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PubMed1994

The human Met-ase gene (GZMM): structure, sequence, and close physical linkage to the serine protease gene cluster on 19p13.3.

Cosmid clones containing the genes for the human and murine natural killer cell serine protease Met-ase (gene symbol GZMM; granzyme M) were identified by screening human and murine cosmid libraries with rat Met-ase (RNK-Met-1) cDNA. The human gene has a size of 7.5 kb and an exon-intron structure identical to that of serine protease genes located on human chromosomes 5q11-q12, 14q11.2, and 19p13.3 that are expressed by lymphocytes, mast cells, or myelomonocyte precursors. Using cosmid DNA as a probe for fluorescence in situ hybridization, we identified the chromosomal position of human Met-ase as 19p13.3. Interphase studies with two differentially labeled probes for Met-ase and the azurocidin (AZU1), proteinase 3 (PRTN3), and neutrophil elastase (ELA2) gene cluster revealed that the distance of Met-ase from this gene cluster is in the range of 200 to 500 kb. Using differentially labeled mouse cosmid probes, we also mapped the murine gene for Met-ase to chromosomal band 10C, close to the gene for lamin B2. Thus, the Met-ase, AZU1, PRTN3, and ELA2 genes fall into an established region of homology between mouse chromosomal band 10C and human 19p13.3.

Amino Acid Sequence↗
PubMed1995

A continuous high-resolution physical map spanning 17 megabases of the q12, q13.1, and q13.2 cytogenetic bands of human chromosome 19.

We report the construction of a high-resolution physical map of a 17-Mb region that encompasses the entire q12, q13.1, and q13.2 bands of human chromosome 19. The continuous map extends from a region approximately 400 kb centromeric of the D19S7 marker to the excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1) locus. The ordered clone map has been obtained starting from a foundation of cosmid contigs assembled by automated fingerprinting and localized to the cytogenetic map by fluorescence in situ hybridization (FISH). Clonal continuity of the map has been achieved by binning and linking the premapped cosmid contigs by means of yeast artificial chromosomes (YACs). The map consists of a single contig composed of 169 YAC members (minimal spanning path of 18 YACs) linking 165 cosmid contigs. Eighty percent, or about 13.2 Mb of the entire region spanned by the map, has been resolved to the EcoRI restriction map level. Twenty-nine sequence-tagged sites associated with genetic markers or derived from FISH-mapped cosmids have been placed on the map. In addition to the ERCC1 gene area, the map includes the location of the creatine kinase muscle locus (CKM), imidazoledipetidase (PEPD), glucophosphate isomerase (GPI), myelin-associated glycoprotein (MAG), the apolipoprotein E and C (APOE and APOC) genes, and the ryanodine receptor (RYR1) gene. This type of map provides a source of continuously overlapping DNA segments at a level of resolution two orders of magnitude higher than that obtained using YACs alone. In addition, it provides ready-to-use reagents for detailed analyses at the gene level, FISH studies of chromosomal aberrations, and DNA sequencing.

Chromosome Mapping↗
PubMed1995

A comparative transcriptional map of a region of 250 kb on the human and mouse X chromosome between the G6PD and the FLN1 genes.

The transcriptional organization of the region of the mouse X chromosome between the G6pd and the Fln1 genes was studied in detail, and it was compared with the syntenic region of the human chromosome. A cosmid contig of 250 kb was constructed by screening mouse cosmid libraries with probes for human genes and with whole cosmids. Overlapping cosmids were aligned by comparing EcoRI and rare-cutter restriction enzyme digestions. The gene order and the orientation of transcription were determined by hybridization with fragments from the 5' and 3' moieties of each cDNA. Our work demonstrates that all of the new genes identified in human are present in the mouse. The size of the region, 250 kb, is also very similar, as are gene order and gene organization: the transcriptional organization in "domains" described in human is found to be identical in the mouse. The major difference detected is the much lower content in rare-cutter restriction sites, which is related to the lower G+C and CpG content of mouse DNA. The very high conservation that we have described suggests that a potent selective pressure has contributed to such conservation of gene organization.

Animals↗
PubMed1999

Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates.

The pathogenesis of hairy cell leukemia (HCL) remains largely unknown since no specific genetic lesion has been identified in this disease. Previous cytogenetic analysis from our group has shown that chromosome abnormalities involving the 5q13 band are common in HCL, occurring in approximately 1/3 of patients. The data suggest that the 5q13.3 band is likely to harbor a gene involved in the transformational events of this disease. We have recently found two cosmids flanking the 5q13.3 breakpoint in patients with HCL, and the distance between them is approximately 35 kb, as analyzed by fiber-FISH. The two cosmids have been located between the markers SGC34998 and WI-15505/WI-6897 by radiation hybrid mapping. Five of 11 patients with HCL had a hemizygous deletion of the two cosmids, indicating that the function of a tumor suppressor gene may be lost. With the aim of delineating the critical region of 5q13.3 loss in patients with HCL, we have constructed an integrated contig of YAC, BAC, PAC, P1, and cosmid clones that covers the region. Within this area, three expressed sequences were identified as candidates for the putative 5q13.3 tumor suppressor gene involved in the pathogenesis of HCL.

Amino Acid Sequence↗
PubMed1999

Clustered cadherin genes: a sequence-ready contig for the desmosomal cadherin locus on human chromosome 18.

We describe the assembly of a cosmid and PAC contig of approximately 700 kb on human chromosome 18q12 spanning the DSC and DSG genes coding for the desmocollins and desmogleins. These are members of the cadherin superfamily of calcium-dependent cell adhesion proteins present in the desmosome type of cell junction found especially in epithelial cells. They provide the strong cell-cell adhesion generated by this type of cell junction for which expression of both a desmocollin and a desmoglein is required. In the autoimmune skin diseases pemphigus foliaceous and pemphigus vulgaris (PV), where the autoantigens are, respectively, encoded by the DSG1 and DSG3 genes, severe areas of acantholysis (cell separation), potentially life-threatening in the case of PV, are evident. Dominant mutations in the DSG1 gene causing striate palmoplantar keratoderma result in hyperkeratosis of the skin on the parts of the body where pressure and abrasion are greatest, viz., on the palms and soles. These genes are also candidate tumor suppressor genes in squamous cell carcinomas and other epithelial cancers. We have screened two chromosome 18-specific cosmid libraries by hybridization with previously isolated YAC clones and DSC and DSG cDNAs, and a whole genome PAC library, both by hybridization with the YACs and by screening by PCR using cDNA sequences and YAC end sequence. The contigs were extended by further PCR screens using STSs generated by vectorette walking from the ends of the cosmids and PACs, together with sequence from PAC ends. Despite screening of two libraries, the cosmid contig still had four gaps. The PAC contig filled these gaps and in fact covered the whole locus. The positions of 45 STSs covering the whole of this region are presented. The desmocollin and desmoglein genes, which are about 30-35 kb in size, are quite well separated at approximately 20-30 kb apart and are arranged in two clusters, one DSC cluster and one DSG cluster, which are transcribed outward from the interlocus region. The order of the genes is correlated with the spatial order of gene expression in the developing mouse embryo, and this, and previous transgenic experiments, suggests that long-range genetic elements that coordinate expression of these genes may be present. The complete bacterial clone contig described in this paper is thus a resource not only for future sequencing but also for investigations into the control of expression of these clustered genes.

Bacteriophage P1↗
PubMed2001

Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus).

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.

Animals↗
PubMed1998

Marker rescue of the host range restriction defects of modified vaccinia virus Ankara.

The severely attenuated and host range (HR) restricted modified vaccinia virus Ankara (MVA) was derived by >500 passages in chick embryo fibroblasts, during which multiple deletions and mutations occurred. To determine the basis of the HR defect, we prepared cosmids from the parental vaccinia virus Ankara genome and transfected them into nonpermissive monkey BS-C-1 cells that had been infected with MVA. Recombinant viruses that formed macroscopic plaques were detected after transfections with DNA segments that mapped near the left end of the viral genome. Plaque-forming viruses, generated by transfections with four individual cosmids and one pair of minimally overlapping cosmids, were purified, and their HRs were evaluated in BS-C-1 cells, rabbit RK-13 cells, and human HeLa, MRC-5, and A549 cells. The acquisition of the K1L and SPI-1 HR genes and the repair of large deletions were determined by polymerase chain reaction or pulse-field gel electrophoresis of NotI restriction enzyme digests of genomic DNA. The following results indicated the presence of previously unrecognized vaccinia virus HR genes: (1) the major mutations that restrict HR are within the left end of the MVA genome because the phenotypes of some recombinants approached that of the parental virus, (2) acquisition of the K1L gene correlated with the ability of recombinant viruses to propagate in RK-13 cells but did not enhance replication in human or monkey cell lines, (3) acquisition of the SPI-1 gene correlated with virus propagation in A549 cells but not with the extent of virus spread in monkey or other human cell lines, (4) there are at least two impaired HR genes because rescue occurred with nonoverlapping or minimally overlapping cosmids and recombinant viruses with intermediate HRs were isolated, and (5) at least one of the new HR genes did not map within any of the major deletions because the size of the left terminal NotI fragment was not appreciably altered in some recombinant viruses.

Animals↗
PubMed1987

Molecular cloning of a pair of human pepsinogen A genes which differ by a Glu----Lys mutation in the activation peptide.

Three human cosmid clones containing pepsinogen A (PGA) encoding sequences were isolated from a genomic bank derived from a single individual. One cosmid contains two PGA genes in tandem in a head-to-tail orientation, while the other two cosmids each contain a single PGA gene. The three cosmids were characterized by restriction mapping and sequence analysis (exons 1 and 2 and flanking regions). As judged from these data, three of the four PGA genes isolated appear to be nearly identical, but one of the tandem genes is clearly different from the other genes. The first exon of all four genes codes for the same amino acid sequence. However, in the second exon of one of the tandem genes we found a nucleotide substitution giving rise to a Glu----Lys substitution of the 43rd amino acid residue of the activation peptide, leading to a charge difference of the corresponding isozymogens. The presence of two distinct PGA genes in the isolated gene pair conclusively proves the multigene structure of the PGA system. These genes might be responsible for at least part of the electrophoretic polymorphism at the protein level.

Amino Acid Sequence↗
PubMed1988

Cloning of the trp genes from the archaebacterium Methanococcus voltae: nucleotide sequence of the trpBA genes.

A cosmid bank of Methanococcus voltae DNA was obtained in Escherichia coli after ligation of partially HindIII-digested M. voltae DNA in the HindIII site of the transferable cosmid pVK100. The bank was used to perform complementation experiments with E. coli auxotrophic mutants. Five cosmids complementing trpA shared three adjacent HindIII fragments of 2.1, 2.3 and 14 kb. Two of these cosmids also complemented trpD and carried an additional 4.2 kb HindIII fragment. The trpA- and trpD- complementing regions were more precisely localized using Tn5 mutagenesis. A 1.7 kb PstI fragment, cloned into pUC9 in both orientations, was responsible for the trpA complementation. This fragment was sequenced and an open reading frame (ORF) of 852 nucleotides (ORFtrpA) encoding a 284 amino acid polypeptide of mol. wt. 31,938 was found. The amino acid sequence was compared with that of the alpha subunit of tryptophan synthase (trpA gene product) from nine eubacterial species and to the N-terminal part of the tryptophan synthase of Saccharomyces cerevisiae (TRP5 gene product). Similarity varied from 24% (Brevibacterium lactofermentum) to 35% (S. cerevisiae). The nucleotide sequence of the region upstream from M. voltae ORFtrpA was determined and revealed the presence of an ORF of 1227 nucleotides (ORFtrpB) encoding a 409 amino acid polypeptide of mol. wt. 44,634. The polypeptide sequence was similar to the beta subunit of tryptophan synthase (trpB gene product) from six eubacterial species and to the C-terminal part of the tryptophan synthase of S. cerevisiae. Similarity varied from 49% (S. cerevisiae, B. lactofermentum) to 58% (Pseudomonas aeruginosa). This high conservation supports the hypothesis of a common ancestor for the trpA and trpB genes of archaebacteria, eubacteria and eucaryotes. M. voltae ORFtrpA and ORFtrpB, which are transcribed in the same direction, are separated by a 37 bp AT-rich region. Immediately upstream from ORFtrpB, the 3' end of an ORF homologous to E. coli and Bacillus subtilis trpF was found. As the trpD-complementing region was located upstream from the trpFBA sequenced region, the organization of trp genes in the archaebacterium might thus be trpDFBA. Such an organization resembles that of enteric eubacteria, in which the trpEDCFBA genes are grouped in a single operon. However, M. voltae ORFtrpA and ORFtrpB do not overlap, in contrast with what is found in most eubacteria.

Amino Acid Sequence↗
PubMed1989

Cloning and comparison of A alpha mating-type alleles of the Basidiomycete Schizophyllum commune.

An A alpha mating-type allele (A alpha 4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A alpha 4 allele isolated from the walk was used as a probe to recover the A alpha 1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A alpha allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A alpha encodes a diffusible product. Restriction mapping shows that A alpha 1 and A alpha 4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A alpha 1 or A alpha 4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alpha alleles. A alpha 1 and A alpha 4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.

Alleles↗
PubMed1992

Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus.

A cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that a 1.7 kb BglII-SphI DNA fragment isolated from this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment was 1.4 kilonucleotides in size. Genetic complementation proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.

Aflatoxin B1↗
PubMed1991

Isolation of virus-like (VL30) elements from the Q10 and D regions of the major histocompatibility complex.

Previous studies from our laboratory have described two endogenous provirus-like sequences in a series of cosmids spanning the TL region of the major histocompatibility complex (MHC) of normal C57BL/10 mice. At least one of these viruses shares similarities with VL30 elements. To determine if additional VL30-like retroviral elements are integrated in the MHC, we constructed a cosmid library using DNA from a radiation leukemia virus (RadLV)-transformed cell line derived from C57BL/6 mice. The library was first screened using the H-2III (5') probe, which detects Class I genes of the H-2 complex. In the primary screening 163 H-2III positives were isolated. The H-2III-positive isolates were then hybridized with an AKR-derived virus probe, EcoB/S, which contains sequences from both the pol and the env genes of the virus. Nine virus-positive isolates were detected. Localization of these cosmid isolates containing viral sequences within the H-2 complex was done utilizing low-copy probes and confirmed using previously mapped cosmid isolates from other laboratories. We report here the isolation and characterization of VL30-like elements from the Qa and D regions of the MHC of several inbred mouse strains.

AKR murine leukemia virus↗
PubMed1991

New DNA markers in the Huntington's disease gene candidate region.

The search for the Huntington's disease (HD) gene has prompted construction of a complete long-range restriction map of a 2.5-Mb candidate region, distal to the DNA marker D4S10. To facilitate the procurement of cloned DNA from this candidate region, we have augmented the existing regional mapping panel of somatic cell hybrids with hybrid HHW1071 containing a t(4p16;12) chromosome from a patient with Wolf-Hirschhorn syndrome. This translocation maps between D4S180 and D4S127, subdividing the HD candidate region and setting a proximal limit to the Wolf-Hirschhorn syndrome region. Using the expanded mapping panel, we have regionally assigned 14 independently cloned cosmids, five proximal to the t(4;12) breakpoint in the same region as D4S10 and nine distal to the breakpoint. By a combination of overlap with previously mapped cosmids and pulsed-field gel analysis, each of these cosmids has been positioned on the long-range restriction map of 4p16.3, increasing the clone coverage of the candidate region to approximately 40%. Single-copy probes from mapped cosmids were used to identify eight new DNA polymorphisms spanning the HD candidate region. These new DNA markers should prove valuable for analysis of recombination and linkage disequilibrium in HD, as well as for preclinical diagnosis of the disorder.

Alleles↗
PubMed1996

The human lanosterol synthase gene maps to chromosome 21q22.3.

In order to contribute to the development of the transcriptional map of human chromosome 21 (HC21) we have used exon trapping to identify portions of HC21 genes. Using pools of random HC21-specific cosmids from the LL21NC02-Q library and cosmids from 21q22.3 we have identified five different coding regions with strong homology to the lanosterol synthase genes of rat and yeast. This enzyme catalyzes the cyclization of squalene-2,3-epoxide lanosterol, which is the parental compound of all steroids in mammals. Using somatic cell hybrids and HC21 yeast artificial chromosomes (YACS) and cosmids, we mapped the human lanosterol synthase cDNA gene to 2lq22.3 between markers D21S25 and 21qter. Cosmid Q7G8 from the LL21NC02-Q library and YAC 145D8 from the CEPH HC21 contig contain this human gene. We cloned a portion of the human lanosterol synthase cDNA (almost 85% of the coding region) from a brain cDNA library and determined its nucleotide sequence. The predicted human protein shows 83% identity to its rat and 40% to its yeast homolog. No obvious candidate human disease exists for lanosterol synthase deficiency and the role (if any) of triplication of this gene in the various phenotypes of trisomy 21 is unknown.

Amino Acid Sequence↗
PubMed1996

Isolation of the human chromosome 22q telomere and its application to detection of cryptic chromosomal abnormalities.

A number of human telomeres have been successfully cloned using a modified yeast artificial chromosome (YAC) vector (half-YAC) cloning strategy, but to date, human chromosome 22q has not been identified by this approach. We used an alternative approach of genomic walking, starting from a subtelomeric sequence, Tel-Bam3.4. present on a number of human chromosomes including 22q. This approach was successful in the development of a cosmid contig representing the terminal 140 kb of human chromosome 22q, providing telomeric closure of the genetic and physical maps for 22q. The most distal region of the contig contains subtelomeric repeats which crosshybridize to a number of chromosomes, while the proximal sequences are unique for 22q. The unique sequence cosmid was used as a 22qter-specific probe for fluorescence in situ hybridization (FISH) analysis, which confirmed that this cosmid was distal to the most telomeric marker previously available for chromosome 22. In addition, this cosmid was used to document a 22q terminal deletion that was not detectable by conventional cytogenetic analysis. Unique telomere-specific FISH probes such as this one will have significant diagnostic value in the detection of cryptic deletions and translocations in patients with unexplained mental retardation and other patient populations.

Cerebroside-Sulfatase↗
PubMed1999

Characterization of chromosome 8 aberrations in the prostate cancer cell line LNCaP-FGC and sublines.

In two androgen-dependent (FGC and P70) and two androgen-independent (LNO and R) sublines of the prostate cancer model LNCaP numerical and structural aberrations of chromosome 8 were investigated in detail. The techniques used were whole chromosome paint (WCP) and fluorescence in situ hybridization (FISH) with three cosmid probes mapping to different parts of the p-arm (D8S7 (8p23.3), LPL (8p22) and PLAT (8p11.1)). By WCP all four cell lines showed four copies of chromosome 8 in most cells. However, FISH demonstrated that in all sublines deletions in the 8p region were present. The majority of both FGC and P70 had two copies of cosmids D8S7 and LPL. The cosmid PLAT showed a broader distribution (1-4 copies), especially in P70. Compared with FGC and P70, both LNO and R showed a larger number of copies (3 or 4) of all three cosmid loci. It is discussed that this difference is probably the result of nondisjunction as a reaction to loss of other sequences on 8p, possibly the tumor suppressor gene (TSG) mapping to 8p21. The fact that both sublines LNO and R are androgen-independent raises the possibility of a link between TSG loss on 8p and androgen independence.

Chromosome Aberrations↗
PubMed1997

Identification of new HLA class I region genes by sample sequencing.

Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 - 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 - 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens.

Base Sequence↗
PubMed2000

Fine linkage and physical mapping suggests cross-over suppression with a retroposon insertion at the npc1 mutation.

Mouse Niemann-Pick disease type C1 (npc1), formerly designated spm (sphingomyelinosis), is an autosomal recessive lipid storage disorder. We generated a high-resolution linkage map in the 2.24-cM npc1 critical region by typing eight polymorphic markers in 2322 meioses (948 of these were previously reported). A minimal set of overlapping yeast artificial chromosomes (YACs) had previously been assembled (Hsu and Erickson 2000). The YAC 313-B-8, which covered this whole region, has been used to construct cosmid libraries. Three cosmid contigs were built, and one of them contained the npc1 locus. Two (CA)(n) microsatellites were identified, and the one new one was characterized, from the YAC-derived cosmids. The most proximal cosmid contig overlaps with markers near twirler (Tw). Both the physical map and genetic linkage map have been integrated to study the recombination frequencies in this particular region of the mouse genome, and recombination suppression due to the heterozygous insertion of DNA was suggested.

Animals↗