Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Influenza B virus genome: assignment of viral polypeptides to RNA segments.

It was shown that all eight RNA segments of influenza B viruses are most likely monocistronic and code for eight virus-specific polypeptides. A genetic map of the influenza B virus genome was established, and six polypeptides (P1 protein, nucleoprotein, hemagglutinin, neuraminidase, M protein, and nonstructural protein) were unambiguously assigned to specific RNA segments. Molecular weight estimates of the eight individual genes are obtained by using the glyoxal method. These results suggest that each influenza B virus RNA segment has a greater molecular weight than the influenza A virus RNA segment which codes for the analogous gene product.     

Hybridization selection and cell-free translation of mRNA''s encoded within the inverted terminal repetition of the vaccinia virus genome

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.     

Phosphotransferase-mediated regulation of carbohydrate utilisation in Escherichia coli K12: identification of the products of genes on the specialised transducing phages lambda iex (crr) and lambda gsr (tgs).

The expression of genes adjacent to ptsI was investigated using a series of specialised transducing phages carrying different, overlapping, segments of the cysA-gsr-ptsI-ptsH- iex - cysZ -lig region of the genome of Escherichia coli. The polypeptides were synthesised following the infection of u.v.-irradiated lysogenic and non-lysogenic uvrA recA hosts or a uvrA recA host carrying the lambda cI+ plasmid pKB280 . The polypeptides were identified by SDS-polyacrylamide gel electrophoresis and fluorography. The gsr gene product had a mol. wt. of 23 000. The product of the iex gene was tentatively identified as a protein of mol. wt. of either 33 000 or 21 000. Hpr, the product of the gene ptsH, had a mol. wt. of 9000. The gsr gene appeared to be expressed at a higher level in a non-immune host, which suggests that it was transcribed from lambda promoters. A new lambda host strain, suitable for the detection of small polypeptides (mol. wt. less than 30 000) is described.     

The tomato Cab-4 and Cab-5 genes encode a second type of CAB polypeptides localized in Photosystem II

The photosynthetic apparatus of plant chloroplasts contains two photosystems, termed Photosystem I (PSI) and Photosystem II (PSII). Both PSI and PSII contain several types of chlorophyll a/b-binding (CAB) polypeptides, at least some of which are structurally related. It has been previously shown that multiple genes encoding one type of PSII CAB polypeptides exist in the genome of many higher plants. In tomato, there are at least eight such genes, distributed in three independent loci. Genes encoding a second type of CAB polypeptides have been isolated from several plant species, but the precise location of the gene products has not been determined. Here we show that tomato has two unlinked genes encoding this second type and that this type of CAB polypeptide is also localized in PSII.     

Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis.

Hybrid selected translation was used to map the genome of herpesvirus saimiri, a lymphotropic and oncogenic herpesvirus. RNA extracted from virus-infected cells was hybridized to cloned genomic fragments, and the hybrid selected mRNAs were translated in vitro in a rabbit reticulocyte lysate. Forty-five virus-induced polypeptides were identified and correlated to their coding regions on the herpesvirus saimiri genome. Inhibition of the replication of viral DNA with phosphonoacetic acid showed that 22 of these polypeptides belong to the early group of herpesvirus saimiri gene products.     

Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus.

A gene encoding a polypeptide with homology to mammalian vascular endothelial growth factors (VEGFs) has been discovered in the genome of orf virus (OV), a parapoxvirus that affects sheep and goats and, occasionally, humans. The gene is transcribed abundantly early in infection and is found immediately outside the inverted terminal repeat at the right end of the genome. In the NZ2 strain of OV (OV NZ2), the gene encodes a polypeptide with a molecular size of 14.7 kDa, while in another strain, OV NZ7, there is a variant gene that encodes a polypeptide of 16 kDa. The OV NZ2 and OV NZ7 polypeptides show 22 to 27% and 16 to 23% identity, respectively, to the mammalian VEGFs. The viral polypeptides are only 41.1% identical to each other, and there is little homology between the two genes at the nucleotide level. Another unusual feature of these genes is their G+C content, particularly that of OV NZ7. In a genome that is otherwise 63% G+C, the OV NZ2 gene is 57.2% G+C and the OV NZ7 gene is 39.7% G+C. The OV NZ2 gene, but not the OV NZ7 gene, is homologous to the mammalian VEGF genes at the DNA level, suggesting that the gene has been acquired from a mammalian host and is undergoing genetic drift. The lesions induced in sheep and humans after infection with OV show extensive dermal vascular endothelial proliferation and dilatation, and it is likely that this is a direct effect of the expression of the VEGF-like gene.     

Mapping and identification of the vaccinia virus thymidine kinase gene

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.     

Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus.

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.     

Nucleotide sequence and chromosomal location of Cab11 and Cab12, the genes for the fourth polypeptide of the photosystem I light-harvesting antenna (LHCI)

Tryptic peptide sequences from the 22 kDa polypeptide of tomato LHCI were used to construct a probe for gene cloning. The two genes cloned, cab11 and cab12, encode proteins of 251 and 250 residues that are 88% identical in overall amino acid sequence and 93% identical in the deduced mature protein. Each gene is present in a single copy per haploid genome; cab11 on chromosome 3 and cab12 on chromosome 6, and each has 2 introns located in similar positions to introns in other members of the Chl a/b-binding (CAB) protein gene family. Comparison of the amino acid sequences of LHCI, LHCII, CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma-exposed sequences preceding them. However, near the N-terminus and between the conserved regions, the LHCI polypeptides have sequence motifs which appear to be PSI-specific.     

Sequence homology between the 32K dalton and the D2 chloroplast membrane polypeptides of Chlamydomonas reinhardii

Summary The region of the chloroplast genome of Chlamydomonas reinhardii containing the gene of the thylakoid polypeptide D2 (psbD) has been sequenced. A unique open reading frame of 350 codons exists in this region. Because the first ATG is followed 11 codons downstream by a second one, the D2 polypeptide consists of either 339 or 350 amino acids. Comparison of the sequences of D2 and the 32K dalton polypeptides, both of which are associated with photosystem II, reveals partial homology. Although, the overall homology of these two polypeptides is only 27%, they contain several related regions and their hydropathic profiles are strikingly similar. These data suggest that the two polypeptides may have related functions and/or that their genes may have originated from a common ancestor. Alternatively, convergent evolution of these polypeptides may be due to structural constraints in the thylakoid membrane. Limited sequence homology is also observed between the D2 polypeptide and some of the subunits of the reaction centers of photosynthetic bacteria.     

Polymorphism in the procyclic acidic repetitive protein gene family of Trypanosoma brucei.

The expression of procyclic acidic repetitive protein (PARP) by Trypanosoma brucei is strongly induced during the transition of bloodstream form to cultured procyclic trypomastigotes in vitro. The membrane-associated protein is distinguished by a central domain consisting of tandemly repeated glutamate-proline dipeptides. The trypanosome genome contains eight PARP genes, at least four of which are expressed. A minimum of four distinct PARP mRNA species comprises two classes of PARP mRNA, based upon divergent 3' untranslated region sequences, and these mRNAs encode polypeptides that exhibited an inverse relation between molecular weight and isoelectric point. Comparative analysis of PARP gene structure indicated that these polypeptides differ by variation in size of the dipeptide repeat domain. Comparison of PARP genes and polypeptides of three independent T. brucei isolates suggested that PARP is not a homogeneous species but instead represents a family of polymorphic proteins.     

Antigenicity of the major polypeptides of hepatitis B surface antigen (HBsAg).

The major polypeptides (P-1, P-2, and P-6) of HBsAg were isolated from purified preparations of 22-nm HBsAg particles, iodinated, and analyzed by double-antibody radioimmunoprecipitation assays for the presence of hepatitis B virus (HBV)-specific antigens. Each polypeptide fraction contained both group (a) and subtype (d) specific determinants in common by virtue of their immunoreaction with antiserum to native HBsAg and antisera to the other structural polypeptides. The antigenic and structural similarities of the HBsAg polypeptides establish that they are not each unique gene products of the HBV genome.     

Location on the Escherichia coli genome of a gene specifying O-acetylserine (thiol)-lyase

The plasmid pAB65, derived from a specialized transducing phage carrying DNA from about 52 min on the Escherichia coli genome, coded for two polypeptides of Mr approx. 34 000. The expression of one was regulated by cyst(e)ine and the cysB gene product and the other by the cysB gene product only. One of these polypeptides was a subunit of O-acetylserine (thiol)-lyase (EC 4.2.99.8); the other, associated with the E. coli membrane, was the N-terminus of the product of the lambda ben gene. The pattern of peptide synthesis directed by plasmids carrying smaller DNA fragments indicated that the gene for O-acetylserine (thiol)-lyase was transcribed clockwise. The spectrum, amino acid composition and subunit number of the enzyme were determined. The enzyme appears homologous with the Salmonella typhimurium cysK gene product. This provides further evidence for the inversion of this region of the genome.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Protein coding assignment of avian reovirus strain S1133.

Avian reovirus S1133 encodes 10 primary translation products, 8 of which are structural components of the viral particle and 2 of which are nonstructural proteins. The identity of the gene that codes for each of these polypeptides was determined by in vitro translation of denatured individual genome segments.