Compilation and analysis of Escherichia coli promoter DNA sequences.

The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.     

Rigorous pattern-recognition methods for DNA sequences. Analysis of promoter sequences from Escherichia coli

The basic nature of the sequence features that define a promoter sequence for Escherichia coli RNA polymerase have been established by a variety of biochemical and genetic methods. We have developed rigorous analytical methods for finding unknown patterns that occur imperfectly in a set of several sequences, and have used them to examine a set of bacterial promoters. The algorithm easily discovers the "consensus" sequences for the -10 and -35 regions, which are essentially identical to the results of previous analyses, but requires no prior assumptions about the common patterns. By explicitly specifying the nature of the search for consensus sequences, we give a rigorous definition to this concept that should be widely applicable. We also have provided estimates for the statistical significance of common patterns discovered in sets of sequences. In addition to providing a rigorous basis for defining known consensus regions, we have found additional features in these promoters that may have functional significance. These added features were located on either side of the -35 region. The pattern 5', or upstream, from the -35 region was found using the standard alphabet (A, G, C and T), but the pattern between the -10 and the -35 regions was detectable only in a sub-alphabet. Recent results relating DNA sequence to helix conformation suggest that the former (upstream) pattern may have a functional significance. Possible roles in promoter function are discussed in this light, and an observation of altered promoter function involving the upstream region is reported that appears to support the suggestion of function in at least one case.     

Discriminant analysis of promoter regions in Escherichia coli sequences

We have previously developed a general method based on the statisticaltechnique of discriminant analysis to predict splice junctionsin eukaryotic mRNA sequences [Nakata, K., Kanehisa, M. and DeLisi,C. (1985) Nucleic Acids Res., 13, 5327–5340]. In orderto evaluate further applicability of this method, we now analyzethe promoter region of Escherichia coli sequences. The attributesused for discrimination include the accuracy of consensus sequencepatterns measured by the perceptron algorithm, the thermal stabilitymap, the base composition and the Calladine-Dickerson rulesfor helical twist angle, roll angle, torsion angle and propellertwist angle. When applied to selected E. coli sequences in theGenBank database, the method correctly identifies 75 % of thetrue promoter regions. Received on May 15, 1987; accepted on April 17, 1988     

The evolution of DNA sequences in Escherichia coli

It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.     

Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA

Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.     

The DNA sequences encoding plsB and dgk loci of Escherichia coli

We have determined the sequence of a 3865-base pair DNA segment from Escherichia coli containing plsB, the structural gene for the sn-glycerol-3-phosphate acyltransferase, and the dgk locus, believed to encode diglyceride kinase. The 806-amino acid sequence encoded within the longest open reading frame is in agreement with NH2-terminal sequences of the sn-glycerol-3-phosphate acyltransferase (Green, P., Vanaman, T. C., Modrich, P., and Bell, R. M. (1983) J. Biol. Chem. 258, 10862-10866), indicating that this is the structural gene for this protein. Furthermore, an open reading frame encoding a 122-residue polypeptide consistent with the size of diglyceride kinase has been identified and coincides with the position of dgk determined by deletion analysis.     

Expression of orthopoxvirus DNA sequences in Escherichia coli cells]

We observed the expression of recombinant plasmids genes containing ectromelia virus DNA fragments in E. coli minicells. Using plasmids with vaccinia or ectromelia viruses DNA fragments inserted upstream of lacZ gene we showed that certain orthopoxvirus genome fragments carry out a promoter-like function in bacterial cells.     

Molecular cloning ofTrichophyton mentagrophytes DNA sequences with promoter activity inEscherichia coli

A promoter probe library from the dermatophyte fungusTrichophyton mentagrophytes has been constructed in the pVB32 plasmid vector. Using this library, a set ofT. mentagrophytes DNA sequences with promoter activity inEscherichia coli has been cloned. The size and the resistance phenotype conferred by these DNA fragments varied. Southern blot analysis confirmed that they were derived fromT. mentagrophytes genomic DNA.     

DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.     

Compilation of DNA sequences of Escherichia coli (update 1993).

We have compiled the DNA sequence data for E. coli available from the GENBANK and EMBL data libraries and over a period of several years independently from the literature. This is the fifth listing replacing and increasing the former listings substantially. However, in order to save space this printed version contains DNA sequence information only, if they are publically available in electronic form. The complete compilation including a full set of genetic map data and the E. coli protein index can be obtained in machine readable form from the EMBL data library (ECD release 15) as a part of the CD-ROM issue of the EMBL sequence database, released and updated every three months. After deletion of all detected overlaps a total of 2,353,635 individual bp is found to be determined till the end of April 1993. This corresponds to a total of 49.87% of the entire E. coli chromosome consisting of about 4,720 kbp. This number may actually be higher by 9161 bp derived from other strains of E. coli.