Mitochondrial DNA from various organisms does not contain internally methylated cytosine in -CCGG- sequences.

Mitochondrial DNAs from yeast, Neurospora, rat and calf do not contain internally methylated cytosine in -CCGG- sequences.

Electrophoretic behaviour of yeast mitochondrial translation products.

We have studied the mobility of yeast mitochondrial translation products during electrophoresis on polyacrylamide gels of different composition and found that these polypeptides can be divided into two groups. One, to which subunit II of cytochrome c oxidase belongs, behaves normal as all water-soluble reference proteins. The other, to which cytochrome b and subunits I and III of cytochrome c oxidase belong, shows a free electrophoretic mobility about twice as fast as the first group. Conditions have been found to separate cytochrome c1 from cytochrome b.

Sequence homology of nuclear and mitochondrial DNAs of different yeasts.

1. Both nuclear and mtDNA of four different yeasts show approximately 10% homology as measured by DNA-DNA filter hybridization. These homologous sequences are mainly attributable to the ribosomal cistrons. 2. Melting curve analysis shows that the heterologous mitochondrial DNA-DNA hybrids contain several times more mismatching than the nuclear DNA-DNA hybrids. 3. DNA-rRNA hybridization shows that the sequences of the ribosomal cistrons in both the nuclear and the mitochondrial genome have been conserved during evolution. 4. However, melting curve analysis of the DNA-RNA hybrids shows that the sequence of the nuclear ribosomal cistrons have undergone considerable fewer nucleotide substitutions than their mitochondrial counterparts. 5. The results suggest that the mitochondrial ribosomal cistrons have evolved more rapidly than the nuclear cistrons. This is discussed in the light of theories on the rat of molecular evolutin.

Biosynthesis of polypeptides of cytochrome c oxidase by isolated mitochondria.

Yeast mitochondria, incubated with radioactive amino acids in a "protein-synthesizing mixture" containing an oxidizable substrate and an ATP regenerating system, have been shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to incorporate label into polypeptides equivalent in molecular weight and relative amount ot those made in vivo in the presence of cycloheximide. The ability of these isolated mitochondria to synthesize "native" polypeptides was assessed by examining the incorporation of label into subunits of cytochrome c oxidase (EC 1.9.3.1). An analysis of immunoprecipitates formed by incubating cholate extracts of labeled mitochondria with an antiserum against holocytochrome c oxidase revealed that label was incorporated into three polypeptides of sizes equivalent to those of cytochrome c oxidase subunits I, II, and III, shown from earlier studies in vivo to be translated on mitochondrial ribosomes. Further evidence that these polypeptides made in vitro are "native" and identical to subunits I, II, and III was provided by the observation that labeled polypeptides equivalent in size to subunits I-III- ARE ALSO IMMUNO-PRECIPITATED BY ANTISERUM AGAINST SUBUNITS V plus VII, an antiserum that can precipitate subunits I, II, and III only when they are complexed to the cytoplasmically synthesized subunits, V and VII, of the enzyme. These results suggest that isolated mitochondria are capable of synthesizing three subunits of cytochrome c oxidase and assembling them into a holoenzyme.

The organization of the genes for ribosomal RNA on mitochondrial DNA of Kluyveromyces lactis.

We have constructed a physical map of Kluyveromyces lactis mtDNA using the restriction enzymes HindII and HindIII. In contrast to Saccharomyces, the genes for the large and small ribosomal RNAs are much closer to each other, being separated by a maximal distance of 2,250 base pairs.

Physical mapping of 4S RNA genes on chloroplast DNA of Spirodela oligorhiza.

We have determined the position of Spirodela oligorhiza chloroplast 4S RNA genes on the restriction fragment map of cp DNA, using purified in vitro[(32)P]-labeled 4S RNA. The overall organization of these genes is very similar to the organization of tRNAs on spinach cp DNA (Driesel et al. 1979).

The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression.

The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.

The cytochrome bc1 complex of yeast mitochondria. Isolation and partial characterization of the cytochrome bc1 complex and cytochrome b.

We have isolated the cytochrome bc1 complex and some of its constituent polypeptides from bakers yeast and have studied its spectroscopy, electrophoresis and amino acid analysis. The isolated complex contained 6 mumol of b heme and approximately 3 mumol of c1 heme per g of protein. The electron paramagnetic resonance spectrum was similar to that of the beef-heart preparation. The complex consisted of 7 polypeptides with mobilities on sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to Mr 44,000, 40,000, 32,000, 32,000, 17,000, 14,000 and 11,000. One of the polypeptides with Mr 32,000 was identified on sodium dodecylsulphate gels as cytochrome c1 by porphyrin fluorescence. Cytochrome b was isolated from the complex by treating it with guanidine hydrochloride; it had a purity of 20 mumol per g of protein and consisted of a polypeptide with Mr 32,000 plus two minor bands with Mr 14,000 and 11,000. We have isolated the polypeptide of Mr 32,000 from cytochrome b and the polypeptides of Mr 44,000 and 40,000 ("core proteins") from the complex, both by preparative sodium dodecylsulphate gel electrophoresis and determined their amino acid composition. Only the b polypeptide of Mr 32,000 shows the low proportion of polar amino acid residues that is considered typical of membrane proteins.

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

We have localized the genes for mitochondrial 4S RNA on the physical map of the mtDNA of several Saccharomyces cerevisiae strains by hybridization of iodinated 4S RNA to the restriction fragments obtained with endonucleases HindII + III, EcoRI and HapII. 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 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.