Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the alpha 47 gene.

The five alpha genes, alpha 0, alpha 4, alpha 22, alpha 27, and alpha 47, are the first set of herpes simplex virus 1 genes to be transcribed and expressed in productively infected cells. We report here the construction of a viral recombinant from which all of the coding sequences of the alpha 47 gene were deleted. In addition to the alpha 47 protein, infected cell lysates did not contain detectable amounts of two polypeptide bands with apparent molecular weights of 18,000 and 21,000 which could be specified by a gene whose regulatory domain and 5' transcribed noncoding sequences overlap with the coding sequences of the alpha 47 gene. The alpha 47- virus grew as well as the wild-type parent virus in Vero, baby hamster kidney, and Rat-1 cell lines.     

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.     

A phenylalanine tRNA gene from Neurospora crassa: conservation of secondary structure involving an intervening sequence.

We have isolated and sequenced a tRNAPhe gene from Neurospora crassa. Hybridization analyses suggest that trnaPhe is the only tRNA encoded on the cloned 5 kb DNA fragment. The tRNAPhe gene contains an intervening sequence 16 nucleotides in length located one nucleotide 3' to the anticodon position. The tRNAPhe coding region of Neurospora and yeast are 91% conserved, whereas their intervening sequences are only 50% identical. The pattern of sequence conservation is consistent with a proposed secondary structure for the tRNA precursor in which the anticodon is base paired with the middle of the intervening sequence and the splice points are located in adjacent single-stranded loops. The DNA sequence following the tRNAPhe coding region is similar to sequences following other genes transcribed by RNA polymerase III in that it is AT-rich and includes a tract of A residues in the coding strand. In contrast, the sequence preceding the Neurospora tRNAPhe coding region does not resemble sequences preceding other sequenced tRNA genes.     

Homology between two EBV early genes and HSV ribonucleotide reductase and 38K genes.

Computer-matching of amino acid sequences predicted from the complete EBV DNA sequence against the known HSV gene sequences has revealed significant homology between two EBV reading frames and the HSV1 and HSV2 140K and 38K proteins which are associated with ribonucleotide reductase activity. The two genes are arranged tandemly as in HSV though it appears that, unlike HSV, the two mRNAs are not 3' co-terminal. We have mapped two promoters predicted from the DNA sequence for these genes and shown them to be transcribed at a similar stage in the virus life cycle to that of the HSV genes.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family

Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences.      

The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences.

Ubiquitin coding sequences were isolated from a human genomic library and two cDNA libraries. One human ubiquitin gene consists of 2055 nucleotides and codes for a polyprotein consisting of 685 amino acid residues. The polyprotein contains nine direct repeats of the ubiquitin amino acid sequence and the last ubiquitin sequence is extended with an additional valyl residue at the C-terminal end. No spacer sequences separate the ubiquitin repeats and the coding regions are not interrupted by intervening sequences. This particular gene is transcribed since cDNAs corresponding to the genomic sequence have been isolated. At least two more types of ubiquitin genes are encoded in the human genome, one coding for an ubiquitin monomer while another presumably codes for three or four direct repeats of the ubiquitin sequence. Human DNA contains many copies of the ubiquitin sequence. Ubiquitin is therefore encoded in the human genome as a multigene family.     

Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus

The genome of avian erythroblastosis virus contains two independently expressed genetic loci (v-erbA and v-erbB) whose activities are probably responsible for oncogenesis by the virus. Both loci are closely related to nucleotide sequences found in the DNA and RNA of chickens and other vertebrates. We have isolated and characterized chicken DNA homologous to v-erbA and v-erbB. The two viral genes are represented by separate domains within chicken DNA (c-erbA and c-erbB), which are separated by a minimum of 12 kilobases (kb) of DNA and may not be linked at all. The nucleotide sequences shared by the viral and cellular erb loci are colinear, but the cellular loci are interrupted by multiple intervening sequences of various lengths. Polyribosomes prepared from normal chicken embryos contain two polyadenylated RNAs transcribed from c-erbA and two transcribed from c-erbB. The evident coding regions of these RNAs represent an unusually small fraction of the lengths of the RNAs, as if the 3′ untranslated domains of the RNAs might be exceptionally large (3–11 kb). These findings indicate that the c-erb loci are normal vertebrate genes rather than genes of cryptic endogenous retroviruses, and that they may have a role in the metabolism of normal cells. It appears that the viral erb genes, like most other retrovirus oncogenes, have been copied from cellular genes. In the viral genome, the two genes are devoid of introns, but they remain independently expressed loci, and they remain colinear with the coding domains of their cellular progenitors.     

DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus.

We have determined the sequence of herpes simplex virus type 1 DNA around the previously mapped location of sequences encoding an epitope of glycoprotein gH, and have deduced the structure of the gH gene and the amino acid sequence of gH. The unprocessed polypeptide is predicted to contain 838 amino acids, and to possess an N-terminal signal sequence and a C-terminal transmembrane sequence. Temperature-sensitive mutant tsQ26 maps within the predicted gH coding sequence. Homologous genes were identified in the genomes of two other herpesviruses, namely varicella-zoster virus and Epstein-Barr virus.     

Structure analysis at the ends of the intervening DNA sequences in the chloroplast 23S ribosomal genes of C. reinhardii

All of the chloroplast 23S ribosomal genes of C. reinhardii are interrupted by a 0.87 kb sequence (Rochaix and Malnoë, 1978). We have sequenced the DNA across the two ends of this intervening element. In parallel, we have examined the nucleotide sequences in the corresponding part of the 23S ribosomal RNA. This allowed us to locate precisely the boundaries between the coding (that is, transcribed into mature 23S rRNA) and the noncoding DNA. The results show that the intervening sequence is flanked by two identical sets of 3 bp (5′-CGT) oriented as direct repeats. In addition, a sequence of 5 bp (5′-CGTGA) lies exactly next to one end and is found very close (16 bp) to the other end, in the coding part of the gene. These two sets are also oriented as direct repeats. Finally, sequences near one end of the intervening element are found with a few alterations near the other end, but in an inverted orientation. Possible interpretations of these results are discussed.     

Sequence determination and genetic content of the short unique region in the genome of herpes simplex virus type 1

We have determined the complete DNA sequence of the short unique region in the genome of herpes simplex virus type 1, strain 17, and have interpreted it in terms of messenger RNAs and encoded proteins. The sequence contains variable regions whose length differs between DNA clones. The clones used for most of the analysis gave a short unique length of 12,979 base-pairs. We consider that this region contains 12 genes, which are expressed by mRNAs which have separate promoters, but may share 3'-termination sites, so that all but two mRNAs belong to one of four 3'-coterminal "families": 79% of the sequence is considered to be polypeptide coding. One pair of genes has an extensive out-of-frame overlap of coding sequences. The proteins encoded in the short unique region include two immediate-early species, two virion surface glycoproteins, and a DNA-binding species. Six of the genes have little or no previous characterization. From the nature of the amino acid sequences predicted for their encoded proteins, we deduce that several of these proteins may be membrane-associated.