摘要:

SummaryMessenger RNA in bacteria may be inactivated by several parallel mechanisms acting independently on different target sites. For any species of mRNA the overall rate of inactivation is determined by the sum of the contributions from the different mechanisms. Transcripts may be inactivated directly by endonucleolytic attack or by processive nucleolytic degradation, which may proceed in the 3′–5′ direction and probably also in the 5′‐3′ direction. Moreover, the functional lifetime of many mRNAs may be determined by processes that are not nucleolytic, such as the binding of translational repressors or the formation of secondary structures which prevent initiation of translation. These non‐nucleolytic processes may also determine the chemical stability as chemical degradation frequently appears to be closely coupled to functional inactivation. The relative importance of the different mechanisms in the inactivation of bulk cellular mRNA, as well as the general prospects for engineering of stable mRNAs

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01469.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryRNA metabolism plays a central role in cell growth. It is essential to regulate RNA synthesis, processing, stability and degradation. Conformational changes in RNA are key elements in regulating cellular processes. Recently, an increasing number of putative RNA helicases from different organisms ranging fromEscherichia colito humans and viruses have been identified. They are Involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division. Based on sequence homologies these proteins were grouped in a family, the D‐E‐A‐D box protein family (D‐E‐A‐D = Asp‐Glu‐Ala‐Asp). Some of the better characterized members have been shown to possess ATP‐binding and hydrolysing activities as well as ATP‐dependent RNA helicase activities. Most of the genes encoding such proteins have been isolated from yeast, on which we will focus in this review. From sequence data, three of the members form a subfamily,

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01470.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryCS1 pili are filamentous proteinaceous appendages found on many enterotoxigenicEscherichia coli(ETEC) strains isolated from human diarrhoeal disease. They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells. We have identified a gene,cooB, which lies directly upstream ofcooA, the gene that encodes the major structural CS1 protein. When translatedin vitro, the protein product ofcooBmigrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence. We constructed a mutant allele (cooB‐1) by insertion of the omega fragment, which inhibits transcription and translation, into thecooBgenein vitro.In a derivative of an ETEC strain with thecooB‐1mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, nocooBand a greatly reduced level ofcooAproduct was detectable in total cell extracts. The reduction ofcooAin this strain appears to result from polarity of thecooBmutation because introduction of the wild‐typecooAgenein transcauses production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant. Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes. However, thecooB‐1strain withcooA in transdoes not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili). Electron microscopy reveals that there are no pili present on the cell surface or in the culture medium in which this strain was grown. Thus, although it is not required for stability or transport of the major CS1 pilin protein, CooB is needed for assembly of CooA into pili. The relationship of these gene products to those of other pili is di

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01471.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryASaccharomyces cerevisiaeglutamate auxotroph, lacking NADP‐glutamate dehydrogenase (NADP‐GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP‐GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified fromS. cerevisiaeis made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transformEscherichia coliglutamate auxotrophs. Transformants were only recovered when the recipient strain was anE. coliGDH‐less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in theE. colitransformants is a hybrid comprising the largeE. colisubunit and the smallS. cerevisiae

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01472.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryCloning of an autonomously replicating sequence (ars) from the origin region ofBacillus subtiliswas previously unsuccessful because of the strong incompatibility exerted by sequences located within theoriCregion. Using anarssearching vector which would be selective for drug resistance even at one copy per cell, and by cloning large fragments covering as much as possible of theoriCregion, we have succeeded in isolatingarsfragments from the origin region of the chromosome. The minimum essential fragment contains two DnaA‐box regions (non‐translatable regions containing multiple repeats of DnaA‐box) separated by thednaAgene. Neither one of the DnaA‐box regions by itself showedarsactivity. When constructed asoriCplasmids, thednaAcoding region could be removed without affectingarsactivity. The minimum distance between the two DnaA‐box regions obtained so far is 274 bp. The copy number of theoriCplasmid is estimated as one per replicating chromosome. These plasmids are unstable and tend to be lost or integrated into c

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01473.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTheCorynebacterium glutamicum gdhgene encoding NADP‐dependent glutamate dehydrogenase (GDH) has been isolated by complementation of theEscherichia coli gdhmutant PA340. Thegdhgene was subcloned into theE. coli/C. glutamicumshuttle vector pEK0 and introduced intoC. glutamicum.Recombinant strains showed approximately eightfold higher specific GDH activity (15U mg protein‐1) relative to the wild type (1.8U mg protein‐1). Physiological studies with wild‐type and recombinantC. glutamicumstrains revealed no indication of significant regulation ofgdhexpression. The DNA sequence of 2082 bp, including thegdhgene, 5′‐, and 3′‐flanking regions, was determined. The structural gene consists of 1344 bp and codes for a polypeptide of 448 amino acid residues (Mr49152) showing up to 53.6% identity with reported amino acid sequences of glutamate dehydrogenases from other organisms. Northern blot hybridization revealed a 1.65 kb mRNA transcript, indicating that thegdhgene ofC. glutamicumis monocistronic. Transcription occurred from a G residue located 284bp upstream of the AUG considered to be the translational i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01474.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe prokaryotic ribosomal operon,str, contains open reading frames for the two elongation factors, elongation factor G (EF‐G) and elongation factor Tu (EF‐Tu), and ribosomal proteins 57 and S12. The DNA sequence and predicted amino acid sequence for S7 fromChlamydia trachomatisare presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF‐G is described. Significant amino acid homology is also noted when the amino‐terminal sequence of chlamydial EF‐G is compared with the cytoplasmic tetra‐cycline resistance factors,tetMandtetO, from streptococci andCampylobacter jejuni.Related findings and possible resistance mechanisms for the newly recognized tetracycline‐resistant clinical isolates ofC. trachomatis

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01475.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryAlasB–lacZtranslational fusion (pTS400) was used to examine expression of the elastase gene (lasB) inPseudomonas aeruginosastrain PAO1. Expression from thelasB–lacZfusion was enhanced when PAO1(pTS400) was grown in a defined medium containing elevated levels of zinc (6.0μg ml‐1). Transcript accumulation studies on PAO1(pTS400) and PAO1 showed that the addition of zinc had a slight negative effect onlasBtranscription. These results indicated that zinc regulates the expression of elastase at the translational level. A comparison between zinc regulation and iron regulation was also made. Iron has a negative effect onlasB–lacZexpression. When PAO1(pTS400) was grown in a defined medium with a low iron content (0.1 μg ml‐1) the bacteria still responded to zinc. The independent effects of low iron and high zinc concentrations suggest separate control mechanisms for the two factors. Transcript accumulation studies on PA01 and PAO1(pTS400) indicated that early in the growth curve iron did not influence transcription oftasBorlasB–lacZ.Later in the growth curve a slight increase inlasB–lacZtranscription was observed only in PAO1(pTS400) grown in low iron. These results suggest that the iron regulation oflasBoccurs predominantly at the transtational level. Finally, when PAO1(pTS400) was grown in a complex peptone‐based medium, a high level of transcript accumulation accounted for elastase expression. Alterations of iron and zinc concentrations of this medium did not affect the expression of elastase. These results suggest that there may be additional environmental cues regulating

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01476.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummarySome natural isolates ofEscherichia colihave been shown to produce a unique branched RNA‐linked single‐stranded DNA called msDNA. These bacteria contain a retro‐element called retron consisting of themsr–msdregion and the gene for reverse transcriptase (RT). All threeE. coliretrons characterized to date have been shown to be integrated into a prophage or to be associated with phage‐related genes. In this report, we identified a new msDNA from anE. coliwild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3 kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single‐stranded DNA, which is considered to be branched out from the 18th G residue of a 75‐base RNA molecule by a 2′,5′‐phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA‐Ec107 and retron‐Ec107, respectively. Most significantly, retron‐Ec107 was inserted into theE. coligenome by replacing a 34bp intergenic sequence between thepyrEandttkgenes located at 82 min on theE. colichromosome. Interestingly, the retron contains palindromic structures at both ends and theE. Coli34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron‐

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01477.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryIn the preceding paper, we showed that a new 1.3kb retron (retron‐Ec 107) inEscherichia coliis responsible for the biosynthesis of a branched‐RNA‐linked multicopy single‐stranded DNA (msDNA‐Ec107). Here, we show that this retron occurs in strains from different branches. A, B1, and D of a well‐defined phylogenetic tree of a collection of wildE. coli.Sequence comparisons of the retrons from these three branches were carried out. Sequence homology was well conserved among the strains within the same branch and the retron sequence from branch A was exactly the same with that from branch D, while there were 18 base substitutions between the retrons from branch B1 and A or D, resulting in seven amino acid substitutions in reverse transcriptase. No substitutions were found in the msDNA‐ and msdRNA‐coding regions, and there was no difference in the ability of msDNA production between them. These results suggest that the retron has probably been integrated into at least one of the three branches at an early stage of evolution and subsequently transferred to the other two branches, and also that the msDNA‐producing system has been conserved during evolution with some mutati

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01478.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY