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2.
Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot 总被引:11,自引:0,他引:11
The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence. 相似文献
3.
The plasmids of Deinococcus spp. and the cloning and restriction mapping of the D. radiophilus plasmid pUE1 总被引:2,自引:0,他引:2
Plasmids were found in strains representing all four species of the genus Deinococcus viz. D. radiodurans, D. radiopugnans, D. radiophilus and D. proteolyticus but were not found in the most intensively-investigated strain of the genus, D. radiodurans R1. Their sizes were calculated from electron micrographs. D. radiophilus yielded three size classes of plasmid while D. radiodurans Sark, D. proteolyticus and D. radiopugnans each yielded two. Attempts to cure D. radiophilus and D. radiodurans Sark of any of their plasmids, using a variety of methods, were unsuccessful. A 10.8 kbase pair (kb) plasmid from D. radiophilus, pUE1, was cloned into the PstI site of pAT153 and propagated in Escherichia coli HB101. The recombinant plasmid, pUE109 was subjected to single and double digestion with various restriction endonucleases and its restriction map constructed. The resistance of E. coli HB101 to ultraviolet radiation was not increased when pUE109 was introduced into it. Attempts to transform D. radiodurans with pUE109 failed to detect tetracycline-resistant transformants. 相似文献
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A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases. 相似文献
6.
Yichun Zeng Yi-Ling Hou Wan-Ru Hou Jian Li 《Nucleosides, nucleotides & nucleic acids》2013,32(8):536-551
Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further. 相似文献
7.
《Process Biochemistry》2014,49(1):61-68
Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis. 相似文献
8.
Abstract A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the d-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M r 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli . 相似文献
9.
Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes 总被引:16,自引:0,他引:16
A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease. 相似文献
10.
Cloning of the second adenylate cyclase gene (cya2) from Rhizobium meliloti F34: Sequence similarity to eukaryotic cyclases 总被引:2,自引:0,他引:2
John Archdeacon John Talty Bert Boesten Antoine Danchin Fergal O'Gara 《FEMS microbiology letters》1995,128(2):177-184
Abstract A second adenylate cyclase ( cya2 ) gene was isolated from a Rhizobium meliloti F34 gene bank. Complemented E. coli Acya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP. DNA hybridization studies showed cya2 to be unique to R. meliloti strains. The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids. Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site. Similiar residues were also found in the prokaryotic R. meliloti Cya1 protein. A R. meliloti cyal/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene. 相似文献