Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.     

Distinct cis-acting signals enhance 3'' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae.

The cyc1-512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3' untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1-512 mRNA contained many discrete 3' termini at certain sites, ranging from the wild-type position to over 2000 nucleotides (nt) downstream. The cyc1-512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base-pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG...TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3' endpoints, apparently identical to those seen in the cyc1-512 mRNA. However, several revertants contained minor end points not corresponding to any of the cyc1-512 mRNAs. The capacity of these three signals to form 3' ends was confirmed with sequences constructed by site-directed mutagenesis. We therefore suggest that the production of 3' termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3' mRNA termini, as represented by the cyc1-512 termini. The second type of element, which includes TAG...TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3' preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immediate-early mRNA-2 of herpes simplex viruses types 1 and 2 is unspliced: conserved sequences around the 5' and 3' termini correspond to transcription regulatory signals

Nuclease S1 and exonuclease VII analyses of immediate-early (IE) mRNA-2 of herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) show them to be unspliced and of similar length. The DNA sequences around the 5' and 3' termini have been determined. Comparison of the sequences around the 5' ends reveals several common features. (1) Four discrete blocks of upstream homology which are precisely colinear with respect to the 5' termini of the mRNAs; the blocks include the 'TATA' box, a G-C rich sequence and a sequence (AATTAAATACAT) which may be involved in the coordinate induction of the IE class of genes. (2) Several copies of the sequence CCCCGCCC, found in different upstream positions in HSV-1 and HSV-2, which may be important in the expression of a wide variety of eukaryotic genes. (3) Potential hairpin structures in the region of the 5' termini which are present at similar locations in HSV-1 and HSV-2. Sequence comparison around the 3' termini of IEmRNA-2 reveals high homology at the proposed C-terminus of the polypeptide.     

Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4.

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.     

The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase

The DNA sequence of the gene for the fermentative yeast alcohol dehydrogenase has been determined. The structural gene contains no introns. The amino acid sequence of the protein as determined from the nucleotide sequence disagrees with the published alcohol dehydrogenase isozyme I (ADH-I) sequence for 5 of the 347 amino acid residues. At least one, and perhaps as many as four, of these differences is probably due to ADH-I protein heterogeneity in different yeast strains and not to sequencing errors. S1 nuclease was used to map the 5' and 3' ends of the ADH-I mRNA. There are two discrete, mature 5' ends of the mRNA, mapping 27 and 37 nucleotides upstream of the translation initiating ATG. These two equally prevalent termini are 101 and 91 nucleotides, respectively, downstream from a TATAAA sequence. Analysis of the 3' end of ADH-I mRNA disclosed two minor ends upstream of the major poly(A) addition site. These three ends map 24, 67, and 83 nucleotides, respectively, downstream from the translation-terminating TAA triplet. The sequence AA-TAAG is found 28 to 34 nucleotides upstream of each ADH-I mRNA poly(A) addition site. Sequence comparisons of these three 3' ends with those for four other yeast mRNAs yielded a 13-nucleotide consensus sequence to which TAAATAAGA is central. All of the known yeast poly(A) addition sites map at or near the A residue of a CTA site 25 to 40 nucleotides downstream from this consensus octamer.     

DNA sequence of an immediate-early gene (IEmRNA-5) of herpes simplex virus type I

We describe a 2560 base pair herpes simplex virus type 1 (HSV-1) DNA sequence containing the entire immediate-early mRNA-5 (IEmRNA-5) gene. The 3' and 5' termini of IEmRNA-5 were mapped within this DNA sequence by single-strand specific endonuclease protection experiments. The IEmRNA-5 gene contains DNA sequences from both the unique (Us) and reiterated (TRs/IRs) regions of the HSV-1 DNA short component and is interrupted by a single intron mapping in TRs/IRs. A search of the transcribed DNA sequence revealed no initiator codon within TRs/IRs. The first ATG was located 6 bases into Us sequences and this reading frame (316 codons) was also observed in the 3' transcribed region. The oligonucleotide sequences adjacent to the IEmRNA-5 termini are discussed in relation to those of the HSV-1 thymidine kinase gene and other genes transcribed by RNA polymerase II.