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PubMed1990

Sequence conservation and divergence of hepatitis delta virus RNA.

The complete RNA sequence of the hepatitis delta virus (HDV) obtained from the Nauru Island in the Pacific was determined by cDNA cloning and amplification by polymerase chain reaction (PCR). The sequence showed 14-17% divergence from the two known HDV RNA sequences. There are three highly conserved domains: the region around the autocatalytic cleavage site of the genomic RNA (nucleotides 659 to 772), the region around the autocatalytic cleavage site of the antigenomic-sense RNA (nucleotides 847 to 966), and the region around the middle one-third domain of the open reading frame (ORF) encoding the hepatitis delta antigen on the antigenomic RNA (nucleotides 1267 to 1347). The two autocatalytic activities are required for the cleavage and ligation of HDV RNA during RNA replication. The third conserved domain codes for the RNA-binding domain of HDAg, which specifically interacts with HDV RNA. Three nucleotide changes within the genomic catalytic sequence are present but did not alter the catalytic cleavage activity of the HDV RNA. Microheterogeneity of the RNA sequences was also detected. One of these occurred within the coding region of the delta antigen, creating an amber termination codon in some of the RNA species. Thus, this HDV strain contains two different RNA species, one of which encodes a delta antigen of 214 amino acids and the other 195 amino acids. These two protein species were detected by immunoblotting of the patient's plasma. In contrast to other HDV strains, only three ORFs capable of encoding more than 100 amino acids each are present in this HDV RNA. We recommend that oligonucleotides complementary to the highly conserved sequences should be used as primers for PCR in clinical detection assays of hepatitis delta virus infection.

Adult↗

PubMed1991

Altered translation of the matrix genes in Niigata and Yamagata neurovirulent measles virus strains.

Niigata and Yamagata strains measles virus were isolated from subacute sclerosing panencephalitis patients. These viruses were defective in virion production and expression of matrix (M) protein. The Niigata M protein-coding frame was interrupted by an in-frame termination codon, whereas the Yamagata M gene lacked the normal translational initiation codon. These mutations prevented translation of a normal M protein. However, RNA derived from the cloned Niigata and Yamagata M genes was translatable in vitro into low levels of aberrant proteins that reacted with M-specific antiserum. These proteins were also translated from poly(A)+ RNA from cells infected by Niigata and Yamagata virus strains. The aberrant M protein of Niigata virus was initiated at a downstream AUG codon created by a second mutation. The Yamagata M gene produced two aberrant proteins: one initiated mainly in vitro at an ACG codon, and a second species initiated at a downstream site both in vitro and in vivo. These results define the abnormal translational functions of the Niigata and Yamagata M genes, and further implicate the involvement of M protein defects in chronic central nervous system infections by measles virus.

Animals↗

PubMed1991

Efficient in vitro translation and processing of the rubella virus structural proteins in the presence of microsomes.

In the structural protein open reading frame (SP-ORF) of rubella virus (RUB), the sequences for the three virion proteins occur in the order NH2-C-E2-E1-COOH with hydrophobic, consensus signal sequences preceding the amino termini for each of the two membrane proteins (T. K. Frey and L. D. Marr, 1988 Gene 62, 85-100). In vitro translation in the presence of microsomes of RNA transcripts from a plasmid containing the SP-ORF resulted in production and accurate processing of the three structural proteins. Since in the absence of microsomes the 110-kDa precursor of these proteins is produced, this finding indicated that the cleavage events in processing of the precursor were mediated by signalase. To study the C-E2 processing event, a DNA construct was made which contained the sequences for E2 beginning at the NH2 terminus of the hydrophobic consensus signal and extending through to the NH2 terminus of E1. In vitro translation of transcripts from this construct in the presence of microsomes resulted in accurate processing of E2 confirming that the hydrophobic sequence was a signal sequence and demonstrating it could function externally as well as internally within the 110-kDa precursor. To determine if the E2 signal was maintained on C after cleavage of the precursor by signalase, the SP-ORF plasmid was mutagenized to place translation termination codons at either the NH2 or COOH side of the E2 signal sequence such that C protein lacking or containing the E2 signal would be produced. As expected, the C-minus-signal protein migrated more rapidly in polyacrylamide gels than did the C-plus-signal protein. C translated from the SP-ORF construct as well as authentic C from infected cells comigrated with the C-plus-signal protein, indicating that the E2 signal was not removed. In a corollary study, it was found that RUB C protein was phosphorylated in vivo, although the percentage of the protein phosphorylated was not determined.

Animals↗

PubMed1991

Mutation and replacement of the 16-kDa protein gene in RNA-1 of tobacco rattle virus.

The function of the 16-kDa protein encoded by tobacco rattle virus (TRV) RNA-1 was investigated by a mutational analysis of the 16-kDa protein gene. Transcripts of TRV RNA-1 produced from a full-length cDNA clone of TRV RNA-1 (SYM strain) remained infectious when the 16-kDa protein gene was disrupted by premature termination codons and a deletion which removed 73% of the coding region. A deletion which included the intergenic region between the 29-kDa protein gene and the 16-kDa protein gene, the entire 16-kDa protein coding region, and 57% of the 3' noncoding region was not infectious. Transcripts in which the 16-kDa protein coding region was replaced by the tobacco mosaic virus (TMV) (L strain) coat protein gene were also infectious and expressed TMV coat protein in infected tissue. Inclusion of the TMV origin of assembly sequence in the chimaeric constructs resulted in the accumulation of TMV-like virus particles in infected tissue.

Blotting, Northern↗

PubMed1991

The 5' and 3' limits of transcription in the pseudorabies virus latency associated transcription unit.

While latent in sensory neurons of infected pigs, pseudorabies virus expresses transcripts from a limited genomic area. These RNAs are transcribed from the strand opposite to that which encodes the pseudorabies immediate-early protein. Using a combination of in situ nucleic acid hybridization performed on latently infected pig trigeminal ganglia and DNA sequencing, 5' and 3' limits of transcription for the pseudorabies LAT transcription unit have been defined. The 5' limit of transcription has been localized to a NarI-BamHI subfragment of the BamHI-6 fragment. Several promoter elements in the correct orientation for the transcript are present including consensus TATA and CAAT boxes and an SP1 site. The 3' limit of transcription has been localized to a HindIII-KpnI subfragment of the BamHI-5 fragment which contains a consensus polyadenylation signal and two termination codones in the correct orientation. From these results we conclude that the region of pseudorabies virus DNA which is active during latency can be no longer than 12.6 kb and completely overlaps the gene encoding the pseudorabies immediate-early protein.

Animals↗

PubMed1991

Identification of a binding site in the hepatitis B virus RNA pregenome for the viral Pol gene product.

The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with beta-galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome.

Binding Sites↗

PubMed1991

Adeno-associated viruses having nonsense mutations in the capsid genes: growth in mammalian cells containing an inducible amber suppressor.

When an adeno-associated virus (AAV) genome contained in a recombinant plasmid is transfected into adenovirus-infected cells, infectious AAV particles are efficiently generated. We previously described the construction of a conditional lethal mutant of AAV having an amber termination codon inserted in the rep gene. This mutant was propagated on a monkey kidney cell line (SupD12) having an inducible amber suppressor tRNAser. We now describe the construction and propagation of two additional conditional lethal mutants of AAV having amber codons affecting all three capsid proteins (AAV Capam) or only the VP1 capsid protein (AAV VP1am). Suppression of the amber mutations in the capsid proteins was demonstrated directly by immunoblot analysis. The efficiency of amber suppression on the SupD12 cell was about 6 to 10% for AAV VP1am and 4 to 5% for AAV Capam. The reversion frequency of either mutant was apparently less than 10(-5). On nonsuppressing cells AAV VP1am exhibited an Lip (Inf) phenotype, whereas AAV Capam exhibited a Cap phenotype.

Animals↗

PubMed1992

A possible role for nonsense suppression in the synthesis of a human cytomegalovirus 58-kDa virion protein.

A 1.6-kb late mRNA originating from the HindIII R fragment of human cytomegalovirus (HCMV) encodes a 58-kDa virion phosphoprotein. Data presented support the hypothesis that this protein may be synthesized via the translational readthrough of an opal termination codon separating two open reading frames located in tandem. To our knowledge this is the first report of nonsense suppression as a means of regulating gene expression in HCMV.

Amino Acid Sequence↗

PubMed1992

A symptom variant of beet curly top geminivirus produced by mutation of open reading frame C4.

Two beet curly top virus (BCTV) mutants have been constructed in vitro that contain G-to-T transversions either at nucleotide 2682 or at nucleotide 2802 within the overlapping open reading frames (ORFs) C1 and C4. The mutations introduce termination codons in ORF C4 without affecting the amino acids encoded by ORF C1. When agroinoculated into Nicotiana benthamiana the mutants caused stunting and yellowing of the plant and downward leaf curl but not the vein swelling and upward leaf curl symptoms that are characteristic of wild-type BCTV infection in this host. Levels of viral single- and double-stranded DNA forms were similar in mutant and wild-type infections. Symptoms induced by one such mutant in Nicotiana clevelandii and Datura stramonium were less severe than those in wild-type infections and were again qualitatively distinct. The mutants caused symptomless infections in Beta vulgaris, contrasting with stunting, severe leaf curl, and vein swelling symptoms associated with wild-type infection of this host. The levels of mutant DNA in newly expanding asymptomatic leaves frequently reached those of wild-type virus in leaves showing severe symptoms. The results suggest that ORF C4 encodes a protein that is a major determinant of pathogenesis that might affect the hyperplastic response of the host to BCTV infection.

Cloning, Molecular↗

PubMed1986

Bovine interstitial retinol-binding protein (IRBP)--isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA.

Three clones for b-IRBP were isolated by anti b-IRBP screening of two bovine retina libraries in the expression vector lambda gt11. The cDNA inserts were then used as hybridization probes to screen and isolate three more clones in a bovine retina library in the non-expression vector lambda gt10. The six overlapping clones generated a b-IRBP cDNA sequence of 3400 nucleotides. An open reading frame encoded the complete amino acid sequences of 8 of the 35 b-IRBP tryptic peptides purified in the present study. One tentative glycosylation site was identified. The coding region was followed by TAG translation terminating codon and an untranslated stretch of about 1700 nucleotides that ended in a sequence containing a presumptive AATAAA polyadenylation signal that was 18 nucleotides upstream from a 10 nucleotide oligo(A) tract. The coding region for b-IRBP would be expected to be 3300 bp long, but Northern blot hybridization experiments performed with bovine retina polyadenylated RNA and probes containing part of the coding region established that the mRNA for b-IRBP consisted of a major species of about 6300 bp, and a minor species of 5200 bp. In vitro translation of bovine retina polyadenylated RNA in a rabbit reticulocyte lysate system yielded an immunoreactive protein that was comparable in size with nonglycosylated, mature IRBP, showing that it is not synthesized from a large precursor, and supporting our finding that the mRNA contains an extensive non-coding region.

Amino Acid Sequence↗

PubMed1996

Molecular characterization of the 5.2 KB isoform of the human cyclooxygenase-1 transcript.

Several cDNA clones from human endothelial cells were isolated by expression cloning with the polyclonal antisera against the Ram seminal vesicle cyclooxygenase enzyme (Cox-1). One such clone produced a fusion protein that reacted with two other Cox-1 antiserum (Hla, T., Farrell, M.P., Kumar, A. and Bailey, J.M. (1986) Prostaglandins 32 (6) 829-845). The 2.5 kb cDNA insert was sequenced and contained 60 bp encoding the C-terminal end of the human Cox-1 polypeptide, followed by 2.3 kb of untranslated region. Northern blot analysis of human endothelial cells using the 2.5 kb cDNA insert detected the 2.8 and 5.2 kb Cox-1 transcripts. These data indicated that the isolated clone represented the 5.2 kb isoform of the human Cox-1 mRNA. The presence of the canonical polyadenylation site AAUAAA at 740 bp downstream from the translation termination codon suggests that alternative polyadenylation of the Cox-1 gene gives rise to 5.2 and 2.8 kb isoforms. The sequence of the 3'-UTR of the Cox-1 transcripts is highly divergent from that of the human Cox-2 transcript isoforms, suggesting a distinct function in the regulation of expression at the post-transcriptional and/or translational levels.

Amino Acid Sequence↗

PubMed1979

Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens.

The sequence of about one third of the polyoma virus genome is presented. This sequence covers the origin of replication of two large plaque strains (A2 and A3) of polyoma virus. The two strains differ by 11 bp in the origin region. A model for replication is suggested. The sequence probably also covers the entire coding region of two of the polyoma virus early proteins--small and middle T antigens--as well as part of the coding region for large T antigen. Over a small region of the DNA, all three coding frames contain termination codons, which argues a need for spliced early messenger RNAs. In another region of the DNA, two coding frames can be used. Correlation with protein data suggests that one frame codes for part of middle T antigen and the other for part of large T antigen.

Amino Acid Sequence↗

PubMed1980

The nucleotide sequence of a rabbit beta-globin pseudogene.

We report the nucleotide sequence of a rabbit beta-globin pseudogene, psi beta 2. A comparison the psi beta 2 sequence with that of the rabbit adult beta-globin gene, beta 1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render pse beta 2 unable to encode a functional beta-globin polypeptide. psi beta 2 contains two intervening sequences at the same locations in the globin protein coding sequence as beta 1 and all other sequenced beta-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative pse beta 2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of pse beta 2 and beta 1 and discuss the evolutionary relationship between these two genes.

Animals↗

PubMed1982

Expression of the split gene cob in yeast: evidence for a precursor of a "maturase" protein translated from intron 4 and preceding exons.

Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon. Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences. We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster). Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies. The other two mutations--a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site--affect both the formation of 14 typical translation products and the trans-acting function. These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded "maturase" protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame. We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.

Base Sequence↗

PubMed1983

Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus.

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.

Animals↗

PubMed1987

Translation is required for regulation of histone mRNA degradation.

When DNA synthesis is inhibited, the mRNAs coding for the replication-dependent histone proteins are selectively destabilized. The histone genes have been altered and reintroduced into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene. Two features of the mRNA are necessary for regulation of degradation: first, the hairpin loop must be present at the 3' end of the histone mRNA; and second, the histone mRNA must be capable of being translated to within 300 nucleotides of the 3' end of the RNA. Polyadenylated histone mRNAs are stable, as are histone mRNAs that contain in-frame termination codons early in the coding region or 500 nucleotide 3' untranslated regions with a normal hairpin loop at the 3' end.

Amino Acid Sequence↗

PubMed1990

An extensively edited mitochondrial transcript in kinetoplastids encodes a protein homologous to ATPase subunit 6.

The mitochondrial MURF4 gene of T. brucei has pronounced G versus C strand bias and heterogeneously sized transcripts, characteristic of genes encoding extensively edited transcripts. We find that MURF4 transcripts of T. brucei are extensively edited throughout by the addition and deletion of numerous uridines, creating potential initiation and termination codons and a continuous open reading frame. A potential guide RNA sequence occurs in a minicircle between inverted repeats. The 5' region of L. tarentolae MURF4 transcripts is also extensively edited, with a created initiation codon. The predicted MURF4 amino acid sequences have homology to those of mitochondrial ATP-ase subunit 6 genes from a variety of organisms. In addition, their hydropathic profiles are quite similar to those of other species. We therefore conclude that MURF4 encodes ATPase subunit 6 genes.

Adenosine Triphosphatases↗

PubMed1990

The MURF3 gene of T. brucei contains multiple domains of extensive editing and is homologous to a subunit of NADH dehydrogenase.

Mitochondrial MURF3 transcripts of T. brucei are extensively edited by the addition and deletion of uridines. The editing creates potential initiation and termination codons and a continuous open reading frame. The predicted amino acid sequence has homology to a subunit of NADH dehydrogenase (ND7). ND7 is independently edited in two distinct domains, suggesting two editing initiation sites. Editing in the two domains is differentially regulated: the 5' domain is edited in both bloodstream and procyclic forms but the 3' domain is completely edited only in the bloodstream form. Two potential guide RNA (gRNA) coding sequences were identified in the same minicircle. One is complementary to edited sequence in the 5' domain, the other to edited sequence in the 3' domain.

Amino Acid Sequence↗