, a vaccine may be directed to a maternal protein which when suppressed, may negatively impact the developing fetus.  However it is important to remember that guinea pigs can spontaneously abort so it is good to have proper control arms of a substantial size in order to statistically determine the impact of those spontaneous abortions.

 

 

Placental Transfer of an Adnectin Protein During Organogenesis in Guinea Pigs Using a Radiolabeled Methodology; Lakshmi Sivaraman, Ph.D. (Bristol-Myers Squibb)

Abstract:  Knowledge regarding the placental transfer of large molecular weight therapeutics is important to support the enrollment of women of childbearing potential in clinical trials.  There is limited information in the scientific literature that reports the extent to which the conceptus is exposed to these large molecules during organogenesis.  Placental transfer of large therapeutics has been difficult to quantify, due to limited blood volumes that can be obtained from the embryo, as well as insufficient assay sensitivity.  Thus, it is possible that embryos are exposed to pharmacologically active concentrations after maternal drug exposure. We have adopted a radiolabeled approach to quantitate embryo-fetal exposure of a novel protein therapeutic platform (adnectins). Adnectins are fibronectin-based proteins containing domains engineered to bind to targets of therapeutic interests.

Notes: Adnectins molecular weight is typically less than monoclonal antibodies and while IgG is not transferred in great quantity past the placental barrier there have been studies in human indicating maternal-fetal transfer of monoclonal antibodies.  This is particularly important for two reasons:  the monoclonal interacts with a target important in development, or the fetal immune system could be augmented.  Their work will be published in Drug Metabolism and Disposition.  In general Dr. Siveraman engineered a radiolabel on adnectin and used different detection methods to quantify the fetal exposure to a single maternal dose.  Dr. Siverman was able to detect radiolabel in the fetus however it is not clear whether this is a significant amount.

Reproductive Toxicity Testing for Biological Products in Nonhuman Primates: Evolution and Current Perspectives: Gary J. Chellman, Ph.D., DABT (Charles River Preclinical Services)

Notes:  Dr. Chellman gave a review of the current trends being driven by regulatory agencies with regard to nonhuman primate DART studies of biopharmaceuticals.  He noted that an advantage using nonhuman primates were the close physiologic resemblance to humans and because a large animal could monitor pregnancy over time using ultrasound technology.  In general, Dr. Chellman spoke about new study designs which not only reduce the number of animals required but also significantly reduce costs.  For example, a DART study which cost upward of $750,000 now can be done for as little as $350,000.  Dr. Kary Thompson of Bristol Myers Squibb then gave a talk about use of these new enhanced designs to determine reproductive toxicity issues with ipilimumab (Yervoy).

Other research papers on Pharmaceutical Intelligence and Reproductive Biology, Bio Insrumentation, Endocrinology Genetics were published on this Scientific Web site as follows

Non-small Cell Lung Cancer drugs – where does the Future lie?

Reboot evidence-based medicine and reconsider the randomized, placebo-controlled clinical trial

Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”

Leptin and Puberty

Gene Trap Mutagenesis in Reproductive Research

Genes involved in Male Fertility and Sperm-egg Binding

Hope for Male Contraception: A small molecule that inhibits a protein important for chromatin organization can cause reversible sterility in male mice

Pregnancy with a Leptin-Receptor Mutation

The contribution of comparative genomic hybridization in reproductive medicine

Sperm collide and crawl the walls in chaotic journey to the ovum

Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations

Biosimilars: CMC Issues and Regulatory Requirements

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

Assisted Reproductive Technology Cycles and Cumulative Birth Rates

Innovations in Bio instrumentation in Reproductive Clinical and Male Fertility Labs in the US

Increased risks of obesity and cancer, Decreased risk of type 2 diabetes: The role of Tumor-suppressor phosphatase and tensin homologue (PTEN)

Guidelines for the welfare and use of animals in cancer research

Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”

 

 

Read Full Post »


Reporter and Curator: Dr. Sudipta Saha, Ph.D.

With the completion of the mapping of the human genome, we now have access to all the DNA sequence information responsible for human biology. Together with microarray technology, we are ushering in a new era in reproductive medicine—the era of Reproductive Genomics.

Whole genome microarray analysis of the testis and ovary suggests that a substantial part of the genome is expressed in reproductive tissues and many of them are likely to be important for normal reproduction. Yet adequate expression and functional information is only available for less than 10% of them. Hence, one of the important questions in reproductive studies now is ‘how do we associate function with the genes expressed in reproductive tissues?’ The establishment of mutations in animal models such as the mouse represents one powerful approach to address this question.

Animal models have played critical roles in improving our understanding of mechanisms and pathogenesis of diseases. Mouse knockout models have often provided highly needed functional validation of genes implicated in human diseases. The rapid advance of human genetics in areas such as

  • single nucleotide polymorphisms (SNP) and
  • haplotyping technology

now allows the identification of disease-associated single nucleotide variation at a much faster pace. Functional examination of those candidate genes is needed to determine if those genes or variants are indeed involved in reproductive disease. Generating mutations in murine homologs of candidate genes represents a direct way to determine their roles, and mouse models will further allow the dissection of genetic pathways underlying the disease condition and provide models to test possible drug treatments. Thus, how to generate mouse models efficiently becomes a priority issue in the Genomics era of Reproductive Medicine.

It is known that generating a mouse knockout is no small endeavor, even for a mouse research lab, often requiring specialized expertise and experience in

  • molecular biology,
  • embryonic stem (ES) biology and
  • mouse husbandry.

Therefore, it could be intimidating for people who have little experience in mouse research. Fortunately, there are some technological developments in the mouse community that make the task of generating mouse mutations less intimidating to people unfamiliar with mouse genetics. One of these developments is the effort led by the International Gene Trap Consortium (IGTC) to generate a library of mouse mutant ES cells covering most of the genes in the mouse genome. This method saves researchers the tedious and sometimes challenging tasks of making knockout vectors and screening ES cell colonies and directly provides researchers an ES cell clone carrying the mutation of the gene of interest.

Because gene trapping involves the use of different mechanisms in generating mutations from the traditional knockout method, and its efficacy in targeting reproductive genes which often are expressed in later development or adult has not been fully established, it is necessary to examine the benefits and limitations of this technology, especially in the perspective of reproductive medicine so that reproductive researchers and physicians who are interested in mouse models could become familiar with this technology.

With this in mind, we provide an overview of the gene trapping mutagenesis method and its possible application to Reproductive Medicine. We evaluate gene trapping as a method in terms of its efficiency in comparison with traditional knockout methods and use an in-house software program to screen the IGTC database for existing cell lines with possible mutations in genes expressed in various reproductive tissues. Among over seven thousand genes highly expressed in human ovaries, almost half of them have existing gene trap lines.

Additionally, from 900 human seminal fluid proteins, 43% of them have gene trap hits in their mouse homologs. Our analysis suggests gene trapping is an effective mutagenesis method for identifying the genetic basis of reproductive diseases and many mutations for important reproductive genes are already present in the database. Given the rapid growth of the number of gene trap lines, the continuing evolution of gene trap vectors, and its easy accessibility to scientific communities, gene trapping could provide a fast and efficient way of generating mouse mutation(s) for any one particular gene of interest or multiple genes involved in a pathway at the same time. Consequently, we recommend gene trapping to be considered in the planning of mouse modeling of human reproductive disease and the IGTC be the first stop for people interested in searching for and generating mouse mutations of genes of interest.

Gene trapping is a high-throughput approach of generating mutations in murine ES cells through vectors that simultaneously disrupt and report the expression of the endogenous gene at the point of insertion. First-generation vectors trapped genes that were actively transcribed in undifferentiated ES cells. Depending on the areas in which they integrate, these vectors can be roughly divided into two classes:

  • promoter trap vectors and
  • gene trap vectors.

Promoter trap vectors contain promoterless reporter regions, usually bgeo (a fusion of neomycin phosphotransferase and b-galactosidase), and thus have to be integrated into an exon of a transcriptionally active locus in order for the cell to be selected for neomycin resistance or by LacZ staining. Gene trap vectors demonstrate more utility by their added ability to integrate into an intron. These vectors contain a splice acceptor (SA) site positioned at the 50-end of the reporter gene, allowing the vector to be spliced to the endogenous gene to form a fusion transcript. Later improvements include an internal ribosomal re-entry site (IRES) between the SA site and the reporter gene sequence; as a result, the reporter gene can be translated even when it is not fused to the trapped gene. Second-generation vectors have sought to trap genes that are transcriptionally silent in ES cells. Although these vectors still contain a promoterless reporter gene with a 50 SA sequence, the antibiotic resistance gene is under the control of a constitutive promoter. Consequently, antibiotic selection is independent from the expression of the trapped gene, whereas the expression of the reporter gene is still regulated by the endogenous promoter.

A disadvantage of these vectors is that all integration events give rise to resistant ES cells regardless of whether or not the vector has integrated into a gene locus. To increase trapping efficiency, a new class of polyA gene trap vectors was developed where the polyadenylation signal of the neo gene was replaced by a splice donor sequence, thereby requiring the vector to trap an endogenous polyA signal for expression of neo. These vectors were recently shown to have a bias toward insertion near the 30-end of a gene due to nonsense-mediated mRNA decay of the fusion transcript. An improved polyA trap vector, UPATrap, was developed to overcome this bias using an IRES sequence placed downstream of a marker containing a termination codon. Gene trap vectors are usually introduced by retroviral infection or electroporation of plasmid DNA, with each approach having its own advantages and disadvantages.

While relatively difficult to manipulate, retroviral gene traps display a preference toward insertion at the 50-end of genes, which is advantageous for generating null alleles. Moreover, the multiplicity of infection with retroviruses can be tightly controlled to a single trap event or simultaneous disruption in many genes. However, there may be a possible bias integration toward certain ‘hotspots’ of the genome.

In contrast, plasmid-based gene trap vectors integrate more randomly into the genome. This can, however, potentially result in a functional partial protein and a hypomorphic phenotype. Additionally, plasmid vectors usually result in multiple integrations in 20–50% of cell lines. The most common approach for identifying the gene trap integration site is to use 50 or 30 rapid amplification of cDNA ends (RACE) to amplify the fusion transcript. The sequence provides a DNA tag for the identification of the disrupted gene and can be used for genotypic screens. Mutagenesis screens can also be performed on the basis of gene function or expression, and data from an expression sequence combined with sequence tag information can elucidate novel expression patterns of known genes or to suggest gene function.

Gene trapping has proven to be an efficacious technique in mutagenesis compared with other methods such as

  • spontaneous mutations,
  • fortuitous transgene integration and
  • N-ethyl-N-nitrosurea (ENU) mutagenesis

We have been able to use our SpiderGene program to identify genes in reproductive tissues that are present in the IGTC database and moreover to narrow down those with restricted expression in the testis and ovary. Gene trapping possesses an enormous potential for researchers in the reproductive field seeking to create mouse models for a gene mutation. The improving versatility of gene trap vectors has enabled groups to trap an increasing number of genes in various organisms, including Arabidopsis, Zebra fish and Drosophila.

The gene trap effort has perhaps been the most extensive in the murine genome, with over 57000 cell lines representing more than 40% of the known genome. These large-scale screens will likely achieve the trapping of the entire mouse genome in the coming years, but the power of gene trapping will only be fully demonstrated by its usefulness in investigator-driven focused functional analyses.

In our laboratory, future work will focus on generating knockout mice in order to investigate gene function and to identify gene products that might have therapeutic value in reproduction. As screening efforts continue, gene trapping will continue to be a valuable tool in mouse genomics and will undoubtedly yield new discoveries in Reproductive Physiology and Pathology.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed?term=Gene%20trap%20mutagenesis%3A%20a%20functional%20genomics%20approach%20towards%20reproductive%20research

 

Read Full Post »