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At least 199 records · Page 11Linked to original sources
PubMed1981

Multiple mRNAs are generated from the chicken lysozyme gene.

We have determined the DNA sequence of a 770 bp Pst 1 fragment containing 450 nucleotides of the 5' flanking region of the chicken lysozyme gene. S1-nuclease mapping was performed to localize the 5' end of nuclear RNA containing lysozyme-specific sequences and of the mRNA. We present evidence that the 5' noncoding region of the chicken lysozyme mRNA is heterogeneous in length. The 5' termini of the different nRNAs map 29, 31 and 53 nucleotides upstream from their common initiation codon. The 5' ends of lysozyme-specific nuclear RNAs map at positions similar to that of the mRNA. AT-rich regions and sequences similar to the E. coli RNA polymerase recognition sequence are found around 30 and 70 nucleotides upstream from each of these 5' termini. The AT-rich regions differ, however, from the canonical Goldberg-Hogness box in that they do not contain the extremely conserved TATA sequence motif. Sequence comparison at the 5' end of the lysozyme, conalbumin and ovalbumin genes reveals only one region of partial homology, 140 nucleotides upstream from the mRNA start sites.

Animals↗
PubMed1983

Identification of multiple species of calmodulin messenger RNA using a full length complementary DNA.

Poly(A) RNA from eel electroplax was used to construct a full length cDNA complementary to calmodulin (CaM) mRNA which was cloned in the PstI site of pBR322 DNA. Recombinant plasmids containing sequences complementary to CaM mRNA were identified by hybridization using a 32P-labeled CaM cDNA (pCM109). Nucleotide sequence analysis reveals that the clone from the plasmid pCM116 contains a 5' nontranslated region of 26 nucleotides, the entire coding region, and a 3' nontranslated region of 408 nucleotides. The amino acid sequence deduced from the nucleotide sequence is similar to those previously reported for CaM from other species. Comparison between the nucleotide sequence of the functional domains of the protein shows extensive homology between all four domains. pCM116 was utilized to determine and compare the populations of RNA present in different tissues. In eel electroplax, three species of cytoplasmic RNAs at 820, 1100, and 2000 nucleotides hybridize to the cDNA probe. The nucleus contains an additional CaM RNA molecule of 5500 nucleotides which may represent a primary transcript of the calmodulin gene. Sequence analysis of the 3' noncoding region of pCM116 reveals 3 possible polyadenylation sites (AATAAA) at positions 573, 580, and 855. The mRNA of 820 nucleotides was derived by polyadenylation at the first site whereas the mRNA of 1100 nucleotides was derived by poly(A) addition at position 855. These data are compatible with the idea that at least 2 of the 3 CaM mRNAs in eel electroplax tissue are derived from a single nuclear transcript by differential polyadenylation during processing.

Amino Acid Sequence↗
PubMed1990

Maternal mRNA from clam oocytes can be specifically unmasked in vitro by antisense RNA complementary to the 3'-untranslated region.

Clam oocytes display a striking difference in the pattern of protein synthesis on fertilization, which is maintained when cell-free extracts are assayed in the rabbit reticulocyte lysate. The mRNAs encoding ribonucleotide reductase and cyclin A (the major masked messages in the oocyte extracts) can be translationally activated by gel filtration in 0.5 M KCl, presumably by removal of repressor protein(s). When synthetic RNAs corresponding to different segments of ribonucleotide reductase and cyclin A mRNAs were added to oocyte extracts in 0.5 M KCl in a "competitive unmasking" assay, specific and complete unmasking of these mRNAs occurred on addition of antisense 3'-noncoding transcripts. The unmasking region in ribonucleotide reductase mRNA maps to a region of 134 nucleotides centered one-third of the way down the 3'-noncoding region.

Animals↗
PubMed1992

Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3'-noncoding region.

cDNAs were constructed to encode plus- or minus-sense analogs of gene 9 RNA of porcine rotavirus strain OSU in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was flanked by the 5'-terminal 44 nucleotides (nt) and 3'-terminal 35 nt of the authentic rotavirus gene. Transfection of plus-sense gene-9-CAT RNA into rotavirus-infected cells resulted in its amplification and in the efficient expression of CAT; this was greatly enhanced by the presence of a 5' cap structure. Amplification was ablated by omitting the rotavirus superinfection or by removing the 3'-terminal 35-nt rotavirus sequence from the RNA. This result indicated that amplification depended both on rotavirus proteins supplied in trans and on cis-acting rotavirus sequences. Minus-sense or double-stranded gene-9-CAT RNA was essentially inactive, indicating that synthetic RNAs can be introduced into the rotavirus replicative cycle in vivo only when provided in the plus sense. However, incorporation of the CAT-bearing RNA into infectious rotavirus was not detected. Two heterologous rotaviruses, the simian RRV and chicken Ch2 strains, efficiently complemented the OSU-based gene-9-CAT RNA, even though the Ch2 strain was only 50%-66% related in the noncoding regions. Mutational analysis of the 35-nt 3'-noncoding region showed that the 3'-terminal 12 or 17 nt were sufficient for reduced (12% or 23%, respectively) levels of amplification, whereas inclusion of the 3'-terminal 19 nt fully restored amplification. Thus, the 3'-terminal cis-acting signals required for amplification include the 7-nt-terminal consensus sequence together with 12 nt of adjoining, less-well-conserved sequence.

Animals↗
PubMed2025

Genome imbalance modulates the expression of long non-coding RNAs in maize.

Genome imbalance, resulting from varying the dosage of individual chromosomes (aneuploidy), has a more detrimental effect than changes in complete sets of chromosomes (haploidy/polyploidy). This imbalance is likely due to disruptions in stoichiometry and interactions among macromolecular assemblies. Previous research has shown that aneuploidy causes global modulation of protein-coding genes (PCGs), microRNAs, and transposable elements (TEs), affecting both the varied chromosome (cis-located) and unvaried genome regions (trans-located) across various taxa. While long non-coding RNAs (lncRNAs) are important gene expression regulators, their roles in the context of genomic imbalance remain largely unexplored. In this study, we analyzed and compared the impact of aneuploidy and haploidy/polyploidy on lncRNA expression using RNA-seq data from maize mature leaf tissue. Our results indicate that cis-located lncRNAs are modulated from dosage compensation to a gene dosage effect, while trans-located lncRNAs exhibit trends ranging from an inverse effect to a positive correlation with chromosomal dosage. Remarkably, the ploidy series showed a lesser degree of lncRNA modulation. LncRNAs and TEs display a similar trend of inverse modulation but exhibit greater sensitivity to dosage changes compared to PCGs. The construction of cis-acting and trans-acting lncRNA co-expression networks indicates that lncRNAs likely function as dosage-sensitive regulators of gene expression under conditions of genomic imbalance. Overall, this study not only elucidates the dosage effect of plant lncRNAs but also serves as a valuable resource for exploring potential regulators of PCGs that play significant biological functions.

Zea mays↗
PubMed1989

Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution.

The genomic sequence of cowpea chlorotic mottle virus (CCMV) was completed by sequencing biologically active cDNA clones of CCMV RNA2 (2774 bases) and RNA3 (2173 bases). While only the central core of the encoded 94-kDa CCMV 2a protein contains features conserved among known and putative RNA replication proteins from many viruses, both flanking regions of CCMV 2a show substantial similarity to the corresponding protein of the related brome mosaic virus (BMV). The 3a proteins of CCMV and BMV, implicated as contributors to the distinct host specificities of the two viruses, show lower levels of conservation but are still discernibly related throughout. Major differences occur in the organization of noncoding sequences in CCMV and BMV RNA3. With respect to an otherwise similar region preceding the BMV 3a gene, the CCMV RNA3 5' noncoding sequence contains a clearly bounded 111-base insertion that must reflect a sequence rearrangement in evolution of at least one of the two viruses. The presence of a subgenomic promoter-like sequence near the end of the novel CCMV sequence makes the organization of genes in CCMV RNA3 reminiscent of the 3' end of tobacco mosaic virus RNA, suggesting that CCMV or its 3a gene might have been derived from an ancestor with fewer genomic RNAs. Sequence similarities between the CCMV and BMV RNA3 intercistronic regions include the subgenomic mRNA promoter and an oligo(A), but not an intercistronic segment required for BMV RNA3 amplification, implying that replication signals on the two RNA3s may be organized quite differently.

Amino Acid Sequence↗
PubMed1986

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15 vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200 copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5' noncoding DNA for actin 15 and 0.7 kilobases of 5' noncoding DNA for actin 6.

Actins↗
PubMed1991

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage.

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.

Base Sequence↗
PubMed1996

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

Animals↗
PubMed1992

An antisense promoter of the murine c-myc gene is localized within intron 2.

Previously we have demonstrated the existence of stable transcripts from the noncoding strand of a rearranged c-myc gene in murine plasmacytomas in which the oncogene has translocated to an immunoglobulin constant-region gene element (M. Dean, R. B. Kent, and G. E. Sonenshein, Nature [London] 305:443-446, 1983). The resulting RNAs are chimeric, containing c-myc antisense and immunoglobulin sense sequences. A normal unrearranged murine c-myc gene is transcribed in the antisense orientation throughout much of the gene; however, stable transcripts have not been detected. In this study, using Northern (RNA) blot, S1 nuclease, and primer extension analyses, we have mapped the 5' end of the stable chimeric transcripts to a site 175 bp from the start of exon 3, within intron 2 of the c-myc gene. In vitro transcription assays with constructs containing this site and 400 bp upstream, in the antisense orientation, and nuclear extracts from plasmacytoma cells, as well as a number of cell lines with normal unrearranged c-myc genes, indicated that this promoter was functional. This finding was confirmed in transient transfection assays using the antisense promoter linked to the chloramphenicol acetyltransferase reporter gene. These results suggest that a normal promoter of antisense transcription is used following c-myc gene translocation.

Animals↗
PubMed1990

Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U.

Previous studies have shown that the genome of Sabin type 3 poliovaccine strain (P3/Leon 12a1b) possesses a diminished translation efficiency as compared to genomes of closely related neurovirulent strains, the neurovirulent progenitor (P3/Leon/37), or a revertant (P3/119/70) of the vaccine (Y.V. Svitkin, S.V. Maslova, and V.I. Agol, 1985, Virology 147, 243-252). Here we attempted to evaluate the contribution of each mutation in the genome of the vaccine to this translation deficiency. Recombinants between P3/Leon 12a1b and P3/Leon/37 or P3/119/70 were constructed in vitro and their RNAs were translated in a cell-free system derived from Krebs-2 cells. The results show that of 10 nucleotide differences between the genomes of P3/Leon 12a1b and P3/Leon/37 9 have minor or no effect on translation and that the only mutation of significance is C472----U which is known to reduce the neurovirulence of the virus. Reversion from uridine to cytosine at position 472 in type 3 poliovaccine upon replication in the human gut resulted in an increase of both translation efficiency of polio RNAs and neurovirulence of corresponding strains. The data provide evidence for a common nucleotide sequence regulatory element for protein synthesis of the virus and its neurovirulence. In vitro translation assays may therefore prove to be useful for detection of attenuating mutations in the 5' noncoding region of poliovirus genome. The apparent involvement of the translation mechanism in the expression of neurovirulent or attenuated phenotype of poliovirus is briefly discussed.

Animals↗
PubMedDate not supplied

A sequence downstream of AAUAAA is required for rabbit beta-globin mRNA 3'-end formation.

The sequence AAUAAA, found 11-30 base pairs (bp) upstream of the poly(A) site of most non-histone eukaryotic messenger RNAs (mRNAs) forms an essential part of the recognition site for 3'-end processing of the primary transcript. However, the sequence AATAAA is found in transcribed regions of genes and is differentially utilized in genes containing multiple copies of the sequence within the 3'-noncoding region, suggesting that the hexanucleotide alone does not comprise a complete recognition site. Therefore, it seems likely that additional sequences are required to form a complete recognition site for 3'-end formation. We have investigated the sequence requirements for mRNA 3'-end formation using the rabbit beta-globin gene as a model system. Here we demonstrate that an additional sequence 3' to AAUAAA is required for the correct 3'-end formation of rabbit beta-globin mRNA.

Animals↗
PubMed1981

Sequence and gene organization of mouse mitochondrial DNA.

The complete sequence of the 16,295 bp mouse L cell mitochondrial DNA genome has been determined. Genes for the 12S and 16S ribosomal RNAs; 22 tRNAs; cytochrome c oxidase subunits I, II and III; ATPase subunit 6; cytochrome b; and eight unidentified proteins have been located. The genome displays exceptional economy of organization, with tRNA genes interspersed between rRNA and protein-coding genes with zero or few noncoding nucleotides between coding sequences. Only two significant portions of the genome, the 879 nucleotide displacement-loop region containing the origin of heavy-strand replication and the 32 nucleotide origin of light-strand replication, do not encode a functional RNA species. All of the remaining nucleotide sequence serves as a defined coding function, with the exception of 32 nucleotides, of which 18 occur at the 5' ends of open reading frames. Mouse mitochondrial DNA is unique in that the translational start codon is AUN, with any of the four nucleotides in the third position, whereas the only translational stop codon is the orthodox UAA. The mouse mitochondrial DNA genome is highly homologous in overall sequence and in gene organization to human mitochondrial DNA, with the descending order of conserved regions being tRNA genes; origin of light-strand replication; rRNA genes; known protein-coding genes; unidentified protein-coding genes; displacement-loop region.

Animals↗
PubMed1991

An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells.

Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.

Base Sequence↗
PubMed1988

Multiple species of myeloperoxidase messenger RNAs produced by alternative splicing and differential polyadenylation.

Three clones of full-length cDNA encoding human myeloperoxidase were isolated from a human leukemia HL-60 cell cDNA library in lambda gt10 and characterized. Analysis of the nucleotide sequence of one of the cDNA clones, lambda MP-H17, indicated that the cDNA contained 3207 bp with an open reading frame of 2238 bp, a 5' noncoding region of 159 bp, a 3' noncoding region of 800 bp, and a poly(A) tail of 10 bp. cDNA of the two other clones, lambda MP-H7 and lambda MP-H14, each contained insertions with shorter sequences of 96 and 82 bp, respectively, on the open reading frame of lambda MP-H17 cDNA. A myeloperoxidase genomic clone was isolated, and the structure of its 5' region was determined and compared with the structures of these cDNAs. The comparison revealed that the three cDNAs were derived from myeloperoxidase mRNAs produced by alternative splicing from a transcript of the single gene. Nucleotide sequence analysis of the 3' region of the cDNAs of several clones indicated that the mRNAs were polyadenylated at five different sites. Amino acid sequence determination of the amino-terminal and carboxy-terminal portions of the myeloperoxidase light and heavy chains revealed that, during processing of a precursor polypeptide into the mature protein, the amino-terminal polypeptide, the small peptide between the light and heavy chains, and the carboxy-terminal amino acid were excised.

Amino Acid Sequence↗
PubMed1997

[Functions of the segment-specific noncoding regions of influenza virus genome RNA].

The genome of influenza A viruses consists of eight negative-strand RNA segments. These segments contain the untranslated regions (UTRs), ranging from 20 to 61 nucleotides, at their 3' and 5' ends. The UTRs are composed of the highly conserved terminal nucleotides and the segment-specific nonconserved nucleotides located adjacent the open reading frame of the viral RNAs. Utilizing the virus-like model RNAs, whose nonconserved UTRs were mutated, deleted or replaced with those of other segments, the unique features of the nonconserved UTRs have been elucidated in the steps of transcription, translation, replication and RNA packaging into virus particles. Here I summarize current understanding of the functions of the segment-specific nonconserved UTRs of virus RNA.

Genome, Viral↗
PubMed1997

Plus- and minus-stranded hepatitis G virus RNA in liver tissue and in peripheral blood mononuclear cells.

Hepatitis G virus (HGV), which was recently identified, is a single, plus-stranded RNA virus that is thought to replicate via minus-stranded RNA, but no information is available about the distribution of either plus- (genomic) or minus- (replicative) stranded HGV RNAs in HGV infected patients. We, therefore, tested the serum, liver tissue, and peripheral blood mononuclear cells (PBMCs) of six hepatitis patients with HGV infection for the presence of plus- and minus-stranded HGV RNA. The RT-nested PCR was used with primers derived from 5'-noncoding region of the genome. Before RT-PCR analysis, the 3'-termini of RNA specimens were chemically modified to discriminate between plus- and minus-stranded HGV RNA. Plus-stranded HGV RNA was detected in the serum and liver tissue of all six patients and in the PBMCs of five patients. Minus-stranded RNA was detected in the liver tissue of all six patients, in the serum of two patients, and in the PBMCs of one patient. In summary, the detection of minus-stranded HGV RNA in liver tissue may indicate that HGV replicates in the liver.

Flaviviridae↗
PubMed1993

Repression of the herpes simplex virus 1 alpha 4 gene by its gene product occurs within the context of the viral genome and is associated with all three identified cognate sites.

The infected cell protein 4 (ICP-4), the major regulatory protein encoded by the a4 gene of the herpes simplex virus 1, binds two sites (alpha 4-1 proximal, alpha 4-1 distal) at the 5'-untranscribed domain and at the transcription initiation site (alpha 4-2) of the alpha 4 gene. Chimeric genes consisting of the 5'-untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene were mutagenized to abolish binding of ICP-4 by substitution of bases, including the guanines whose methylation interferes with binding of the protein, and recombined into the viral genome. The cytoplasmic RNAs extracted from infected cells treated with cycloheximide, from untreated infected cells maintained for 4 or 8 hr, and from cells infected first with a virus deleted in the alpha 22 gene and 3 hr later with the test viruses were tested in RNase protection assay for amounts of the chimeric gene RNA relative to amounts of alpha 22 gene RNA. We report the following: (i) Mutation of the alpha 4-2 binding site resulted in a 5-to 6-fold higher accumulation of chimeric gene RNA at 4 hr and as much as 15-fold higher accumulation by 8 hr after infection. (ii) Mutations of alpha 4-1 sites by themselves had no effect on RNA accumulation. However, mutagenesis of all three sites significantly increased mRNA amounts above the levels seen in cells infected with alpha 4-2 site mutants. (iii) The mutations have no effect on accumulation of alpha 4 mRNA in the absence of ICP-4 synthesis and, therefore, the mutations had no effect on RNA stability or transcription rate. (iv) Accumulation of alpha 4 mRNA relative to that of alpha 22 mRNA is highest in the presence of cycloheximide and decreases with time after infection. We conclude that ICP-4 autoregulates the transcription of its own gene in infected cells and that binding of ICP-4 to three sites in its promoter is additive in its effects on this process.

Animals↗