摘要:

SummaryIn response to recommendations from the Steering Committees responsible for co‐ordination of World Health Organization programmes for research on the immunology of leprosy (IMMLEP) and tuberculosis (IMMTUB), a list was prepared summarizing the properties of mycobacterial proteins currently under investigation with respect to their immunological activities. After consultation with more than 40 laboratories world‐wide this list was extended to form the compilation shown below and is intended to provide a comprehensive and convenient reference for future studies in this fi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01994.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThis review explores the underlying biochemical and genetic principles leading to the formation of hybrid anthraquinones by recombinant anthracycline‐producing streptomycetes transformed with genes encoding the early steps in actinorhodin biosynthesis. Experiments indicate that simple aromatic polyketides are probably synthesized using very similar mechanisms, allowing for the interspecies cloning of polyketide synthase genes for the potential production of novel aromatic polyketide structure

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01995.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryBy screening aMycobacterium lepraeλgt11 genomic DNA library with leprosy‐patient sera we have previously identified 50 recombinant clones that expressed novelM. lepraeantigens (Sathishet al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express aM. lepraehomologue of the fibronectin‐binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327‐amino‐acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of theM. lepraeantigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome ofM. leprae.The amino acid sequence displays 75–85% sequence identity with components of the antigen 85 complex fromM. tuberculosis, M. bovisBCG andM. kansasii.Furthermore, antibodies to the antigen 85 complex ofM. tuberculosisandM. bovisBCG reacted with two fusion proteins containing the amino acid regions 55–266 and 265–327 of theM. lepraeprotein. TheM. leprae30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55–266 and 265–327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin‐binding sites on theM. lepraeprotein. These data indicate that this 30/31 kDa protein is not only important in the immune response againstM. leprae, but may also have a biological role in the interaction of this bacillus

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01996.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe direct visualization of plasmid DNA insideEscherichia colicells is demonstrated using phase‐fluorescence microscopy of DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01997.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA white, prototrophicCandida albicansstrain, heterozygous for theADE2gene (ade2/ADE2), was treated with the antimitotic agent methyl benzimidazole carbamate, and yielded red, adenine‐requiring colonies at a rate of 4 × 10‐3, an order of magnitude higher than the spontaneous rate of Ade‐colony formation. These red Ade‐colonies were small, growing at approximately half the rate of the parent strain, and gave rise to large red colonies spontaneously. When the chromosomes of the small red colonies were separated by pulsed‐field gel electrophoresis, the band hybridizing with theADE2gene was diminished in staining intensity by half relative to the parent and large red‐colony strains. Restriction fragment‐length polymorphism analysis and auxotrophic mutant spectra after mutagenesis suggested that the small red Ade‐strains were monosomic aneuploids lacking one of a pair of chromosome homologues, while the large red strains had regained a homologue, presumably via a second non‐disjunction event. Parasexual genetic analysis of two of the auxotrophs isolated from a putative aneuploid suggested that both mutations were linked to theADE2gene. These experiments suggest that targeted chromosome loss and monosomic, aneuploid strains have the potential to extend the scope of genetic analysis in this diploid

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01998.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA single gene (plcA) was cloned from a cosmid library ofErwinia chrysanthemiEC16 DNA that encoded an extracellular phospholipase. The gene was subcloned and DNA sequence data showed the presence of a single open reading frame encoding a protein with a predicted size of 39kDa. The coding region was G+C‐rich and the protein had a predicted basic isoelectric point. The protein showed no significant homology with others in the PIR library, including other phospholipases. When overexpressed inEscherichia colicells, theplcAgene directed production of ac.39kDa protein that was largely localized in the periplasm, but itsN‐terminal amino acid sequence was that of the native protein predicted from DNA sequence data. Unlike the wild‐type bacterium, anE. chrysanthemiEC16 marker exchange mutant of theplcAgene did not secrete extracellular phospholipase activity into the medium. However, no detectable change was observed in terms of the virulence of the mutant strain on potato tubers or chrysanthemum

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01999.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe hybrid prokaryotic lipo‐β‐lactamase mature and precursor proteins spontaneously form an intramolecular disulphide bond when oxidizedin vitro.When expressed inSaccharomyces cerevisiae (in vivo)the lipo‐β‐lactamase precursor is in a reduced form whereas the majority of the mature protein is oxidized. The results indicate that in yeast, the lipo‐β‐lactamase precursor is first processed (the signal peptide is removed) and then oxidized to form a disulphide bond in the mature protein. Reduced‐mature lipo‐β‐lactamase was found to reach the yeast periplasm and the process depends on endoplasmic reticulum (ER) entry even though the protein is not oxidized. This result is remarkable since in eukaryotes, disulphide bond formation occurs in the ER. Oxidized mature lipo‐β‐lactamase can also be released from the sphaeroplast into the yeast periplasm. Mutant lipo‐β‐lactamase genes in which cysteine residue 131 was changed to either tyrosine or threonine, were efficiently processed and secreted in yeast, which is consistent with the finding that reduced‐mature non‐mutant lipo‐β‐lactamase can be secreted. We discuss the possibility that the folding mechanism of lipo‐β‐lactamase in vitro may be fundamentally different from the pro

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02000.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryPilin variation inNeisseria gonorrhoeaedepends on a family of variant genes that undergo homologous, intragenic recombination. This work focuses on the repertoire of silent variant pilin genes in strain MS11, which contribute to the extensive variation of the expressed gene copy. A total of 17 silent copies were identified, which are, to varying degrees, truncated at their 5′ coding region and grouped in seven distinctpilloci. Most silent copies belong to locipilS1, pilS2andpilS6, which contain six, two and three silent copies, respectively, tandemly arranged. ThepilS5andpilS7loci each contain only a single copy. In addition, two silent copies are associated with each of the twopilEloci. By comparison with sequences present in the expressed gene of other variants of the same strain, it is suggested that each silent locus is capable of donating variant sequences into the expression locus and, thus, each silent copy can contribute to the variability of pilin expression. Often, concomitant with changes in the expressed copy, the silent copies of thepilE1locus undergo recombinations as well. Analyses of unrelated clinical isolates ofN. gonorrhoeaereveal homologies of hypervariant pilin sequences with those present in strain MS11, suggesting a limited diversity of such sequences within the gonococcal population and the existence of substantial functional constraints on the variability of pilin and pili. The data further indicate that hypervariant pilin sequences are subject to horizontal exchange and interstrain recombinatio

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02001.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTwo nitrate reductases, NRA and NRZ, are present inEscherichia coli.These isoenzymes have the same αβγ, subunits composition and have similar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the α subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane‐bound heterologous enzyme. One such heterologous enzyme, αAβZγZ, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heterologous nitrate reductases could be formedin vivois discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02002.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTwo membrane‐bound nitrate reductases, NRA and NRZ, exist inEscherichia coil.Both isoenzymes are composed of three structural subunits, α, β and γ encoded bynarG/narZ, narH/narYandnarl/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, δ, which is not part of the final enzyme. A strain which is devoid of, or does not express, thenargenes, was used to investigate the role of the δ and γ polypeptides in the formation and/or processing of the nitrate reductase. When only the α and γ polypeptides are produced, an (αβ) complex exists which is inactive and soluble. When the α, β and δ and polypeptides are produced, the (αβ) complex is active with artificial donors such as benzyl viologen but is soluble. When the α, β, and δ polypeptides are produced, the (αβ) complex is inactive but partially binds the membrane. It was concluded that the γ polypeptide is involved in the binding of the (αβ) complex to the membrane while the δ polypeptide is indispensable for the (αβ) nitrate reductase activity. The activation by the δ polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (αβ) complex was shown to contain the four iron–sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the δ polypeptide promotes the correct assembly of the α and β subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the δ or γ polypeptide encoded by onenaroperon can be substituted succesfully for by its respective counterpart from the othernaroperon to give an active membrane bound

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02003.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY