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PubMed1989

Interferons as gene activators: a cluster of six interferon-activatable genes is linked to the erythroid alpha-spectrin locus on murine chromosome 1.

Several interferon-activatable murine genes were mapped to murine chromosomes by hybridizing cDNA probes to Southern blots of genomic DNA samples from a panel of mouse-hamster somatic cell hybrid lines. The 12 gene is located on chromosome 12 and it specifies a 3.6-kb mRNA. The 204 gene (specifying a 2.5-kb mRNA), and three genes of the 203 gene family (hybridizing to five mRNAs of sizes between 2 and 4.5 kb), together with the 202 gene (specifying a 2-kb mRNA) are located on murine chromosome 1. By restriction fragment length polymorphism analysis of DNA samples prepared from a panel of recombinant inbred mouse lines (C57BL/6J D DBA/2J) and from 85 [C3H/HeJ-gld/gld x Mus spretus) F1 X C3H/HeJ-gld/gld] backcross mice we established a close linkage of the 202, 203, and 204 genes to the erythroid alpha-spectrin gene (Spna-1) on distal murine chromosome 1. Cosmids containing the 202, 203, and 204 genes were isolated from a library derived from AKR mouse DNA. Southern blot analysis of such cosmids revealed: (a) hybridization of a partial 203 cDNA to three genes of the 203 gene family; (b) cross-hybridization of the 202 and 204 genes with one another and with a third gene (designated as 201 gene), and (c) a close linkage of genes of the 203 family with the 201, 202, and 204 genes. These results indicate the existence of a cluster of at least six closely linked, interferon-activatable genes on distal murine chromosome 1 in the vicinity of the Spna-1 locus and also of the Minor lymphocyte stimulating locus (Mlsa).

Animals↗
PubMed1993

Picogram detection of stable dye-DNA intercalation complexes with two-color laser-excited confocal fluorescence gel scanner.

The stable complexes between highly fluorescent, polyfunctional intercalators and dsDNA can be used to detect dsDNA in agarose gels at picogram levels and for multicolor detection of multiplexed dsDNA fragments. Development of additional DNA-binding fluorophores with appropriate spectroscopic properties will expand the range of applications. In principle, the DNA-dye intercalation complexes represent a more sensitive alternative to an established approach to fluorescent labeling and detection of restriction fragments by ligation to single-stranded short oligonucleotides labeled with different fluorochromes, followed by separation on denaturing polyacrylamide gels. The latter technique gives near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases, and has been successfully applied to cosmid mapping. Detection of DNA fragments as intercalation complexes requires that the separations be performed on agarose gels under nondenaturing conditions. Such conditions have been used for extensive mapping of yeast cosmids with postelectrophoresis staining with ethidium bromide. For the patterns on agarose gels, the magnitude of the "error window," which specifies how similar two fragments must be before the corresponding fragments in different digests are paired, was reported to be strongly size dependent. The error window was expanded by a factor of 1.3 for fragments from 400 to 600 bp, 1.2 for fragments from 600 to 800 bp, and 1.1 for fragments from 800 to 1000 bp. Moreover, it was necessary to introduce corrections for systematic differences between size estimates taken from two different gels. For the multiplexing procedure described here, the size estimates for fragments from 600 bp to over 23 kbp were in close agreement with actual sizes as determined from DNA sequence (Table I), and certainly within the error windows given above. The multiplexing procedure should also minimize errors introduced by gel-to-gel variations in mobility, because the standard and unknowns are always run in the same lanes. Kohara et al. established a physical map of almost the entire Escherichia coli chromosome by analysis of a large genomic library. In this case, partial restriction digests were used to generate patterns of fragments and the mapping was performed by agarose gel electrophoresis. The disadvantage of this approach is that fewer fragments are generated. However, this is compensated for by the fact that partial digests reveal the order of the fragments produced and thus greatly increase the amount of information relevant to the question of overlap between different DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzothiazoles↗
PubMed1982

Clusters of genes encoding mouse transplantation antigens.

We constructed a cosmid library from BALB/c mouse sperm DNA and isolated 64 cosmid clones with cDNA probes for transplantation antigens (class I molecules). Of these clones, 54 mapped into 13 gene clusters containing 36 distinct class I genes and encompassing 837 kilobases of DNA. One gene cluster mapped to the L region and a second cluster with seven genes to the Qa-2,3 region of the major histocompatibility complex. Restriction map and Southern blot analyses suggest that there are subgroups of class I genes. Using a 5' flanking sequence of the L gene as a hybridization probe, we show the L gene to be present in mouse strains expressing this antigen but deleted or mutated in strains failing to express it. Our data suggest that gene duplication and deletion presumably by homologous but unequal crossing-over has altered the size and organization of the class I clusters in different mouse strains and probably is an important mechanism for generating polymorphism in these genes. Analysis of the 36 class I genes with cDNA probes specific for the 5' and 3' ends shows that the exon encoding the third external domain is far more conserved than those encoding the first and second external domains of the transplantation antigen. These differences in variability have interesting functional implications.

Animals↗
PubMed1994

Isolation of the gene for McLeod syndrome that encodes a novel membrane transport protein.

McLeod syndrome is an X-linked multisystem disorder characterized by abnormalities in the neuromuscular and hematopoietic systems. We have assembled a cosmid contig of 360 kb that encompasses the McLeod gene locus. A 50 kb deletion was detected by screening DNA from patients with radiolabeled whole cosmids, and two transcription units were identified within this deletion. The mRNA expression pattern of one of them, designated as XK, correlates closely to the McLeod phenotype. XK encodes a novel protein with structural characteristics of prokaryotic and eukaryotic membrane transport proteins. Nucleotide sequence analysis of XK from two unrelated McLeod patients has identified point mutations at conserved splice donor and acceptor sites. These findings provide direct evidence that XK is responsible for McLeod syndrome.

Amino Acid Sequence↗
PubMed1986

Expression of F plasmid traT: independence of traY----Z promoter and traJ control.

We have shown, using an F-derived Tra+ cosmid in conjunction with the infected-cell translational system and a time-course study, that one of the surface exclusion genes, traT, can be expressed independently of the promoter of the traY----Z operon, PYZ, and in the absence of a normal quantity of traJ gene product. Studies with deleted derivatives of the cosmid pRS2405 confirmed this independence and also indicated that expression of traD can be independent of PYZ. We propose that the expression of traT by these deleted plasmids is directed from a traJ-independent promoter, PT, located adjacent to traT.

Bacterial Proteins↗
PubMed1986

The presence of repeated DNA sequences and a partial restriction map of the pSym of Rhizobium fredii USDA193.

The large, 350-kb Sym (symbiotic) plasmid pRjaUSDA193 of Rhizobium fredii was examined to determine the frequency of repeated sequences present and to produce a physical and genetic map of a large region of the plasmid. A novel hybridization method, the Southern Cross, revealed that the plasmid pRjaUSDA193 contained many repeated sequences and assisted in restriction enzyme mapping of a 100-kb region containing nod genes. A cosmid clone bank was prepared with the broad-host-range cosmid pVK102. The restriction enzymes HindIII, HpaI, and KpnI were used to construct a physical map of overlapping clones. Labeled nod gene sequences were used to determine their location in the mapped region.

Chromosome Mapping↗
PubMed1994

Analysis of cells harboring a putative DNA repair gene reveals a lack of evidence for a second independent xeroderma pigmentosum group A correcting gene.

The UV hypersensitivity of xeroderma pigmentosum (XP) complementation group A cells is restored to near-normal by transfection of the XPA gene located on human chromosome 9. However, it has been reported that a cosmid related to a cDNA on chromosome 8 is also able to partially correct the UV sensitivity of XP-A cells. We describe here an investigation of a representative cosmid transfectant, denoted 2-0-A2. Whole cell extracts prepared from 2-0-A2 cells carried out DNA repair synthesis in vitro that was in the normal range, consistent with their UV-resistant phenotype. Immunoblotting indicated that 2-0-A2 cells expressed full-length XPA protein. This was unexpected because the 2-0-A2 cell line was thought to have been isolated by transfection of a cell line derived from patient XP2OS, and a known homozygous mutation in XP2OS prevents expression of XPA gene product. This mutation creates an AlwNI restriction endonuclease cleavage site in XPA and was not present in 2-0-A2. These results prompted an RFLP analysis which revealed that the 2-0-A2 cell line was not derived from XP2OS but from another line that fails to express XPA protein, XP12BE. It appears that the significant UV-resistance and DNA repair capacity of 2-0-A2 can be ascribed to the re-expression of XPA in XP12BE, and it is unnecessary to postulate a second XP-A complementing gene to explain the results.

Base Sequence↗
PubMed1995

Isolation and characterization of the gene encoding the surface membrane 3'-nucleotidase/nuclease of Leishmania donovani.

Leishmania donovani and related trypanosomatid protozoa possess an externally oriented surface membrane enzyme capable of hydrolyzing both 3'-nucleotides and nucleic acids. By virtue of these activities, this 3'-nucleotidase/nuclease (3'-NT/Nu), previously shown to be analogous to fungal and plant class-I single-strand-specific nucleases, is thought to play a critical role in the salvage of purines, essential for the survival of these organisms. The 43-kDa 3'-NT/Nu was purified from L. donovani promastigotes and trypsin treated. Four of the released tryptic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleotide primers used for PCR amplification of an approx. 300-bp DNA fragment. This fragment was cloned, sequenced and used to probe a genomic L. donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb SmaI fragment, isolated from a cosmid clone, revealed an open reading frame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept-4 were mapped onto the ORF-deduced protein sequence. Peptides corresponding to Pept-1 to Pept-4 were synthesized and used to immunize rabbits. The resulting anti-peptide antibodies recognized the 43-kDa protein on Western blots and immunoprecipitated the native 3'-nucleotidase activity from L. donovani membrane extracts. Further, the ORF-deduced protein shared significant sequence identity with the S1 and P1 fungal nucleases of Aspergillus oryzae and Penicillium citrinum, respectively. Cumulatively, these results demonstrated that the ORF corresponded to a gene for the L. donovani 3'-nucleotidase/nuclease. In Northern blots a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a single 2.5-kb messenger RNA. Results of Southern blot analyses were consistent with the 3'-NT/Nu being encoded by a single copy gene. These data constitute the first report of the gene for this unique trypanosomatid surface membrane enzyme.

Amino Acid Sequence↗
PubMed1995

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Animals↗
PubMed1986

Cloning and developmental expression of the murine neurofilament gene family.

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal alpha-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.

Animals↗
PubMed1983

Molecular analysis of the HLA-D region genes.

Analysis of the amino acid sequence of the HLA-DR alpha chain and the structure of the gene encoding it have shown strong homologies to membrane proximal domains of the DR beta chain and HLA-ABC, as well as to beta 2-microglobulin. Weaker, but nevertheless apparent homologies to C kappa and C lambda immunoglobulin domains support the interpretation that these genes diverged in evolution from a primordial immunoglobulin-like domain. Cosmid clones containing sequences related to the DR alpha chain have been isolated. These sequences probably represent the SB and DC alpha genes. In addition to the alpha "genes," some of the cosmids also contain sequences related to the DR beta chain suggesting that the human HLA-D region alpha and beta genes are interspersed.

Amino Acid Sequence↗
PubMed1991

Characterization of an expressible nonclassical class I HLA gene.

Screening of a human cosmid library representing genomic DNA from an individual homozygous for the HLA-DR2 B7 A2 haplotype yielded 109 class I HLA-specific clones. One cosmid clone, Ice 6.23, had a full-length nonclassical class I gene within a 5.4-kb HindIII fragment. The Ice 6.23-5.4H gene was cloned into the unique NotI site of an expression vector pSV2.Not, a derivative of pSV2neo, which was constructed to contain a second SV40 early region promoter adjacent to an introduced NotI site. The resulting construct was transfected into the P815-B2M cell line, a derivative of the mouse mastocytoma P815 (HTR) line which expressed human beta2-microglobulin following stable transfection with a cloned human beta2-microglobulin gene. Following transfection the Ice 6.23-5.4 H gene was found to be expressed at both the mRNA and cell surface product levels. DNA sequencing of this gene suggests that it is allelic to the HLA-6.0 gene clone (HLA-G) of Geraghty et al. (Proceedings of the National Academy of Sciences USA, 84:9145, 1987); thereby revealing a HindIII restriction fragment length polymorphism at the HLA-G locus. An extraordinarily high degree of sequence similarity (99.92%) between these two genes, which derive from unrelated HLA haplotypes, suggests strong conservative selection pressure at the HLA-G locus. A flanking single copy sequence probe 4 kb distant from the Ice 6.23-5.4H gene was used to generate long-range restriction mapping at the HLA-G locus.

Amino Acid Sequence↗
PubMed1993

Mapping of the ben, ant and cat genes of Pseudomonas aeruginosa and evolutionary relationship of the ben region of P. aeruginosa and P. putida.

Genes responsible for the utilization of benzoate, anthranilate or catechol (ben, ant, cat) of Pseudomonas aeruginosa PAO were mapped precisely using a cosmid clone carrying all these genes. Genes were localized either by subcloning and complementation or by Tn5 mutagenesis and mapping of the Tn5 insertions. To achieve this, a novel Tn5 mutagenesis procedure was developed by constructing a Tn5 insertion derivative of the Escherichia coli strain S17-1. Preliminary mapping of the ben cat genes of P. putida PPN was accomplished by complementation using a PPN cosmid bank. Sequence homology was demonstrated by Southern hybridization between the ben regions of both P. aeruginosa and P. putida, implying an evolutionary relationship of this chromosomal region of these two pseudomonads.

Chromosome Mapping↗
PubMed1982

Cloning of Pseudomonas plasmid pMG7 and its restriction-modification system in Escherichia coli.

Plasmid pMG7 of Pseudomonas aeruginosa codes for a type II DNA restriction-modification (r-m) system, PaeR7. This plasmid has not been observed to transfer to Escherichia coli either by conjugation or by transformation. We have cloned BglII linears (42 kb) and the BamHI large fragment (37 kb) of pMG7 into cosmid pHC79 (6.4 kb) and introduced the recombinant molecules into E. coli by in vitro packaging. Several clones were obtained which demonstrated in vivo restriction of phage phi 80. One of these clones, GT138, was further tested and showed in vivo modification of phi 80. Extracts from two clones, GT138 and GT125, yielded a restriction endonuclease activity which produced fragments of phi 80 DNA identical to those produced by PaeR7. Cosmid cloning should be useful for obtaining substantial yields of large fragments of plasmids that are difficult to purify in their native strains.

Cloning, Molecular↗
PubMed1982

Selective deletion of large segments of Balbiani ring DNA during molecular cloning.

Transcription units in Balbiani ring 1 (BR1) and Balbiani ring 2 (BR2) of Chironomus tenans salivary glands are comprised of about 40 kb of repetitive DNA sequences organized in a satellite-like array. Because of this sequence organization, it was possible to prepare 30 to 40-kb target DNA fragments for cloning by performing limit restriction endonuclease digestion of high-Mr genomic DNA. These fragments were ligated to cohesive termini of the linearized cosmid, pHC79, packaged in vitro, and used to transduce Escherichia coli. Alternatively, target fragments were randomly sheared to a mean length of 8-10 kb, annealed to plasmid pBR322 using homopolymeric tails, and used for bacterial transformation. Recombinant cosmids and plasmids generally contained inserts which were proportional to the length of target fragments used in ligation reactions. However, the subset of recombinants that hybridized to 32P-labeled 75S RNA (highly enriched in BR1 + BR2 sequences) had disproportionately smaller inserts. With the exception of one metastable clone with a 2.1-kb insert, all others had inserts of 0.8 or 0.4 kb. Similar results were obtained in host cells that were recA- or recBC-. The most likely conclusion is that repetitive BR sequences are highly unstable during replication in E. coli and are selectively deleted.

Animals↗
PubMed1987

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Antigens, Bacterial↗