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PubMed1988

Insulin-like growth factor II messenger ribonucleic acids are synthesized in the choroid plexus of the rat brain.

Previous studies demonstrating the presence of immunoreactive insulin-like growth factors (IGFs) and their receptors in the brain suggest a role of the IGFs in the central nervous system. IGF-II has been implicated as the predominant IGF in brain of mature animals based on studies of immunoreactive peptide and of IGF-II mRNAs. To obtain information about the sites of synthesis of IGF-II in adult rat brain, a 32P-labeled 31 base long synthetic oligodeoxyribonucleotide complementary in sequence to trailer peptide coding sequences in rat IGF-II mRNA (IGF-II 31 mer) was hybridized with coronal sections of fixed rat brain. The IGF-II 31 mer showed specific hybridization with the choroid plexus throughout rat brain, whereas in other brain regions, structures or cells, hybridization was not discernibly above background. These findings suggest that the choroid plexus is a primary site of synthesis of IGF-II, a probable source of IGF-II in cerebrospinal fluid, and a potential source of IGF-II for actions on target cells within the adult rat brain.

Animals↗

PubMed1999

Sequence Analysis of the Trailer Region of Wheat Rosette Stunt Virus Genomic RNA.

According to a highly conserved 8 amino acid sequence of L proteins in Rhabdoviridae, a 24-mer degenerate oligonucleotide primer was designed to amplify the 5' terminus of the genomic RNA of wheat rosette stunt virus (WRSV), a plant rhabdovirus, by anchored PCR. The complete nucleotide sequence of the 5' trailer in WRSV genomic RNA was determined by deoxynucleotide sequencing of cloned cDNA derived from the anchored PCR products.

Journal Article↗

PubMed1991

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.

Base Sequence↗

PubMed1988

Post-translational processing of thyrotropin-releasing hormone precursor in rat brain: identification of 3 novel peptides derived from proTRH.

The sequence of rat hypothalamic pro-thyrotropin releasing hormone, deduced by sequencing of cDNA, in addition to 5 TRH progenitor sequences contains leader, trailer and 4 intervening sequences separated by paired basic amino acid sequences. We have developed radioimmunoassays to synthetic peptides corresponding to portions of these cryptic proTRH sequences and have used these assays to identify and partially characterize proTRH peptides, distinct from TRH, in extracts of rat brain. Two of these peptides correspond closely in size to one intervening sequence and the carboxy-terminal sequence of proTRH. Three other peptides correspond to the intact amino-terminal leader sequence and two peptides formed by a further cleavage of the leader sequence at an internal paired basic amino acid sequence.

Animals↗

PubMed1983

The terminal RNA stem-loop structure and 80 bp of spacer DNA are required for the formation of 3' termini of sea urchin H2A mRNA.

We have studied the exact sequence requirement for the formation of 3' termini of the sea urchin H2A mRNA in frog oocyte injection experiments. Point mutations destroying the symmetry of the inverted DNA repeat in the mRNA trailer coding sequences prevent the generation of genuine 3' termini. Mutants containing complementary base changes are pseudorevertants and allow the production of H2A mRNA with faithful 3' termini at wild-type levels. Our transcription analyses show that it is primarily the sequence of the transcribed strand that decides whether or not true 3' mRNA termini are produced. This is evidence that an RNA stem-loop structure, rather than a DNA cruciform, is essential for this process. Spacer sequences are absolutely required, because in their absence only H2A mRNA with spacer transcript extensions are found. Once the canonical CAAGAAAGA and flanking sequences are linked to the H2A gene, H2A messenger is synthesized at a suboptimal rate, which can be increased to wild-type levels by the addition of 80 bp of the spacer immediately adjacent to the H2A gene.

Animals↗

PubMed2000

Sequence determination and molecular analysis of two strains of bovine parainfluenza virus type 3 that are attenuated for primates.

The Kansas/15626/84 (Ka) and Shipping Fever (SF) strains of bovine parainfluenza virus type 3 (BPIV3) replicate less efficiently than human PIV3 (HPIV3) in the upper and lower respiratory tract of rhesus monkeys, and BPIV3 Ka is also highly attenuated in humans and is in clinical trials as a candidate vaccine against HPIV3. To initiate an investigation of the genetic basis of the observed attenuation phenotype of BPIV3 in primates, the complete genomic sequences of Ka and SF genomes were determined and compared to those of BPIV3 strain 910N and two HPIV3 strains, JS and Wash/47885/57. There is a high degree of identity between the five PIV3 viruses in their 55 nucleotide (nt) leader (83.6%) and 44 nt trailer (93.2%) sequences. The five viruses display amino acid sequence identity ranging from 58.6% for the phosphoprotein to 89.7% for the matrix protein. Interestingly, the majority of amino acid residues found to be variable at a given position in a five-way protein alignment are nonetheless identical within the viruses of either host species (BPIV3 or HPIV3). These host-specific residues might be products of distinct selective pressures on BPIV3 and HPIV3 during evolution in their respective hosts. These host-specific sequences likely include ones which are responsible for the host range differences, such as the efficient growth of BPIV3 in bovines compared to its restricted growth in primates. It should now be possible using the techniques of reverse genetics to import sequences from BPIV3 into HPIV3 and identify those nt or protein sequences which attenuate HPIV3 for primates. This information should be useful in understanding virus-host interactions and in the development of vaccines to protect against HPIV3-induced disease.

Animals↗

PubMed1995

The structure of the bovine cathepsin B gene. Genetic variability in the 3' untranslated region.

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15-kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathepsin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu-like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3' end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6-kb. Polyadenylation at the third polyadenylation site generates a 3.2-kb mRNA, only expressed at low levels and in a developmental and tissue-specific manner. Due to micro-heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR-amplified genomic fragments revealed that, in contrast to the protein-encoding region, genetic variability exists in the 3' untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribonuclease-protection assays. We finally emphasize that while exon-exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.

Amino Acid Sequence↗

PubMed1994

Genomic organization of lettuce necrotic yellows rhabdovirus.

We have mapped the genome of lettuce necrotic yellows virus (LNYV), the type member of the genus cytorhabdovirus of the family Rhabdoviridae. We have cloned and sequenced all intergenic regions and the 3' leader and 5' trailer of the negative-sense, single-stranded RNA genome of LNYV. The LNYV genome appears to contain six genes, the five expected genes coding for the virion proteins, and a sixth gene of unknown function, as for sonchus yellow net virus (SYNV), a member of the genus nucleorhabdovirus. The proposed LNYV genomic map is 3'-N-4a-4b-M-G-L-5', where N is the nucleocapsid protein gene; 4a and 4b are two genes, one of which codes for the proposed phosphoprotein P and the other for a putative protein of unknown function; M is the proposed matrix protein gene; G is the proposed glycoprotein gene; and L is the proposed transcriptase gene. The different LNYV intergenic regions have highly conserved consensus sequences, which could be divided into three components: the sequences corresponding to the 3' end of the mRNAs, intergenic sequences of variable length, and the sequences corresponding to the 5' end of the mRNAs. A leader sequence of 84 nucleotides (nt) at the 3' end of the LNYV genomic RNA preceeded the N gene. A trailer sequence of 187 nt at the 5' end of the genomic RNA followed the L gene. A comparison between LNYV leader and trailer sequences revealed complementary 3' and 5' ends, which could give rise to a putative "panhandle" structure with a two bases overhang in the leader sequence. We have compared these sequences to the corresponding sequences of SYNV as well as to vesicular stomatitis virus (VSV) and rabies virus (RV), the type members of the vesiculovirus and lyssavirus genera, respectively, of animal rhabdoviruses. Homologies were found in the intergenic regions between LNYV, SYNV, VSV, and RV, at the 3' ends of the mRNAs. LNYV intergenic sequences were of variable lengths, as were those found in RV. The consensus sequences found at the 5' ends of LNYV mRNAs differed from the highly conserved consensus transcription start sequence UUGU/A found in SYNV, VSV, and RV. Conserved sequences were also found in the first 30 nt of the leader and the last 30 nt of the trailer, between LNYV, SYNV, VSV, and RV.

Base Sequence↗

PubMed1999

Characterization of the L gene and 5' trailer region of Ebola virus.

The nucleotide sequences of the L gene and 5' trailer region of Ebola virus strain Mayinga (subtype Zaire) have been determined, thus completing the sequence of the Ebola virus genome. The putative transcription start signal of the L gene was identical to the determined 5' terminus of the L mRNA (5' GAGGAAGAUUAA) and showed a high degree of similarity to the corresponding regions of other Ebola virus genes. The 3' end of the L mRNA terminated with 5' AUUAUAAAAAA, a sequence which is distinct from the proposed transcription termination signals of other genes. The 5' trailer sequence of the Ebola virus genomic RNA consisted of 676 nt and revealed a self-complementary sequence at the extreme end which may play an important role in virus replication. The L gene contained a single ORF encoding a polypeptide of 2212 aa. The deduced amino acid sequence showed identities of about 73 and 44% to the L proteins of Ebola virus strain Maleo (subtype Sudan) and Marburg virus, respectively. Sequence comparison studies of the Ebola virus L proteins with several corresponding proteins of other non-segmented, negative-strand RNA viruses, including Marburg viruses, confirmed a close relationship between filoviruses and members of the Paramyxovirinae. The presence of several conserved linear domains commonly found within L proteins of other members of the order Mononegavirales identified this protein as the RNA-dependent RNA polymerase of Ebola virus.

Amino Acid Sequence↗

PubMed1985

Developmental and tissue-specific expression of a family of transcripts related to rat insulin-like growth factor II mRNA.

We have constructed a cDNA library from the mRNA of a rat liver cell line (BRL-3A) and characterized cDNA clones encoding the protein precursor of the rat insulin-like growth factor II (pre-pro-rIGF-II). This precursor, inferred from the nucleotide sequence, consists of a signal peptide, the rIGF-II sequence, and a trailer polypeptide of unknown significance. The characterized cDNA sequence (1016 nt) is part of a 3.4 kb mRNA species. Northern analysis reveals that a probe containing the extreme 5' noncoding region hybridizes to a second RNA (1.6 kb), while a probe corresponding to the 5' noncoding region proximal to the coding region hybridizes to two other RNA species (1.75 and 1.1 kb). All four RNAs are differentially expressed in all of the neonatal tissues that were examined, while the 3.4 kb pre-pro-rIGF-II mRNA and the 1.1 kb transcript are absent from adult tissues.

Amino Acid Sequence↗

PubMed2006

The properties of recombinant Sendai virus having the P gene of Sendai virus pi strain derived from BHK cells persistently infected with Sendai virus.

We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38 degrees C than at 32 degrees C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38 degrees C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38 degrees C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-beta at 32 and 38 degrees C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.

Cell Line↗

PubMed1992

Regenerating good sense: RNA editing and trans splicing in plant mitochondria.

The protein products of plant mitochondrial genes cannot be predicted accurately from genomic sequences, since RNA editing modifies almost all mRNA sequences post-transcriptionally. Furthermore, RNA editing alters leader, trailer and intron sequences, and may be required for processing of these sequences. For several plant mitochondrial transcripts, processing includes trans splicing, which connects exons scattered throughout the genome. The mature transcripts are assembled via split group II intron sequences.

Introns↗

PubMed1992

Nucleotide sequence analysis of the simian virus 41 gene encoding the large (L) protein and construction of a phylogenetic tree for the L proteins of paramyxoviruses.

The complete nucleotide sequence of the simian virus 41 (SV41) large (L) protein gene was determined. The L gene spanned 6883 nucleotides including a putative trailer RNA, and the L mRNA contained a single large open reading frame encoding a polypeptide of 2269 amino acids. Dot-matrix comparisons under stringent conditions identified domains highly conserved among paramyxoviruses. Domain 3 is the most highly conserved, and has been hypothesized to be the RNA polymerase active site. A phylogenetic tree was constructed from the sequences of the L proteins of seven paramyxoviruses. SV41 was most closely related to human parainfluenza virus type 2 (HPIV-2), and SV41, HPIV-2 and SV5 form a subgroup. The intergenic sequences at the nucleocapsid protein-phosphoprotein and haemagglutinin-neuraminidase-L protein gene junctions, and the 5' trailer sequence of SV41 were also determined, and it was shown that the first 13 nucleotides of the 5' trailer sequence are complementary to those of the 3' leader sequence. The intergenic, and gene-start and -end sequences of SV41, HPIV-2 and SV5 are shown.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequences of the spacer-1, spacer-2 and trailer regions of the rrn operons and secondary structures of precursor 23S rRNAs and precursor 5S rRNAs of slow-growing mycobacteria.

The single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 16S, 23S and 5S rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 16S rRNA and 23S rRNA genes and the spacer-2 region separating the 23S rRNA and 5S rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, 'Mycobacterium lufu' and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 5S rRNA gene, the trailer region and the downstream region of the rrn operon of Mycobacterium tuberculosis were cloned and sequenced. These data, together with those obtained previously for Mycobacterium leprae, were used to identify putative antitermination signals and RNase III processing sites within the spacer-1 region. Notable features include two adjacent potential Box B elements and a Box A element. The latter is located within a sequence of 46 nucleotides which is very highly conserved among the slow-growers which were examined. The conserved sequence has the capacity to interact through base-pairing with part of the spacer-2 region. Secondary structures for mycobacterial precursor 23S rRNA and for precursor 5S rRNA were devised, based on sequence homologies and homologous nucleotide substitutions. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. A putative motif for the intrinsic termination of transcription was identified approximately 33 bp downstream from the 3'-end of the 5S rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species.

Base Composition↗

PubMed2000

Molecular characterization of guinea pig-adapted variants of Ebola virus.

Serial passage of initially nonlethal Ebola virus (EBOV) in outbred guinea pigs resulted in the selection of variants with high pathogenicity. Nucleotide sequence analysis of the complete genome of the guinea pig-adapted variant 8mc revealed that it differed from wild-type virus by eight mutations. No mutations were identified in nontranscribed regions, including leader, trailer, and intragenic sequences. Among noncoding regions the only base change was found in the VP30 gene. Two silent base changes were found in the open reading frame (ORF) encoding NP protein. Nucleotide changes resulting in single-amino-acid exchanges were identified in both NP and L genes. Three other mutations found in VP24 caused amino acid substitutions, which are responsible for larger structural changes of this protein, as indicated by an alteration in electrophoretic mobility. A highly pathogenic EBOV variant K5 from another passaging series showed an amino acid substitution at nearly the same location in the VP24 gene, suggesting the importance of this protein in the adaptation process. In addition, sequence variability of the GP gene was found when plaque-purified clones of EBOV-8mc were analyzed. Three of five viral clones showed insertion of one uridine residue at the GP gene-editing site, which led to a significant change in the expression of virus glycoproteins. This observation suggests that the editing site is a hot spot for insertion and deletion of nucleotides, not only at the level of transcription but also of genome replication. Irrespective of the number of uridine residues at the editing site, all plaque-purified clones of EBOV variant 8mc resembled each other in their pathogenicity for guinea pigs, indicating either the absence or only supportive role of mutations in the GP gene on the adaptation process.

Amino Acid Substitution↗

PubMed2003

Macronuclear molecules encoding actins in spirotrichs.

The nucleotide sequences of 16 newly reported and 8 previously reported actin-encoding macronuclear DNA molecules in spirotrichs have been compared. As described for the eight previously reported molecules, the first 50 bases (noncoding) inside the telomere at both 5' strands in additional actin molecules are purine-rich. This anomalous base composition might serve as a signal to identify macronuclear molecules in micronuclear DNA during development. The 50-base segment upstream of the ATG in the 5' leaders of the actin molecules contains extensive, conserved sequence motifs that are possibly promoter elements. The 3' noncoding trailers contain virtually no conserved sequence motifs. With one exception, the 3' trailers contain a second stop codon (TGA) 36 bases on average downstream of the primary stop codon. Excluding Moneuplotes crassus, amino acid identities in actin I range from 78 to 100%, with variations distributed nonrandomly along the sequence. Phylogenetic trees based on the actin nucleotide sequences of 22 spirotrichs define the evolutionary relationships of their actin-encoding molecules. The actin phylogeny, while well supported by posterior probabilities, does not always coincide with the phylogeny defined in rDNA analyses or classical taxonomic classifications.

3' Untranslated Regions↗

PubMedDate not supplied

Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

In order to determine the effect of the nucleotide composition of the 5'-untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiation site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady-state levels of PGK mRNA and protein were determined. We found translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long polyA or polyC tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a poly U tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long-range base-pairing between the leader and sequences elsewhere in the coding or 3'-non-coding regions of the messenger. In agreement with this hypothesis, a five-fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base-pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon. The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found that leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequences in naturally occurring yeast mRNAs. Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, nor replacement of this sequence by homopolymer tracts had any effect on translational efficiency.

Amino Acid Sequence↗

PubMed1991

Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs.

To investigate whether RNA editing in plant mitochondria modifies structural RNAs as well as protein-coding RNAs we compared the genomic-encoded information with the respective transcripts of several genes in Oenothera. The genes analysed are the 5S, 18S and 26 S rRNAs, the alpha-subunit of ATPase (atpA), cytochrome b (cytb), orfB, which is located upstream of cytochrome oxidase subunit III, and the respective leader, trailer and spacer sequences. All open reading frames were found to be edited to some degree. The atpA coding region has the least edited mRNA in Oenothera mitochondria, with only four nucleotides altered in the 1533 nucleotide open reading frame. From this analysis we conclude that frequent RNA editing is indicative of functional protein coding regions in plant mitochondria. The extensive editing in orfB, for example, suggests that this orf codes for a mitochondrial protein. No RNA editing event was found in the 5S rRNA or in the 1824 nucleotides analysed of the 18S rRNA, but two nucleotides were found to be altered in the 1970 nucleotides compared for the 26S rRNA. One nucleotide alteration has changed C to U, the other in reverse U to C. However, only one of five cDNA clones covering this region shows the modifications, similar to many silent editing events in open reading frames. RNA editing in the structural RNAs thus does not seem to be essential for their function in the mitochondrial ribosome.

Adenosine Triphosphatases↗