cDNA clones that represent various portions of the coronavirus mouse hepatitis virus strain A59 genome RNA have been constructed. cDNAs were synthesized by transcription of genome RNA by using either oligo(dT)12.18 or random oligomers of calf thymus DNA as primers. These cDNAs were converted into double-stranded DNA and cloned into pBR322 by standard techniques. The resulting cloned viral DNA fragments were mapped to viral genes by hybridization with Northern blots of intracellular RNA from mouse hepatitis virus strain A59-infected cells. These cDNA clones map in six of the seven viral genes. Clone g344, 1.8 kilobases, is the largest and encompasses gene 5 (which encodes a nonstructural protein) and gene 6 (which encodes the El viral glycoprotein) as well as the intergenic regions preceding genes 5, 6, and 7. Sequencing of parts of this cloned DNA show that these three intergenic regions contain a common 11-nucleotide sequence. This sequence shares homology with the 3' end of the viral mRNA leader sequence. Thus, this common intergenic sequence may contain a binding site for a leader RNA that hybridizes to negative-strand viral RNA at the beginning of each gene to prime mRNA synthesis. The different degrees of homology between the leader and its putative binding site may influence the differential rates of transcription of the various viral mRNAs.
The complete nucleotide sequence of nonstructural gene 5 of coronavirus mouse hepatitis virus (MHV) strain A59 has been determined. This sequence contains two potential open reading frames which overlap by five nucleotides. The putative protein products predicted from the sequence are a basic 13,000-Da polypeptide and a 9600-Da polypeptide containing an unusually long hydrophobic amino terminus. RNAs transcribed in vitro from DNAs containing each of the open reading frames in pGEM vectors direct the synthesis in vitro of polypeptides of the sizes predicted by the sequence. An RNA transcript containing both of the open reading frames directs the synthesis primarily of the polypeptide corresponding to the downstream open reading frame. These data suggest that MHV-A59 messenger RNA 5 potentially encodes two proteins and may be preferentially translated from an internal AUG initiation codon.
Mouse hepatitis virus (MHV) gene 5 contains two open reading frames. We have expressed the second open reading frame of this gene (gene 5 ORF 2) in an Escherichia coli expression system. This system utilized a plasmid which contained the promoter and the first 36 codons of the recA gene fused in frame with the MHV gene 5 ORF 2, which is fused in turn to the beta-galactosidase gene. The protein product of this gene fusion was used to raise antibody to gene 5 ORF 2. The specificity of the antibody was verified by immunoprecipitation of the in vitro transcribed and translated protein product of gene 5 ORF 2. The second reading frame of MHV gene 5 was shown to be expressed during the course of infection by immunocytochemistry and radioimmunoprecipitation using the antibody raised against the E. coli fusion protein and by two-dimensional gel electrophoresis.
The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 508 ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein Lll. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount ofN-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with eitherN-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction ofN-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.
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