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Radiation hybrid maps were constructed for river buffalo and cattle Y chromosomes. A total of 41 cattle-derived Y-chromosome molecular markers were selected and tested with 2 previously described 5,000-rad whole-genome radiation hybrid (RH) panels (river buffalo – BBURH5000 and cattle – BTARH5000) for generation of maps. Among the initial 41 selected markers, a subset of 26 markers generated PCR products suitable for scoring with the BBURH5000 panel. Of these, 19 markers (73%) were distributed in 1 linkage group spanning 341.3 cR. Retention frequencies (RF) for individual markers ranged from 17.8% for SMCY to 56.7% for BTY1, with an average RF of 37.6%. From the selected markers, 37 generated reliable scores using the BTARH5000 panel. The newly constructed BTAY RH map contains 28 markers distributed within 1 linkage group. Twenty-four of these markers had been previously mapped on BTAY using a 7,000-rad cattle-hamster WG-RH panel and 4 markers were mapped for the first time (ZFY, SeqRep, RepSeqS4 and BTY1). The length of the BTAY RH map was estimated to be 602.4 cR. Retention frequencies for individual mapped markers ranged from 10% (INRA126) to 63.3% (SeqRep), with an average RF of 35.3%. RH marker positions along the Y chromosome were compared between BBUY and BTAY, which revealed differences in the order of some of the markers. The BBUY pseudoautosomal region (PAR) is delineated by 3 BTAY PAR markers (MAF45, TGLA325 and UMN2008). These markers are telomeric in both species but are not found in the same order. Here we have demonstrated the effective use of bovine Y chromosome markers for the development of the first BBUY RH map. Likewise, these set of markers can be used for comparative assessment of Y chromosomes in other members of the Bovidae family.

Nuclear transcription factor Y, beta (NFYB) was evaluated as candidate gene for congenital splay leg in piglets based on data from differential display and QTL analysis. We mapped NFYB to pig chromosome 5 (SSC5). By assigning further five porcine genes from the corresponding region on human chromosome (HSA) 12q23.3→ q24.11 to SSC5 and 14 we could confine an evolutionary breakpoint from an interval of more than 10 Mb to less than 400 kb. Comparative sequence analysis of the coding region of NFYB in healthy and splay leg piglets revealed no polymorphism. Inter-species conservation of the codons ranges from 87% to 95% between pig, human, cow, dog, rat and mouse, respectively. The expression of NFYB in M. biceps femoris was not different between healthy and splay leg piglets. However, healthy male piglets had a significantly higher expression than females. Our results exclude NFYB as candidate gene for congenital splay leg but provide a basis for selection of further candidates for the disease from SSC5.

Genes coding for sarcomeric proteins may play a key role in muscle mass accretion and meat production. Screening a skeletal muscle cDNA library we isolated two partial sequences coding for the sarcomeric myopalladin and titin genes. In the present work we identified three SNPs in the 3′ untranslated region, two at the myopalladin locus and one at the titin locus. Myopalladin was mapped on porcine chromosome (SSC) 14 using a somatic cell hybrid panel, a radiation hybrid panel and by linkage mapping. The linkage mapping of titin confirmed the position on SSC15. Then we analysed the allelic distribution of the alleles at both loci in six different porcine breeds. The analysis of the allele frequencies for these two loci in extremely divergent groups of pigs selected according to lean cuts (LC) and average daily gain (ADG) approached the significance level for myopalladin and LC trait. Further studies are needed to test the presence of a putative effect of myopalladin on lean meat content.

ZOO-FISH mapping shows human chromosomes 1, 9 and 10 share regions of homology with pig chromosome 10 (SSC10). A more refined comparative map of SSC10 has been developed to help identify positional candidate genes for QTL on SSC10 from human genome sequence. Genes from relevant chromosomal regions of the public human genome sequence were used to BLAST porcine EST databases. Primers were designed from the matching porcine ESTs to assign them to porcine chromosomes using the INRA somatic cell hybrid panel (INRA-SCHP) and the INRA-University of Minnesota Radiation Hybrid Panel (IMpRH). Twenty-eight genes from HSA1, 9 and 10 were physically mapped: fifteen to SSC10 (ACO1, ATP5C1, BMI1, CYB5R1, DCTN3, DNAJA1, EPHX1, GALT, GDI2, HSPC177, OPRS1, NUDT2, PHYH, RGS2, VIM), eleven to SSC1 (ADFP, ALDHIB1, CLTA, CMG1, HARC, PLAA, STOML2, RRP40, TESK1, VCP and VLDLR) and two to SSC4 (ALDH9A1 and TNRC4). Two anonymous markers were also physically mapped to SSC10 (SWR1849 and S0070) to better connect the physical and linkage maps. These assignments have further refined the comparative map between SSC1, 4 and 10 and HSA1, 9 and 10.

Several genes (PRKAA2, PRKAB1, PRKAB2, PRKAG3, GAA, GYS1, PYGM, ALDOA, GPI, LDHA, PGAM2 and PKM2), chosen according to their role in the regulation of the energy balance and in the glycogen metabolism and glycolysis of the skeletal muscle, were studied. Eleven single nucleotide polymorphisms (SNPs) were identified in six of these genes (PRKAB1, GAA, PYGM, LDHA, PGAM2 and PKM2). Allele frequencies were analyzed in seven different pig breeds for these loci and for a polymorphism already described for GPI and for three polymorphic sites already reported at the PRKAG3 locus (T30N, G52S and I199V). Linkage mapping assigned PYGM and LDHA to porcine chromosome (SSC) 2, PKM2 to SSC7, GAA to SSC12, PRKAB1 to SSC14 and PGAM2 to SSC18. Physical mapping, obtained by somatic cell hybrid panel analysis, confirmed the linkage assignments of PRKAB1 and GAA and localized ALDOA, PRKAB2 and GYS1 to SSC3, SSC4 and SSC6, respectively. Pigs selected for the association study, for which several meat quality traits were measured, were first genotyped at the PRKAG3 R200Q polymorphic site (RN locus), in order to exclude carriers of the 200Q allele, and then were genotyped for all the mutations considered in this work. Significant associations (P ≤ 0.001) were observed for the PRKAG3 T30N and G52S polymorphic sites with meat colour (L* at 24 h post mortem). PGAM2 and PKM2 were significantly associated (P = 0.01) with drip loss percentage and glycogen content at one hour post mortem, respectively.

Our on-going goal is to improve and update the comparative genome organization between cattle and man but also among the most detailed mammalian species genomes i.e. cattle, mouse, rat and pig. In this work, we localized 195 genes in cattle and checked all human/bovine non-concordant localizations found in the literature. Next, we compiled all the genes mapped in cattle, goat, sheep and pig (2,166) for which the human ortholog with its chromosomal position is known, added corresponding data in mouse and rat, and ordered the genes relatively to the human genome sequence. We estimate that our compilation provides bovine mapping information for about 89% of the human autosomes. Thus, a near complete, overall and detailed picture of the number, distribution and extent of bovine conserved syntenies (regardless of gene order) on human R-banded autosomes is proposed as well as a comparison with mouse, rat and pig genomes.

Previously, we showed that CYP1A1 expression can be induced by omeprazole (OP) in the human cell line HepG2, but not in the mouse cell line Hepa-1. Now we show induction of CYP1A1 by α-naphthoflavone (αNF) in Hepa-1 cells. This induction was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by the aromatic hydrocarbon (Ah)-receptor antagonist PD98059, suggesting the presence of a ligand-independent signal-transduction pathway in the mouse cell line too. We utilized the lack of CYP1A1 induction by OP in Hepa-1 cells to map a putative human gene for OP-respon- siveness in cell hybrids produced by fusion of Hepa-1 and HepG2 cells. OP-induced CYP1A1 expression was detected in four out of the 32 Hepa-1 × HepG2 cell hybrids analyzed. To help identify the gene locus, a radiation-hybrid cell (E11) was constructed. Use of reverse-fluorescence in situ hybridization revealed that these five cell lines commonly retained human chromosome 10p. These results suggest that the human gene for OP-responsiveness is present on chromosome 10p.

The gene for S-adenosylhomocysteine hydrolase (AHCY) is involved in the regulation of cellular methylation reactions. Here we report the cloning, sequencing, and chromosomal assignment of the porcine AHCY gene. The gene consists of 10 exons spanning approximately 19 kb of genomic DNA. It encodes a protein of 432 amino acids that shows about 96% identity to the orthologous S-adenosylhomocysteine hydrolases from human, rat, and mouse. The porcine AHCY gene is located very close to the agouti signaling protein gene (ASIP) on SSC17q21. The chromosomal localization was subsequently confirmed by RH mapping and the genetic mapping of an intragenic microsatellite that maps to 57 cM on the linkage map of SSC17.

A comparative genome map is necessary for the implementation of comparative positional candidate gene cloning in cattle. We have developed a medium density comparative gene map of bovine chromosome 25 (BTA25). A radiation hybrid (RH) panel was used to map nine microsatellites and nine genes. Eight of the nine comparative loci were also mapped by FISH. These results were combined with data from published articles to create a comprehensive comparative map of BTA25 with human chromosomes 7 (HSA7) and 16 (HSA16). This map should facilitate the cloning of genes of interest on bovine chromosome 25.

NEKs (NIMA-related kinases) are a group of protein kinases sharing high amino acid sequence identities with NIMA (never in mitosis gene a) which control mitosis in Aspergillus nidulans. We have cloned a cDNA for human NEK3, a novel human gene structurally related to NIMA, by RT-PCR. Its open reading frame encodes a protein of 489 amino acid residues with the calculated molecular mass of 56.0 kDa and a predicted pI of 6.58. Phylogenetic analysis suggests that mouse and human NEK3s constitute a subfamily within the NIMA family of protein kinases. The expression pattern of NEK3 was studied by RT-PCR and a high level of expression was detected in testis, ovary, and brain, with low-level expression being detected in most of the tissues studied. NEK3 mRNA was detected in all the proliferating cell lines studied, and the amount did not change during the cell cycle. The human NEK3 gene was assigned to human chromosome 13 by somatic cell hybrids and 13q14.2 by radiation hybrid mapping.

We have identified a chromosome duplication in the pericentromeric region of human chromosome 11 located in 11p11 and 11q14. A detailed physical map of each duplicated region was generated to describe the nature of the duplication, the involvement at the centromere and to resolve the correct maps. All clones were evaluated to ensure they were representative of their genetic origin. The order of clones, based on their marker content, as well as the distance covered was determined by SEGMAP. Each duplication encompasses more than 1 Mb of DNA and appears to be chromosome 11 specific. Ten STS markers were mapped within each duplication. Comparative sequence analysis along the duplication identified 35 nucleotide changes in 2,036 bp between the two copies, suggesting the duplication occurred over 14 million years ago. A suggested organization of the pericentromeric region, including the duplications and alpha-related repetitive sequences, is presented.

We have generated pig–mouse somatic cell hybrids by fusing normal pig fibroblasts with an anchorage independent mouse cell line GM05267. High quality G-banding analysis was applied to a set of 18 hybrid cell lines derived from 15 independent hybrids and chromosomes were identified. Cytogenetic analysis showed that all hybrids contained one or several pig chromosomes with normal morphology devoid of any structural changes. Out of 18 hybrids tested for colony formation in soft agar, 15 were suppressed for anchorage independence while the remaining three were not suppressed. Correlation of the cellular phenotype with the pig chromosome content of the hybrids suggests that the suppressor function for anchorage independence is located on pig chromosome (SSC) 14. We have previously shown that a suppressor gene for anchorage independence (SAI1) is located on rat chromosome (RNO) 5 and another suppressor gene for the same phenotype is located on human chromosome (HSA) 9. Given the genetic homology of both RNO5 and HSA9 with two pig chromosomes including SSC14, the third suppressor gene we have mapped on SSC14 may well be a functional homologue of the previously identified rat and human genes.

ERV9 is a class I family of human endogenous retroviral sequences. Somatic cell hybrid genomic hybridization experiments using a mono-chromosomal panel indicate the presence of approximately 120 ERV9 loci in the human genome distributed on most chromosomes. Fluorescence in situ hybridization (FISH) using an ERV9 cDNA probe containing gag, pol and env sequences, verified this observation and a consistent signal was found at the chromosome region 11q13.3→q13.5. By analysis of a panel of radiation hybrids, an ERV9 locus was mapped to within a 300-kbp region at the chromosome site 11q13. The marker cCLGW567 and the locus MAP3K11/D11S546 centromeric and telomeric flanked it, respectively. Northern blot analysis, using an ERV9 LTR probe, indicated that most normal tissues examined expressed low abundant ERV9 LTR driven mRNAs of various sizes. The most prominent expression was found in adrenal glands and testis. However, the level of expression varied in the same tissues among different individuals indicating that ERV9 mRNA expression probably is inducible in certain tissues or at various cell stages.

The human UBE2L6 gene encodes UbcH8Kumar, a ubiquitin-conjugating enzyme (E2) highly simliar in primary structure to UbcH7 which is encoded by UBE2L3. Like UBC4 and UBC5 in yeast, these proteins demonstrate functional redundancy. Herein we report the intron/exon structure of UBE2L6. Comparison of the genomic organization of UBE2L6 with UBE2L3 demonstrates that these genes remain highly conserved at the genomic as well as at the protein level. We also describe the chromosomal localization of UBE2L6, which maps to chromosome 11q12.

We have cloned a novel, widely expressed human gene, ZNF237, that shows extensive similarity to the N-terminal region of ZNF198. Two alternatively spliced regions were identified by RT-PCR; the major splice variant is predicted to encode a 383 amino acid protein that contains a single diverged MYM domain. ZNF237 maps to 13q11→q12, immediately proximal to ZNF198.

Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence. In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases. We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2. Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21. We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21. The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis.

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) human chromosome (HSA), derived from human fibroblast strain GM0705, into the Syrian hamster cell line BHK-191-5C produced only near-tetraploid hybrids, although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells. However, the tumorigenicity and the anchorage independence could be suppressed in the near-tetraploid hybrids with one copy of the der(9)t(X;9) chromosome. The introduction of an HSA X chromosome did not suppress either of these phenotypes. We concluded that in addition to two suppressor genes, one for tumorigenicity and another for anchorage independence, HSA 9 might carry a third gene capable of inhibiting cellular growth in vitro, which had dosage effects. In the present study, keeping one copy of the der(9)t(X;9) chromosome, we have increased the hamster background chromosome number beyond hexaploid level by fusing two microcell-generated hybrid cell lines, where both malignant and anchorage-independent phenotypes were suppressed, with the parental malignant BHK-191-5C cell line. Tests with nude mice showed that hybrids containing one copy of the der(9)t(X;9) chromosome against the increased background of chromosomes of malignant parental origin were still suppressed for both phenotypes. These results suggest that the suppressor genes for malignancy and for anchorage independence have no dosage effects, in contrast to the suppressor gene(s) for cellular growth.

TBC1D1 is the founding member of a family of related proteins with homology to t̲re-2/UPS6, b̲UB2, and c̲dc16 and containing the tbc box motif of 180–220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1→4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1→4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.

We have identified a new human gene, FTCD, which maps to chromosome 21q22.3 and encodes the enzyme formiminotransferase cyclodeaminase, an intermediate metabolism enzyme that links histidine catabolism to folate metabolism. The major cDNA encodes a protein containing 541 amino acid residues and shows 84% identity with porcine FTCD. Several other cDNAs have been isolated, which may result from alternative splicing events and have the potential to code for three different protein isoforms. The gene is highly expressed in human fetal and adult liver. The two FTCD protein domains show high sequence similarity to two distinct open reading frames from eubacterial genomes, suggesting that eukaryotic FTCD appeared through a gene fusion event. Defects in the glutamate formiminotransferase pathway have been documented, and the deficiency is presumed to be inherited as an autosomal recessive trait. The sequence reported here may be helpful in identifying the primary defect in glutamate formiminotransferase deficiency and establishing a molecular diagnosis.

As part of a project involving large-scale sequencing of clones randomly selected from a human cDNA library, we isolated a novel human gene, termed TM7SF3 (transmembrane 7 superfamily member 3). Its open reading frame encodes a 570 amino acid protein containing seven putative transmembrane domains. The transcript of this gene was expressed in all human tissues examined, but most abundantlyin kidney. Immunocytochemical analysis demonstrated subcellular localization of TM7SF3 protein at the plasma membrane. We determined the chromosome location of TM7SF3 as 12q11.2→q12 by a combination of fluorescence in situ hybridization and radiation hybrid mapping.

ZNF41 belongs to a cluster of human zinc finger genes residing within a gene-rich region at Xp11.23. ZNF41 encodes a KRAB/FPB (Krüppel-associated/finger preceding box) domain, a potent transcription repression motif present in hundreds of vertebrate zinc finger protein genes, composed of two protein modules, A and B. Three introns, placed at identical positions in paralogous genes, interrupt four exons encoding the ZNF41 N-terminal amino acids, the KRAB/FPB-A and KRAB/FPB-B modules, and the remaining coding region adjoined to the C-terminal zinc finger domain. Since the KRAB/FPB-A and KRAB/FPB-B modules are encoded by dedicated exons in ZNF41 and paralogous genes, exon skipping may lead to differential usage of these modules in alternative gene products. RT-PCR analysis of ZNF41 mRNAs showed that, while skipping of the KRAB/FPB-A and/or KRAB/FPB-B exons was not detected, the use of alternative donor/acceptor sites upstream of the KRAB/FPB-A exon generates multiple ZNF41 transcripts potentially encoding polypeptides differing in the N-terminal region and expressed in different tissues. The expression pattern in cell hybrids containing either active or inactive X chromosomes indicates that ZNF41, which resides within a region of the X chromosome that includes genes that are both subject to and escape X-inactivation, is susceptible to X-chromosome inactivation.

The realization of physical and genetic maps of the chicken genome is dependent on progress in cytogenetic knowledge of its karyotype. To help achieve this goal, we constructed amplified representative DNA samples of the chicken chromosomes 1, 2, 3, 4, 5, 6, 7, 8, Z, W and of the terminal heterochromatin part of Zq by chromosome microdissection and DOP-PCR amplification. These chromosome DNA samples, which represent about 75% of the chicken genome, were used to generate whole chromosome painting probes for FISH. The direct application of these chromosome specific probes is dual FISH localization and characterization of panels of chicken interspecific somatic hybrids. We discuss some aspects of the chicken genome and its repeated sequences.

We have previously shown that microcell-mediated transfer of a der(9)t(X,9) chromosome, containing an almost complete human chromosome (HSA) 9 derived from the human fibroblast strain GM0705, into the Syrian hamster (Mesocricetus auratus) cell line BHK-191-5C suppressed the anchorage independence and tumorigenicity of the hybrids. Transfer of a normal HSA X did not have any effect on these phenotypes. Although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells, all hybrids retaining the der(9) chromosome were near-tetraploid, in contrast to hybrids retaining a normal X chromosome. In the present study, we have generated microcell hybrids by transferring another der(9)t(X,9) chromosome derived from the human fibroblast strain GM01429. This derivative chromosome contained a deletion on the short arm of HSA 9 and was also missing the distal part of the long arm of HSA 9 due to the involvement in a reciprocal (constitutive) translocation of this chromosome with HSA X. Cytogenetic analysis showed that all hybrid clones were near-tetraploid, confirming our previous finding. We also observed that the introduction of the deleted der(9) chromosome forced the hybrids to lose Syrian hamster chromosome 10. A soft agar test and nude mice assay indicated that none of the hybrids was suppressed for either anchorage independent growth or tumor formation. These data suggest that there is an antagonistic relationship between growth-promoting genes and antiproliferative genes. The observed dosage effects of both growth-promoting and growth-suppressing genes indicate that cellular growth may be a quantitative trait. Copyright (C) 1999 S. Karger AG, Basel.

By protein interaction screening using a radioactive LMO2 protein probe we have isolated a LIM domain binding protein. The gene shows high homology to independently isolated genes from mouse, Xenopus and Drosophila called Ldb1/Nli/Clim-2, Xldb1 and Chip, respectively. The human and mouse genes differ by only two amino acids, suggesting that the gene that we have isolated is the human homologue. Here we describe the genomic organization, alternative transcript forms and the chromosome mapping of the human gene LDB1 (alias NLI). The gene is spread over at least 12 kb and has 11 exons. Preceding the described ATG initiation site in the mouse a highly conserved region between mouse, chicken and human was detected with a second possible in frame initiation site coding for further 36 amino acids. An alternative splice site adding six nucleotides corresponding to the addition of two amino acids at the end of exon 10 was found. The gene was mapped to chromosome 10q24→q25 by in situ hybridization, a region frequently deleted in many types of cancer. Fine mapping with a radiation hybrid panel localized the gene in the interval between the markers D10S603 and D10S540.

A cDNA encoding a third member of human class III ubiquitin-conjugating enzymes (E2s), UBE2E3, was cloned from a human gastric adenocarcinoma cDNA library. The deduced 207-amino acid protein shares over 94% amino acid identity with the UBC domains of class III E2s, UbcH6, UBE2E2, UbcM2, UbcM3, and UbcD2. But the N-terminal extension exhibited little homology among these, except for UbcM2, which showed 100% identity, and which is thought to be a mouse counterpart. Northern hybridization analysis exhibited a strong 1.9-kb band of UBE2E3 in skeletal muscle. Recombinant fusion protein of GST-UBE2E3 was found to form a thioester bond with ubiquitin (Ub) in an E1-dependent manner, demonstrating that the cDNA encodes a functional E2. In addition, a UBE2E3 mutant of cysteine-145 to serine failed in UBE2E3-Ub complex formation, indicating that the cysteine is essential for E2 function. Using FISH and PCR analysis of radiation hybrid and somatic cell hybrid panels the UBE2E3 gene was mapped to human chromosome 2q32.1 and showed strong linkage to SHGC-8506 (LOD = 11.52) between D2S1302 and D2S364.

Gene specific PCR primers were constructed for five mouse and three bovine CXC chemokine genes. The mouse genes were assigned using SSCP analyses of the Jackson BSS backcross panel to two groups on chromosome 5. One group containing Gro1 and Mip2 cosegregated with reference markers Alb1 and Btc, and was positioned 2.2 cM proximal to a group comprising Ifi10, Mig, and Scyb5. The bovine genes IL8, GRO1, and GRO3, mapped using bovine × hamster somatic cell hybrids, were all found to be located on chromosome 6. The locations of these genes in these two animal species are consistent with the positions in humans (4q13→q21), and previous syntenic relationships among these three mammals.

Several human malignancies frequently exhibit deletions or rearrangements of the distal short arm of chromosome 1 (1p36), and a number of genetic diseases also map to this region. The carbonic anhydrase (CA6) and α-enolase (ENO1) genes, previously mapped to 1p36, were physically linked in yeast- and P1-artificial chromosome (YAC and PAC) contigs. PACs from the contig were mapped to 1p36.2 by fluorescence in situ hybridization. The ESTs D1S2068, D1S274E, D1S3275, and stSG4370 were also placed in the same contig. The physical map was integrated with the genetic map of chromosome 1 by assignment of genetic markers D1S160, D1S1615, and D1S503 to the contig. Sequencing of the EST clone representing D1S274E indicated that it was derived from the same transcript as D1S2068E and corresponded to the SLC2A5 (GLUT5) gene, previously assigned to 1p31. Reassignment of SLC2A5 to 1p36.2 was confirmed by somatic cell and radiation hybrid mapping panels and was consistent with previous EST mapping data. Sequencing of the EST clone for D1S274E revealed the presence of intronic sequences, suggesting that the clone was derived from an unprocessed message. The presence of unprocessed and/or alternatively spliced EST clones has potential ramifications for EST-based genomic projects. This information should facilitate the mapping of tumor suppressor and genetic disease loci that have been localized to this region.

Comparative mapping of Ateles paniscus chamek and man indicated that four human 3p markers are syntenic in this karyotypically rearranged neotropical primate. The evolutionary conservation of this gene cluster includes three adjacent human shortest regions of overlap (SROs): 3p21.1 (ACY1), 3p21.3→p21.2 (CACNA1D), and 3p21.3 (ZNF64). A fourth syntenic marker (ATP2B2), at a more distal human SRO (3p26→p25), indicated that human 3pter→p14 is evolutionarily conserved in Ateles chromosome 3 (APC 3). Conversely, allocations of two human 3q markers (AGTRl and IL12A) clearly excluded APC 3. Finally, allocation of the major histocompatibility complex class I genes further confirmed human 6p-6q dissociations in Ateles.

Cri-du-chat syndrome is associated with a deletion of the short arm of chromosome 5. Through the phenotypic and molecular analyses of individuals with a subset of the features associated with the syndrome, the genes involved in the syndrome have been mapped to two distinct critical regions. Deletion of a critical region in 5p15.2 results in the distinct facial features associated with the syndrome as well as the severe mental and developmental delay, while a deletion of 5pl 5.3 is associated only with the characteristic cat-like cry, the key diagnostic feature of the syndrome. Therefore, subtle differences in the extent of the 5p deletion can have a profound affect on the prognosis of the patient. In order to more easily differentiate between deletions that lead to the cri-du-chat syndrome phenotype and deletions that lead only to the isolated cat-like cry, we have constructed YAC contigs that span both critical regions. The YAC clones have been used to isolate cosmids mapping to each critical region and cosmids that lie just within the two critical region boundaries have been identified. We report here on the use of these cosmids as probes for fluorescent in situ hybridization experiments on interphase nuclei as a means of more accurately differentiating between small 5p deletions that coincide with a complete cri-du-chat syndrome phenotype and the severe mental and developmental delay that is associated with it and deletions that only delete the distal critical region that coincide with the isolated cat-like cry and a much improved prognosis.

Human chromosomes 1, 2, 3, and 11 were tagged with pSV2 neo and transferred via microcell fusion from mouse A9 human monochromosomal hybrids to a chicken pre-B cell line, DT40, proficient for homologous recombination. Hybrids containing two copies of human chromosome 11 were transfected with targeting vectors containing a mammalian selectable gene with either the Dl 1S16 or HRAS genomic sequences corresponding to two different chromosome 11 loci. Analysis of stable transfectants showed a high frequency (∼80%) of targeted integration of these constructs into each of the homologous loci of human chromosome 11 in DT40 hybrids. The results suggest that any human genomic sequences on human chromosomes transferred into DT40 cells could be targeted at high frequency, thereby allowing for subsequent modification of human genes and chromosomes.

A somatic cell hybrid panel was constructed consisting of seven hybrids with translocation breakpoints spanning the region 17q23→q25. Hybrid clones carrying the long-arm derivative of chromosome 17 in the absence of the normal chromosome 17 and of the derivative 17 were initially identified by PCR typing for a proximal and distal 17q marker. The translocation breakpoints of the hybrids were then mapped in more detail by PCR analysis for a number of microsatellite markers from chromosome 17q as well as for five gene loci (CACNLG, GH1, SOX9, TIMP2, TK1) previously mapped to the region 17q23→q25. In addition, the locus for GDIA1 was mapped by FISH to 17q25.3 and fine mapped with the help of the hybrid panel. These seven new hybrids complement the existing somatic cell hybrid panel for the long arm of chromosome 17q.

We previously described a patient with a de novo constitutional translocation, t(l;22)(p22;ql 1.2), who developed a malignant ependymoma at age 5, and we proposed that the translocation predisposed the child to the development of the tumor. As a step toward isolation of a putative cancer gene, we have characterized the breakpoints of the (1;22) translocation at the molecular level. The chromosome 22 breakpoint has been narrowed to a region between ARVCF and D22S264. The chromosome 1 breakpoint has been mapped onto a doubly-linked Whitehead YAC contig by PCR analysis of the STS contents of the patient’s derivative chromosomes isolated in somatic cell hybrids. Loss-of-heterozygosity (LOH) studies of the patient’s ependymoma and of sporadic ependymomas showed no evidence of consistent loss in the breakpoint regions, suggesting that activation of an oncogene, rather than inactivation of a tumor suppressor gene, is the more likefv molecular mechanism involved in this case. The gene xor Edg-1, a neurally expressed, seven-segment transmembrane receptor, maps to the region of the chromosome 1 breakpoint but does not appear to be interrupted by the translocation. Molecular characterization of the breakpoint regions reported here represents an important step in the identification of the gene(s) affected by this translocation.

Cytogenetic and molecular studies have implicated one or more tumor suppressor genes on the long arm of human chromosome 11 in the malignant progression of several human solid tumors, including malignant melanoma and carcinomas of the breast, cervix, ovary, and lung. Microcell-mediated chromosome transfer of an intact copy of chromosome 11 into tumor cell lines has provided additional evidence of tumor suppressor gene function in melanoma, breast cancer, and cervical cancer. However, sublocalization of the region(s) conferring the tumor suppressive effect has been difficult. To facilitate mapping of tumor suppressor gene(s) on chromosome 11, we have generated a panel of 25 mouse donor cell lines containing neo-taggedfragments of human chromosome 11q which can be transferred into cell lines to test for tumor suppressor activity. The chromosome fragments in these cell lines have been characterized by fluorescence in situ hybridization with probes to human DNA and to the centromere of chromosome 11, and also by analysis of microsatellite markers spanning chromosome 11. Finally, to demonstrate the usefulness of these cell lines as donors for microcell-mediated chromosome transfer, two fragments were transferred into the human melanoma cell line UACC 903. This panel of selectable subchromosomal fragments, derived from the long arm of human chromosome 11, will be useful for the regional localization of tumor suppressors and other genes by means of functional assays.

One hundred and one DNA markers previously assigned to the short arm of the human X chromosome were localized on a hybrid mapping panel consisting of ten radiation-reduced, and four classical somatic cell hybrids. Of the 101 DNA markers, 16 are genes, two are pseudogenes, 13 are expressed sequence tags, 32 are simple tandem repeats (STRs), four are restriction fragment length polymorphisms, one is a variable number of tandem repeats, and 33 are sequence tagged sites (STSs). Three of these markers, two STSs and one STR, were generated from the products of an inter-Alu PCR library of a radiation-reduced hybrid containing Xp11.4→p11.22 as its only human DNA content. A second STR was isolated from a region-specific cosmid containing the gene ZNF21. The 101 DNA markers fell into 22 bins based on their retention on the hybrids of this panel, which, in combination with YAC contig data, could be further resolved into 24 bins. This hybrid map of Xpl 1 has an average resolution of approximately 0.8 Mb.

CTAG was initially cloned from an esophageal squamous cell carcinoma cDNA expression library by immunoscreening with autologous patient’s serum. CTAG mRNA is expressed in a proportion of human cancers in a lineage-nonspecific fashion, whereas its expression in normal tissues is restricted to testis and ovary only. This expression pattern suggests that the CTAG product (NY-ESO-1) is an aberrantly activated tumor antigen and can potentially be an antigenic target for tumor vaccination. In the present study, we isolated human genomic clones of CTAG and established its genomic organization. By somatic cell hybrid studies and fluorescence in-situ hybridization, we localized this gene to chromosome Xq28, a region that also contains members of MAGE, a gene family that encodes several immunogenic tumor antigens with the characteristic cancer/testis expression pattern.

We report the existence of a new family of swine centromeric satellite DNA composed of a 51-bp repeat unit, most specifically found on pig chromosome 11 centromere and with less specificity at the centromeric region of other meta-and submetacentric chromosomes. This satellite DNA family, which has no homologies with the Mel and Ac2 families published previously, was named Mc2. We designed a specific primer set for PCR amplification of this centromeric satellite DNA. Specificity of amplification was checked by using a porcine somatic cell hybrid panel and by FISH. Furthermore, the development of a PCR-RFLP marker of Mc2 repetition allowed its genetic mapping on the PiGMaP reference families panel. The centromere of chromosome 11 was thus integrated to the genetic map previously published.

Dr1 is a nuclear protein of 19 kDa that exists in the nucleoplasm as a homotetramer. By binding to TBP (the DNA-binding subunit of TFIID, and also a subunit of SL1 and TFIIIB), the protein blocks class II and class III preinitiation complex assembly, thus repressing the activity of the corresponding promoters. Since transcription of class I genes is unaffected by Drl, it has been proposed that the protein may coordinate the expression of class I, class II and class III genes. By somatic cell genetics and fluorescence in situ hybridization, we have localized the gene (DRl), present in the genome of higher eukaryotes as a single copy, to human chromosome region 1p21→p13. The nucleotide sequence conservation of the coding segment of the gene, as determined by Noah’s ark blot analysis, and its ubiquitous transcription suggest that Drl has an important biological role, which could be related to the negative control of cell proliferation.

Somatic cell hybrids were obtained by cell fusions between Lck-positive EL4 mouse T lymphoma cells and Lck-negative B82 mouse fibroblasts or S194 mouse plasmacytoma cells to examine negative control of lck gene expression in the resulting hybrids. Western blot analysis using a monoclonal antibody against the Lck protein showed a marked decrease in p56lck expression in B82 × EL4 (BEL) hybrids. In contrast to BEL hybrids, the level of p56lck was not changed significantly in S194 × EL4 (SEL) hybrids and was approximately one-half of that seen in EL4 cells. Diminished expression of the Lck protein in BEL hybrids paralleled downregulation of lck mRNA, which was exclusively transcribed from the distal promoter in EL4 cells. It is unlikely that the suppression was simply a consequence of chromosome segregation critical for lck gene expression, since BEL hybrids retained the EL4-derived lck gene and most of the chromosomes from both parental cells. The results from treatment of BEL hybrids with actinomycin D or cycloheximide suggested that suppression of lck gene expression in the hybrids might not be due to posttranscriptional control. DNA methylation status in the lck distal promoter and the coding regions did not appear to correlate with the expression of the gene. Our results suggest that negative control of lck gene expression differs between fibroblasts and B cells, in that lck gene expression in T cells can be shut down by transfer of a putative repressor factor or factors in fibroblasts but not in B cells.

We have mapped in the pig (Sus scrofa) the genes for zona pellucida glycoprotein 3 (ZP3), erythropoietin (EPO), and malate dehydrogenase 2 (MDH2) by somatic cell hybrid analysis in the pig genome. Previously, the gene for the T-cell receptor β cluster (TCRB) was assigned to SSC 18 and that for interleukin 6 (IL6) to SSC 9. However, statistical analysis of mapping data for ZP3, EPO, and MDH2 did not discriminate between SSC 9 and SSC 3. Porcine-specific PCR primers for ZP3 and IL6 were used to isolate probes from porcine YAC libraries. These two genes were mapped by FISH to porcine chromosome regions 3pter→p15 (ZP3) and 9q14→q15 (IL6). Comparative mapping considerations permit the tentative assignment of porcine EPO and MDH2 to 3pter→p15.

The Wilms’ tumour suppressor 1 gene (WT1) encodes a zinc finger transcription factor critical for normal urogenital development. We have previously isolated a DNA fragment, +P5 (D1S3309E), to which all WT1 protein isoforms bind. Using PCR of a human × rodent somatic cell hybrid mapping panel, together with two-color fluorescence in situ hybridisation of +P5-containing cosmids and previously localised human chromosome 1q cosmids, we have mapped the +P5 fragment to chromosome 1q21→q22.

A cDNA representing the high-affinity gonadotropin-releasing hormone (GnRH) receptor has been molecularly cloned from the human pituitary gland, a breast tumor cell line (MCF 7), and an ovarian tumor. The nucleotide sequence of this cDNA was determined, and its expression in various human tumors and tumor cell lines was demonstrated. In this study, we localized the gene encoding the GnRH receptor to human chromosome 4, using polymerase chain reaction (PCR) analysis of genomic DNA from human × hamster somatic cell hybrids. The gene was sublocalized to chromosome band 4q13 using fluorescence in situ hybridization with the GnRH receptor gene (GNRHR).

Using a chromosomally characterized minipanel of sheep x hamster cell hybrids, five new loci, including carbonic anhydrase II (CA2), calbindin 1 (28 kDa) (CALB1), corticotropin releasing hormone (CRH), cytochrome P450 11B subfamily XIB (steroid-11-beta-hydroxylase), polypeptide 1 (CYP11B1), and interleukin 7 (IL7), have been assigned to sheep chromosome 9. A homolog of CA2 was detected on sheep chromosome 1. CRH was regionally localized to sheep 9q23-->q28 by in situ hybridization. This study assigns chromosome 9 as the sheep equivalent of cattle chromosome 14 and indicates that CALB1, CYP11B1, and IL7, which have not been mapped on the cattle genome, are likely to be present on cattle chromosome 14. It also shows by comparative genome analysis that a large segment of human chromosome 8q is highly conserved in sheep chromosome 9 and cattle chromosome 14. Based on these data, we propose that sheep chromosome 9 be recognised as the equivalent of cattle chromosome 14.

Radiation-induced somatic cell hybrids containing fragments of the human X chromosome were constructed. A panel of 17 hybrids was selected with the help of known markers in the Xp22 region. These hybrids identified 11 different breakpoints between Xp22.2 and Xp21.3. Eight markers were located in eight of the nine corresponding intervals, resulting in the following physical map: tel DXS89-DXS278-DXS85-(DXS1224, DXS16)-(GLRA2, DXS987)-DXS207-(DXS-197, DXS1053)-(DXS43, DXS1195)-(DXS1229, DXS-999)-(DXS1052, DXS92, DXS274)-(DXS41, DXS1226)-DXS1198-DXS28...cen.

Identification and localization of genes that encode regulators of transcription could provide landmarks for functional analysis of the human genome. Toward this goal, we examined a panel of somatic cell hybrids and assigned the gene (TCF12) encoding the helix-loop-helix transcription factors 4 (HTF4) to chromosome 15. Fluorescence in situ hybridization further localized TCF12 to chromosome 15q21. Northern analysis revealed that the relative abundance of HTF4 gene transcripts is not constant but varies depending on the human cell-line or tissue examined.