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Biomedical subjects

C Machida

Publications and source records attributed to C Machida.

26 records · Page 2Linked to original sources
PubMed1984

Insertion element IS1 encodes two structural genes required for its transposition.

The nucleotide sequence analysis of insertion element IS1 has shown that IS1 could have as many as six translational reading frames encoding possible proteins. In order to determine which reading frames are actual structural genes responsible for IS1-mediated recombination, we introduced base substitution mutations including nonsense mutations into all of the potential reading frames and examined the ability of these IS1 mutants to mediate cointegration between two plasmids. The results reveal that IS1 has two structural genes (termed insA and insB), which are required for plasmid cointegration mediated by IS1.

Base Sequence↗
PubMed1984

Both inverted repeat sequences located at the ends of IS1 provide promoter functions.

Escherichia coli RNA polymerase was found to bind specifically to restriction fragments containing either end of IS1. DNase I footprint analyses indicate that RNA polymerase protects approximately 70 base-pairs at each end of IS1, including the left or right terminal inverted repeat sequences in IS1 (termed insL or insR, respectively) as well as some non-IS1 sequence directly adjacent to each end of IS1. Analysis of transcripts from the left terminal region of IS1 shows that the insL sequence contains a promoter (named insPL), and that RNA synthesis initiates apparently at one in a stretch of five adenylate residues within insL and continues toward the interior region of IS1. Interestingly, most of the resulting transcripts contain polyuridylate residues (more than 5 U residues) at their 5'-ends. Analysis of transcripts from the right terminal region of IS1 indicates that the insR sequence also contains a promoter (named insPR). RNA synthesis initiates specifically at an adenylate residue within insR and continues toward the interior region of IS1, i.e. in the opposite direction to RNA synthesis initiating at insPL, which is present at the other end of IS1. We propose that insPL is used to make the messenger RNA for the IS1-encoded genes insA and insB, while insPR might be used to synthesize an anti-mRNA and thereby negatively regulate insPL.

Base Sequence↗
PubMed1983

Repression of cointegration ability of insertion element IS1 by transcriptional readthrough from flanking regions.

We describe a repression mechanism in which read-through messages transcribed from a gene into an IS1 sequence inhibit its ability to mediate plasmid cointegration. This mechanism was derived from the demonstration that removal of the promoter region of the chloramphenicol resistance gene in transposon Tn9, or introduction of a strong transcription terminator of phage T7 downstream of the chloramphenicol resistance gene, increases the cointegration ability of the downstream IS1 sequence when in a particular orientation. The cointegration ability of an IS1 sequence downstream of the chloramphenicol resistance gene but in an orientation opposite that of the above-mentioned IS1 sequence also can be repressed. Analysis of transcripts synthesized in vitro showed that the transcripts of the chloramphenicol resistance gene were read through into the IS1 sequence located downstream of the gene in either orientation. Repression of this type may be one mechanism that controls the rate of transposition of the IS1 element, which apparently does not encode a structural gene for repressor.

Chloramphenicol↗
PubMed1983

Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3.

We have identified the Escherichia coli RNA polymerase-binding sites and the transcription initiation sites in the transposon Tn3. Results from nitrocellulose filter-binding assays indicate that there are two regions within Tn3 capable of forming stable binary complexes with RNA polymerase. The two regions are a 208-bp region containing the N-terminal coding sequence of the transposase (tnpA) and repressor (tnpR) genes, and a 332-bp region containing the N-terminal coding sequence for the beta-lactamase (bla) gene. DNase I footprint analysis of the 208-bp and 332-bp fragments further defined an extended region of protection, approx. 110 bp long, located between the transposase and repressor coding regions, and an 80-bp region of protection near the N-terminal coding sequence of the beta-lactamase gene. In vitro transcription studies with fragments containing these protected regions allowed us to determine the precise transcription initiation sites for the transposase, repressor, and beta-lactamase mRNAs. The transposase and repressor mRNAs are transcribed divergently and their transcription initiation sites are separated by 80 bp. The -35 homology regions for the transposase and repressor promoters are separated by 10 bp and the -10 homology region of the transposase promoter is coincident with the recombination site (res) for the site-specific recombinase activity (resolvase) of the repressor protein, which is required for resolution of Tn3 cointegrates. We discuss the significance of this complex divergently transcribed promoter region with respect to regulation of Tn3 transposition and we propose a model for coordinated regulation of the tnpA and tnpR genes. We also compare the Tn3 tnpA-tnpR intercistronic region with that of the closely related transposon gamma delta.

Binding Sites↗
PubMed1983

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.

Animals↗
PubMed1982

A novel type of transposon generated by insertion element IS102 present in a pSC101 derivative.

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM103 with plasmid Col E1. The resulting cointegrate contains either of these pYM103 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS102 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.

Bacteriocin Plasmids↗
PubMed1982

Factors determining frequency of plasmid cointegration mediated by insertion sequence IS1.

We demonstrate that mutants with deletions at either end of the insertion sequence IS1 lose the ability to mediate cointegration of two plasmids, whereas mutants with deletions or an insertion within IS1 can mediate cointegration at a reduced frequency. These results, together with the nucleotide sequence analysis of the IS1 mutants, indicate that the two ends of IS1 (insL and insR) and two genes (insA and insB) that are encoded by IS1 are required for cointegration. Using a plasmid carrying two copies of IS1, we found that the individual IS1s mediate cointegration at different characteristic frequencies, and that each of two parts of plasmid DNA segments flanked by the two IS1s is a transposon, mediating plasmid cointegration at a unique frequency. When one IS1 was replaced with a mutant IS1, the remaining wild-type IS1 complemented the cointegration ability of the mutant IS1 as well as a resulting mutant transposon that was then flanked by a wild-type IS1 and a mutant IS1. The efficiency of this complementation reflected the characteristic ability of an individual IS1 present on the plasmid to promote cointegration. The results suggest that the IS1-encoded proteins are produced in different amounts, depending on the location of IS1 in the plasmid, and that these amounts determine the efficiency of complementation of the cointegration ability of a mutant IS1 as well as a mutant transposon. However, the location of an individual IS1 itself can also determine the frequency of cointegration in the presence of a given amount of the IS1 proteins. On the basis of the observation that the cointegration ability of a mutant IS1 is less efficiently complemented than is the ability of a mutant transposon, we also suggest that the IS1-encoded proteins can function in trans, but act preferentially on the IS1 or transposon sequence from which they are produced in promoting cointegration.

Codon↗