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PubMed1991

Somatic cell mapping and restriction fragment length polymorphism analysis of bovine insulin-like growth factor I.

The DNA isolated from cow-hamster hybrid somatic cells segregating bovine chromosomes was analyzed by Southern blotting and hybridization with a heterologous [32P]-labeled porcine cDNA probe encoding insulin-like growth factor I (IGF-I). Thirteen of 25 cow-hamster hybrid cell lines exhibited the bovine-specific IGF-I fragment. Analysis for the retention or loss of bovine IGF-I with markers previously screened against the same panel of hybrid cells revealed a 100% concordance with lactate dehydrogenase B of bovine syntenic group U3 located on bovine chromosome 5. Restriction fragment length analyses of genomic DNA from animals representing five breeds (Angus, Polled Hereford, Simmental, Gelbvieh, and Belgian Blue) and from seven half-sib Angus calves indicated that polymorphisms for the genomic composition of the bovine IGF-I gene may exist in cattle populations.

Animals↗
PubMed2004

Isolation of Nanjing local strains of HHV-7 and their biological and immunological characteristics.

To investigate the biological and immunological characteristics of the Nanjing local strains of HHV-7, four strains of herpesvirus were isolated from saliva specimens of one healthy individual and three children suffering from a kidney disease in Nanjing. The viruses were identified by transmission electron microscopy (EM), indirect immunofluorescence assay (IFA) with a specific monoclonal antibody; nested polymerase chain reaction, restriction mapping and DNA sequencing. The virus-infected cells showed the typical cytophathic effect (CPE) under microscopy and could be detected by IFA with the human herpesvirus-7 (HHV-7) specific antibody. Under EM, herpesvirus-like and virions capsids could be found in their cytoplasm or nucleoplasm. HHV-7 DNA fragments amplified from infected cells by nested PCR were confirmed by restriction mapping and DNA sequencing. Similarly to DC strain, an known HHV-7 strain used in the present study as the positive control, the virus could be inactivated by ultraviolet irradiation for 10 min, heated at 45 centigrade degree for 30 min, pH<5 or>9 at 4 centigrade degree for 2 h and ether or chloroform for 10 h. The virus induced the production of TNF-alpha, IL-10 and IL-12 p 70 while inhibited IFN-alpha secretion, increased the percentage of CD2(+) cells while decreased that of CD4(+) or CD 45 RA(+) cells. The results indicate that the viruses isolated in Nanjing are HHV-7, which has similar biological characteristic to the known HHV-7 strain, DC. Infection with HHV-7 in vitro could affect immune function of lymphocytes by disturbing cytokine production and CD antigen expression.

Adult↗
PubMed1979

Restriction-fragment map of the temperate Bacillus subtilis bacteriophage SPO2.

The endonucleases BglI, BglII, EcoRI, SalI, SmaI, and XbaI were used to fragment the phage SPO2 DNA. Electrophoretic analysis using ethidiumbromide agarose gels showed the phage to have nine BglI sites, one BglII site, four EcoRI sites, one SalI site, one SmaI site, and six XbaI sites. Using partial digestions, multiple endonuclease digestion, and autoradiography the fragments were sized and ordered into a circular map of 23 Md. Such an analysis locates the endonuclease sites, indicates which endonucleases are potentially useful in cloning with SPO2, and allows insertions and/or deletions in the SPO2 DNA to be characterized.

Bacillus subtilis↗
PubMed1984

Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.

We have isolated a clone containing a gene fragment coding for a 64-kilodalton glycoprotein that is the major late antigen of human cytomegalovirus (HCMV). Based upon the amino acid sequence of a tryptic peptide of this glycoprotein (HCMVgp64), two sets of mixed-sequence probes, one consisting of a mixture of 16 heptadecadeoxyribonucleotides and the other a mixture of 32 icosadeoxyribonucleotides, were synthesized. A subgenomic library of HCMV (Towne strain) DNA was constructed in plasmid pBR327 and transformants were screened with 32P-labeled aliquots of these synthetic oligodeoxyribonucleotide probes. Two clones among 15,000 gave strong positive signals. Plasmid DNA was isolated from the positive clones and characterized by restriction mapping and Southern blot analysis using both probes. The plasmid DNA contained a 2.3-kilobase insert, which yielded an 800-base-pair and a 1500-base-pair fragment after Sau3A digestion. Only the 800-base-pair fragment hybridized to the mixed probes, and DNA sequence analysis revealed that it contains nucleotide sequences compatible with amino acid sequences of tryptic peptides of HCMVgp64. Restriction mapping studies of HCMV DNA using this 32P-labeled 800-base-pair cloned DNA have allowed us to locate this gene fragment in the long unique region of HCMV (Towne strain) genome at approximately equal to 0.5-0.51 map unit.

Antigens, Viral↗
PubMed1996

A physical and genetic map of Neisseria meningitidis B1940.

A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one- and two-dimensional pulsed-field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes Nhel (16 sites), Sgfl (13 sites), Sfil (11 sites) and l-Ceul (4 sites), as well as a partial restriction map for the restriction enzyme Spel (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11-N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.

Chromosome Mapping↗
PubMed1991

Molecular cloning of the gene coding for the human T cell differentiation antigen CD7.

The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites -122 base pairs (bp) to -38 bp from ATG translation initiation site were demonstrated. The promoter region had high G + C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs.

Amino Acid Sequence↗
PubMed1986

[Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286].

The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol. Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established. Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7. Some nah genes were localized. An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped. Possible occurrence of a regulatory R locus in this region is suggested.

Chromosome Mapping↗
PubMed1988

Somatic cell mapping and restriction fragment analysis of bovine genes for fibronectin and gamma crystallin.

DNAs from cow-hamster and cow-mouse somatic hybrid cells segregating bovine chromosomes have been analyzed by Southern blotting and hybridization with human fibronectin and gamma crystallin probes. Concordancy of retention of these bovine genes was compared to cattle isozyme loci representing previously described syntenic groups. Bovine fibronectin (FNI) and gamma crystallin (CRYG) fragments were concordant with each other and with isocitrate dehydrogenase 1 (IDH1), representing the bovine syntenic group U17. The syntenic relationship of these genes is conserved on human chromosome 2q and also on mouse chromosome 1. In addition, bovine RFLPs were identified with both fibronectin and gamma crystallin probes. These polymorphisms will be used to study recombination between the syntenic loci in pedigreed herds and to mark a segment of the bovine genome that is likely homologous to the Lsh region of mouse chromosome 1, which confers resistance in mice to several intracellular parasites.

Animals↗
PubMed1979

Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.

Chromosome Mapping↗
PubMed1981

Physical mapping of the genomes of lytic and temperate forms of phage theta.

Restriction maps of genomes of the lytic form and diverse temperate mutants of phage theta of Bacillus licheniformis were constructed. Most temperate mutants produced fragmentation patterns identical to that of the parent lytic form, theta c: in other mutants the only detectable change in the map was the deletion of a Bg/II restriction endonuclease site at 46.5% genome length. In the genomes of two other temperate mutants, theta 1 and theta 2, the central part of the genome was replaced by a piece of DNA of equal length, but with a different distribution of restriction sites; the maps of the two mutants failed to reveal any similarity in the location of restriction sites in the inserted DNA. It seems that any alteration comprising the locus around the coordinate 46.5% of the theta c genome, brings about a transition from the lytic to temperate phenotype, indicating the position of a regulatory gene responsible for positive control of phage replication.

Bacillus↗
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↗
PubMed1994

Homogeneity of the major outer membrane protein gene of feline Chlamydia psittaci.

Thirteen feline Chlamydia psittaci strains isolated in various countries over a 50-year period were examined by restriction mapping of the major outer membrane protein (MOMP) gene obtained after DNA amplification by the polymerase chain reaction and compared with avian, ovine and guinea pig inclusion conjunctivitis (GPIC) C psittaci strains. All the feline isolates produced a unique AluI pattern distinct from the other C psittaci strains and were characterised by a typical fragment doublet of 185/180 base pairs. Such a homogeneity argues for their genetic relatedness and suggests their clonal origin. These data demonstrate the usefulness of MOMP-gene restriction mapping in C psittaci typing.

Animals↗
PubMed1987

Isolation and characterization of genes for blood proteins.

The expression vector lambda gt11Amp3 has been used to construct a cDNA library from rat liver polyadenylated RNA. Clones expressing antigenic determinants for rat albumin, transferrin, transthyretin, apolipoprotein E and apolipoprotein AII have been identified. Albumin clones containing cDNA inserts ranging from 0.9 kb to 1.9 kb were further identified by restriction mapping and nucleic acid sequencing. The largest insert contained the entire coding sequence for albumin. Characterization of the expressed proteins by acrylamide gel electrophoresis followed by immunological detection indicated that the proteins were produced as hybrids linked to the bacterial beta-galactosidase. A cDNA library for human liver polyadenylated RNA has also been constructed. Clones expressing antigenic determinants for human serum albumin, transferrin and apolipoproteins AI, AII, AIV and E have been isolated and their identity established by nucleotide sequencing and restriction mapping. Both rat and human serum protein cDNA clones are currently being used to study the tissue specific expression of serum proteins and for the isolation and characterization of the corresponding genes.

Animals↗
PubMed1986

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain.

A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5-20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic alpha-glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and alpha-glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.

Cloning, Molecular↗
PubMed2007

[Construction of the expression vector pET32a/His MBL-CLR and its expression in E.coli].

AIM: To construct pET32a/His MBL-CLR recombinant prokaryotic expression plasmid and to express mannan-binding lectin-CLR (MBL-CLR) protein in E.coli METHODS: The human MBL-CLR gene was amplified by PCR from pGEM-MBL plasmid, and was inserted into prokaryotic expression vector pET32a. After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBL-CLR was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDS-PAGE, Western blot and indirect enzyme-linked immunosorbent assay (ELISA) using the antibody from BALB/c mice immunized with the recombinant human MBL protein. RESULTS: The cDNA fragment of 180 bp was amplified from pGEM-MBL plasmid and the recombinant expression vector pET32/His MBL-CLR was constructed. The recombinant plasmid was consistent with those expected by restriction maps and sequence. Three components of relative molecular mass 30,000, 60,000 and 120,000 in the purified recombinant product were detected by SDS-PAGE and all the components could be recognized by anti-6His antibody in Western blot assay. The three components were correspondingly with the band of the monomer and oligomer of the fusion protein. The purified recombinant product could react with the antibody against the recombinant human MBL protein in the indirect ELISA. CONCLUSION: The prokaryotic expression strains that efficiently express recombinant human MBL-CLR and the recombinant human MBL-CLR-Trx fusion protein were obtained successfully, which will help the further structure-function research of MBL molecule.

Animals↗
PubMed1984

Integrated DNA sequences in three streptomycetes form related autonomous plasmids after transfer to Streptomyces lividans.

When Streptomyces parvulus ATCC 12434 was crossed with a plasmid-free S. lividans 66 derivative, some S. lividans exconjugants contained plasmid DNA, pIJ110 (13.6 kb). In a similar way, pIJ408 (15.05 kb) was found after mating S. glaucescens ETH 22794 with S. lividans. CCC DNA was not visualized in the donor strains. pIJ110 and pIJ408 each originates from a larger replicon, probably the chromosome, of S. parvulus or S. glaucescens. Restriction maps of pIJ110 and pIJ408, each for 10 enzymes, were derived. Derivatives of each plasmid were constructed carrying antibiotic-resistance markers (thiostrepton or viomycin) in a nonessential region and each plasmid was cloned into an Escherichia coli plasmid vector (pBR327 or pBR325). pIJ110 and pIJ408 resemble, in their origin, the previously known SLP1 plasmids (such as SLP1.2) which come from integrated sequences in the chromosome of S. coelicolor A3(2). pIJ110 and pIJ408, like SLP1.2, are self-transmissible, elicit the so-called lethal zygosis reaction (pock formation) and mobilize chromosomal markers. The three plasmids, in spite of their very different restriction maps, were found to be related: SLP1.2 and pIJ110 were strongly incompatible, showed complete resistance to each other's lethal zygosis reaction, and shared a segment of DNA with a considerable degree of cross-hybridization; pIJ110 and pIJ408 were weakly incompatible and showed partial resistance to lethal zygosis and a weak DNA cross-hybridization; pIJ408 and SLP1.2 were only distantly related on these criteria. pIJ110, pIJ408, and SLP1.2 hybridized with varying degrees of homology in Southern transfer experiments to DNA from 7 out of 13 of an arbitrary collection of wild-type streptomycetes. Integrated sequences capable of forming plasmids after transfer to S. lividans may therefore be widespread in the genus Streptomyces.

Base Sequence↗
PubMed1993

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains.

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype-specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei O polysaccharide is covalently bound to LPS cores of both the K-12 and R1 types, but neither to Salmonella (Ra-type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1-type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfasonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by R1-type LPS. Co-expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of rfaR1 DNA are required for the expression of complete phase-I-like S. sonnei LPS in E. coli K-12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae. S. sonnei O antigen expressed in a V. cholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.

Animals↗
PubMed1988

Molecular events during B lymphocyte differentiation. Induction of endogenous mouse mammary tumor proviral envelope transcripts after B cell stimulation.

We have identified a gene whose expression appears to be associated with a late stage in the differentiation of B lymphocytes into antibody secreting cells, as shown by using the inducible B cell lymphoma, CH12. Restriction mapping and partial sequencing of a cDNA clone isolated by subtraction analysis demonstrated that the clone, SC34, represents an envelope (env) gene transcript of a mouse mammary tumor virus (MMTV). In CH12 cells and in normal B cells, levels of MMTV RNA were increased after stimulation with LPS. The env transcript was the predominant MMTV RNA species and increased more dramatically than did levels of the genomic transcript. In differentiating CH12 cells, env transcripts increased as much as 20-fold above levels found in replicating, antibody nonsecreting CH12 cells. The major increase in expression appeared to be associated with B cell differentiation and not replication. By Southern blot analysis, only bands characteristic of endogenous proviruses were found in CH12, indicating that viral sequences were not amplified in this cell line. Restriction mapping indicated that the SC34 cDNA clone was a product of the Mtv-9 locus. Mtv-9 previously was shown to encode a complete MMTV provirus on chromosome 12, on which Ig heavy chain genes also are located. Increases in MMTV transcripts followed distinct kinetics and were quantitatively different from changes in immunoglobulin gene products. The expression of env RNA appears to more accurately reflect differentiation to antibody secretion in CH12 cells than does the expression of immunoglobulin gene transcripts.

Animals↗