摘要:

SummaryDNA‐binding proteins can be converted into site‐specific nucleases by linking them to the chemical nuclease 1,10‐phenanthroline‐copper. This can be readily accomplished by converting a minor groove‐proximal amino acid to a cysteine residue using site‐directed mutagenesis and then chemically modifying the sulphydryl group with 5‐iodoacetamido‐1,10‐ phenanthroline‐copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high‐resolution nuclear magnetic resonance and X‐ray crystal structures, and to locate binding sites of

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01022.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryMolecular analysis reveals a surprising sharing of short gene segments among a variety of large double‐stranded DNA bacteriophages of enteric bacteria. Ancestral genomes from otherwise unrelated phages, including λ Mu, P1, P2 and T4, must have exchanged parts of their tail‐fibre genes, Individual genes appear as mosaics with parts derived from a common gene pool. Therefore, horizontal gene transfer emerges as a major factor in the evolution of a specific part of phage genomes. Current concepts of homologous recombination cannot account for the formation of such chimeric genes and the recombinational mechanisms responsible are not known. However, recombination sites for DNA invertases and recombination site‐like sequences are present at the boundaries of gene segments conferring the specificity for the host receptor. This, together with the properties of the DNA inversion mechanism, suggests that these site‐specific recombination enzymes could be responsible for the exchange of host‐range de

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01023.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryMammalian alkaline phosphatases are 20‐30‐fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25–30% absolutely conserved. In the active‐site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes, in theEscherichia colienzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site‐specific mutagenesis, genetic and X‐ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline p

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01024.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryProtein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product, in a post‐translational event analogous to the removal of an intron from rRNA. Protein splicing introns, or‘inteins’appear to be a novel type of genetic element capable of mediating gene conversion of an‘intein‐less’allele, and hence promoting their own dissemination. The mechanism by which protein splicing is achieved is probably entirety encoded within the internal protein sequence, or intein, and does not require other accessory molecules. Although the concept of protein splicing inteins as selfish genetic elements of no immediate consequence to the host organism has emerged, this interpretation is questioned by recent evidence that in at least one example there appears to have been selection for prote

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01025.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA number of lines of evidence suggest that theN‐terminal sub‐domain of the DNA gyrase B protein contains the binding site for the coumarin antibiotics. We have engineered a clone which encodes a 24 kDa protein which represents this domain. Bacteria which overproduce this protein show an elevated level of resistance to coumarins, suggestive of binding of the 24 kDa protein to the drugsIn vivo. In vitrowe find that the 24 kDa protein does not interact with the gyrase A or B proteins or with DNA, and fails to hydrolyse ATP or show significant binding to ATP, ADP or ADPNP. However, we show that the 24 kDa protein binds coumarin drugs as tightly as the Intact B protein. A number of experiments suggest that the Interaction of the coumarins with the protein is predominantly hydrophobic in nat

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01026.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryChanges in expression of ribosomal protein genes during growth and stationary phase ofStreptomyces coelicolorA3(2) in liquid medium were studied. Proteins being synthesized were pulse‐labelled with [35S]‐methionine, separated by two‐dimensional poly‐acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues ofEscherichia coliribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins andEscherichia coliβ‐galactosldase. The genes (rplJandrplL)encoding the L10 and L7/L12 proteins were contained in a 1.2 kbBamHlfragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10‐L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case forE. coliand other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kbB

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01027.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryMany strains of enterotoxigenicEscherichia coli(ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple Identical protein subunits encoded bycooA.We have sequenced the DNA directly downstream ofcooAand identified two open reading frames,cooCandcooD, transcribed in the same direction ascooBandcooA.FollowingcooDIs DNA homologous to an insertion sequence, socooB, A, C and D appear to encode all the information needed forE. coliK‐12 to synthesize CS1 pili. Complementation analysis of mutants cloned inE. coliK‐12 and constructed in an ETEC‐derived strain indicates thatcooCandcooDare not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, likecooB, both are needed for assembly ofcooAinto

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01028.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryDensity‐dependent expression of luminescence inVibrio harveyiis regulated by the concentration of extracellular signal molecules (autoinducers) In the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density‐dependent response. TransposonTn5Insertions in the recombinant clone were isolated, and the mutations were transferred to the genome ofV. harveyifor examination of mutant phenotypes. Expression of luminescence inV. harveyistrains with transposon insertions in one locus,luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild‐type bacteria. Sequence analysis ofluxOrevealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two‐component, signal transduction proteins. The constitutive phenotype of LuxO∼ mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalli

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01029.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe genecbhAfrom the cellulolytic bacteriumCellulomonas fimiencodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: anN‐terminal catalytic domain, three repeated sequences of 95 amino acids, and a C‐terminal cellulose‐binding domain typical of otherC. fimiglycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungusTrichoderma reesel.CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme f

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01030.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThermally induced expression of the heat‐shock genegroELis subject to post‐transcriptional regulation inStreptomyces albus.WhenS. albuscells were shifted from 30°C to 41°C, synthesis of three GroEL‐like proteins was induced from two genes transcribed from associated promoters P1 and P2. Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes Indicated constitutive expression independent of heat shock. In contrast,neoexpression was thermally inducible as a GroELI‐APH translational fusion protein. Furthermore, expression of thegroEL1‐neogene was heat Inducible even after thegroEL1promoter region was replaced by a heteroiogous non‐heat‐inducible promoter such as theEscherichia coli lacpromoter. Finally, synthesis of GroE proteins, as well as the GroEL‐APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin. Post‐transcriptional regulatory signals needed for heat‐induced GroEL1 synthesis were mapped within of theg

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb01031.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY