In vitro synthesis of a unique RNA species by a T particle of vesicular stomatitis virus.

A T particle of vesicular stomatitis virus, containing most of the L-gene region, has been isolated. In vitro, these T particles synthesize exclusively a small adenine-rich RNA that is complementary to the T-particle genome. Partial sequence analysis of this small RNA indicates that it is an RNA of unique sequence with a length of approximately 45 nucleotides.     

A specific internal RNA polymerase recognition site of VSV RNA is involved in the generation of DI particles.

The 3′ terminal sequences of four different DI particle RNAs ranging in size from 10S to 30S have been determined directly using rapid RNA sequencing methods or deduced, in the case of the fourth DI RNA, from the complementary sequence of a small RNA transcribed from this part of the genome (Schubert et al., 1978). One DI particle (DI 011) contains covalently linked genomic and antigenomic RNA. The 5′ end of this RNA is identical to that of VSV RNA, as determined by annealing for at least 1 kb, as well as to the other DI particle RNAs used in this study. The 3′ ends of the other three DI particle RNAs are exact copies of the common 5′ terminal sequence for 48 nucleotides in two cases and 45 nucleotides in the third. Beyond these complementary regions the sequences are different for each DI RNA. The fact that these regions differ in length by only three nucleotides, despite the wide differences in the overall size of the DI particle RNAs, indicates that if these DIs were formed by the copy-back mechanisms similar to those proposed by Leppert, Kort and Kolakofsky (1977) and Huang (1977), a specific recognition site for the RNA polymerase must be involved in copying the 5′ terminus. We determined the 5′ terminal sequence from position 43–48 at the end of the complementary region and found it to be 5′-GGUCUU-3′. This hexamer is also part of other highly conserved terminal RNA polymerase initiation sites (Keene et al., 1978; Keene, Schubert and Lazzarini, 1979) and may be a specific internal RNA polymerase recognition site. We conclude that this sequence is one of the elements involved in the genesis of DI particle chromosomes containing short complementary sequences at their termini. The ability of the polymerase to resume synthesis at or near a specific recognition site is discussed.     

The complete sequence and coding content of snowshoe hare bunyavirus small (S) viral RNA species.

The complete sequence of the small (S) viral RNA species of snowshoe hare (SSH) bunyavirus has been determined, principally from a DNA copy of the RNA cloned in the E.coli plasmid pBr322. The viral S RNA (negative sense strand) is 982 nucleotides long (3.3 x 10(5) daltons) with complementary 5' and 3' end sequences. It has a base composition of 30.5%U, 25.8%A, 24.9%C and 18.7%G. In the viral complementary (plus sense) strand there are two overlapping open reading frames initiated by methionine codons. One reading frame codes for a 26.8 x 10(3) dalton protein, the other for a 10.5 x 10(3) dalton protein. The larger gene product is presumably related to the viral nucleoprotein (N) that is coded by the S RNA (Gentsch and Bishop (1978) J. Virol. 28, 417-419). The smaller gene product is probably related to the recently identified S RNA coded nonstructural protein (NSS) induced in virus infected cells (Fuller and Bishop (1982) J. Virol. 41, 643-648).     

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 X 106. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 X 106, 0.52 X 106, and 0.37 X 106, respectively. Hybridization experiments show that both the 31S and 12-18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.     

The N, O-diacetylmuramidase of Chalaropsis species. V. The complete amino acid sequence.

The complete amino acid sequence of lysozyme Ch has been established by a combination of automated and manual Edman degradation and carboxypeptidase digestion.(see article)There is a single disulfide bond in the center of the molecule. The enzyme has 211 residues with a calculated molecular weight of 22,415. Lysozyme Ch has an amino acid sequence that is totally different from all other lysozymes whose sequences are known.     

The complete genome sequence of J virus reveals a unique genome structure in the family Paramyxoviridae

J virus (J-V) was isolated from feral mice (Mus musculus) trapped in Queensland, Australia, during the early 1970s. Although studies undertaken at the time revealed that J-V was a new paramyxovirus, it remained unclassified beyond the family level. The complete genome sequence of J-V has now been determined, revealing a genome structure unique within the family Paramyxoviridae. At 18,954 nucleotides (nt), the J-V genome is the largest paramyxovirus genome sequenced to date, containing eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The two genes located between the fusion (F) and attachment (G) protein genes, which have been named the small hydrophobic (SH) protein gene and the transmembrane (TM) protein gene, encode putative proteins of 69 and 258 amino acids, respectively. The 4,401-nt J-V G gene, much larger than other paramyxovirus attachment protein genes sequenced to date, encodes a putative attachment protein of 709 amino acids and distally contains a second open reading frame (ORF) of 2,115 nt, referred to as ORF-X. Taken together, these novel features represent the most significant divergence to date from the common six-gene genome structure of Paramyxovirinae. Although genome analysis has confirmed that J-V can be classified as a member of the subfamily Paramyxovirinae, it cannot be assigned to any of the five existing genera within this subfamily. Interestingly, a recently isolated paramyxovirus appears to be closely related to J-V, and preliminary phylogenetic analyses based on putative matrix protein sequences indicate that these two viruses will likely represent a new genus within the subfamily Paramyxovirinae.     

The complete nucleotide sequence of the group II RNA coliphage GA

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.     

Transduction of a human RNA sequence by poliovirus.

Cells infected with poliovirus express a virally encoded polyprotein which undergoes self-mediated cleavage into structural and nonstructural viral proteins. Most of these cleavages are catalyzed by the 3C proteolytic domain of the polyprotein. Polyprotein synthesized in vitro from an RNA template containing a three-nucleotide insertion in 3C underwent proteolytic processing at all but one of the 3C-dependent cleavage sites. When transfected into HeLa cells, this RNA template displayed a lethal phenotype. We report here the isolation of two pseudorevertant progeny strains with restored protein-processing phenotypes, one of which appears to have arisen by transduction of a stretch of nucleotides from human 28S rRNA.     

The 7S RNA from tomato leaf tissue resembles a signal recognition particle RNA and exhibits a remarkable sequence complementarity to viroids.

From tomato leaf tissue we sequenced and characterized a 7S RNA which consists of 299 nucleotides with either two or three additional uridine nucleotides at its 3'-terminus. About 56% of the nucleotides of this higher plant 7S RNA are in nearly identical positions as those of the human 7SL RNA which is an integral component of the signal recognition particle (SRP) that mediates protein translocation. Computer modelling and digestion studies with nucleases led to a secondary structure model for tomato 7S RNA, the overall shape of which is very similar to that of the human 7SL (SRP) RNA. This structural similarity strongly suggests that tomato 7S RNA is actually an SRP RNA and an integral part of the plant SRP, and that the protein translocation system of higher plants is very similar to the one operating in mammalian cells. Tomato SRP RNA contains a stretch of 36-53 nucleotides which exhibit a high degree of sequence complementarity to five viroid 'species' that cause disease in tomato. In the case of potato spindle tuber viroid and citrus exocortis viroid this complementarity spans the lower strand of the region, the nucleotides of which are known to modulate virulence. This extensive sequence complementarity could lead to a thermodynamically favoured base-pairing in vivo which renders the tomato SRP RNA a possible host target with which viroids could interact and thus incite disease.     

A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro.

Purified virions of vesicular stomatitis virus (VSV) are capable of synthesizing two distinct types of virus-specific RNA in vitro. The first consists of several viral mRNAs which have been previously shown to contain the blocked 5' terminal sequence GpppApApCpApGp and 3' terminal poly(A). The second type of RNA has an unblocked 5' terminus and does not contain poly(A) stretches long enough to bind to oligo (dT)-cellulose columns. It migrates in 20% polyacrylamide gels as a single homogeneous peak with an estimated chain length of 68 nucleotides. Base analysis demonstrated that this small RNA molecule is composed of 48% AMP, 20% CMP, 11% GMP, and 21% UMP. The 5' terminal sequence of the small RNA is ppApCpGp, which appears to be complementary to the 3' terminal sequence of the VSV genome RNA (...PypGpU). These results indicate that this small RNA molecule probably represents the intitiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.     

Determining a unique defining DNA sequence for yeast species using hashing techniques

MOTIVATION: Yeasts are often still identified with physiological growth tests, which are both time consuming and unsuitable for detection of a mixture of organisms. Hence, there is a need for molecular methods to identify yeast species. RESULTS: A hashing technique has been developed to search for unique DNA sequences in 702 26S rRNA genes. A unique DNA sequence has been found for almost every yeast species described to date. The locations of the unique defining sequences are in accordance with the variability map of large subunit ribosomal RNA and provide detail of the evolution of the D1/D2 region. This approach will be applicable to the rapid identification of unique sequences in other DNA sequence sets. AVAILABILITY: Freely available upon request from the authors. Supplementary information: Results are available at http://www.sys.uea.ac.uk/~jjw/project/paper     

Three unique viral RNA species of snowshoe hare and La Crosse bunyaviruses.

Two-dimensional gel electrophoreses of RNase T1-derived oligonucleotides of the three individual RNA segments of the bunyavirus snowshow hare virus indicate that its three RNA segments possess distinct nucleotide sequences. The fingerprints of the RNA species of snowshoe hare virus differ from those of the antigenically closely related La Crosse virus. Three viral RNA species have been identified in preparations of Melao and Trivittatus as well as snowshoe hare, Lumbo, and La Crosse bunyaviruses.     

The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae.

The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam-Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the 18 S RNA was deduced. Extensive homology with many eucaryotic as well as E. coli ribosomal small subunit rRNA (S-rRNA) has been observed in the 3'-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence has been conserved in evolution.     

Two unique RNA species of the nodavirus black beetle virus

Two-dimensional fingerprinting of RNase T₁-derived oligonucleotides of the two individual RNA segments of the Nodavirus black beetle virus indicates that each RNA species possesses a distinct nucleotide sequence. Species 1 RNA has a genome complexity of approximately 3,000 nucleotides, and species 2 RNA is composed of approximately 1,500 nucleotides. Submolar amounts of oligonucleotides apparently derived from a third virus-specific RNA were also detected in black beetle virus RNA preparations.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans

The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans, has been determined. Its coding region is estimated to be 1,487 base pairs long, which is nearly identical to those reported for chloroplast 16S rRNA genes and is about 4% shorter than that of the Escherichia coli gene. The 16S rRNA sequence of A. nidulans has 83% homology with that of tobacco chloroplast and 74% homology with that of E. coli. Possible stem and loop structures of A. nidulans 16S rRNA sequences resemble more closely those of chloroplast 16S rRNAs than those of E. coli 16S rRNA. These observations support the endosymbiotic theory of chloroplast origin.     

Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.