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Biomedical subjects

D W Mount

Publications and source records attributed to D W Mount.

At least 55 records · Page 3Linked to original sources
PubMed1984

Microcomputer programs for back translation of protein to DNA sequences and analysis of ambiguous DNA sequences.

Three computer programs are described which may be used to translate a DNA sequence into a protein sequence, back translate the protein sequence into an ambiguous DNA sequence, and then do pattern searching in the ambiguous sequence. The programs are written in the C programming language, have been compiled to run on a microcomputer under the CP/M 80 operating system, and may be copied in binary format through a modem. They are also to become available for the IBM/PC.

Amino Acid Sequence↗
PubMed1984

Creating new restriction sites by silent changes in coding sequences.

We present methods for identifying a useful type of DNA site--one that can be mutated to create a new restriction site within a coding region without changing the amino acid sequence. These "latent sites" are abundant--silent mutations creating one of 44 different 6-bp or 8-bp recognition sites were found at relatively high density, roughly one latent site per 9 bp, in the eleven genes tested. Our analysis suggests that site-directed mutagenesis can be used to refashion coding sequences at will for flexible analysis.

Base Sequence↗
PubMed1984

Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13.

A derivative of bacteriophage M13mp8 , designated M13mp8 /P, was prepared in which the promoter and NH2-terminal codons of bacterial genes may be fused to a portion of beta-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes and to determine the mutational changes by dideoxy sequence analysis. The feasibility of the method was demonstrated by isolation of a large number of mutations in the regulatory regions of two genes, lexA and recA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. Our results suggest that this approach will be generally useful for mutational analysis of transcriptional and translational regulatory elements.

Bacterial Proteins↗
PubMed1982

Microcomputer programs for DNA sequence analysis.

Computer programs are described which allow (a) analysis of DNA sequences to be performed on a laboratory microcomputer or (b) transfer of DNA sequences between a laboratory microcomputer and another computer system, such as a DNA library. The sequence analysis programs are interactive, do not require prior experience with computers and in many other respects resemble programs which have been written for larger computer systems (1-7). The user enters sequence data into a text file, accesses this file with the programs, and is then able to (a) search for restriction enzyme sites or other specified sequences, (b) translate in one or more reading frames in one or both directions in order to find open reading frames, or (c) determine codon usage in the sequence in one or more given reading frames. The results are given in table format and a restriction map is generated. The modem program permits collection of large amounts of data from a sequence library into a permanent file on the microcomputer disc system, or transfer of laboratory data in the reverse direction to a remote computer system.

Base Sequence↗
PubMed1982

Cell survival, UV-reactivation and induction of prophage lambda in Escherichia coli K12 overproducing RecA protein.

The effect of the cellular level of RecA protein on the ability of E. coli K12 bacteria to (i) survive UV-irradiation (ii) promote UV-reactivation of UV-damaged phage lambda (iii) induce prophage lambda was determined in bacterial mutants with discrete increasing levels of RecA protein. The various levels of RecA protein were obtained by combining lexA and recA alleles. Except for the double mutant lexA3 recAo98, whose repair ability was 25% less than that observed in wild type bacteria, bacterial survival was proportional to the level of RecA protein measured after 90 min of incubation. In lexA3 recAo98 bacteria, RecA protein, at a constitutive high basal level, failed to compensate totally for the lack of LexA repressor cleavage; UV-reactivation of UV-damaged phage lambda was not restored; yet, prophage lambda was induced with 35% efficiency. Efficient UV-induction of prophage lambda is linked to the induction of lexA-controlled host processes that repair the UV-damaged prophage.

Bacterial Proteins↗
PubMed1982

Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K-12.

The recA gene of E. coli is regulated by a specific repressor, the lexA protein, which binds to an operator in the recA regulatory region. We describe in this paper the isolation and characterization of a mutant thought to carry an operator-constitutive mutation in the recA gene. This mutation has the following properties: 1) It partially suppresses the UV sensitivity of lexA- strains. 2) It maps near the recA gene. 3) It allows constitutive high-level synthesis of recA protein in both lexA- and lexA+ backgrounds. 4) It allows constitutive synthesis of the recA messenger RNA. 5) It is cis-acting. The mutation does not restore induced cellular mutagenesis in a lexA- background. The expression of induced repair and mutagenesis of UV irradiated phage lambda or the regulation of the lexA gene is not affected by the presence of the mutation in either a lexA+ or lexA- strain. These observations confirm other findings that high levels of recA protein synthesis per se is not sufficient for the expression of UV inducible functions and that the lexA protein represses other genes besides the recA gene.

Autoradiography↗
PubMed1981

Nucleotide sequence of the lexA gene of Escherichia coli K-12.

A number of E. coli genes exhibit increased expression when the cellular DNA is damaged. In undamaged cells, lexA repressor limits the extent of their transcription, whereas, in damaged cells, the repressor is cleaved by a cellular protease, the product of the recA gene. We have sequenced 943 base pairs of cloned E. coli DNA containing the lexA gene. A regulatory region has been identified, followed by a translational open reading frame which encodes a polypeptide of 202 amino acids with a molecular weight of 22,300. The protein contains a single alanyl-glycyl peptide near its middle. This peptide is also found in certain phage repressors which are cleaved by the recA protease and has been shown to be the site of cleavage in these repressors. We have determined the nucleotide sequence of a portion of the lexA3 gene, whose product is 100-fold less susceptible to recA protease than the wild type repressor. We report a single base change (G to A) which alters the unique alanine-glycine sequence to alanine-aspartic acid.

Amino Acid Sequence↗
PubMed1981

Purified lexA protein is a repressor of the recA and lexA genes.

Escherichia coli shows a pleiotropic response (the SOS response) to treatments that damage DNA or inhibit DNA replication. Previous evidence has suggested that the product of the lexA gene is involved in regulating the SOS response, perhaps as a repressor, and that it is sensitive to the recA protease. We show here that lexA protein is a repressor of at least two genes, recA and lexA. Purified protein bound specifically to the regulatory regions of the two genes, as judged by DNase I protection experiments, and it specifically inhibited in vitro transcription of both genes. The binding sites in recA and lexA were found to be about 20 base pairs (bp) and 40 bp long, respectively. The 40-bp sequence in lexA was composed of two adjacent 20-bp sequences, which had considerable homology to one another and to the corresponding recA sequence. These 20-bp sequences, which we term "SOS boxes," show considerable inverted repeat structure as well. These features suggest that each box represents a single repressor binding site. Finally, we found that purified lexA protein was a substrate for the recA protease in a reaction requiring ATP or an analogue, adenosine 5'-[gamma-thio]triphosphate, and denatured DNA.

Bacterial Proteins↗
PubMed1980

Cleavage of the Escherichia coli lexA protein by the recA protease.

The recA and lexA proteins of EScherichia coli are involved in a complex regulatory circuit that allows the expression of a diverse set of functions after DNA damage or inhibition of DNA replication. Exponentially growing cells contain a low level of recA protein, and genetic evidence suggests that lexA protein is involved in its regulation, perhaps as a simple repressor. Recent models for recA derepression after DNA damage have suggested that an early event in this process is the proteolytic cleavage of lexA protein, leading to high-level expression of recA. We present several lines of evidence that the specific protease activity of the recA protein, previously described with the lambda repressor as substrate, is capable of cleaving the wild-type lexA+ protein. First, lexA protein can be cleaved in vitro under the same conditions as prevously described for lambda repressor cleavage in a reaction requring both recA protease and ATP or an analogue, adenosine 5'-[lambda-thio]-triphosphate. Second, lexA protein can be observed in vivo as a physical entity after infection with lambda lexA+ transducing phage of host strains containing ittle or no active protease, but not in strains containing high levels of active protease. Finally, infection of host cells containing active protease with a lambda lexA+ transducing phage does not lead to repression of recA, but does so in cells lacking active protease. In all of these conditions the mutant lexA3 protein is largely resistant to inactivation or cleavage; this resistance can explain the dominant phenotype of lexA3 over lexA+. We discuss models for recA derepression and re-establishment of repression which propose that modulation of the protease activity of recA protein regulates both of these transitions.

Adenosine Triphosphatases↗
PubMed1979

Isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12.

We describe the isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12. These mutations, designated spr(Am), were isolated and characterized in a lexA tif sfi genetic background. They abolished the sensitivity of the strain to UV light and resulted in high rates of synthesis of recA protein. Phage lambda+ failed to lysogenize the strains as observed with similar strains carrying non-amber spr mutations described previously, thereby indicating a constitutive expression of the phage induction pathway. Introduction of an amber suppressor mutation into a strain bearing the spr(Am) mutation restored expression of the LexA mutant phenotype. We conclude that spr mutations either inactivate or prevent synthesis of the lexA gene product and that loss of this product results in constitutive expression of the E. coli induction system in the tif sfi genetic background.

Bacterial Proteins↗
PubMed1979

Distribution of cell lengths in cultures of a lexA mutant of Escherichia coli K-12.

Distributions of cell lengths in lexA+ and lexA mutant cultures during normal growth and under thymidine starvation conditions are presented. During normal growth lexA mutant cells were slightly shorter, on the average, than were lexA+ cells. lexA mutant cells were also shorter in comparison with lexA+ cells after a period of thymidine starvation. These results are consistent with the hypothesis that the lexA gene is involved in the coordination of cell division with DNA repair.

Cell Division↗