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M N Hayashi

Publications and source records attributed to M N Hayashi.

12 recordsLinked to original sources
PubMed1989

mRNA stabilizing signals encoded in the genome of the bacteriophage phi x174.

In Escherichia coli cells infected with bacteriophage phi x174, mRNAs initiated by promoters PB and PD terminate after genes J, F, G, or H (TJ, TF, TG, or TH). These RNAs are relatively stable and contain mRNA-stabilizing signals at their 3' ends. These signals were cloned after gene D of phi x174 in an expression vector plasmid. The cloned signals stabilize mRNA of the upstream gene D and the stabilized mRNA is translationally functional. When these signals are inserted in reverse, no stabilizing effect on mRNA is observed indicating that the correct sequences at the 3' ends of transcripts determine their stability. When a stabilizing signal (+) and a mutated stabilizing signal (-) which has reduced stabilizing activity are tandemly inserted after gene D, two sets of 3' termini of the transcript are observed indicating that both signals also function as terminators. The amount of gpD synthesized from these constructs varies depending upon the relative positions of the (+) or (-) signals after gene D. The stabilizing function seems to act by preventing mRNA degradation from the 3' to 5' direction. Several common features of these stabilizers are described.

Bacteriophage phi X 174↗
PubMed1985

Cloned DNA sequences that determine mRNA stability of bacteriophage phi X174 in vivo are functional.

The stability of two species of phi X174 polycistronic mRNA in vivo can be altered by mutating sequences existing immediately upstream of a termination site. The wild type phage contains an mRNA stabilizing sequence ((+) sequence), while the same sequence mutated by insertion ((-) sequence) reduces the stability of the mRNAs. These two sequences were cloned at the 3' ends of gene D or gene B of phi X174 in a pBR322 derivative plasmid. The cloned sequences were functional. The (+) sequence stabilized gene B or gene D mRNA; half-lives of these mRNAs were 7 to 8 min. When the (+) sequence is eliminated ((o) sequence) or replaced with the (-) sequence, the half-lives of the mRNA were reduced to about 1 to 2 min. The stabilization of mRNAs caused an increased production of these proteins.

Bacteriophage phi X 174↗
PubMed1983

Role for the J-F intercistronic region of bacteriophages phi X174 and G4 in stability of mRNA.

A hairpin-like secondary structure in the intercistronic region between genes J and F of bacteriophages, phi X174 and G4 has been postulated to act as a transcription termination signal. We analyzed the in vivo transcripts of both phages and mutants derived from them with modifications of this hairpin structure. The phi X174 mutants appeared to fall into two groups with respect to the stability of two mRNA species. Class 1 mutants showed an mRNA profile very similar to the parental strain, whereas class 2 mutants lacked two major mRNA species normally terminated near the J-F region. The G4 mutants behaved like class 2 mutants of phi X174. Analysis of the stability of phi X174 mRNA revealed that messages specific for the genes upstream of the hairpin turn over more rapidly in class 2 mutants than in class 1 mutants. In class 1 mutants, the mRNA decay rates are similar but not identical to those of the wild-type strain. These data suggest a role for the nucleotide sequence within the J-F intercistronic region in mRNA degradation. They further imply that transcription termination occurs downstream from this site.

Bacteriophage phi X 174↗
PubMed1981

Bacteriophage phi X174-specific mRNA synthesis in cells deficient in termination factor rho activity.

A previous report (Hayashi et al., Proc. Natl. Acad. Sci. U.S.A. 73:3519-3523, 1976) indicated that in vivo bacteriophage phi X174 mRNA's terminate after genes J, F, G, and H. However, termination at these sites is not stringent. To determine whether termination of phi X174 transcription depends on rho factor activity, we introduced a temperature-sensitive rho mutation (nitA) into a phi X174-sensitive host cell line and determined termination sites in wild-type and nitA cells. We found that (i) normal phi X174 terminators were recognized in phi X174-infected nitA cells, (ii) the rho mutation relieved polar effects caused by nonsense mutations in the phage genome or by chloramphenicol treatment of the host cells, and (iii) polarity was not caused by premature termination of transcription at the site of the polar mutation. RNA synthesis continued beyond the site to the first rho-sensitive site.

Bacteriophage phi X 174↗
PubMed1974

Fragment maps of phiX-174 replicative DNA produced by restriction enzymes from haemophilus aphirophilus and haemophilus influenzae H-I.

phiX-174 replicative form (RF) DNA was cleaved by restriction enzymes from Haemophilus aphirophilus (Hap) and H. influenzae (H-I strain Osaka) (HinH). Five fragments were produced by Hap and eight by HinH. The positions of all of the Hap fragments and six of the HinH fragments were mapped on the genome by heteroduplex transfection and DNA-DNA hybridization methods. In addition, fragment sizes of phiX-174 RF DNA and S13 RF DNA by Hap and HinH were compared. Considerable differences of the size of the fragments produced from these closely related phage genomes were observed.

Chromosome Mapping↗
PubMed1972

Isolation of phi X174 specific messenger ribonucleic acids in vivo and identification of their 5' terminal nucleotides.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hybridization in formamide at low temperature was applied to hybridization of PhiX174 replicative form DNA and in vivo PhiX174 specific messenger RNA (mRNA) with some modification. We found that PhiX174 mRNA up to molecular weight 1.2 x 10(6) could be hybridized to and eluted from DNA without apparent breakage of phosphodiester bonds and the 5' terminal guanosine triphosphate and adenosine triphosphate of the RNA. By alkali hydrolysis of the purified in vivo PhiX174 mRNA and subsequent thin-layer chromatography of the digest, we isolated the 5' terminal nucleotides and identified them as 2'- or 3'-monophosphate guanosine 5'-triphosphate (pppGp) and 2'- or 3'-monophosphate adenosine 5'-triphosphate (pppAp). By comparing the in vitro and in vivo synthesized PhiX174 mRNA, a difference in the pppAp-pppGp ratio was observed. In the in vitro RNA, this ratio was 1.5, whereas in the in vivo RNA it was 5.5.

Adenosine Monophosphate↗