Organization of tRNA and rRNA genes in N. crassa mitochondria: intervening sequence in the large rRNA gene and strand distribution of the RNA genes.

Through analysis of cloned fragments of N. crassa mitochondrial DNA, we have derived a physical map for the region of the mitochondrial genome which encodes the ribosomal RNAs and most of the tRNAs. We have located RNA genes on this map by hybridization of purified 32P end-labeled RNA probes, and our findings are as follows. First, the gene for the large ribosomal RNA contains an intervening sequence of approximately 2000 bp. Second, the genes for the small and large ribosomal RNAs are not adjacent, as previously reported, and the region between them contains a number of tRNA genes, including that for the mitochondrial tRNATyr, which is located close to the small rRNA gene on the same strand of the mitochondrial DNA. Third, there is a second cluster of tRNA genes on the mitochondrial DNA following the large ribosomal RNA gene, but there is no evidence for the presence of tRNA genes in the intervening sequence of the large ribosomal RNA. Fourth, hybridization of labeled ribosomal and transfer RNAs to the separated strands of a cloned 16 kbp DNA fragment covering this region indicates that the two ribosomal RNAs and most, if not all, of the mitochondrial tRNAs are encoded on one strand of the mitochondrial DNA.     

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.     

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

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.     

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton     

Mapping of rRNA genes in an inverted repeat in Nicotiana tabacum chloroplast DNA.

Nicotiana tabacum chloroplast DNA contains two copies each of 16S and 23S rRNA genes. These genes are located in an inverted order as determined from restriction fragment mapping and Southern hybridization to restriction fragments. The position of these genes on the N. tabacum chloroplast DNA molecule has been established relative to a complete map of SalI and SMaI restriction enzyme cleavage sites.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

Fine structure physical mapping of 4S RNA genes on mitochondrial DNA of Saccharomyces cerevisiae.

Summary We have localized the genes for mitochondrial 4S RNA on the physical map of themtDNA of severalSaccharomyces cerevisiae strains by hybridization of iodinated 4S RNA to the restriction fragments obtained with endonucleasesHindII+III,EcoRI andHapII. The data indicate that 5–8 of the 4S RNA genes are dispersed over a large area of the genome whereas the rest (about 18 genes) is located within an area of about 9000 bp in length (about 12% of the genome) between the markers for chloramphenicol and paromomycin resistance (RIB 1 and PAR 1 loci). Within this region a cluster is present of 5 genes on a DNA fragment of 460 bp.Abbreviations Used mtDNA mitochondrial DNA - mtRNA mitochondrial RNA - rRNA ribosomal RNA - tRNA transfer RNA - C, E, P and O cytoplasmically-inherited resistance markers for chloramphenicol, erythromycin, paromomycin and oligomycin, respectively - SSC 150 mM sodium chloride, 15 mM sodium citrate (pH 7.0) - SDS sodium dodecylsulphate - EDTA (sodium)ethylenediaminetetraacetate; TEMED - N,N,N N-tetramethylethylenediamine; (k)bp, (kilo)base pairs - EthBr ethidium bromide     

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI.

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.     

Laboratory of Physiological Chemistry, State University Groningen, Netherlands.

To obtain more information about the arrangement of Hind III restriction fragments in the tRNA-rRNA region of the Neurospora crassa mitochondrial (mt) DNA we have cleaved the mtDNA with Hpa I and Hind II. We could construct additional cleavage maps for these enzymes. Hybridization of rRNAs to Hind II fragments confirmed the existence of an intervening region of about 2,300 basepairs in the 24S rRNA (Hahn et al., Cell, in press). About seven tRNA genes, among which the genes for tRNA1Ser and tRNAMetM, are located in a segment of about 5,000 bp separating the 24S and 17S rRNA genes. Another cluster of 14 tRNA genes is found adjacent to the other end of the 24S gene. The genes for tRNALeu1 and tRNAMetF are located in this cluster.     

Analysis of sequence homology between human and mouse mitochondrial DNA

The base sequence homology between human and mouse mitochondrial DNA has been investigated by hybridization of highly labelled mitochondrial DNA probes with restriction fragments of mitochondrial DNA blotted according to the Southern technique. By this analysis, the homologous regions have been found to be widely distributed along the mitochondrial genome. Competition hybridization experiments with unlabelled HeLa mitochondrial RNAs have shown that most of the cross-hybridization involves the ribosomal and 4 S RNA genes.     

Organization and closing of mitochondrial deoxyribonucleic acid from Paramecium tetraaurelia and Paramecium primaurelia.

Previously we showed that the mitochondrial deoxyribonucleic acid (DNA) from Paramecium aurelia consists of a linear genome and that replication of this genome is initiated at one terminus and proceeds unidirectionally to the other terminus. Analyses of mitochondria from four closely related species (1, 4, 5, and 7) indicated that the species 1, 5, and 7 DNAs are essentially completely homologous but that the species 4 mitochondrial DNA is only 40 to 50% homologous with that from species 1. The major regions of homology are those containing the genes for ribosomal ribonucleic acid (RNA). To understand the replication and organization of the linear mitochondrial genome better, we compared species 1 (Paramecium primaurelia) and 4 (Paramecium tetraaurelia) DNAs with regard to restriction fragment mapping and homology between initiation regions; we also identified the sites of the genes for ribosomal RNA. In general, the structures of the species 1 and 4 mitochondrial genomes were quite similar. Each ribosomal RNA gene was present in one copy per genome, with the large ribosomal RNA gene located near the terminal region of replication and the small ribosomal RNA gene located more centrally. These two genes were separated by about 10 kilobases in the species 1 genome and by about 12 kilobases in the species 4 genome. In contrast to our previous findings, by using nonstringent hybridization conditions we detected homology between the species 1 and 4 DNA fragments containing the initiation regions. We constructed recombinant DNA clones for many fragments, especially those containing the initiation region and the ribosomal RNA genes. We also constructed restriction enzyme maps for six enzymes for both P. primaurelia and P. tetraaurelia.