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1.
Nucleic Acids Res ; 28(15): 2959-68, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10908360

ABSTRACT

A U3 snoRNA gene isolated from a Chlamydomonas reinhardtii (CRE:) genomic library contains putative pol III-specific transcription signals similar to those of RNA polymerase III-specific small nuclear (sn)RNA genes of higher plants. The 222 nt long CRE: U3 snoRNA was immunoprecipitated by anti-gamma-mpppN antisera, but not by anti-m(2,2,7)G antibodies, supporting the notion that it is a RNA polymerase III transcript. Tagged CRE: U3 snoRNA gene constructs were expressed in CRE: cells. Results of chemical and enzymatic structure probing of CRE: U3 snoRNA in solution and of DMS modification of CRE: U3 snoRNA under in vivo conditions revealed that the two-hairpin structure of the 5'-domain that is found in solution is no longer detected under in vivo conditions. The observed differences can be explained by the formation of several base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. A model that involves five intermolecular helices is proposed.


Subject(s)
Chlamydomonas reinhardtii/genetics , RNA, Protozoan/chemistry , RNA, Small Nucleolar/chemistry , Animals , Base Pairing , Base Sequence , Gene Expression , Immunosorbent Techniques , Models, Molecular , Molecular Sequence Data , Mutagenesis , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA Polymerase III/metabolism , RNA, Protozoan/isolation & purification , RNA, Small Nucleolar/analysis , RNA, Small Nucleolar/genetics , Sequence Alignment , Solutions , Transcription, Genetic
2.
Biochimie ; 79(7): 387-95, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9352088

ABSTRACT

The spliceosomal UsnRNAs U2, U4 and U6 from the green alga Chlamydomonas reinhardtii (Cre) were sequenced using a combination of RNA and cDNA sequencing methods and were compared to other sequenced UsnRNAs. The lengths of Cre U6 and Cre U2 RNAs are similar to those of their higher plant equivalents. Cre U4 RNA is shorter (139 nt) than its counterpart from higher plants (150-154 nt), and contains stem IV and loop D which are absent, with the exception of the Tetrahymena U4 RNA, from the U4 RNAs of other unicellular organisms studied to date. Base-pairing interactions between U6 and U4 RNAs and between U6 and U2 RNAs, identical to those described for mammalian and yeast systems, are structurally feasible in the Cre system. In addition, based on comparative analyses of the predicted U4/U6 RNA duplex from various species, an evolutionary conserved third putative U6-U4 interaction was found. Interestingly, it can also be formed with the recently discovered U6atac and U4atac RNAs. This is a strong support in favor of the possible biological significance of this third putative interaction. Based on comparative analysis, an extension of the earlier described U6-U2 interaction patterns is also proposed.


Subject(s)
Chlamydomonas reinhardtii/genetics , Conserved Sequence , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Alternative Splicing , Animals , Base Sequence , Evolution, Molecular , Humans , Molecular Sequence Data , Nucleic Acid Conformation , RNA/chemistry , RNA/isolation & purification , RNA, Small Nuclear/isolation & purification , RNA, Small Nuclear/metabolism , Rats , Sequence Analysis, RNA , Spliceosomes
3.
Biochimie ; 78(6): 425-35, 1996.
Article in English | MEDLINE | ID: mdl-8915532

ABSTRACT

The 5' terminal sequence of U1 snRNA that base-pairs with the intron 5' splice site in the course of spliceosome assembly was considered to be universally conserved. A study of the P polycephalum U1 snRNA at both RNA and gene levels shows that there are exceptions to this rule: the P polycephalum U1 snRNA has a U to A substitution at position 5, that is partially compensated by a high frequency of T residue at position +4 of introns. In contrast to the yeast genome, the P polycephalum genome contains several U1 snRNA coding sequences (about 20). They either encode the U1A snRNA expressed in microplasmodia or correspond to the previously cloned U1B coding sequence. Both coding sequences show the U5A substitution. The ratio of U1A versus U1B coding sequences is of about 3. A U1A gene was cloned. The 60 nt region upstream of the coding sequence has the same sequence as in the U1B gene. The U1B gene is probably expressed at another stage of the P polycephalum life cycle.


Subject(s)
Physarum polycephalum/chemistry , RNA, Small Nuclear/chemistry , Amino Acid Sequence , Animals , Blotting, Southern , Cloning, Molecular , DNA Primers , DNA, Fungal/chemistry , DNA, Fungal/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression/genetics , Introns/genetics , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA Splicing/genetics , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Small Nuclear/genetics , Sequence Alignment , Sequence Analysis
8.
Nucleic Acids Res ; 18(8): 1941-9, 1990 Apr 25.
Article in English | MEDLINE | ID: mdl-2336383

ABSTRACT

By screening a tomato genomic library with a tomato U3 RNA probe, we detected a U3 genomic locus whose coding region was determined by primer extension (5' end) and direct RNA sequencing of purified U3 RNA from tomato (3' end). Tomato U3 RNA is 216 nucleotides long, contains all the four evolutionarily highly conserved sequence blocks (Boxes A to D), has at its 5' end a cap not precipitable with anti-m3G antibodies and can be folded into a peculiar secondary structure with two stem-loops at its 5' end. A tagged derivative of the U3 gene was faithfully expressed in transgenic tobacco plants. In the 5' flanking region both plant-specific UsnRNA transcription signals [the TATA-like sequence and the upstream sequence element (USE)] were present, but were positioned closer to each other and also to the cap site in the U3 gene than in the genes for the plant spliceosomal UsnRNAs studied so far. The 3' flanking region of the tomato U3 gene lacked the consensus sequence of the putative termination signal established for the plant spliceosomal UsnRNA genes and contained a pyrimidine-rich tract (R1) followed by four tandemly repeated U3 pseudogenes (U3.1 ps to U3.4 ps) flanked by slightly altered forms (R2 to R5) of R1 and most probably generated by DNA-mediated events. Our results are in line with the conjecture that the enzyme transcribing the tomato U3 gene has different structural requirements for transcriptional activity than the enzyme transcribing plant U1, U2 and U5 genes.


Subject(s)
Nicotiana/genetics , Plants, Toxic , Plants/genetics , Pseudogenes , RNA, Small Nuclear/genetics , Transcription, Genetic , Base Sequence , DNA/genetics , Exons , Genes , Molecular Sequence Data , Multigene Family , Nucleic Acid Conformation , Restriction Mapping
12.
Nucleic Acids Res ; 17(15): 6319-37, 1989 Aug 11.
Article in English | MEDLINE | ID: mdl-2528122

ABSTRACT

From a tomato genomic library we isolated and characterized eight U1 RNA gene candidates (U1.1 to U1.8) all of which possessed the canonical plant U-snRNA transcription signals in their 5' and 3' flanking regions and exhibited nucleotide sequence conservation in the 5' splice site recognition sequence, in the Sm antigen binding site and in Loops B, C, D as well as in Stems III and IV of their coding region. Deviations from the U1 RNA consensus sequence were mainly localized to Loop A and Stems I and II, suggesting that the putative transcripts of the tomato U1.1-U1.8 genes would differ from each other in their capacity of binding to the U1 RNA-specific snRNP proteins.


Subject(s)
Nucleic Acid Conformation , Plants/genetics , Ribonucleoproteins/genetics , Transcription, Genetic , Animals , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Genetic Variation , Humans , Molecular Sequence Data , Nucleic Acid Hybridization , RNA Probes , Ribonucleoproteins, Small Nuclear , Sequence Homology, Nucleic Acid
14.
Nucleic Acids Res ; 17(4): 1445-58, 1989 Feb 25.
Article in English | MEDLINE | ID: mdl-2522185

ABSTRACT

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.


Subject(s)
Plants/analysis , Ribonucleoproteins/isolation & purification , Chromatography, Affinity , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Fabaceae/analysis , Immunoblotting , Indicators and Reagents , Molecular Weight , Plants, Medicinal , RNA, Small Nuclear/isolation & purification , Ribonucleoproteins, Small Nuclear
16.
Plant Mol Biol ; 12(6): 709-11, 1989 Jun.
Article in English | MEDLINE | ID: mdl-24271203
17.
Biochim Biophys Acta ; 950(3): 455-8, 1988 Sep 07.
Article in English | MEDLINE | ID: mdl-3167064

ABSTRACT

Viroids were found to exhibit a structural periodicity characterized by repeat units of a length of 11 or 12 (potato spindle tuber viroid group and coconut cadang-cadang viroid), 60 (apple scar skin viroid) and 80 (avocado sunblotch viroid) nucleotide residues, respectively. It is suggested that structural periodicity of viroids is an indication of their protein-binding ability.


Subject(s)
Plant Viruses/genetics , Viroids/genetics , Base Sequence , Genes, Viral , Molecular Sequence Data , RNA, Viral/genetics , Species Specificity
19.
Nucleic Acids Res ; 16(12): 5407-26, 1988 Jun 24.
Article in English | MEDLINE | ID: mdl-3387237

ABSTRACT

U4 RNA, which is known to play an indispensable role in pre-mRNA splicing, is present in plant nuclei, has a canonical m3 2,2,7 G cap at its 5' end and is associated with U6 RNA in snRNP particles. It occurs in broad bean in the form of a number of sequence variants. Two of these were sequenced: U4A RNA is 154 and U4B RNA is 152 nucleotides long. Sequence similarity of broad bean U4B RNA is 94 per cent to broad bean U4A RNA, 65 per cent to rat U4A RNA, 61 per cent to Drosophila U4A RNA and 50 per cent to snR14, the U4 RNA equivalent of the yeast Saccharomyces cerevisiae. Sequence conservation is much more pronounced in the 5' half of the molecule than in its 3' half. The secondary structure of both variants of broad bean U4 RNA perfectly fits with that of all other U4 RNAs sequenced so far. Nucleotide changes between broad bean U4A and U4B RNAs are restricted to molecular regions that affect the thermodynamic stability of these molecules. A model is proposed for the base pairing interaction of broad bean U4 RNA with broad bean U6 RNA. This is the first report on the structure of a plant U4 RNA.


Subject(s)
Plants/genetics , RNA, Small Nuclear/genetics , Animals , Antibodies, Monoclonal , Antigen-Antibody Complex/analysis , Base Sequence , Cell Nucleus/analysis , Fabaceae/genetics , Genetic Variation , Molecular Sequence Data , Nucleic Acid Conformation , Plants, Medicinal , RNA, Small Nuclear/isolation & purification , Species Specificity
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