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Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co‐factor, as a candidate tumor suppressor gene (TSG) in high‐grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell‐mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12‐q12.1) into the OV‐90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV‐90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular‐bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV‐90 was achieved using a lentivirus‐based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3‐expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p

A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration.

Eric Shoubridge and colleagues report the identification of a mutation in the CCDC44 gene that is causal in a Leigh syndrome pedigree. The CCDC44 gene product, TACO1, is involved in mitochondrial translation and is the first specific mitochondrial translational activator identified in mammals. Defects in mitochondrial translation are among the most common causes of mitochondrial disease1, but the mechanisms that regulate mitochondrial translation remain largely unknown. In the yeast Saccharomyces cerevisiae, all mitochondrial mRNAs require specific translational activators, which recognize sequences in 5′ UTRs and mediate translation2. As mammalian mitochondrial mRNAs do not have significant 5′ UTRs3, alternate mechanisms must exist to promote translation. We identified a specific defect in the synthesis of the mitochondrial DNA (mtDNA)-encoded COX I subunit in a pedigree segregating late-onset Leigh syndrome and cytochrome c oxidase (COX) deficiency. We mapped the defect to chromosome 17q by functional complementation and identified a homozygous single-base-pair insertion in CCDC44, encoding a member of a large family of hypothetical proteins containing a conserved DUF28 domain. CCDC44, renamed TACO1 for translational activator of COX I, shares a notable degree of structural similarity with bacterial homologs4, and our findings suggest that it is one of a family of specific mammalian mitochondrial translational activators.

The genetic analysis of nontumorigenic radiation hybrids generated by transfer of chromosome 3 fragments into the tumorigenic OV-90 ovarian cancer cell line identified the 3p12.3-pcen region as a candidate tumor suppressor gene (TSG) locus. In the present study, polymorphic microsatellite repeat analysis of the hybrids further defined the 3p12.3-pcen interval to a 16.1 Mb common region containing 12 known or hypothetical genes: 3ptel-ROBO2-ROBO1-GBE1-CADM2-VGLL3-CHMP2B-POU1F1-HTR1F-CGGBP1-ZNF654-C3orf38-EPHA3-3pcen. Seven of these genes, ROBO1, GBE1, VGLL3, CHMP2B, CGGBP1, ZNF654, and C3orf38, exhibited gene expression in the hybrids, placing them as top TSG candidates for further analysis. The expression of all but one (VGLL3) of these genes was also detected in the parental OV-90 cell line. Mutations were not identified in a comparative sequence analysis of the predicted protein coding regions of these candidates in OV-90 and donor normal chromosome 3 contig. However, the nondeleterious sequence variants identified in the transcribed regions distinguished parent of origin alleles for ROBO1, VGLL3, CHMP2B, and CGGBP1 and cDNA sequencing of the hybrids revealed biallelic expression of these genes. Interestingly, underexpression of VGLL3 and ZNF654 were observed in malignant ovarian tumor samples as compared with primary cultures of normal ovarian surface epithelial cells or benign ovarian tumors, and this occurred regardless of allelic content of 3p12.3-pcen. The results taken together suggest that dysregulation of VGLL3 and/or ZNF654 expression may have affected pathways important in ovarian tumorigenesis which was offset by the transfer of chromosome 3 fragments in OV-90, a cell line hemizygous for 3p.

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was approximately 33 kb/cR. We estimate the potential resolution of the RH panel to be approximately 186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.

Purpose: Cluster-of-differentiation antigen 9 (CD9) protein, a member of the tetraspanin family, has been implicated in carcinogenesis of various human tumors. Although decreased expression of the CD82 tetraspanin protein, a close CD9 relative, is associated with prostate cancer progression, CD9 expression has not been analyzed in this malignancy. Experimental Design: CD9 expression in human prostatic adenocarcinoma was analyzed by immunohistochemistry on 167 primary tumors and 88 lymph node or bone metastases. CD9 cDNA was sequenced from two human prostate cancer cell lines, prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia (PIN), and normal prostatic tissues. Results: Although CD9 was detected in the epithelium of normal prostatic tissues, reduced or loss of CD9 expression within neoplastic cells was observed in 24% of 107 clinically localized primary adenocarcinomas, 85% of 60 clinically advanced primary adenocarcinomas, 85% of 65 lymph node metastases, and 65% of 23 bone metastases. Difference in CD9 expression between clinically localized and advanced diseases was highly significant (P < 1 × 10−7). Whereas there was no alteration of CD9 cDNA in normal tissues, all PC-3–derived cell lines, one PIN, and four prostatic adenocarcinomas harbored deletions in their CD9 cDNAs. Recurring CD9 point mutations were also found in PC-3M-LN4 cells, one PIN, and seven prostatic adenocarcinomas. Conclusions: CD9 expression is significantly reduced and even lost during prostate cancer progression. Moreover, deletions and mutations of the CD9 mRNA may be associated with loss of protein expression observed in tumor cells. Our data suggest that CD9 inactivation may play an important role in prostate cancer progression.

Although prostate cancer is still the most diagnosed cancer in men, most genes implicated in its progression are yet to be identified. Chromosome abnormalities have been detected in human prostate tumors, many of them associated with prostate cancer progression. Indeed, alterations (including deletions or amplifications) of more than 15 human chromosomes have been reported in prostate cancer. We hypothesized that transferring normal human chromosomes into human prostate cancer cells would interfere with their tumorigenic and/or metastatic properties. We used microcell-mediated chromosome transfer to introduce human chromosomes 10, 12, 17, and 18 into highly tumorigenic (PC-3M-Pro4) and highly metastatic (PC-3M-LN4) PC-3-derived cell lines. We tested the in vitro and in vivo properties of these hybrids. Introducing chromosome 18 into the PC-3M-LN4 prostate cancer cell line greatly reduced its tumorigenic phenotype. We observed retarded growth in soft agar, decreased invasiveness through Matrigel, and delayed tumor growth into nude mice, both subcutaneously and orthotopically. This phenotype is associated with a marker in the 18q21 region. Combined with the loss of human chromosome 18 regions often seen in patients with advanced prostate cancer, our results show that chromosome 18 encodes one or more tumor-suppressor genes whose inactivation contributes to prostate cancer progression. © 2005 Wiley-Liss, Inc.

The zebrafish is an excellent genetic system for the study of vertebrate development and disease. In an effort to provide a rapid and robust tool for zebrafish gene mapping, a panel of radiation hybrids (RH) was produced by fusion of irradiated zebrafish AB9 cells with mouse B78 cells. The overall retention of zebrafish sequences in the 93 RH cell lines that constitute the LN54 panel is 22%. Characterization of the LN54 panel with 849 simple sequence length polymorphism markers, 84 cloned genes and 122 expressed sequence tags allowed the production of an RH map whose total size was 11,501 centiRays. From this value, we estimated the average breakpoint frequency of the LN54 RH panel to correspond to 1 centiRay = 148 kilobase. Placement of a group of 235 unbiased markers on the RH map suggests that the map generated for the LN54 panel, at present, covers 88% of the zebrafish genome. Comparison of marker positions in RH and meiotic maps indicated a 96% concordance. Mapping expressed sequence tags and cloned genes by using the LN54 panel should prove to be a valuable method for the identification of candidate genes for specific mutations in zebrafish.

To facilitate the identification of the gene responsible for Clouston hidrotic ectodermal dysplasia (HED), we used a chromosome 13-specific radiation hybrid panel to map 54 loci in the HED candidate region. The marker retention data were analyzed using RHMAP version 3. The 54 markers have an average retention frequency of 31.6% with decreasing retention as a function of distance from the centromere. Two-point analysis identified three linkage groups with a threshold lod score of 4.00; one linkage group consisted of 49 loci including the centromeric marker D13Z1 and the telomeric flanking marker for the HED candidate region D13S143. Assuming a centromeric retention model, multipoint maximum likelihood analysis of these 49 loci except D13Z1 provided a 1000:1 framework map ordering 29 loci with 21 unique map positions and ∼2000 times more likely than the next order. Loci that could not be ordered with this level of support were positioned within a range of adjacent intervals. This map spans 347 cR 9000 , has an average resolution of 17.3 cR 9000 , and includes 3 genes (TUBA2, GJβ2, and FGF-9), 18 ESTs, 19 polymorphic loci, and 8 single-copy DNA segments. Comparison of our RH map to a YAC contig showed an inconsistency in order involving a reversed interval of 6 loci. Fiber-FISH and FISH on interphase nuclei analyses with PACs isolated from this region supported our order. We also describe the isolation of 8 new chromosome 13q polymorphic (CA) n markers that have an average PIC value of 0.67. These data and mapping reagents will facilitate the isolation of disease genes from this region.

Leigh Syndrome (LS) is a severe neurological disorder characterized by bilaterally symmetrical necrotic lesions in subcortical brain regions that is commonly associated with systemic cytochrome c oxidase (COX) deficiency. COX deficiency is an autosomal recessive trait and most patients belong to a single genetic complementation group. DNA sequence analysis of the genes encoding the structural subunits of the COX complex has failed to identify a pathogenic mutation. Using microcell-mediated chromosome transfer, we mapped the gene defect in this disorder to chromosome 9q34 by complementation of the respiratory chain deficiency in patient fibroblasts. Analysis of a candidate gene (SURF1) of unknown function revealed several mutations, all of which predict a truncated protein. These data suggest a role for SURF1 in the biogenesis of the COX complex and define a new class of gene defects causing human neurodegenerative disease.

Whole-cell fusion between zebrafish fibroblast-like ZF4 cells and mouse B78 melanoma cells resulted in hybrids containing one or a few zebrafish chromosome segments in a murine chromosomal background. Fluorescence in situ hybridization to hybrid cell metaphases with a zebrafish genomic DNA probe revealed that many hybrids contained zebrafish chromosome segments that were either inserted or translocated to a mouse chromosome, whereas other hybrids contained zebrafish chromosomes with no evidence of insertion or translocation. We have assigned hybrids to 17 linkage groups of the genetic map of the zebrafish genome. Our results demonstrate the feasibility of producing somatic cell hybrids between distantly related species. Zebrafish/mouse cell hybrids will provide a useful tool for the physical mapping of the zebrafish genome and for the cloning of genes affected in zebrafish mutants.

A family of short interspersed repetitive elements (SINEs), designated mermaid, is present in the genomes of fish, amphibian and primates, but absent in the mouse genome. We have demonstrated that the sequences of the mermaid family are highly polymorphic in the zebrafish genome as in the human genome. We have also shown that the mermaid sequence can be used to recover zebrafish specific DNA from zebrafish–mouse cell hybrids by using mermaid -specific oligonucleotides as PCR primers. Thus, the mermaid family serves as a valuable genetic tool for the zebrafish genome mapping.

In vitro exposure of tumorigenic cell lines to the chemotherapeutic agent PALA (N-(phosphonoacetyl)-L-aspartate) usually results in cell death (shown here to be apoptosis), followed by clonal growth of rare survivors. On the other hand, normal diploid cells respond to PALA by arresting in G1 and G2 of the cell cycle. It was previously suggested that growth control mechanisms might exist to prevent cells from entering S phase under toxic conditions and that genes involved in such mechanisms were mutated or deleted in tumor cells. Interestingly, the tumor suppressor gene p53, a putative G1 control gene, was shown to mediate PALA-induced growth arrest. However, growth arrest occurs in cells that lack wild-type p53, suggesting that other genes are involved as well. To identify these genes, we have generated whole cell hybrids between mouse melanoma and normal human fibroblast cells. At early passage, a whole cell hybrid (BHF12) responds to PALA with growth arrest, while at later passage, the same hybrid undergoes apoptosis. To determine which human chromosomes are required for the PALA-induced growth arrest phenotype, we isolated subclones of the hybrid and tested them for their PALA response. FISH (fluorescence in situ hybridization) and PCR (polymerase chain reaction) amplification have been used to identify the human chromosome content of BHF12 and its subclones. Several human chromosomes, in addition to chromosome 17 (the location of p53), are consistently associated with the growth arrest phenotype. These findings provide evidence that one or more genes in addition to p53 may be involved in the suppression of a drug-induced apoptosis pathway and may lie on one or more of these chromosomes.Key words: apoptosis, growth arrest, N-(phosphonoacetyl)-L-aspartate, somatic cell hybrids.

Review on CD9 (CD9 molecule), with data on DNA, on the protein encoded, and where the gene is implicated.

Human genes expressed in interspecific somatic cell hybrids can be cloned specifically by subtractive cDNA hybridization. This approach is based on the observation that cDNA fragments from noncoding segments of mature human transcripts do not form stable heteroduplexes with their rodent homologues under high-stringency hybridization conditions. Thus, small, oligo-dT primed cDNAs from a rat/human hybrid retaining a fragment of human chromosome 17 were enriched for human sequences by hybridization with RNA from a sister clone containing a smaller human chromosome fragment. The enriched probe was used to screen a human cDNA library, and nine expressed genes from within the non-overlap region were obtained. This method should be useful for cloning active human genes from defined chromosome segments.

Despite progress in diagnosis and treatment, prostate cancer is still one of the most frequent lethal diseases in men in Western countries. Today, an increasing number of prostate cancers are detected through elevated serum prostate-specific antigen (PSA) levels. PSA detection is very sensitive. Determining free versus total PSA serum level has enabled the achievement of better specificity (Balk et al. 2003), however, this tool remains imperfect. Many patients undergo unnecessary diagnostic procedures, experiencing physiological and psychological stress. Similarly, current techniques such as imaging and biopsies are not optimal thus, hopes are high for the discovery of new molecular markers for prostate cancer. Studying prostate cancer progression may reveal new insights into the molecular mechanisms of cancer development to help improve prevention, provide better tools for diagnosis, as well as for prognosis and treatment.

The genetic analysis of nontumorigenic radiation hybrids generated by transfer of chromosome 3 fragments into the tumorigenic OV-90 ovarian cancer cell line identified the 3p12.3-pcen region as a candidate tumor suppressor gene (TSG) locus. In the present study, polymorphic microsatellite repeat analysis of the hybrids further defined the 3p12.3-pcen interval to a 16.1 Mb common region containing 12 known or hypothetical genes: 3ptel-ROBO2-ROBO1-GBE1-CADM2-VGLL3-CHMP2B-POU1F1-HTR1F-CGGBP1-ZNF654-C3orf38-EPHA3-3pcen. Seven of these genes, ROBO1, GBE1, VGLL3, CHMP2B, CGGBP1, ZNF654, and C3orf38, exhibited gene expression in the hybrids, placing them as top TSG candidates for further analysis. The expression of all but one (VGLL3) of these genes was also detected in the parental OV-90 cell line. Mutations were not identified in a comparative sequence analysis of the predicted protein coding regions of these candidates in OV-90 and donor normal chromosome 3 contig. However, the nondeleterious sequence variants identified in the transcribed regions distinguished parent of origin alleles for ROBO1, VGLL3, CHMP2B, and CGGBP1 and cDNA sequencing of the hybrids revealed biallelic expression of these genes. Interestingly, underexpression of VGLL3 and ZNF654 were observed in malignant ovarian tumor samples as compared with primary cultures of normal ovarian surface epithelial cells or benign ovarian tumors, and this occurred regardless of allelic content of 3p12.3-pcen. The results taken together suggest that dysregulation of VGLL3 and/or ZNF654 expression may have affected pathways important in ovarian tumorigenesis which was offset by the transfer of chromosome 3 fragments in OV-90, a cell line hemizygous for 3p.

Multiple chromosome 3p tumor suppressor genes (TSG) have been proposed in the pathogenesis of ovarian cancer based on complex patterns of 3p loss. To attain functional evidence in support of TSGs and identify candidate regions, we applied a chromosome transfer method involving cell fusions of the tumorigenic OV90 human ovarian cancer cell line, monoallelic for 3p and an irradiated mouse cell line containing a human chromosome 3 in order to derive OV90 hybrids containing normal 3p fragments. The resulting hybrids showed complete or incomplete suppression of tumorigenicity in nude mouse xenograft assays, and varied in their ability to form colonies in soft agarose and three-dimensional spheroids in a manner consistent with alteration of their in vivo tumorigenic phenotypes. Expression microarray analysis identified a set of common differentially expressed genes, such as SPARC, DAB2 and VEGF, some of which have been shown implicated in ovarian cancer. Genotyping assays revealed that they harbored normal 3p fragments, some of which overlapped candidate TSG regions (3p25-p26, 3p24 and 3p14-pcen) identified previously in loss of heterozygosity analyses of ovarian cancers. However, only the 3p12-pcen region was acquired in common by all hybrids where expression microarray analysis identified differentially expressed genes. The correlation of 3p12-pcen transfer and tumor suppression with a concerted re-programming of the cellular transcriptome suggest that the putative TSG may have affected key underlying events in ovarian cancer.

Expression of tetraspanin CD9 protein is downregulated in many tumors. CD9 over-expression also reduces the tumorigenicity of some human cancer cells. Here, we determined if exogenous expression of CD9 affects the properties of human prostate cancer cells. Highly metastatic prostate cancer cells PC-3M-LN4 over-expressing exogenous CD9 were orthotopically injected into the prostate of nude mice. CD9 expression was determined in tumors using PCR and Western immunoblotting techniques. Over-expression of CD9 increased invasiveness of prostate cancer cells in vitro. Animals injected with either parental PC-3M-LN4 or CD9-transfected cells developed tumor and harbored lymph node metastasis. There was no statistical difference in tumor growth between the two cell lines. CD9 did not suppress tumorigenic or metastatic properties of PC-3M-LN4 cells. Our data contrast with published results in other tumor types and likely indicate that other proteins (CD9 partners) are needed for CD9 full anti-tumorigenic action.

Purpose: An ideal vaccine therapy for tumors should activate both effector and memory immune responses against tumor-specific antigens. Here we investigated the effect of CpG oligodeoxynucleotides (CpG-ODN) for their ability to potentiate the activity of tumor antigen–pulsed bone marrow–derived dendritic cells (DC) in a vaccine model for the treatment of murine renal cell carcinoma (RENCA).Experimental Design: First we evaluated the effects of a murine renal cell carcinoma (RENCA) on immune cell activity in a mouse model using in vitro assays for T-cell proliferation and natural killer cell activation. To overcome the immune suppression of the tumor, we s.c. injected groups of 10 mice with dendritic cells and tumor cells. We compared the effect of different conditioning regimens of the DCs with RENCA antigen and/or CpG-ODNs before injection by measuring tumor size twice a week.Results: Tumor growth was shown to negatively affect spleen cell and T-cell proliferation, IFN-γ production, natural killer cell activity, and NF-κB activation in T cells. In this model, we have shown that RENCA-pulsed CpG-ODN-treated DCs were able not only to significantly reduce tumor growth but also to prevent tumor implantation in 60% of mice. Tumor-free mice were resistant to tumor challenge and the immunity conferred by the vaccine was transferable and tumor specific.Conclusions: This data show that RENCA down-modulates the immune response, and DC vaccine therapy, in conjunction with CpG-ODN, can restore tumor-specific immunity.

The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up-regulated during “initiation” (1 day) and “outgrowth and differentiation” (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT-PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration. Developmental Dynamics 231:527–541, 2004. © 2004 Wiley-Liss, Inc.

Background The pufferfish Fugu rubripes (Fugu) with its compact genome is increasingly recognized as an important vertebrate model for comparative genomic studies. In particular, large regions of conserved synteny between human and Fugu genomes indicate its utility to identify disease-causing genes. The human chromosome 12p12 is frequently deleted in various hematological malignancies and solid tumors, but the actual tumor suppressor gene remains unidentified. Results We investigated approximately 200 kb of the genomic region surrounding the ETV6 locus in Fugu (fETV6) in order to find conserved functional features, such as genes or regulatory regions, that could give insight into the nature of the genes targeted by deletions in human cancer cells. Seven genes were identified near the fETV6 locus. We found that the synteny with human chromosome 12 was conserved, but extensive genomic rearrangements occurred between the Fugu and human ETV6 loci. Conclusion This comparative analysis led to the identification of previously uncharacterized genes in the human genome and some potentially important regulatory sequences as well. This is a good indication that the analysis of the compact Fugu genome will be valuable to identify functional features that have been conserved throughout the evolution of vertebrates.

To increase the density of a gene map of the zebrafish, Danio rerio, we have placed 3119 expressed sequence tags (ESTs) and cDNA sequences on the LN54 radiation hybrid (RH) panel. The ESTs and genes mapped here join 748 SSLp markers and 459 previously mapped genes and ESTs, bringing the total number of markers on the LN54 RH panel to 4226. Addition of these new markers brings the total LN54 map size to 14,372 cR, with 118 kb/cR. The distribution of ESTs according to linkage groups shows relatively little variation (minimum, 73; maximum, 201). This observation, combined with a relatively uniform size for zebrafish chromosomes, as previously indicated by karyotyping, indicates that there are no especially gene-rich or gene-poor chromosomes in this species. We developed an algorithm to provide a semiautomatic method for the selection of additional framework markers for the LN54 map. This algorithm increased the total number of framework markers to 1150 and permitted the mapping of a high percentage of sequences that could not be placed on a previous version of the LN54 map. The increased concentration of expressed sequences on the LN54 map of the zebrafish genome will facilitate the molecular characterization of mutations in this species.

We have characterized a collection of zebrafish/mouse somatic cell hybrids with 211 genes and markers chosen from the 25 zebrafish linkage groups. Most of the zebrafish genome is represented in this collection with 88% of genes/markers present in at least one hybrid cell line. Although most hybrids contain chromosomal fragments, there are a few instances where a complete or nearly complete zebrafish chromosome has been maintained in a mouse background, based on multiple markers covering the entire chromosome. In addition to their use in mapping studies, this collection of somatic cell hybrids should constitute an important tool as a source of specific chromosome fragments and for assessing the function of genome regions.

In our cloning strategy to identify tyrosine kinases implicated in the regulation of prostate growth, the dog fer cDNA was obtained and shown to be highly homologous to known fer cDNAs. Using a polyclonal Fer antibody directed against a C-terminal peptide, we studied its associations with cortactin, β-catenin and p120Cas in human prostate carcinoma PC-3 cells. In contrast to previous reports, no interactions were observed. To assess its functional role, fer cDNA constructs were transfected in PC-3 cells. Antisense clones exhibiting a marked diminution of Fer expression had a reduced growth rate (doubling time of 29 vs. 42 h) and were unable to form colonies in soft agar. In agreement with these results, Fer protein expression was linked to human prostatic proliferative diseases, with enhanced levels in extracts from cancer tissues as compared to those from normal and hyperplastic ones, and was also expressed in the human prostate carcinoma cell lines DU145 and LNCaP. In the dog model, Fer expression was up-regulated in dividing versus resting prostate epithelial cells in vitro, and also in vivo when basal cell hyperplasia and metaplasia was induced by estrogen after castration. Minimal effects were observed when renewing the luminal epithelium with androgens. Taken together, these results show that Fer expression is associated with prostate cell proliferation and enhanced in prostate cancer.

We have constructed a zebrafish genetic linkage map consisting of 705 simple sequence-length polymorphism markers (SSLPs). The map covers 2350 centimorgans (cM) of the zebrafish genome with an average resolution of 3.3 cM. It is a complete map in genetic mapping terms (there is one linkage group for each of the 25 chromosomes), and it has been confirmed by somatic-cell hybrids and centromere-mapping using half-tetrad analysis. The markers are highly polymorphic in the zebrafish strains used for genetic crosses and provide a means to compare genetic segregation of developmental mutations between laboratories. These markers will provide an initial infrastructure for the positional cloning of the nearly 600 zebrafish genes identified as crucial to vertebrate development,and will become the anchor for the physical map of the zebrafish genome.

Publisher Summary This chapter overviews the techniques pertinent to the production and the characterization of zebrafish/mouse cell hybrids. Transfer of zebrafish chromosomes to cells of a foreign species has been accomplished successfully with three different fish cell lines-ZF4, LFF, and AB9—as donor cells, and with the mouse B78 cells or hamster Wg3H cells as recipients. The mouse B78 melanoma cell line was chosen as recipient in whole-cell fusion experiments because it was previously shown to fuse very easily with other cell lines. Furthermore, the cells have a fast growth rate and are easy to maintain, characteristics that were often retained after fusion. Zebrafish chromosomes are tagged with the neo resistance gene. Mouse B78 cells are not deficient in any enzyme—therefore, the aminoglycoside phosphotransferase (neo) gene, conferring resistance to the aminoglycoside drug G418, is introduced into zebrafish chromosomes to select against unfused recipient mouse cells and to ensure retention of zebrafish chromosomes in hybrids.

The zebrafish, Danio rerio, is becoming an increasingly popular model for the study of vertebrate development. Indeed, the biology of the fish offers great advantages for such studies. The life cycle of the zebrafish is relatively short (2-3 months) and the embryos develop outside the mother, facilitating the visualization of any mutated phenotype. At present, more than 1000 embryonic mutations have been reported. However, until recently, there was no physical or genetic map for this organism. In an effort to generate such a map, we have produced and characterized a panel of zebrafish-mouse cell hybrids. We have used whole-cell fusion to transfer zebrafish chromosomes from two different zebrafish cell lines into mouse recipient cells, thus generating more than 100 hybrids. Using fluorescence in situ hybridization and polymerase chain reaction analysis, we have determined the zebrafish chromosome composition of these hybrids. Here we report that elements from the 25 linkage groups of the zebrafish genome are present in our hybrids. These hybrids could identify the chromosomal location of genes affected in zebrafish mutants.

We have discovered a family of short interspersed repetitive elements (SINEs) that are present in the genomes of fish, amphibian and primates. The family of the SINEs, designated mermaid, is distinctive in each species except for a conserved region of approximately 80 bp. Some members of the mermaid family were found in transposon-like repetitive elements, including Tc1-like elements which are also distributed in the genomes of fish and amphibian. This raises the possibility of horizontal transfer of the mermaid family between vertebrates via transposons.

Tissue-specific extinguisher 1 (TSE1) is a trans-acting locus on human chromosome 17 that down-regulates expression of seven liver genes in hepatoma x fibroblast hybrids. To study the mechanism by which TSE1 functions, we used subtractive cDNA hybridization to clone transcripts encoded within a 2-4 Mb segment of chromosome 17 that includes TSE1. High resolution mapping within this region indicated that 8 of 9 different human cDNAs so obtained were distinct from TSE1. The remaining cDNA clone mapped concordantly with TSE1 in a panel of fragment-containing hybrids. DNA sequencing indicated that this cDNA encoded regulatory subunit RI alpha of cAMP-dependent protein kinase, and RI alpha mRNA levels correlated with TSE1 activity in various hybrid lines. Stable transfection of wild-type or cAMP-binding mutant RI alpha alleles into hepatoma recipients produced an extinction phenotype indistinguishable from that encoded by human TSE1. We conclude that TSE1 encodes a regulatory subunit of protein kinase A whose activity differs in different cell types.

At least one third of all cases of epithelial ovarian cancer are associated with the production of ascites, although its effect on tumor cell microenvironment remains poorly understood. This study addresses the effect of the heterologous acellular fraction of ovarian cancer-derived ascites on a cell line (OV-90) derived from the chemotherapy-naive ovarian cancer patient. Ascites were assayed for their effect on cell invasion, growth, and spheroid formation. When compared to either no serum or 5% serum, ascites fell into one of two categories: stimulatory or inhibitory. RNA from OV-90 cells exposed to selected ascites were arrayed on an Affymetrix HG-U133A GeneChip. A supervised analysis identified a number of differentially expressed genes and quantitative polymerase chain reaction validation based on OV-90 cells exposed to 54 independent ascites demonstrated that stimulatory ascites affected the expression of ISGF3G, TRIB1, MKP1, RGS4, PLEC1, and MOSPD1 genes. In addition, TRIB1 expression was shown to independently correlate with prognosis when its expression was ascertained in an independent set of primary cultures established from ovarian ascites. The data support the validity of the strategy to uncover molecular events that are associated with tumor cell behavior and highlight the impact of ascites on the cellular and molecular parameters of ovarian cancer.

Three major regions of DNase I hypersensitivity (DH) were found in alpha 1-fetoprotein (AFP) chromatin of rat liver. DH site I is located at the transcription initiation site and associated with ongoing AFP transcription. DH site II is located 2.5 kb upstream from the cap site: it is developmental stage-dependent but dissociable from ongoing AFP transcription. DH site III, 3.7 kb upstream from the cap site, behaves as hepatocyte-constitutive. DH sites are present in similar regions of liver albumin chromatin. Dexamethasone-induced AFP gene repression is accompanied by the selective loss of AFP DH site I, a likely result of glucocorticoid receptors binding to a DNA recognition sequence located 5'-adjacent to DH site I. Sl nuclease-hypersensitive sites were found on naked superhelical AFP and albumin DNA, but do not appear to contribute DH sites in liver chromatin. The extent of hypomethylation of HpaII sites at the 5'-end of the AFP gene correlates positively with the level of potential and actual expression of the gene. We conclude that developmental and hormonal regulation of the AFP gene is confined within congruent to 4 kb of 5'-flanking DNA, and we discuss possible hierarchical interactions among DH sites, in relation to DNA methylation and replication.

Mutations were introduced in 7 kilobases of 5'-flanking rat ..cap alpha../sub 1/-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps withmore » promoter-activating element DE.« less