摘要:

SummaryThe Cdc7 protein kinase is the product of an essential cell cycle gene, and Is involved in three aspects of DNA metabolism: mitotic DNA replication, meiotic DNA recombination, and replication‐dependent DNA repair. The mechanism by which Cdc7 regulates each of its cellular functions is an issue of considerable interest. Recently, much of the research regarding the regulation of cell cycle progression has focused on the regulatory action of cyclins on their catalytic counterparts. We propose that the function of Cdc7 in ceil cycle progression is mediated in a similar manner, in that Dbf4, a protein whose transcript level is known to fluctuate in the cell cycle, is essential for Cdc7 kinase activity. The periodic association of Dbf4 with Cdc7 may account for the regulation of Cdc7 kinase function and progression through the cell cycl

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00358.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe cold‐shock response ofEscherichia colidescribes a specific pattern of gene expression In response to abrupt shifts to tower temperatures. This pattern includes the induction of cold‐shock proteins, synthesis of proteins involved in transcription and translation, and repression of heat‐shock proteins. The identified cold‐shock proteins are involved in various cellular functions from supercoiling of DNA to Initiation of translation. The major cold‐shock protein, CspA, has high sequence similarity with three otherE. coliproteins ‐ CspB, CspC, and CspD. Using translationallacZfusions,cspBwas found to be cold‐shock inducible at the level of transcription likecspA, whilecspCandcspDwere not. The Csp proteins, which share sequence similarity with other prokaryotic proteins and with the‘cold‐shock domain’of eukaryotic Y‐box proteins, may have a function in activating transcription or unwinding or masking RNA molecules. Because the cold‐shock response can also be induced by the addition of certain inhibitors of translation, it has been proposed that the state of the ribosome is the physiological sensor for the induction. In addition toE. coll, cold‐shock proteins have also been found in other prokaryotic

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00359.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryHybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino‐terminai OmpA signal sequence was fused with TEM β‐lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein ofEscherichia colias an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β‐lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β‐lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino‐terminal signal sequence. Introduction of two lysine residues at the carboxy‐terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β‐actamase domain in the cytoplasm. When three more lysine residues were added to the amino‐terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino‐terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00360.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThecspAis a gene ofEscherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino add residues has high sequence similarity with eukaryotic Y‐box DNA‐binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containingcspA.DNA sequencing of these clones confirmed that the two genes are highly homologous tocspA.One designatedcspBis mapped at 35 min on theE. colichromosome and encodes a 71‐residue protein with 79% identity to CspA, while the other,cspC, is mapped at 40 min and encodes a 69‐residue protein with 70% identity. In addition, a DNA sequence upstream of theclpAgene at 19 mm published elsewhere contains an open reading frame for a 74‐residue protein with 45% identity to CspA. Allcspgenes were fused in the coding regions with thelacZgene, and the expression of β‐galactosidase was examined for these hybrid genes upon cold shock. A similar cold‐shock induction tocspAwas observed forcspBbut notcspCandcspD.These results Indicate thatE. colihas a family of thecspAgene, some of which are induced

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00361.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryHomology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) Includes (i) two metal‐resistance efflux pumps inAlcaligenes eutrophus(CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots byRhizobium meliloti(NoIGHI), and (iii) a cell division protein inEscherichia coli(EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co‐operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram‐negative bacterial cell env

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00362.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryWe report the cloning of the RNase P RNA genes from the primary aetiological agent of porcine pneumonia,Mycoplasma hyopneumoniae, and the closely related commensal,Mycoplasma flocculare.The monocistronic genes each have promoters with AT‐rich ‐35 regions and Rho‐independent‐like transcription terminators which are retained in the RNase P RNA. Both of these RNase P RNA variants are shown to be catalytically activein vitroin spite of a low overall GC content (30%). Our results suggest a new example of a stable mini‐helix in the conserved core of the mycoplasmal RNase P RNAs. Deletion of the corresponding structural element in Escherichia coli RNase P RNA (M1 RNA) generated an RNase P RNA with an impaired substrate interaction. Displacement of this structural element with the mycoplasmal mini‐helix resulted in an enzyme with a phenotype similar to that of wild‐type M1 RNA. in addition, this structural element is important for lead ion‐induced cleavage at specific

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00363.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryTheEscherichia coliDnaK homoiogue inVibriosp. strain S14 was shown to possess chaperone function for translocation during carbon starvation. This was demonstrated by using the method of co‐immunoprecipitation. DnaK co‐precipitated with the carbon starvation‐specific periplasmic space protein Csp5 three hours after the onset of carbon starvation. Pulse‐chasing of the protein with radiolabelled methlonine followed by the addition of an excess of unlabelled methionine demonstrated that the Csp5 protein was translocated across the inner membrane. Only the cytoplasmic unprocessed precursor form of Csp5 co‐precipitated with DnaK. The non‐covalent binding between the two proteins was found to be ATP‐dependent, as the addition of ATP released the interaction between DnaK and the precursor form of Csp5, as was shown both on silver‐stained SDS‐poly‐acrylamide gels and by Western blot analysis. We suggest that DnaK maintains the carbon starvatlon‐Inducible protein Csp5 in a translocation‐competen

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00364.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryIn the obligate Intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of σ73to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immuno‐precipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as Is true for the σ subunit, in R. prowazekii and Escherichia coli the β’and β subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of σ73to core polymerase inR prowazekiiwould be 0.1. This is in striking contrast to E. Coli where the ratio is typically 0.4. it remains to be established whether this low sigma saturation results In a limitation of active RNA polymerase in R. prowazekii and contributes to its slo

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00365.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of therepAgene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds iR1 and the RepA monomers that bind the RS sequences.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00366.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryWithin the capsule gene complex (cps) ofNeisseria meningitidisB a 5.5 kb DNA fragment encodes proteins with strong homologies to enzymes of the lipopolysaccharide biosynthetic pathway ofSalmonella typhimuriumandEscherichia coli, GalE, RfbB, RfbC and RfbD. A meningococcal galE mutant expressed a truncated lipooligosaccharide (LOS), which terminated at the glucose residue between inner and outer core, and a second gaiE gene present outside the cps cluster was found to be transcriptionally and functionally inactive and, thus, unable to complement this defect. Because of the defect in the outer core, the LOS of the galE‐defective meningococcal mutant was not sialylated. In contrast, carbohydrate analysis of the LOS of an rfb‐defective meningococcal mutant revealed no difference from the LOS of the wild‐type strain, suggesting that the rfb genes are inactive. This was supported by Northern blot analysis, which showed that expression of the rfb gene products was transcriptionally regulated. The inability of the meningococcal galE mutant, which cannot sialylate the LOS, allowed us to investigate the significance of LOS sialylation in relation to the presence of the polysialic acid capsule. Sialylated LOS, but not the polysialic acid capsule, is necessary to confer complete serum resistance on the meningococcus by inhibition of the alternative complement pa

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00367.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY