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J R Mao

Publications and source records attributed to J R Mao.

10 recordsLinked to original sources
PubMed1997

In vivo production of oligodeoxyribonucleotides of specific sequences: application to antisense DNA.

Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.

Animals↗
PubMed1997

msDNA-Ec48, the smallest multicopy single-stranded DNA from Escherichia coli.

Previously we have reported a novel bacterial reverse transcriptase (RT) from Escherichia coli ECOR58 strains in which the YXDD box was replaced with LVDD (J.-R. Mao, S. Inouye, and M. Inouye, Biochem. Biophys. Res. Commun. 227:489-493, 1996). Here we determined the structure of the multicopy single-stranded DNA (msDNA) produced by the RT. The msDNA was found to consist of a single-stranded DNA of 48 nucleotides in length, the shortest msDNA thus far identified from natural sources. The msDNA, the RT, and the retron are designated msDNA-Ec48, RT-Ec48, and retron-Ec48, respectively. On the basis of the structure of the msr gene, the RNA molecule of msDNA-Ec48 is predicted to be composed of 119 ribonucleotides; it is the longest RNA among the known msDNAs. Analysis of the DNA sequences flanking the retron indicates that retron-Ec48 is associated with a prophage related to phages P2 and P4.

Amino Acid Sequence↗
PubMed1996

Enhancement of frame-shift mutation by the overproduction of msDNA in Escherichia coli.

A minor population of wild Escherichia coli strains contain retroelements called retrons, which produce a peculiar satellite DNA, multicopy single-stranded DNA (msDNA). It has been reported that mismatched base pairs in the secondary structure formed in msDNA are mutagenic in E. coli[Maas et al.(1994) Mol.Microbiol. 14,437-441; Maas et al. (1996) Mol. Microbiol, 19, 505-509]. We reexamined this proposal by converting mismatched base pairs to matched base pairs using a single msDNA species, msDNA-Ec86, or by deleting mismatched regions using msDNA-Ec73. We also examined the effect of reverse transcriptases (RT) without msDNA production on mutagenesis. All the constructs are under the lpp/lac promoter-operator control so that their mutagenic effects can be tested in the absence and the presence of a lac inducer. It was found that when the production of msDNA-Ec86 or Ec73 was induced, reversion frequencies from Lac- to Lac+ significantly increased in the case of a Lac- mutation caused by a frame-shift mutation, but much less by a substitution mutation. The removal of mismatched base pairs eliminated the high mutation frequencies, and the inducible expression of RT alone was not mutagenic. These results are consistent with the hypothesis of Maas and his associates that mismatched base pairs in msDNA sequester a cellular mismatch repair system, resulting in the increase of frame-shift mutations.

Base Composition↗
PubMed1996

An unusual bacterial reverse transcriptase having LVDD in the YXDD box from Escherichia coli.

A minor population of wild strains of Escherichia coli contains a gene for reverse transcriptase (RT) which is responsible for the synthesis of multicopy single-stranded DNA (msDNA), a branched DNA-RNA complex. A DNA fragment capable of synthesizing msDNA was cloned from strain ECOR-58, one of the 72 wild strains in the ECOR collection. The complete open reading frame of a novel reverse transcriptase, designated ECOR-58 RT, was identified. ECOR-58 RT consisted of 408 amino acid residues, and its 227-residue polymerase domain from residue 43 to 269 showed significant homologies to all the other bacterial RTs so far identified. Most significantly, its YXDD box, the most highly conserved sequence in all RTs from prokaryotes to eukaryotes, was found to be replaced with LVDD. ECOR-58 RT was found to be most distantly related in a phylogenetic tree to all 9 other bacterial RTs so far identified.

Amino Acid Sequence↗
PubMed1995

Gene regulation by antisense DNA produced in vivo.

Antisense technology has been widely used for regulating gene expression. Single-stranded RNA or DNA complementary to a target mRNA can inhibit the translation of the mRNA. Antisense RNA is produced in vivo, while antisense DNA is chemically synthesized as an oligonucleotide, which is extracellularly added to the cells. To maintain the effect of antisense DNA, a synthetic oligonucleotide has to be constantly added to the system. An advantage of antisense DNA over antisense RNA is that the target mRNA hybridized with the antisense DNA can be specifically digested by ribonuclease H. Here, we attempted to produce in vivo short single-stranded DNAs complementary to a specific mRNA. We demonstrate that such antisense oligodeoxyribonucleotide of a desired sequence can be produced in Escherichia coli using a retron, a bacterial retroelement, as a vector and that the antisense DNA thus produced in vivo can effectively inhibit the expression of a specific E. coli gene, such as the gene for the major outer membrane lipoprotein.

Bacterial Outer Membrane Proteins↗
PubMed1991

Regional changes in spinal cord glucose metabolism in a rat model of painful neuropathy.

Spinal cord patterns of metabolic activity in a model of neuropathic pain were assessed in unanesthetized rats by the [14C]-2-deoxyglucose (2-DG) technique. Rats used in this procedure had demonstrable thermal hyperalgesia ipsilateral to sciatic nerve ligation and ipsilateral hindpaws that were lifted in a guarded position. The latter indicated possible spontaneous pain. Sciatic nerve ligation produced significant increases in glucose utilization in the dorsal and ventral horns of both sides, with greater activity present on the ipsilateral as compared to the contralateral side. Peak activity was in laminae V-VI, a region involved in nociceptive processing. Thus, a chronic increase in neuronal activity in these regions may reflect spontaneous neuropathic pain.

Animals↗
PubMedDate not supplied

Changes of reactions of neurones in dorsal raphe nucleus and locus coeruleus to electroacupuncture by hypothalamic arcuate nucleus stimulation.

In this experiment the role of the hypothalamic arcuate nucleus (ARC) in acupuncture analgesia and its mechanisms were studied with behavioural and electrophysiological methods. After ARC stimulation the analgesic effect of acupuncture was enhanced significantly and the responses of neurones to electroacupuncture were increased in the dorsal raphe nucleus (DR) and reduced in the locus coeruleus (LC), which could be reversed by intraperitoneal injection of naloxone. The results indicate that ARC might participate in acupuncture analgesia via changing the responses of DR and LC neurones to electroacupuncture, a process in which opiate-like substances (probably beta-endorphin) are involved.

Action Potentials↗