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1.
Use of a synthetic DNA oligonucleotide to probe the precision of RNA splicing in a yeast mitochondrial petite mutant. 总被引:2,自引:1,他引:1
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H F Tabak J van der Laan K A Osinga J P Schouten J H van Boom G H Veeneman 《Nucleic acids research》1981,9(18):4475-4483
In some strains of Saccharomyces cerevisiae the mitochondrial gene coding for 21S rRNA is interrupted by an intron of 1143 bp. This intron contains a reading frame for 235 amino acids: Unassigned Reading Frame (URF). In order to check whether expression of this URF is required for proper splicing of precursors to 21S rRNA, the precision of RNA splicing was analysed in a petite mutant, where no mitochondrial protein synthesis is possible anymore. We have devised a new assay to monitor the precision of the splicing event. The method is of general application, provided that the sequence of the splice boundaries is known. In the case of the 21S rRNA it involves the synthesis of the DNA oligonucleotide d(CGATCCCTATTGTC( complementary to the 5' d(CGATCCCTAT) and 3' d(TGTC) borders flanking the intron in the 21S rRNA gene. The oligonucleotide is labelled with 32p at the 5'-end, hybridised to RNA and subsequently subjected to digestion with S1 nuclease. Resistance to digestion will only be observed if the correct splice-junction is made. The petite mutant we have studied contains a 21S rRNA with the same migration behaviour as wildtype 21S rRNA. In RNA blotting experiments, using an intron specific hybridisation probe, the same intermediates in splicing are found both in wild type and petite mutant. Finally the synthetic oligonucleotide hybridises to petite 21S rRNA and its thermal dissociation behaviour is indistinguishable from a hybrid formed with wildtype 21S rRNA. We conclude that expression of the URF, present in the intron of the 21S rRNA gene, is not required for processing and correct splicing of 21S ribosomal precursor RNA. 相似文献
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Isolation and Properties of a New Species of Ribonucleic Acid Synthesized in Sporulating Cells of Saccharomyces cerevisiae 总被引:11,自引:6,他引:5
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A new species of ribonucleic acid (RNA) was detected in sporulating culture of Saccharomyces cerevisiae. This RNA was isolated by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis and partially characterized. It has a sedimentation coefficient of approximately 20S and a nucleotide composition distinct from other known RNA species in yeast, and it hybridizes with nuclear but not with mitochondrial deoxyribonucleic acid. 相似文献
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E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene. 相似文献
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Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae. 总被引:33,自引:5,他引:28
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S Nagata K Nagashima Y Tsunetsugu-Yokota K Fujimura M Miyazaki Y Kaziro 《The EMBO journal》1984,3(8):1825-1830
Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively. 相似文献
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The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST. 总被引:18,自引:0,他引:18
R W Goldbach P Borst J E Bollen-de Boer E F van Bruggen 《Biochimica et biophysica acta》1978,521(1):169-186
1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated. 相似文献
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Candida pintolopesii 108-1 is an indigenous yeast which colonizes the surface of the secreting gastric mucosa of mice. We have been exploring the aerobic respiratory capacities of this organism in reference to its capacity to colonize the stomach surface, an environment that could contain little oxygen for microbial growth. In this paper, we report mitochondrial DNA and membranes in cells of a strain of this yeast isolated from the gastric epithelium of a mouse and compare the findings with those made by other investigators in studies of Saccharomyces cerevisiae. Putative mitochondrial DNA was isolated from crude lysates of C. pintolopesii and S. cerevisiae as fluorescing bands in CsCl gradients containing 4',6-diamidino-2-phenylindole. The DNA from C. pintolopesii hybridized with a 32P-labeled DNA probe for the 21S rRNA gene encoded by mitochondrial DNA in S. cerevisiae. Postvital cell staining with 4',6-diamidino-2-phenylindole and rhodamine 123 revealed mitochondrial DNA and membranes, respectively, in the cytoplasm of intact C. pintolopesii cells. The staining patterns were generally similar to those reported for S. cerevisiae. Finally, structures similar to those reported to be mitochondria in electron micrographs of S. cerevisiae were seen in preparations of C. pintolopesii cells examined by transmission electron microscopy. These data confirm findings from studies of its respiratory capacity published earlier that a strain of C. pintolopesii isolated directly from its native habitat has functional mitochondria. 相似文献
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Mitochondrial DNA in Candida pintolopesii, a yeast indigenous to the surface of the secreting epithelium of the murine stomach.
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Candida pintolopesii 108-1 is an indigenous yeast which colonizes the surface of the secreting gastric mucosa of mice. We have been exploring the aerobic respiratory capacities of this organism in reference to its capacity to colonize the stomach surface, an environment that could contain little oxygen for microbial growth. In this paper, we report mitochondrial DNA and membranes in cells of a strain of this yeast isolated from the gastric epithelium of a mouse and compare the findings with those made by other investigators in studies of Saccharomyces cerevisiae. Putative mitochondrial DNA was isolated from crude lysates of C. pintolopesii and S. cerevisiae as fluorescing bands in CsCl gradients containing 4',6-diamidino-2-phenylindole. The DNA from C. pintolopesii hybridized with a 32P-labeled DNA probe for the 21S rRNA gene encoded by mitochondrial DNA in S. cerevisiae. Postvital cell staining with 4',6-diamidino-2-phenylindole and rhodamine 123 revealed mitochondrial DNA and membranes, respectively, in the cytoplasm of intact C. pintolopesii cells. The staining patterns were generally similar to those reported for S. cerevisiae. Finally, structures similar to those reported to be mitochondria in electron micrographs of S. cerevisiae were seen in preparations of C. pintolopesii cells examined by transmission electron microscopy. These data confirm findings from studies of its respiratory capacity published earlier that a strain of C. pintolopesii isolated directly from its native habitat has functional mitochondria. 相似文献
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Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs. 总被引:2,自引:0,他引:2
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Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals. 相似文献
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The RNA from the mitochondrial fraction of animal cells contains a polyadenylic acid sequence, approximately 55 nucleotides in length, which migrates at about 4 S in gel electrophoresis and which is attached to high molecular weight RNA. The experiments reported here indicate that: (a) the 4 S poly(A) sequence is found only in the mitochondrial fraction; (b) the RNA containing 4 S poly(A) is located within structures (presumably mitochondria) which protect it from pancreatic ribonuclease; (c) no RNA containing the longer poly(A) of nuclear origin appears to be located in mitochondria; (d) the 4 S poly(A), but not the longer poly(A), is attached to RNA which hybridizes to mitochondrial DNA; and (e) this poly(A) sequence is located at the 3′ end of the RNA molecule.The poly(A)-containing RNA can be isolated by affinity to oligodeoxyribothymidylic acid cellulose and resolved into approximately eight distinct species by acrylamide gel electrophoresis. These may correspond to individual mitochondrial messenger RNA molecules. 相似文献
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A fine mapping study of the ribosomal RNA region of HeLa cell mitochondrial DNA has been carried out by using as an approach the protection by hybridized 12 S and 16 S rRNA of the complementary sequences in DNA against digestion with the single strand-specific Aspergillus nuclease S1 or Escherichia coli exonuclease VII. No inserts have been detected in the main body of the 12 S and 16 S rRNA cistrons, in contrast to the situation described in the large mitochondrial ribosomal RNA gene of some strains of yeast and of Neurospora crassa. Furthermore, it has been possible to assign more precisely than previously the positions of the 5′ and 3′-ends of the 12 S rRNA and 16 S rRNA genes in the HpaII restriction map of HeLa cell mitochondrial DNA. 相似文献
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We have screened numerous different yeast species for the presence of sequences homologous to the intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (intron r1) and found them in all Kluyveromyces species, some of the Saccharomyces species and none of the other yeasts tested. We have determined the nucleotide sequence of the r1-intron in K. thermotolerans and compared it with that of S. cerevisiae. The two introns are inserted at the same position within the 21S rRNA gene. They contain homologous internal open reading frames (ORFs) initiated at the same AUG codon which can be aligned over their entire length. Several silent multi-substitutions indicate that these intronic ORFs represent selectively conserved functional genes. Other intron segments, on the contrary, reveal short blocks of extensive homology separated by non-homologous stretches and/or additions-deletions. Comparison of our two yeast r1-introns with equivalent introns of N. crassa and A. nidulans mitochondria reveals that introns with very similar RNA secondary structures can accommodate different types of ORFs. 相似文献
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C Heyting F C Meijlink M P Verbeet J P Sanders J L Bos P Borst 《Molecular & general genetics : MGG》1979,168(3):231-246
1. We have used restriction enzyme analysis of petite mtDNAs to construct a detailed physical map of the 21S region on the mtDNA of the Saccharomyces cerevisiae strain JS1-3D. The map covers a segment of about 20,000 bp, on which the recognition sites of the enzymes HapII, HindII, HindIII, Sa1I, XhoI and HhaI have been localized (22 sites in total). This map has been checked in various ways against the independently constructed overall physical map of the mtDNA of strain JS1-3D. In addition, we have constructed a physical map with a resolution of about 200 bp of a HapII fragment of 1850 bp long, which carries the loci omega, RIB-1 and probably RIB-2. 2. The 21S rRNA hybridizes with the five adjacent HindII + III fragments TD9, DT19, TD15, DT14 and TT1, which lie in that order on the physical map of the 21S region. Of these, the two non-adjacent fragments TD9 and DT14 show a much stronger hybridization with 21S rRNA than DT19, TD15, and TT1. 3. The fragment DD5 (= DT19 + TD15) and part of DT14 belong to a sequence of about 1000 bp, which is absent from Saccharomyces carlsbergensis mtDNA. Although DD5 and DT14 show (very weak, respectively stronger) hybridization with 21S rRNA, the 1000 bp insert probably does not code for the 21S rRNA: the 21S rRNA of S. carlsbergensis comigrates with the 21S rRNA of JS1-3D on polyacrylamide gels under denaturing conditions. 4. Fragment DT14 hybridizes with the HindII + III fragment TD9, which shows the strongest hybridization with 21S rRNA. The presence of these sequence homologies has hampered the precise mapping of the 21S rRNA cistron. Our results are compatible, however, with the hypothesis that the sequences, coding for 21S rRNA, are located on HindII + III fragments that are not adjacent on JS1-3D mtDNA, namely TD9, DT14 and TT1. 相似文献