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PubMed1990

Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male sterile lines of maize.

Maize mitochondrial (mt) tRNA genes were localized on the mt master circles of two fertile lines (WF9-N and B37-N) and of one cytoplasmic male sterile line (B37-cmsT) of maize. The three genomes contain 16 tRNA genes with 14 different anticodons which correspond to 13 amino acids. Out of these 16 tRNA genes, 6 show a high degree of homology with the corresponding chloroplast (cp) tRNA genes and were shown to originate from cp DNA insertions and to be expressed in the mitochondria. The organization of the mt tRNA genes in both fertile lines is similar. The same genes are found, in the same environment, as judged from the restriction maps, in fertile and male sterile lines that have the same nuclear background, but the relative organization of the mt tRNA genes on the master circle is completely different.

Genes, Plant↗
PubMed1990

[Directed cloning of the HLA-B27 gene isolated from a patient with ankylosing spondylitis (Bechterew's disease)].

The authors have used the modified method of the direct gene cloning suggested by Nichols et al to isolate HLA-B27 gene from a patient suffering from ankylosing spondylitis. Five restriction enzymes (ClaI, HindIII, SnaBI, PvuI, SalGI) which had no recognition sites within the 6.0 kb EcoRI-BamHI-DNA fragment supposedly containing the HLA-B27 gene have been chosen by blotting-hybridization of the restriction fragments of the patients DNA with HLA-B27-specific probe. The 6.0 +/- 0.5 kb DNA fragments were isolated and cloned after the DNA treatment by 300 micrograms of all of these restriction enzymes. The obtained mini-library containing 280 recombinants has been screened with the use of HLA-B27 specific oligonucleotide probe. The clone PB27-2 has been isolated the restriction map of which is identical to HLA-B27k. The authors are planning to determine the sequence of the isolated gene in order to find a possible structural defect in it.

Cloning, Molecular↗
PubMed1991

Cloning and characterization of the ColE7 plasmid.

The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.

Amino Acid Sequence↗
PubMed1995

The physical map of the human RET proto-oncogene.

The RET proto-oncogene, a transmembrane tyrosine kinase receptor, is involved in the development of at least five different disease phenotypes. RET is activated through somatic rearrangements in a number of cases of papillary thyroid carcinoma while germ-line point mutations are associated with three inherited cancer syndromes MEN 2A, MEN 2B and FMTC. Moreover, point mutations or heterozygous deletions of RET are found in the dominant form of Hirschsprung disease or congenital colonic aganglionosis. We cloned the entire RET genomic sequence in a contig of cosmids encompassing 150 kb, from the CA repeat sTCL-2 to the region upstream the RET promoter, and established the position of the 20 exons of the RET gene with respect to a detailed restriction map based on eight endonucleases. A new highly polymorphic CA repeat sequence was identified within intron 5 of RET (RET-INT5). Finally the orientation of RET on chromosome 10q11.2 made it possible to orientate three other genes rearranged with RET in papillary thyroid carcinomas, namely H4/D10S170 on 10q21, R1 alpha on 17q23 and RFG2/Ele1 on 10q11.2.

Animals↗
PubMed1996

Genetic differentiation in the rice blast fungus revealed by the distribution of the Fosbury retrotransposon.

Repetitive DNA sequences genetically differentiate certain host-specific forms of the plant pathogenic fungus Pyricularia grisea. For example, high copy numbers of a sequence designated MGR586 are conserved in isolates that infect rice. In this report, we describe the molecular characterization of another repetitive DNA sequence designated fosbury. Restriction mapping and DNA sequence analysis show that fosbury is a member of a long terminalrepeat (LTR)-containing retrotransposon family, and gel blot hybridization analysis suggests that, like MGR586, fosbury is preferentially found in isolates that infect rice. This supports the view that rice pathogens comprise a genetically distinct form of P. grisea. We also investigated the distribution of fosbury and MGR586 and found that these elements are sometimes associated in the genome, and gel blot hybridization analysis shows that restriction sites flanking both of these elements resolve rice pathogens into similar clonal lineage groups. We conclude that the transposition of repetitive DNA sequences plays an important role in generating DNA fingerprint variation in the rice blast fungus.

Ascomycota↗
PubMed2000

Mechanism of phage PS166-mediated biotype conversion in Vibrio cholerae: role of the hlyA locus.

Temperate phage PS166 lysogens of Vibrio eltor MAK757 biotype eltor belong to two major categories. Seventy percent of the lysogens acquire auxotrophy for glycine and histidine and maintain their parental biotype. About 10% of the lysogens become Cys(-) or Cys(-) Met(-) and are converted to the classical biotype with complete changes in all biotype-specific determinants. PCR and RFLP analysis revealed that in the latter lysogens, the phage genome integrated at the hlyA locus, whereas the same locus remained unaffected in lysogens that retained their parental biotype. These results suggest that the two types of lysogens arose due to integration of the phage genome at two different locations on the chromosome. A restriction map of the phage genome was constructed using AvaII and BglII. An 800-bp BglII fragment carrying the attP site, located at one of the termini of the phage genome, was used to distinguish the two classes of lysogen.

Bacterial Proteins↗
PubMed1990

Isolation of a second cryptic plasmid from Mycoplasma mycoides subsp. mycoides.

Plasmids have rarely been detected in organisms constituting the genus Mycoplasma. Recently, the isolation of a cryptic plasmid from Mycoplasma mycoides subsp. mycoides has been described, and we report here the isolation of a second cryptic plasmid from this species. Restriction map and Southern blot analyses show that the second plasmid is distinct from the previously described plasmid, although a limited region of homology was detected. The availability of mycoplasmal cryptic plasmids may lead to the development of cloning vectors that replicate in these organisms.

Genetic Vectors↗
PubMed2002

Chromosomal sequences from Klebsiella pneumoniae flank the SHV-5 extended-spectrum beta-lactamase gene in pACM1.

The nucleotide sequence was determined for Anon 13, a 1250-bp SmaI fragment located approximately 2.8 kb downstream from bla(SHV-5) in pACM1. Anon 13 is 99% identical to a segment of the unpublished sequence of the Klebsiella pneumoniae chromosome. Genes of the K. pneumoniae sequence are undefined, but conceptual amino acid translations of two ORFs in Anon 13 are homologous to L-fuculose-1-phosphate aldolase (FucA) and a conserved hypothetical protein present in the chromosomes of several species of bacteria. In addition, restriction mapping indicates that the region of homology between the K. pneumoniae chromosome and pACM1 is as least 7.9 kb and includes both Anon 13 and bla(SHV). These observations demonstrate the chromosomal origin of the bla(SHV-5) on pACM1.

Chromosomes, Bacterial↗
PubMed1990

Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Structure and chromosomal localization of the gene for the oligodendrocyte-myelin glycoprotein.

Utilizing a cDNA clone encoding the oligodendrocyte-myelin glycoprotein (OMgp) to screen a human genomic DNA library, we have obtained a clone that contains the OMgp gene. The genomic clone was restriction mapped and the OMgp gene and its 5' and 3' flanking regions were sequenced. A single intron is found in the 5' untranslated region of the gene, while the coding region is uninterrupted by an intron. This placement of a single intron in the OMgp gene is identical to that of the gene for the alpha-chain of platelet glycoprotein Ib, which, along with OMgp, belongs to a family of proteins sharing two distinct structural domains: an NH2-terminal cysteine-rich domain and an adjacent domain of tandem leucine-rich repeats. Hence, it is possible that this family of proteins is not only related in terms of primary structure, but also through similar gene structure. Sequence comparison of the 5' and 3' flanking regions did not reveal striking similarities to other DNA sequences, and no obvious promoter elements were noted. By hybridization of the genomic clone to metaphase cells, we have localized the human OMgp gene to chromosome 17 bands q11-12, a region to which the neurofibromatosis type 1 gene has been previously mapped.

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↗
PubMed1994

The human stromelysin promoter contains a previously unreported 1.0-kb sequence.

Cloning and characterization of the promoter region controlling the gene encoding human stromelysin (Str) has been previously reported [Quinones et al., J. Biol. Chem. 264 (1989) 8339-8344]. We have characterized independently isolated genomic clones of the STR promoter, designated pSKStrB and 682, that are considerably different from the published sequence. Although the sequences up to an XbaI site at -480 of the 5' regions are identical, a novel 1.0-kb segment exists upstream from -480. This sequence is absent from the published clone, but its presence in the genomic DNA from twelve individuals has been confirmed by both PCR analysis and restriction mapping. Upstream of the novel 1-kb segment, the sequence of the published clone reappears, but in pSKStrB exists in inverse orientation.

Base Sequence↗
PubMed1990

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.

Antigens, Bacterial↗
PubMed1991

Cloning and expression of penicillin G acylase gene in Bacillus megaterium.

Bacillus megaterium BM1, which produces penicillin G acylase (PGA), has been isolated. Gene encoding for PGA was cloned into E. coli MC1061 using pBR322 as the vector, obtaining a recombinant plasmid pBmPA4 containing 9.9 kb inserted DNA. Restriction map of the plasmid was analyzed. A pBmPA5 containing 4.9 kb was gained by deletion in vitro. Both pBmPA4 and pBmPA5 clones can be expressed in E.coli MC1061, and their expressions were induced by phenylacetic acid.

Bacillus megaterium↗
PubMed1990

Tissue-specific regulation of cytochrome P-450 dependent testosterone 15 alpha-hydroxylase.

Testosterone 15 alpha-hydroxylase activity in kidney microsomes is higher in male mice than in female mice, while in the liver the activity is higher in females than in males. Cytochrome P-450 15 alpha, a specific form of cytochrome P-450 having testosterone 15 alpha-hydroxylase activity, accounts for virtually all of the testosterone 15 alpha-hydroxylase activity in female kidney microsomes, while other isozymes of testosterone 15 alpha-hydroxylase are present in male kidney microsomes. In female kidney, P-450 15 alpha expression is regulated by a single sex-dependent locus, called Rsh for "regulation of steroid hydroxylase." The higher level of P-450 15 alpha expression in male kidneys is dependent on androgens. Of all mice strains, 129/J seems to be the least dependent on androgens to maintain a high expression of P-450 15 alpha in male kidneys. Castration of male mice lowers kidney levels of P-450 15 alpha but in the liver, P-450 15 alpha levels rise after castration. This reciprocal regulation of P-450 15 alpha genes in liver and kidney was investigated by isolating cDNA clones encoding P-450 15 alpha from liver and kidney cDNA libraries. Two highly homologous cDNA clones encoding P-450 15 alpha designated type I and type II were identified, and levels of type I and type II mRNA in liver and kidney were determined by differential restriction mapping of double-stranded cDNA prepared from mRNA from these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Physical linkage of the human growth hormone gene cluster and the CD79b (Ig beta/B29) gene.

We have previously characterized a locus control region for the GH1 gene consisting of four DNase I hypersensitive sites (HS) located between 14.5 and 32 kb 5' to the GH1 gene transcription start site. Sequence analysis of the region between the GH1 gene and its most proximal HS (HSI) revealed a perfect match to the B-lymphocyte-specific CD79b gene. Restriction mapping and hybridization analysis of YAC and cosmid clones confirmed the close linkage of the CD79b gene to the hGH gene cluster and facilitated the assembly of a 100-kb physical map linking the hGH locus, the CD79b gene, and the more distant muscle-specific sodium channel alpha-subunit (SCN4A) gene.

Antigens, CD↗
PubMed1990

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1.

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with lambda NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was expressed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded beta-casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plasmid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.

Cloning, Molecular↗
PubMed1990

Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes.

Mammalian DNA segments cloned as yeast artificial chromosomes (YACs) can be manipulated by DNA-mediated transformation when placed in an appropriate yeast genetic background. A "fragmenting vector" has been developed that can introduce a yeast telomere and selectable marker into human-derived YACs at specific sites by means of homologous recombination, deleting all sequences distal to the recombination site. A powerful application of the method uses a human Alu family repeat sequence to target recombination to multiple independent sites on a human-derived YAC. Sets of deletion derivatives generated by this procedure greatly facilitate restriction mapping of large genomic segments. Targeting recombination with single copy sequences, such as cDNAs, will have many additional applications. This approach establishes a paradigm for manipulation and characterization of mammalian DNA segments cloned as YACs.

Chromosome Deletion↗