Human BAC Clones Research Articles

PhaseDancer: a novel targeted assembler of segmental duplications unravels the complexity of the human chromosome 2 fusion going from 48 to 46 chromosomes in hominin evolution

Resolving complex genomic regions rich in segmental duplications (SDs) is challenging due to the high error rate of long-read sequencing. Here, we describe a targeted approach with a novel genome assembler PhaseDancer that extends SD-rich regions of interest iteratively. We validate its robustness and efficiency using a golden-standard set of human BAC clones and in silico-generated SDs with predefined evolutionary scenarios. PhaseDancer enables extension of the incomplete complex SD-rich subtelomeric regions of Great Ape chromosomes orthologous to the human chromosome 2 (HSA2) fusion site, informing a model of HSA2 formation and unravelling the evolution of human and Great Ape genomes.

Rapid emergence of independent \u201cchromosomal lineages\u201d in silvered-leaf monkey triggered by Y/autosome translocation

Sex/autosome translocations are rare events. The only known example in catarrhines is in the silvered-leaf monkey. Here the Y chromosome was reciprocally translocated with chromosome 1. The rearrangement produced an X1X2Y1Y2 sex chromosome system. At least three chromosomal variants of the intact chromosome 1 are known to exist. We characterized in high resolution the translocation products (Y1 and Y2) and the polymorphic forms of the intact chromosome 1 with a panel of more than 150 human BAC clones. We showed that the translocation products were extremely rearranged, in contrast to the high level of marker order conservation of the other silvered-leaf monkey chromosomes. Surprisingly, each translocation product appeared to form independent “chromosome lineages”; each having a myriad of distinct rearrangements. We reconstructed the evolutionary history of the translocation products by comparing the homologous chromosomes of two other colobine species: the African mantled guereza and the Indian langur. The results showed a massive reuse of breakpoints: only 12, out of the 40 breaks occurred in domains never reused in other rearrangements, while, strikingly, some domains were used up to four times. Such frequent breakpoint reuse if proved to be a general phenomenon has profound implications for mechanisms of chromosome evolution.

Genomic leftovers: identifying novel microsatellites, over-represented motifs and functional elements in the human genome.

The human genome is 99% complete. This study contributes to filling the 1% gap by enriching previously unknown repeat regions called microsatellites (MST). We devised a Global MST Enrichment (GME) kit to enrich and nextgen sequence 2 colorectal cell lines and 16 normal human samples to illustrate its utility in identifying contigs from reads that do not map to the genome reference. The analysis of these samples yielded 790 novel extra-referential concordant contigs that are observed in more than one sample. We searched for evidence of functional elements in the concordant contigs in two ways: (1) BLAST-ing each contig against normal RNA-Seq samples, (2) Checking for predicted functional elements using GlimmerHMM. Of the 790 concordant contigs, 37 had an exact match to at least one RNA-Seq read; 15 aligned to more than 100 RNA-Seq reads. Of the 249 concordant contigs predicted by GlimmerHMM to have functional elements, 6 had at least one exact RNA-Seq match. BLAST-ing these novel contigs against all publically available sequences confirmed that they were found in human and chimpanzee BAC and FOSMID clones sequenced as part of the original human genome project. These extra-referential contigs predominantly contained pentameric repeats, especially two motifs: AATGG and GTGGA.

Abstract A06: Interspecific recombination between orthologous human and mouse BAC clones in E. coli: Exploring scalable humanization of cancer-relevant genes in the mouse genome

Abstract Our interests lie in humanizing specific regions of the mouse genome using large segments of human DNA with extents of 10s to 100s of kilobase pairs. To that end, we have undertaken a series of projects to transfer large segments of human DNA, from human BAC clones, into the orthologous region of corresponding mouse BAC clones using— (1), traditional recombineering methods; (2), recombinase-mediated cassette exchange (RCME); and (3), a novel technology that we call trans- or trimolecular-recombineering. In each instance the goal is to place human DNA within the larger context of homologous BAC-derived mouse arms in E. coli. Completed constructs will be used to drive homologous recombination in mouse embryonic stem (ES) cells with positive selection imparted at each end of the transferred human segment using dual, recombinase recognition site-flanked, selectable marker cassettes. Properly targeted ES cells will be used to develop viable mouse strains carrying the humanized segments. Selection cassettes will be removed by crossing to mouse strains with germline expression of the appropriate recombinases. All experiments have been designed to maintain a fixed genetic background from community-standard C57BL/6 strains. Our poster presentation details the specifics of our projects and describes progress to date. This research is supported by National Cancer Institute Grant Number 5P30CA034196-28. Citation Format: Tiffany Leidy-Davis, David E. Bergstrom. Interspecific recombination between orthologous human and mouse BAC clones in E. coli: Exploring scalable humanization of cancer-relevant genes in the mouse genome. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A06.

Expression of the G72/G30 gene in transgenic mice induces behavioral changes

The G72/G30 gene complex is a candidate gene for schizophrenia and bipolar disorder. However, G72 and G30 mRNAs are expressed at very low levels in human brain, with only rare splicing forms observed. We report here G72/G30 expression profiles and behavioral changes in a G72/G30 transgenic mouse model. A human BAC clone containing the G72/G30 genomic region was used to establish the transgenic mouse model, on which gene expression studies, Western blot and behavioral tests were performed. Relative to their minimal expression in humans, G72 and G30 mRNAs were highly expressed in the transgenic mice, and had a more complex splicing pattern. The highest G72 transcript levels were found in testis, followed by cerebral cortex, with very low or undetectable levels in other tissues. No LG72 (the long putative isoform of G72) protein was detected in the transgenic mice. Whole-genome expression profiling identified 361 genes differentially-expressed in transgenic mice compared to wild-type, including genes previously implicated in neurological and psychological disorders. Relative to wild-type mice, the transgenic mice exhibited fewer stereotypic movements in the open field test, higher baseline startle responses in the course of the prepulse inhibition test, and lower hedonic responses in the sucrose preference test. The transcriptome profile changes and multiple mouse behavioral effects suggest that the G72 gene may play a role in modulating behaviors relevant to psychiatric disorders.

Chromosomal evolution of the PKD1 gene family in primates.

BackgroundThe autosomal dominant polycystic kidney disease (ADPKD) is mostly caused by mutations in the PKD1 (polycystic kidney disease 1) gene located in 16p13.3. Moreover, there are six pseudogenes of PKD1 that are located proximal to the master gene in 16p13.1. In contrast, no pseudogene could be detected in the mouse genome, only a single copy gene on chromosome 17. The question arises how the human situation originated phylogenetically. To address this question we applied comparative FISH-mapping of a human PKD1-containing genomic BAC clone and a PKD1-cDNA clone to chromosomes of a variety of primate species and the dog as a non-primate outgroup species.ResultsComparative FISH with the PKD1-cDNA clone clearly shows that in all primate species studied distinct single signals map in subtelomeric chromosomal positions orthologous to the short arm of human chromosome 16 harbouring the master PKD1 gene. Only in human and African great apes, but not in orangutan, FISH with both BAC and cDNA clones reveals additional signal clusters located proximal of and clearly separated from the PKD1 master genes indicating the chromosomal position of PKD1 pseudogenes in 16p of these species, respectively. Indeed, this is in accordance with sequencing data in human, chimpanzee and orangutan. Apart from the master PKD1 gene, six pseudogenes are identified in both, human and chimpanzee, while only a single-copy gene is present in the whole-genome sequence of orangutan. The phylogenetic reconstruction of the PKD1-tree reveals that all human pseudogenes are closely related to the human PKD1 gene, and all chimpanzee pseudogenes are closely related to the chimpanzee PKD1 gene. However, our statistical analyses provide strong indication that gene conversion events may have occurred within the PKD1 family members of human and chimpanzee, respectively.ConclusionPKD1 must have undergone amplification very recently in hominid evolution. Duplicative transposition of the PKD1 gene and further amplification and evolution of the PKD1 pseudogenes may have arisen in a common ancestor of Homo, Pan and Gorilla ~8 MYA. Reticulate evolutionary processes such as gene conversion and non-allelic homologous recombination (NAHR) may have resulted in concerted evolution of PKD1 family members in human and chimpanzee and, thus, simulate an independent evolution of the PKD1 pseudogenes from their master PKD1 genes in human and chimpanzee.

Tracking the complex flow of chromosome rearrangements from the Hominoidea Ancestor to extant Hylobates and Nomascus Gibbons by high-resolution synteny mapping

In this study we characterized the extension, reciprocal arrangement, and orientation of syntenic chromosomal segments in the lar gibbon (Hylobates lar, HLA) by hybridization of a panel of approximately 1000 human BAC clones. Each lar gibbon rearrangement was defined by a splitting BAC clone or by two overlapping clones flanking the breakpoint. A reconstruction of the synteny arrangement of the last common ancestor of all living lesser apes was made by combining these data with previous results in Nomascus leucogenys, Hoolock hoolock, and Symphalangus syndactylus. The definition of the ancestral synteny organization facilitated tracking the cascade of chromosomal changes from the Hominoidea ancestor to the present day karyotype of Hylobates and Nomascus. Each chromosomal rearrangement could be placed within an approximate phylogenetic and temporal framework. We identified 12 lar-specific rearrangements and five previously undescribed rearrangements that occurred in the Hylobatidae ancestor. The majority of the chromosomal differences between lar gibbons and humans are due to rearrangements that occurred in the Hylobatidae ancestor (38 events), consistent with the hypothesis that the genus Hylobates is the most recently evolved lesser ape genus. The rates of rearrangements in gibbons are 10 to 20 times higher than the mammalian default rate. Segmental duplication may be a driving force in gibbon chromosome evolution, because a consistent number of rearrangements involves pericentromeric regions (10 events) and centromere inactivation (seven events). Both phenomena can be reasonably supposed to have strongly contributed to the euchromatic dispersal of segmental duplications typical of pericentromeric regions. This hypothesis can be more fully tested when the sequence of this gibbon species becomes available. The detailed synteny map provided here will, in turn, substantially facilitate sequence assembly efforts.

Transcriptional activity and Sp 1/3 transcription factor binding to the P1 promoter sequences of the human AβH‐J‐J locus

Alternative splicing of the locus AbetaH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase, humbug and junctate (truncated homologs of aspartyl (asparaginyl) beta-hydroxylase with a role in calcium regulation), and junctin (a structural protein of the sarcoplasmic reticulum membrane). Aspartyl (asparaginyl) beta-hydroxylase and humbug are overexpressed in a broad range of malignant neoplasms. We have previously reported the gene structure of this locus, showing the presence of two putative promoters, P1 and P2, and characterized the P2 sequences, directing tissue-specific transcription of junctin, aspartyl (asparaginyl) beta-hydroxylase and junctate. In addition, aspartyl (asparaginyl) beta-hydroxylase and humbug are expressed from exon 1 by the P1 promoter. The present study identifies and functionally characterizes the P1 promoter activity of the AbetaH-J-J locus. We demonstrate that mRNAs from the P1 promoter are actively transcribed in all the human tissues and cell lines analyzed, and define the transcription start point in HeLa and RD cells. To investigate the transcription mechanism we cloned 1.7 kb upstream of exon 1 from a human BAC clone, and produced progressively deleted reporter constructs. Our results showed that: (a) the 1.7 kb fragment was a powerful activator of the reporter gene in human hepatoblastoma (HepG2) and human embryonic rhabdomyosarcoma (RD) cell lines; (b) 512 bp upstream of the transcription start site were essential for maximal promoter activity; and (c) progressive deletions from -512 resulted in gradually decreased reporter expression. The region responsible for maximal transcription contains at least 12 GC boxes homologous to binding sequences of specific transcription factor 1 (Sp1); by electrophoretic mobility shift assay and supershift analysis, we identified three GC-rich elements that bind Sp transcription factor family nuclear factors with very high efficiency. A functional role of Sp transcription factors in upregulating P1-directed transcription was demonstrated by analysis of the effects of: (a) in vitro mutagenesis of the Sp1 transcription factor binding sites; (b) transfection with Sp transcription factor 1/3 expression vectors; and (c) treatment with decoy oligonucleotides targeting Sp transcription factors. In addition, Sp1 and Sp3 transcription factor chromatin immunoprecipitation demonstrated in vivo binding of these proteins to P1 promoter. Our results suggest that Sp transcription factors positively regulate the core of the P1 promoter, and the comparison of the two promoters of the AbetaH-J-J locus demonstrates that they are very different with regard to transcriptional efficiency and ability to direct tissue-specific transcription.

Comparative mapping of mink (Mustela vision) chromosome 8p: Localization of three human BAC clones

Using fluorescent in situ hybridization (FISH), three human BAC clones, localized in the terminal region of human chromosome 17p (HSA17p13; 1.44--3.68 Mp), were mapped to chromosome 8p of American mink (MVI8p). It was demonstrated that in MVI8p the region, homeologous to HSA17p13, was split into three fragments, which were detected within terminal, pericentric, and probably nucleolus-organizing regions. Using human BAC clones as heterologous markers for mapping of the gene sequences to the chromosomes of American mink, regional localization of eight sequences (PRPF8, SLC43A2, and RILP in MVI8p25; C17orf31 in MVI8p21-22; and CTNS, TAX1BP3, and P2RX5 in MVI8p11) was deduced.

Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms

A strategy is presented to select, pool and spot human BAC clones on an array in such a way that each spot contains five well performing BAC clones, covering one chromosome arm. A mini-array of 240 spots was prepared representing all human chromosome arms in a 5-fold as well as some controls, and used for comparative genomic hybridization (CGH) of 10 cell lines with aneusomies frequently found in clinical cytogenetics and oncology. Spot-to-spot variation within five replicates was below 6% and all expected abnormalities were detected 100% correctly. Sensitivity was such that replacing one BAC clone in a given spot of five by a BAC clone from another chromosome, thus resulting in a change in ratio of 20%, was reproducibly detected. Incubation time of the mini-array was varied and the fluorescently labelled target DNA was diluted. Typically, aneusomies could be detected using 30 ng of non-amplified random primed labelled DNA amounts in a 4 h hybridization reaction. Potential application of these mini-arrays for genomic profiling of disseminated tumour cells or of blastomeres for preimplantation genetic diagnosis, using specially designed DNA amplification methods, are discussed.

Measurement and relevance of neuroblastoma DNA copy number changes in the post-genome era

The completion of the human genome sequence and the development of high throughput technology present exciting opportunities for the study of cancer cells. High-resolution analysis of chromosomal aberrations provides a global framework for understanding complex patterns in cancer cells, allowing us to ask hypothesis-driven questions. Genome-wide analysis of amplification and deletion of genomic regions is a critical step to resolving the mechanisms of neuroblastoma tumorigenesis. We used a high-resolution aCGH system that has over 4000 human BAC clones, resulting in an average coverage of 1 Mb across the genome, to define whole genome copy number aberrations (CNAs) in a panel of human neuroblastoma-derived cell lines. By combining the aCGH data with meticulous regional validation studies, we showed that array CGH could reliably detect known aberrations including single copy gain or loss, that data correlate well with standard techniques used for the detection of these genetic changes, and that this technique can be used to identify novel regions of genomic imbalance.

Comparative cytogenetics of human chromosome 3q21.3 reveals a hot spot for ectopic recombination in hominoid evolution

Fluorescence in situ hybridization mapping of fully integrated human BAC clones to primate chromosomes, combined with precise breakpoint localization by PCR analysis of flow-sorted chromosomes, was used to analyze the evolutionary rearrangements of the human 3q21.3-syntenic region in orangutan, siamang gibbon, and silvered-leaf monkey. Three independent evolutionary breakpoints were localized within a 230-kb segment contained in BACs RP11-93K22 and RP11-77P16. Approximately 200 kb of the human 3q21.3 sequence was not present on the homologous orangutan, siamang, and Old World monkey chromosomes, suggesting a genomic DNA insertion into the breakpoint region in the lineage leading to humans and African great apes. The breakpoints in the orangutan and siamang genomes were narrowed down to 12- and 20-kb DNA segments, respectively, which are enriched with endogenous retrovirus long terminal repeats and other repetitive elements. The inserted DNA segment represents part of an ancestral duplication. Paralogous sequence blocks were found at human 3q21, approximately 4 Mb proximal to the evolutionary breakpoint cluster region; at human 3p12.3, which contains an independent orangutan-specific breakpoint; and at the subtelomeric and pericentromeric regions of multiple human and orangutan chromosomes. The evolutionary breakpoint regions between human chromosome 3 and orangutan 2 as well their paralogous segments in the human genome coincide with breaks of chromosomal synteny in the mouse, rat, and/or chicken genomes. Collectively our data reveal reuse of the same short recombinogenic DNA segments in primate and vertebrate evolution, supporting a nonrandom breakage model of genome evolution.

CDNA-based comparative genomic hybridization (cCGH) analysis of genomic aberrations of pancreatic cancer cells

4122 Introduction: Pancreatic carcinoma (PanCa) is the fourth leading cause of cancer mortality in US with a 5-year survival rate of 5%. urrent treatments have only a minimal survival benefit. In this study, we are using cDNA-based comparative genomic hybridization (cCGH) technologies to search for common genomic alternations in human PanCa cell lines. Methods: Genomic DNA from seven human PanCa cell lines was extracted and labeled with Cyanine 3-dUTP. Normal human genomic DNA isolated from leukocytes of healthy male and female volunteers was labeled with Cyanine 5-dUTP. The labeled probe mixtures were then hybridized with 10K (5K in replicates) cDNA slides prepared by the UT MD Anderson Cancer Genomics Core Laboratory. The gain or loss of individual genes after background subtraction and appropriate normalization. The identified genomic alternations were then verified by florescent in situ hybridization (FISH) analysis in pancreatic cells and biopsy tissues with human BAC clone DNA and the centromere con...

CDNA-based comparative genomic hybridization (cCGH) analysis of genomic aberrations of pancreatic cancer cells

4122 Introduction: Pancreatic carcinoma (PanCa) is the fourth leading cause of cancer mortality in US with a 5-year survival rate of 5%. urrent treatments have only a minimal survival benefit. In this study, we are using cDNA-based comparative genomic hybridization (cCGH) technologies to search for common genomic alternations in human PanCa cell lines. Methods: Genomic DNA from seven human PanCa cell lines was extracted and labeled with Cyanine 3-dUTP. Normal human genomic DNA isolated from leukocytes of healthy male and female volunteers was labeled with Cyanine 5-dUTP. The labeled probe mixtures were then hybridized with 10K (5K in replicates) cDNA slides prepared by the UT MD Anderson Cancer Genomics Core Laboratory. The gain or loss of individual genes after background subtraction and appropriate normalization. The identified genomic alternations were then verified by florescent in situ hybridization (FISH) analysis in pancreatic cells and biopsy tissues with human BAC clone DNA and the centromere controls. Results: We successfully performed cCGH analysis using 7 pancreatic cancer cell lines. Our data revealed that human hepatocyte nuclear factor-6 (HNF6), telomeric repeat binding factor 2 (TRF2), reticulocalbin 2 EF-hand calcium binding domain (RCN2), and bromodomain testis-specific (BRDT) were amplified by more than 6 copies and fucose-1-phosphate guanyltransferase (FPGT) lost two endogenous copies in 43 to 71% of the PanCa cell lines studied (see the attached table). The genomic amplifications of HNF6 and TRF2 have been validated by FISH assays. We are validating FISH analysis for RCN3, BRDT and FPGT and with PanCa tissues from primary and metastatic tumors. Conclusions: We identified 4 genes with 6+fold gain and one gene with complete loss in PanCa cell lines. Two of these genes have been validated by the FISH analysis. Using similar analyses with patient tissues, we are searching for predictive and prognostic genomic markers and new therapeutic targets for improving PanCa survival (Supported by a pilot study grant and a T32 training grant from UT MD Anderson Cancer Center and NCI). Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration M.D. Anderson Cancer Center; National Cancer Institute

The Structure of a Conserved Region of Porcine Genome, Represented in Human Genome by Chromosome 17

Radiation mapping of nine genes (H3F3B, HLR1, MYL4, STAT5B, THRA1, TOP2A, MCP1, NF1, and MPO) to porcine chromosome 12 was carried out. Also, subchromosomal location of the NF1 gene along with the two loci containing the DNA sequences homologous to the DNA of the two human BAC clones was determined. The NF1 position was ascertained via microdissection of chromosome 12 with subsequent PCR amplification of the gene fragment with specific primers. BAC clones were mapped using FISH. Comparative analysis of the gene order in porcine chromosome 12 and in the homologous human chromosome 17 was performed. It was demonstrated that the gene orders in these chromosomes differed relative to the position of the MPO gene.

Cloning and characterization of the promoter region of human LPTS/PinX1 gene

The gene for LPTS/PinX1 encodes a potent telomerase inhibitor and suppresses tumor cell growth. In order to investigate the transcriptional regulation of this gene, we isolated its 5′-flanking region from the human genomic BAC clone and identified a major transcriptional initiation site. The sequence of the 5′-flanking region is GC-rich, lacks canonical TATA box, but contains potential binding sites for a variety of transcription factors. The deletion analysis indicated that the proximal 100 bp (from nt −66 to +34) is essential for minimal promoter activity and the regions of promoter from nt −1272 to −573 and nt −330 to −66 are required for maximal expression of the LPTS/PinX1 gene. Four DNase I hypersensitive sites (DHS1–4) mapping to the regions of transcription initiation and promoter in LPTS/Pinx1 gene were also revealed.

Conservation of aphidicolin-induced fragile sites in Papionini (Primates) species and humans

Fragile sites are considered structural features of mammalian chromosomes and a commonly repeated hypothesis is that they are evolutionarily conserved. We tested this hypothesis by establishing the subchromosomal homology of regions harbouring fragile sites in the chromosomes of humans, Macaca fascicularis (MFA) and Mandrillus sphinx (MSP). We delineated the interspecific homology of chromosome bands expressing aphidicolin-induced fragile sites of homologues to human chromosomes 1, 3, 5, 7, 18 and X by the comparative FISH of human BAC and YAC clones. Notably, two YAC clones known to span human chromosome regions containing fragile sites were shown to also span fragile sites in macaques and mandrills. The present comparative BAC/YAC mapping data represent, up to now, the most precise evidence of fragile site conservation during primate evolution.

Targeted modification of a human β-globin locus BAC clone using GET Recombination and an I- Scei counterselection cassette

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human β-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I- SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I- SceI site in the counterselection cassette is cut by I- SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.

DNA modification and functional delivery into human cells using Escherichia coli DH10B.

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a approximately 200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd(-) rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.

Isolation, characterization and expression of the human Factor In the Germline alpha (FIGLA) gene in ovarian follicles and oocytes.

The Factor In the Germline alpha (FIGalpha) transcription factor regulates expression of the zona pellucida proteins ZP1, ZP2 and ZP3 and is essential for folliculogenesis in the mouse. Using the published mouse Figla sequence, BLAST searches identified a human chromosome 2 BAC clone with high sequence identity. Using PCR primers derived from this clone, amplicons derived from ovarian follicles and mature oocytes revealed 100% identity with the appropriate human BAC clone, the expected homology with the mouse Figla gene sequence, and homology on translation with the FIGalpha protein identified in the Japanese rice fish, medaka (Oryzias latipes). PCR expression profiling of this transcript revealed FIGLA mRNA expression in cDNA derived from ovarian follicles (5/5 samples from the primordial through to the secondary stage) mature oocytes (6/9 samples), and less frequently in preimplantation embryos (2/7 samples). Subsequent BLAST searches revealed the predicted full length coding sequence of the human FIGalpha protein which demonstrates 68 and 25% similarity overall to mouse and medaka proteins respectively, with 96 and 57% identity respectively within the basic helix-loop-helix region. This confirms our identification of the human homologue for this gene which maps to chromosome 2p12. Further work is required to understand its role in normal human oocyte development and the potential involvement in human infertility.