Biomedical subjects

C G Miyada

Publications and source records attributed to C G Miyada.

24 records · Page 2Linked to original sources

PubMed1983

Nucleotide sequence for the catalytic domain of colicin E3 and its immunity protein. Evidence for a third gene overlapping colicin.

We have determined the nucleotide sequence of a segment of Co1E3 DNA coding for the carboxyl-terminal, catalytic peptide of colicin E3 and for the immunity protein. The end of the colicin E3 gene is separated from the beginning of the immunity gene by a nine-basepair intercistronic region, suggesting the two genes are expressed as a single transcriptional unit. The immunity gene is expressed, however, in E. coli strains containing Co1E3-pBR322 hybrid plasmids deleted for the 5'-end of the colicin gene. The DNA sequence also contains an unexpected open reading frame (ORF). This ORF is contained within the colicin gene and is in the +1 reading frame with respect to that gene. Plasmids containing the ORF, directed the synthesis of an 11 kilodalton protein in a cell-free, transcription-translation system.

Amino Acid Sequence↗

PubMed1983

Five mutations in the promoter region of the araBAD operon of Escherichia coli B/r.

Five mutations that result in reduced expression of the araBAD operon were cloned onto the plasmid pBR322. The position of each mutation was determined by DNA sequence analysis. Three of the mutations were located in the RNA polymerase binding site of the araBAD promoter. The first, ara-1016, was a one-base-pair deletion at position -35; the second, ara-1036, was a transversion at position -13; the third, ara-1027, was a nine-base-pair deletion from +5 to +13. S1 nuclease mapping showed that mutations ara-1016 and ara-1036 greatly reduced transcription and that mutation ara-1027 had little, if any, effect on transcription. Two other mutations resulted from the transposition of the insertion element, IS1, downstream from the transcriptional start site of the operon. Molecular mechanisms for all of the mutations are discussed.

Arabinose↗

PubMed1982

The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r.

Two oligodeoxyribonucleotides were chemically synthesized and used to specifically mutate the regulatory region of the araBAD operon in Escherichia coli B/r. One oligodeoxyribonucleotide introduced a 3-bp deletion in the araC activator binding site, the other a 3-bp deletion in the CRP-cAMP binding site. The mutations were introduced onto an ara insert cloned in an M13 vector using the synthetic oligodeoxyribonucleotides as primers and the (+) strand of an M13 mp2::ara hybrid phage as a template in an in vitro polymerization reaction. Hybridizations using the original synthetic oligodeoxyribonucleotide as a radioactive probe identified phage containing the desired deletion. The mutant ara inserts were subcloned into a stable plasmid for functional analysis. Transcription studies performed on strains containing the mutant ara plasmids demonstrated that both mutations reduced the amount of araBA mRNA synthesized in the presence of L-arabinose.

Arabinose↗

PubMed1980

DNA sequence of the araC regulatory gene from Escherichia coli B/r.

The DNA sequence of the araC regulatory gene from Escherichia coli B/r has been determined by the base-specific chemical cleavage reactions of Maxam and Gilbert. An open reading frame is found which codes for a protein of 292 amino acids. A nonsense mutation, araC5, is shown to result from a G to A transition at nucleotide 429 converting the tryptophan codon TGG to the amber codon TAG. A deletion which does not recombine with any known point mutation in araC, delta(araCO)719, removes all but the last 22 codons of the gene.

Amino Acid Sequence↗

PubMed1980

The araC regulatory gene mRNA contains a leader sequence.

An estimation of the size of the araC gene in Escherichila coli B/r was made by sub-cloning restriction fragments of the araC-containing hybrid plasmid pTB1 into the plasmid pBR322. Plasmids which contained a functional araC gene were identified by genetic complementation tests. DNA sequence analysis of the promoter-proximal region of the araC gene revealed that araC mRNA contains a 150 nucleotide leader.

Arabinose↗

PubMed1980

Construction of pBR322-ara hybrid plasmids by in vivo recombination.

In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analyses demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%--3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araC mutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.

Arabinose↗