Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Culture Media , Electrophoresis, Gel, Two-Dimensional/methods , Isotope Labeling/methods , Bacillus subtilis/chemistry , Bacillus subtilis/growth & development , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Mutation , Sulfur RadioisotopesABSTRACT
Aspergillus niger var. awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of the niaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in both A. niger var. awamori and A. niger. A single copy of Vader was detected from only one of the two laboratory strains of A. nidulans which were also examined. Insertion of the Vader element into the niaD gene of A. niger var. awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements at niaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. Mutants niaD392 and niaD436 reverted at a frequency of 9x10(-3) and 4x10(-2), respectively. Two of the mutants, niaD587 and niaD410, reverted at a lower frequency of 6x10(-4).
Subject(s)
Aspergillus niger/genetics , DNA Transposable Elements , Base Sequence , Blotting, Southern , Gene Dosage , Molecular Sequence Data , Mutagenesis , Nitrate Reductase , Nitrate Reductases/analysis , Nitrate Reductases/genetics , Polymerase Chain Reaction , Selection, Genetic , Sequence Analysis, DNA , Species SpecificityABSTRACT
Escherichia coli strains carrying the protease III structural gene (ptr) on a plasmid secreted the protein into the growth medium. Plasmid-encoded beta-lactamase and chloramphenicol acetyl transferase, which served as periplasmic and cytoplasmic markers during cell fractionation, were not released into the growth medium. There appeared to be some strain dependence on the proficiency of the secretion system. Protease III was not detectably processed upon export through the outer cell membrane.
Subject(s)
Endopeptidases/metabolism , Escherichia coli/enzymology , Metalloendopeptidases , Blotting, Western , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Endopeptidases/genetics , Escherichia coli/genetics , Escherichia coli/growth & development , Genes, Bacterial , Plasmids/genetics , Protein Sorting Signals/genetics , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
The product of the altered mRNA stability (ams) gene of Escherichia coli is involved in decay of mRNA. The complete nucleotide sequence of a 4-kilobase BamHI restriction fragment containing the ams coding sequence was determined. Transcription of the ams gene was analyzed by high resolution S1 mapping. A promoter was found with a homology score of 58% 361 nucleotides upstream from the start codon of ams. The ams structural gene consists of an open reading frame of 2,445 nucleotides. The protein predicted from this open reading frame has a molecular mass of 91,327 Da, which is significantly smaller than that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Confirmation of the ams coding sequence was obtained by comparing the predicted amino acid sequence with that derived by amino-terminal analysis of gel-purified Ams protein. The predicted protein sequence of the ams gene was screened against translations of the GenBank DNA sequence data base. A homology of 18% over a region of 315 amino acids of the carboxyl terminus of the Ams product was found to MRP3, a mitochondrial ribosomal protein from Neurospora crassa. A smaller region of homology (29% in 86 residues) was found to the human U1 small nuclear ribonucleoparticle 70,000-Da protein.
Subject(s)
Escherichia coli/genetics , Fungal Proteins/genetics , Pyruvate Dehydrogenase Complex , RNA, Bacterial/genetics , RNA, Messenger/genetics , Ribosomal Proteins/genetics , Sequence Homology, Nucleic Acid , Transcription, Genetic , Amino Acid Sequence , Cloning, Molecular , Codon , Dihydrolipoyllysine-Residue Acetyltransferase , Genes, Bacterial , Molecular Sequence Data , Neurospora crassa , Nucleic Acid Conformation , Restriction MappingABSTRACT
Regulation of bgl operon expression in E. coli occurs by a mechanism involving antitermination of transcription at two termination sites within the operon. The bglG gene product is absolutely required for this process. Here we provide evidence that BglG is an RNA binding protein that recognizes a specific sequence located just upstream of each of the terminators. The sequence was delimited using a series of specific oligonucleotide probes. Mutational analysis of this sequence indicates that the protein requires a specific RNA secondary structure for recognition. We propose that BglG prevents transcription termination by binding to nascent RNA and blocking formation of the terminator structure.
Subject(s)
Bacterial Proteins/genetics , Carrier Proteins/genetics , Escherichia coli/genetics , Genes, Regulator , Operon , Terminator Regions, Genetic , Transcription, Genetic , Bacterial Proteins/metabolism , Base Composition , Base Sequence , Binding Sites , Carrier Proteins/metabolism , Escherichia coli/metabolism , Genetic Vectors , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotide Probes , Plasmids , Protein Sorting Signals/genetics , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA-Binding ProteinsABSTRACT
A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by Tn1000 insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , RNA, Messenger/metabolism , Blotting, Southern , Cloning, Molecular , DNA Mutational Analysis , DNA Transposable Elements , DNA, Bacterial/genetics , Genetic Complementation Test , Molecular Weight , Nucleic Acid Hybridization , RNA, Bacterial/metabolism , Restriction Mapping , Ribonucleases/geneticsABSTRACT
A simple method for RNA transcript mapping has been developed that combines the use of 35S-labeled M13 DNA probes and the presence of high concentrations of sodium trichloroacetate in the hybridization buffer. These hybridization conditions permit the use of M13 probes without purification from the template. The dideoxy sequencing ladders used for sizing the protected DNA fragments are obtained from the same M13 templates utilized to synthesize the DNA probes. The method was tested by analyzing the transcripts controlled by lac, ptr and trxA promoters. Comparison of the results with previously published data obtained with the conventional S1 nuclease mapping technique indicated that the present method is just as precise and at least 50 times more sensitive. Clones constructed for sequencing a gene of interest can be used directly to identify transcriptional start points.
Subject(s)
Coliphages/genetics , DNA, Viral/genetics , Escherichia coli/genetics , Nucleic Acid Hybridization/drug effects , Transcription, Genetic , Trichloroacetic Acid/pharmacology , Base Sequence , DNA, Viral/drug effects , Molecular Sequence Data , Nucleotide Mapping , Promoter Regions, Genetic , Sulfur RadioisotopesABSTRACT
The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.
