The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

Biosynthesis of inositol in yeast. Primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the INO1 locus

A biochemical, molecular, and genetic analysis of the Saccharomyces cerevisiae INO1 gene and its product, L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been carried out. The sequence of the entire INO1 gene and surrounding regions has been determined. Computer analysis of the DNA sequence revealed four potential peptides. The largest open reading frame of 553 amino acids predicted a peptide with a molecular weight of 62,842. The amino acid composition and amino terminus of purified L-myo-inositol-1-phosphate synthase were chemically determined and compared to the amino acid composition and amino terminus of the protein predicted from the DNA sequence of the large open reading frame. This analysis established that the large open reading frame encodes L-myo-inositol-1-phosphate synthase. The largest of several small open reading frames adjacent to INO1 predicted a protein of 133 amino acids with a molecular weight of 15,182 and features which suggested that the encoded protein may be membrane-associated. A gene disruption was constructed at INO1 by eliminating a portion of the coding sequence and replacing it with another sequence. Strains carrying the gene disruption failed to express any protein cross-reactive to antibody directed against L-myo-inositol-1-phosphate synthase. Although auxotrophic for inositol, strains carrying the gene disruption were completely viable when supplemented with inositol. In a similar fashion, a gene disruption was constructed in the chromosomal locus of the 133-amino acid open reading frame. This mutation did not affect viability but did cause inositol to be excreted from the cell.     

Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope.

A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.     

Structure of rodent helix-destabilizing protein revealed by cDNA cloning

A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively.     

Structure and evolution of the repetitive gene encoding streptococcal protein G

The complete sequence of the structural gene encoding the immunoglobulin G binding protein from Streptococcus G148 has been determined, as well as its 5' and 3' flanking sequences. The sequence reveals an open reading frame encoding a putative preprotein with a relative molecular mass of 63294. N-Terminal sequencing of the mature protein, spontaneously released from streptococcal cells, demonstrates that the signal peptide consists of 33 amino acids. The DNA sequence reveals extensive internal homologies similar to other cell-wall-bound receptors from gram-positive bacteria. Comparisons with a related gene previously isolated from another strain of streptococci revealed large differences in size, due to variations in the number of internal repeats. The structure of the gene suggests an evolution through multiple duplications.     

Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor.

The nucleotide sequence of black beetle virion (BBV) RNA2 has been determined. RNA2 is 1399 b long. Its 5' terminus is capped. Its 3' terminus has an unidentified moiety that renders the RNA resistent to polyadenylation and ligation. The first AUG codon at base 23 is followed by an open reading frame for a protein 407 amino acids long, the predicted size of coat protein precursor. A second open reading frame for a putative protein 72 amino acid residues long begins at base 1110. No other large open reading frames exist. The 5' half of the RNA can be folded into a long, imperfect hairpin of high predicted stability. The 3' half of the RNA can fold into a complex set of multiply bifurcated stem and loop regions.     

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.     

Identification of hormonogenic domains in the carboxyl terminal region of bovine thyroglobulin

A segment of 986 nucleotides corresponding to the 3' end of the 8.5 kb bovine thyroglobulin (Tg) mRNA has been sequenced. An open reading frame of 302 codons was found, ending with TGA and preceding an 80 nucleotide long 3' untranslated sequence. The encoded protein sequence provided the first data on the carboxyl terminal portion of Tg. Lysine was identified as the last residue. Comparison of the amino acid sequence with that of peptides known to contain thyroid hormones in the mature protein, lead to the identification of three regions involved in thyroid hormone formation. Two closely linked thyroxine- forming sites were found 182 and 196 amino acids from the carboxyl terminus respectively. The antepenultimate amino acid of the protein corresponded to the recently described triiodothyronine-forming site. Together with the previous localization of the main thyroxine-containing peptide at the amino terminus, the present results provide a map of all hormonogenic sites identified to date in Tg.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

A novel,anchorless streptococcal surface protein that binds to human immunoglobulins

We have characterized a novel surface protein from urea extract of whole cells of group A Streptococcus pyogenes (GAS). A major protein band (35kD) was found to hybridize with human IgG by Western blotting. A search of the N-terminal amino acid sequence of this protein by using the GAS genome sequence database revealed an open reading frame that encoded a 38-kDa protein with a signal peptide sequence. We have named this protein streptococcal immunoglobulin-binding protein 35 (Sib35). It was found to be an anchorless protein with no LPXTG motif, distinct from the M protein superfamily exhibiting immunoglobulin-binding activity, and partially secreted in the culture supernatant. Recombinant Sib35 was also shown to bind human IgA and IgM. The sib35 gene was found in all GAS strains examined, but not in oral, group B, C, or G streptococcal strains. These results suggest that Sib35 is a unique immunoglobulin-binding protein in GAS.     

Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein.

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.