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At least 793 records · Page 44Linked to original sources
PubMed1986

Characterization of a human gene inducible by alpha- and beta-interferons and its expression in mouse cells.

An intact interferon-inducible gene has been isolated from a cosmid library of human genomic DNA. The gene (designated 6-16) encodes a mRNA of approximately 1 kb which is induced well by alpha- and beta- but poorly by gamma-interferons. Genomic and cDNA sequences indicate that the gene contains five exons, and that the mRNA encodes a hydrophobic polypeptide of 130 amino acids with a putative NH2-terminal signal sequence. The 5' end has been identified by primer extension. The corresponding genomic DNA contains a TATA box 20 nucleotides upstream of the putative transcription initiation site. After transfection of the human genomic cosmid into mouse Ltk- cells, human 6-16 mRNA is expressed in response to mouse alpha- and beta- but not gamma-interferons with the same kinetics and dose-response as in the human cells. No such expression is observed in response to human interferons. It can be concluded that the human cosmid DNA contains all of the sequences necessary for alpha- and beta-interferon-induced gene expression and that the mechanisms governing such expression are conserved between murine and human cells.

Amino Acid Sequence↗
PubMed1988

Dispersed localization of D segments in the human immunoglobulin heavy-chain locus.

We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.

Amino Acid Sequence↗
PubMed1988

A molecular map of the chicken major histocompatibility complex: the class II beta genes are closely linked to the class I genes and the nucleolar organizer.

We have cloned in a cosmid vector four DNA clusters covering 320 kb of the chicken MHC (B complex), including five class II (B-L) beta genes defining two related isotypic families. Additional B complex genes have been revealed using tissue-specific cDNA probes. A cosmid fragment has been used to isolate a cDNA for a class I (B-F) transcript. This transcript, that is by far the most divergent known member of the class I gene family, hybridized to six B-F genes present in the cosmids. One of the clusters was shown to contain two rRNA transcriptional units from the nucleolar organizer region (NOR), marking the telomeric boundary of the B complex. None of the other B complex genes hybridizes to, or has the transcriptional characteristics of mammalian MHC class II alpha or class III genes. The map we have obtained shows that the B complex does not contain well defined class I and class II regions since B-F and B-L beta genes are closely associated with unrelated genes. Moreover, class II beta genes are very closely linked to class I genes in two clusters, and to the NOR in a third one.

Amino Acid Sequence↗
PubMed1993

Fifty sequenced-tagged sites on human chromosome 11.

Fifty novel sequenced-tagged sites (STSs) were identified from cosmid clones mapped to human chromosome 11. DNA sequences were determined for one or both cloning ends of 69 cosmid markers that had each been localized to 1 of 24 subchromosomal regions by means of hybridization to somatic cell hybrid panels. Proper primer sequences and appropriate conditions for a polymerase chain reaction (PCR) were determined for each marker. Twenty-one of the cosmids were not suitable for generating STSs, mainly because both of their ends contained repetitive elements such as Alu and L1 sequences; however, some were inappropriate because the sizes of their PCR products from human DNA, used as template, were same as those from yeast DNA. Finally, 50 STSs were established from 48 clones: 20 were derived from markers localized on the short arm and 30 from the long arm. These STSs can serve as new reagents for investigating human DNA in somatic cell hybrids and for isolating yeast artificial chromosomes to anchor large DNA contigs and fine-scale physical maps of chromosome 11.

Base Sequence↗
PubMed1993

TSPY-related sequences represent a microheterogeneous gene family organized as constitutive elements in DYZ5 tandem repeat units on the human Y chromosome.

TSPY (testis-specific protein, Y-encoded) is encoded by members of a Y-chromosome-specific sequence family. We show here that TSPY elements are part of the DYZ5 repeat unit. We have established a cosmid library of Y-chromosomal DNA derived from the hybrid cell line 3E7 and isolated eight cosmids representing 15 TSPY elements. Interindividual variability with respect to TSPY copy number was observed. One cosmid clone was investigated in detail by subcloning and sequencing. Sequence microheterogeneity was identified among both transcribed and nontranscribed TSPY elements. Transcript variability is not restricted to single basepair exchanges. We present data for the existence of at least three differently sized TSPY transcripts, caused by different patterns of splicing.

Base Sequence↗
PubMed1994

Twenty-one polymorphic markers from human chromosome 12 for integration of genetic and physical maps.

Twenty-one physically mapped, polymorphic markers have been developed from a chromosome 12-specific cosmid library. The markers consist of CA repeat-containing sequence-tagged sites (STSs) derived from cosmid clones mapped by fluorescence in situ hybridization (FISH). Three methods for determining the sequence flanking CA microsatellites were used, including one using degenerate primer sets for direct sequence analysis. Oligonucleotide primer pairs suitable for use in polymerase chain reaction (PCR) were selected from the sequences flanking the CA microsatellite and were tested for their ability to generate unique PCR products. The informativeness of these STSs as genetic markers was determined by typing 10 unrelated individuals who are part of the Centre d'Etude du Polymorphisme Humaine (EPH) pedigrees. Eleven of the 21 FISH-mapped, polymorphic STSs are heterozygous in 7 or more of the individuals tested. Since these markers are derived from physically mapped cosmids, genetic linkage analysis with them will facilitate the integration of the developing physical and genetic maps of chromosome 12.

Alleles↗
PubMed1995

Isolation and regional assignment of human chromosome 12p cDNAs.

We have characterized 117 cDNAs isolated by direct cDNA selection using pools of human chromosome 12p cosmids. Sequencing revealed that 41 clones did overlap with other cDNAs. Of the remaining 76 cDNA sequences, 11 matched previously identified human chromosome 12p genes and 3 matched previously determined cDNA sequences, including the retinoblastoma binding protein 2 (RBBP2), the cyclin-dependent kinase inhibitor KIP1, and an expressed sequence tag. For each of the 76 cDNAs specific selection by a genomic cosmid clone was confirmed. STSs were developed for all cosmids, among them 3 polymorphic simple sequence repeats associated with, respectively, the TNFR related protein, CD27, and SCNN1. Regional assignment of the STSs by PCR analysis with somatic cell hybrids and fluorescence in situ hybridization showed that the majority of the loci map to chromosome 12p13, similar to the distribution of the known 12p genes. Evidence was found for the duplication on 12p of a region containing a polymorphic simple sequence repeat and sequences of two different cDNAs.

Base Sequence↗
PubMed1996

Sequence analysis in the olfactory receptor gene cluster on human chromosome 17: recombinatorial events affecting receptor diversity.

A cosmid clone covering a region of high olfactory receptor (OR) gene density inside the OR gene cluster on human chromosome 17 (17p13.3) was subjected to shotgun automated DNA sequencing. The resulting 40-kb sequence revealed three known OR coding regions, as well as a new OR pseudogene (OR17-25), fused to one of the previously identified OR genes (OR17-24). The suggested mechanism for the generation of this doublet structure involves an initial duplication mediated by flanking repeats and a subsequent deletion via nonhomologous recombination. Sequence analysis further suggests that the two other OR genes present in the cosmid (OR17-40 and OR17-228) may have evolved by ancient tandem duplication of an 11-kb fragment, mediated by recombination between mammalian-wide interspersed repeats. The duplicated genes appear to be complete and potentially functional. Their conserved structure reveals a long upstream intron and a previously uncharacterized 5' noncoding exon. No additional genes could be discerned in the cosmid, suggesting that the cluster may be part of a dedicated OR subgenome.

Base Sequence↗
PubMed1997

Isolation of novel genes from the CMT1A duplication/HNPP deletion critical region in 17p11.2-p12.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a 1.5-Mb tandem DNA duplication in chromosome 17p11.2-p12, while hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a 1.5-Mb deletion at this locus. The 1.5-Mb CMT1A monomer unit duplicated in CMT1A and deleted in HNPP is flanked by low-copy repeats termed CMT1A-REPs. Both diseases appear to be caused by an altered copy number of the peripheral myelin protein 22 gene (PMP22), which lies within the critical region. To identify additional genes rapidly, we used a cosmid contig of this region and reciprocal probing of arrayed chromosome 17-specific cosmid and cDNA libraries. Three cDNA clones were identified within the CMT1A duplication/HNPP deletion region and one just proximal to the critical region. The cDNA for human heme A:farnesyltransferase (COX10) mapped 10 kb centromeric to the distal CMT1A-REP. The other two cDNA clones from within the critical interval mapped to cosmid 126D1 at the mfd41 (D17S261) DNA marker, and their conceptual translation showed homology to 60S ribosomal protein L9 (RPL9) and chromosomal protein RMSA-1 (RMSA-1). A gene that is homologous to human peroxisome proliferator activated receptor alpha (hPPARA) was identified near the proximal CMT1A-REP.

Alkyl and Aryl Transferases↗
PubMed1997

Transcript mapping in a 46-kb sequenced region at the core of 12q13.3 amplification in human cancers.

We used a combination of sequence analysis and exon trapping in an effort to determine the complete transcript map for a cosmid (6E5) derived from 12q13.3, a region of DNA sequence amplification in human cancers. This cosmid, previously known to contain three genes (CDK4, SAS, and OS9), was sequenced, and that information was used for computer-assisted analysis. In addition, 6E5 was subjected to both internal and 3'-terminal exon-trapping protocols, and the results of these studies were used to guide cDNA cloning experiments. These studies demonstrate that this cosmid is derived from a remarkably gene-dense region and add two new transcripts (KIAA0167 and 6E5.2) to the list of sequences that are expressed in tumors bearing amplification of this region.

Amino Acid Sequence↗
PubMed1997

High-resolution physical map of the immunoglobulin lambda variant gene cluster assembled by quantitative DNA fiber mapping.

Quantitative DNA fiber mapping (QDFM) allows rapid construction of near-kilobase-resolution physical maps by hybridizing specific probes to individual stretched DNA molecules. We evaluated the utility of QDFM for the large-scale physical mapping of a rather unstable, repeat-rich 850-kb region encompassing the immunoglobulin lambda variant (IGLV) gene segments. We mapped a minimal tiling path composed of 32 cosmid clones to three partially overlapping yeast artificial chromosome (YAC) clones and determined the physical size of each clone, the extent of overlap between clones, and contig orientation, as well as the sizes of gaps between adjacent contigs. Regions of germline DNA for which we had no YAC coverage were characterized by cosmid to cosmid hybridizations. Compared to other methods commonly used for physical map assembly, QDFM is a rapid, versatile technique delivering unambiguous data necessary for map closure and preparation of sequence-ready minimal tiling paths.

Chromosome Mapping↗
PubMed1998

Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms.

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.

Cell Line↗
PubMed1994

Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay.

A highly sensitive genetic screen for the detection of cloned genes coding for iron-regulated and iron-storage/binding proteins was developed. The Fur titration assay (FURTA) enabled identification of cloned iron-regulated genes from different Gram-positive and Gram-negative bacteria such as: Bacillus subtilis, Escherichia coli, Pantoea agglomerans, Pseudomonas putida, Salmonella typhimurium, Serratia marcescens and Yersinia enterocolitica. An ordered E. coli cosmid library was screened for either new genes containing Fur-box nucleotide sequences or genes coding for iron-storage/binding proteins. Among 150 cosmids covering approximately 85% of the E. coli genome, 24 cosmids were identified as positive by FURTA. Nine of them contained new E. coli Fur-regulated genes and/or iron-storage/binding genes since they mapped at loci different to any of the known Fur-box containing genes. A new E. coli gene encoding a 8.7 kDa high-potential iron-sulfur-like protein was identified, cloned and sequenced. The Fur titration assay was also used to probe in vivo interaction between Fur repressor and different synthetic plasmid-located Fur-boxes. Non-optimal base-pairs in one half of the Fur-box nucleotide sequence led to a dramatic decrease of Fur repressor affinity. A synthetic Fur-box with changes in both Fur-box halves was no longer bound by the Fur repressor complex in vivo.

Amino Acid Sequence↗
PubMed1994

Functional mapping of regions of the Autographa californica nuclear polyhedrosis viral genome required for DNA replication.

Previous results showed that plasmids containing one of the eight putative origins (ori's) of Autographa californica nuclear polyhedrosis virus (AcMNPV) are replicated after transfection into Spodoptera frugiperda cells if essential trans-acting factors are supplied by AcMNPV infection (Kool et al., Virology, 192, 94-101, 1993a; Kool et al., J. Gen. Virol., in press, 1993b; Leisy and Rohrmann, Virology, 196, 722-730, 1993). In this report a transient complementation assay is described in which four cotransfected cosmid clones, instead of AcMNPV infection, provided essential trans-acting factors for plasmid DNA replication. In this assay plasmid replication was found to be independent of the presence, in cis, of a viral ori. No replication of plasmids occurred when one of the cosmids was omitted from the transfection mixture. This result indicated that this assay is a valid approach for identification of AcMNPV replication genes. We further used the assay to define essential regions in the four required cosmids. Six regions of the AcMNPV genome, EcoRI-I (map unit 0.3-5.8), EcoRI-O (map unit 6.9-8.7), SstI-F (map unit 38.9-45.0), EcoRI-D (map unit 59.9-68.3), a BamHI-SstII fragment of BamHI-B (map unit 84.3-89.7), and EcoRI-B (map unit 90.0-100), with at least seven genes, were found to be essential for plasmid DNA replication. These regions contain the putative DNA polymerase gene (SstI-F), the helicase-like gene (EcoRI-D), and the region where most of the trans-activating immediate--early genes of AcMNPV are located (EcoRI-B). For SstI-F it was shown that this region contains besides the DNA polymerase gene at least one other replication gene. These results show that it will now be possible to define the set of AcMNPV genes necessary and sufficient for DNA replication.

Animals↗
PubMed1994

The extent of the human germline T-cell receptor V beta gene segment repertoire.

An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: 1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and 2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV germline repertoire encodes at least 63 TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.

Base Sequence↗
PubMed1986

Clustered GATA repeats (Bkm sequences) on the human Y chromosome.

Sixty eight individual clones of a human Y chromosome cosmid library were screened for the presence of GATA repeats, the major component of Bkm-related DNA sequences. Nine cosmid clones were found to cross-hybridize. The sequence organization of the repetitive base quadruplet GATA was analyzed using synthetic oligonucleotide probes. Subclones of GATA-positive cosmid clones were used for chromosomal localization of the Y-derived DNA sequences thus revealing male-specificity or male-female homology.

Cosmids↗
PubMed1988

Genomic structure and evolution of the human pepsinogen A multigene family.

A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0-16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.

Biological Evolution↗
PubMed1988

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum.

We utilized diaminopimelate-lysine mutants of Escherichia coli K12 to clone the genes specifically involved in the Corynebacterium glutamicum diaminopimelate-lysine anabolic pathway. From a cosmid genomic bank of C. glutamicum strain AS019, we isolated cosmids pSM71, pSM61 and pSM531, that are respectively able to complement dapA/dapB, dapD, and lysA mutants of E. coli. DNA hybridization analysis indicates that these complementing genes are located on the chromosome of C. glutamicum in at least three separate transcription units. Subcloning of parental cosmids in dapA, dapD, and lysA mutants of E. coli localized these genes, respectively, within 1.4, 3.4, and 1.8 kb fragments, cloned in an E. coli/C. glutamicum shuttle vector. Enzymatic analysis in C. glutamicum identified the dapA-complementing gene as L-2,3-dihydrodipicolinate synthetase (dapA), and the lysA-complementing gene as meso-diaminopimelate decarboxylase (lysA). In contrast, complementation of E. coli dapD8, presumably lacking L-delta 1-tetrahydrodipicolinate synthetase (dapD), led us to clone a diaminopimelate-lysine anabolic gene of C. glutamicum which does not exist in E. coli: meso-diaminopimelate dehydrogenase. Although meso-diaminopimelate is crucial in lysine formation and in cell wall biosynthesis, expression of the genomic copies of the cloned genes, which encode activities involved at key branching points of the diaminopimelate-lysine pathway of C. glutamicum, appears constitutive with regard to the addition of diaminopimelate and/or lysine during cell growth.

Amino Acids, Diamino↗