ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00096.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryWe have cloned three genes (amy1, amy2andamy3) encoding α‐amylase in the filamentous fungusAspergillus oryzae.The established overall sequences have a very high degree of homology, showing divergences mainly in the 3’‐untranslated regions. The positions and the sequences of the eight introns were found to be absolutely identical in the three genes. The sequence analysis of the 5’‐regions revealed presumptive TATA, CAAT and GC boxes. Primer extension analysis was performed to determine the transcription start. We were able to detect mRNAs fromamy1andamy3but not fromamy2with gene‐specific oligonucleotide probes complementary to the 3’‐n

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00097.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe arsenical resistance (ars) operon of the conjugative R‐factor R773 encodes an ATP‐driven anion extrusion pump, producing bacterial resistance to arsenicals. There are three structural genes, of which the product of the middle gene,arsB, has not previously been identified. From nucleotide sequence data, the ArsB protein is predicted to be a 45577 Dalton hydrophobic protein. A mini‐Mu transposition procedure was used to construct anarsB‐lacZgene fusion, producing a hybrid ArsB‐β‐galactosidase protein which was localized in the inner membrane. The operon was cloned into a T7 RNA polymerase expression vector. In addition to the previously identified ArsA and ArsC proteins, the cells synthesized an inner membrane protein with an apparent mass of 36 kD identified as the

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00098.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe region required for regulation of a previously characterized arginine‐regulatable promoter upstream from theargCgene in theargCAEBD‐cpa‐argFcluster ofBacillus subtiliswas defined by integration ofargC‐lacZtranslational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between theargCregulatory region and the well‐characterizedEscherichia coliarginine operators (ARG boxes). This similarity was shown to be functionalin vivoin that theB. subtilisrepressor regulated theE. coliarginine genes, but theE. colirepressor, even when encoded by a multicopy plasmid, could not repress theB. subtilis argCpromoter.In vitrobinding studies using purified repressors on DNA fragments encoding operators from bothE. coliandB. subtilisdemonstrated interactions by both

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00099.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryOne limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on differentEscherichia coliplasmids and usingMycobacterium smegmatisas the host. In both cases we found that the originalE. coliplasmid is capable of being replicated inM. smegmatis, yielding chloramphenicol‐resistant colonies. One such plasmid has been recovered from aM. smegmatistransformant and used to re‐transform bothM. smegmatisandE. colito chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial spec

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00100.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryOligonucleotides that correspond to regions of the penicillin‐binding protein 2 gene (penA) that differ between penicillin‐sensitive and penicillin‐resistant strains have been used as probes to classify thepenAgenes in a collection of penicillin‐resistant gonococci isolated in Britain. 44/47 of those gonococcal strains that had minimal inhibitory concentrations of ≥0.25 μg benzylpenicillin per ml contained extensively alteredpenAgenes which appeared to be very similar (or identical) to one or other of the two classes of alteredpenAgenes that have been described previously. Since these two classes of alteredpenAgenes are related, it appears that the great majority of the alteredpenAgenes of non‐β‐lactamase‐producing penicillin‐resistant gonococci have a clonal origin. The other three penicillin‐resistant strains had alteredpenAgenes that were different to those described previously. A crucial step in the development of the altered forms of PBP2 with decreased affinity for penicillin appears to have been the insertion of an extra codon within the transpeptidase domain of thepenAgene. This insertion was found in thepenAgene of all gonococci with minimal inhibitory concentrations of>0.016μg benzylpenicillin per ml but was not found in any strains with minimal inhibitory concentrati

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00101.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe pathogenicNeisseria, N. gonorrhoeaeandN. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)‐binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5‐residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the prot

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00102.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryH.8 is a virulence‐associated, surface‐exposed, immunogenic macromolecule composed of lipid and protein, common toNeisseria gonorrhoeaeandNeisseria meningitidis.The H.8 DNA sequence predicted a 6.9 kD peptide comprising 14 tandemly repeated pentameric sequences. Ten were identical: Pro, Ala, Ala, Glu, Ala. Also predicted was a lipoprotein leader consensus sequence which probably specified acylation since theEscherichia coli‐expressed protein was tightly associated with lipid. Lipid appeared to contribute significantly to H.8 antigen's electrophoretic mobility. This is the first description of a prokaryotic outer membrane protein composed solely of tandem repeats. Furthermore, DNA encoding this repeat appears to have been duplicated and translocated into another neisserial gene encoding an a

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00103.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryCommon epitopes accessible to antibody on purified macromolecules or structurally altered gonococci may not be accessible to antibody when those macro‐molecules are in their native state on the surface of intact organisms. To determine the immunologic accessibility of cyanogen bromide fragment 2 (CNBr2), a portion of the gonococcal pilin molecule that is common to all gonococcal strains on the surface of viable gonococci, probes composed of specific CNBr2 antibodies linked to gold spheres were manufactured. When whole piliated gonococci were exposed to these anti‐CNBr2 immunological probes and examined using transmission electron microscopy, no significant marking of native pili was evident. These probes, however, detected CNBr2 in purified form. The epitopes encompassed within the CNBr2 portion of pili appear to be inaccessible to anti‐CNBr2 probes within native gonococcal

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00104.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryAs an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes inStaphylococcus aureusand other Gram‐positive bacteria. The use of a restriction‐deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) inS. aureusNCTC 8325‐4. Plasmid pPQ126 (13.6 kb) is a novel, temperature‐sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30°C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551orTn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39°C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination‐deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30°C), and excision results in derivatives of pPQ126 that harbour DNA of chromo

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00105.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY