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C Machida

Publications and source records attributed to C Machida.

18 recordsLinked to original sources
PubMed1995

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana.

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

Arabidopsis↗
PubMed1995

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs).

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.

Amino Acid Sequence↗
PubMed1995

DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target.

Transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid is transferred from Agrobacterium tumefaciens to plant cells and is stably integrated into the plant nuclear genome. By the inverse polymerase chain reaction DNA fragments were amplified that contained the T-DNA/plant DNA junctions from the total DNA of a transgenic tobacco plant that had a single copy of the T-DNA in a repetitive region of its genome. A DNA fragment containing the target site was amplified from the total DNA of non-transformed tobacco by the polymerase chain reaction using high-stringency conditions. Comparison of the nucleotide sequence of the target site with those of the T-DNA/plant DNA junctions revealed that various duplications of short stretches of nucleotide sequences around the target and in the incoming T-DNA had accompanied the integration of the T-DNA. A deletion of 16 bp at the target site was also found and the target site was similar, in terms of nucleotide sequence, to regions around the breakpoints of the T-DNA. This finding provides a clear example of the occurrence of complex rearrangements during the integration of T-DNA.

Agrobacterium tumefaciens↗
PubMed1991

Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

Base Sequence↗
PubMed1989

Regulation of IS1 transposition by the insA gene product.

The IS1 element contains two adjacent genes called insA and insB, both required for IS1 transposition and IS1-mediated plasmid cointegration. These two genes are transcribed polycistronically from the promoter in the left terminal inverted repeat of IS1 (insL). We constructed overexpression systems of these genes with the tac promoter, which are regulated by an exogenous inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG). Then we have examined, under various conditions of induction with IPTG, how overexpression of these genes affects IS1 transposition, using an assay based on plasmid cointegration. When the insA and insB genes were organized identically to the wild-type IS1 genes and simultaneously expressed using low concentrations of IPTG, activity of a mutant IS1 in cis was restored, but not in trans. Higher IPTG concentrations resulted in lower transposition activity. Expression in trans of insA and insB results in a 50 to 100-fold reduction of the frequency of cointegration mediated by wild-type IS1. Such a reduction is also observed when only the insA gene is overexpressed in trans. Overexpression of either mutant insA or insB does not affect the cointegration event. Tests with the insA-lacZ fusion gene showed that the InsA product inhibits the expression of IS1 genes directed by its own promoter in insL. These results suggest that the InsA product regulates IS1 transposition by inhibiting expression of IS1 transposition genes in addition to acting as part of a transposase complex.

Bacterial Proteins↗
PubMed1988

Right-hand border regions of octopine T-DNA are recognized by RNA polymerase of Agrobacterium as well as by VirD1 and VirD2 proteins.

The T-DNA of octopine Ti plasmid of Agrobacterium tumefaciens contains TL- and TR-DNA regions each bounded by 25 base-pair-repeats (designated A, B, C and D from left to right). Short DNA segments containing the borders B, C and D were found to function as promoter when placed in the rightward orientation upstream of promoter-less lacZ. Promoter consensus sequence of Agrobacterium were found within these border repeats and in their adjacent regions. The expression of lacZ was low when the segments contained the overdrive, a sequence known to enhance T-DNA transfer. Simultaneous overproduction of VirD1 and D2 proteins, endonuclease acting on the border repeats, interfered with the promoter functions of the border segments. In spite of their activity under these conditions, the border regions do not seem to be involved in the gene expression, because they are not followed by appropriate open reading frames. We propose that RNA polymerase of Agrobacterium competes with VirD products for T-DNA borders and thereby affects the transfer of T-DNA.

Arginine↗
PubMed1987

Base substitutions in transposable element IS1 cause DNA duplication of variable length at the target site for plasmid co-integration.

We demonstrate that base substitutions in the IS1 sequence affect the length of the nucleotide sequence which is duplicated during IS1-mediated co-integration. IS1K, an IS1 variant present in the Escherichia coli chromosome, has seven base substitutions in its sequence as compared with that of IS1R derived from the plasmid R100. All substitutions are located in the internal region of IS1K. We have constructed plasmids containing IS1R, IS1K and hybrids between them: one contains four base substitutions causing an amino acid substitution in the insA gene and the other has three substitutions producing an amino acid substitution in the insB gene. We have isolated co-integrate plasmids formed by each IS1 and analysed nucleotide sequences of the target sites duplicated at the co-integration junctions. The results show that IS1K generates duplications of 8 or 14 bp as well as 9 bp, while IS1R exclusively generates the 9-bp duplications. Both hybrid IS1s also create 8- or 7-bp target duplications in addition to 9-bp duplications. These results indicate that the base substitutions in either insA or insB are sufficient for the occurrence of unusual target duplications, suggesting that both genes are involved in the target duplication.

Base Sequence↗
PubMed1987

The next frontier in the molecular biology of the opioid system. The opioid receptors.

The analgesic and euphoric properties of some plant alkaloids such as morphine have been known and exploited for centuries. In contrast, only during the last twenty years have we begun to unravel the molecular basis by which opiates exert their effects, mechanisms important to our general understanding of the nervous system. The analgesic response to opiates is the result of a cascade of biochemical events that are triggered by the interaction of the opiate with specific macromolecular components found on the membranes of nervous system tissues, the opioid receptors. The endogenous ligands of these receptors are small peptides, the opioid peptides. Although much has been learned about the structures and the mode of synthesis of the opioid peptides, little is understood about the structure of their receptors. The application of molecular genetic techniques was of great importance to the studies of the opioid peptides. It is now expected that this same technology will unravel the physical mysteries of the opioid receptors.

Animals↗
PubMed1986

Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses.

We previously isolated spontaneous env gene mutants of Friend spleen focus-forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys-180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic-related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function.

Amino Acid Sequence↗
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↗