Analysis of chloroplast and mitochondrial DNAs in asymmetric somatic hybrids between tobacco and carrot

Summary Chloroplast and mitochondrial DNAs have been examined by comparison of restriction enzyme patterns in asymmetric hybrid plants, resulting from the fusion between leaf mesophyll protoplasts of Nicotiana tabacum (Solanaceae), and irradiated cell culture protoplasts of Daucus carota (Umbellifereae). These somatic hybrids with normal tobacco morphology were selected as a consequence of the transfer of methotrexate and 5-methyltryptophan resistance from carrot to tobacco. The restriction patterns of chloroplast DNAs in somatic hybrids were indistinguishable from the tobacco parent. However, we found somatic hybrids with mitochondrial DNA significantly different from either parent, as judged by analysis of fragment distribution after restriction enzyme digestion. The possible formation of altered mitochondrial DNA molecules as the result of parasexual hybrid production between two phylogenetically highly divergent plant species will be discussed.     

Organelle DNA compositions and isoenzyme expression in an interspecific somatic hybrid of Daucus

Summary Cell suspension cultures of Daucus carota, D. capillifolius and a somatic hybrid of these lines were analyzed to determine their chloroplast and mitochondrial DNA compositions. The plastid DNAs (pDNA) from the somatic hybrid and D. carota were identical and were different from that of D. capillifolius when analyzed on agarose electrophoretic gels after digestion by the restriction endonuclease HpaII. The endonuclease restriction patterns of the mitochondrial DNAs (mtDNA) from each cell line were different. Although the restriction pattern of the mtDNA from the somatic hybrid contained fragments in common with one or both parents, unique fragments not found in the restriction pattern of either parent were also present.The amounts and feedback regulation of aspartokinase, homoserine dehydrogenase and dihydrodipicolinic acid synthase were quantified to define the effects of somatic hybridization upon the pathway leading to the biosynthesis of lysine, threonine, methionine and isoleucine. Regulation of each enzyme by end product inhibitors was not altered in the somatic hybrid, but levels of each enzyme appeared to be increased. However, isoenzyme analysis indicated two major forms of homoserine dehydrogenase were present in the hybrid, including one unique form not present in either parent.     

Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus

The gene encoding the phosphotransferase enzyme that modifies hygromycin B in its producing organism $\text{Streptomyces hygroscopicus}$, has been cloned in the $\text{Streptomyces}$ vector pIJ41. Two plasmids, pFM4 and pFM6, containing 2.1 and 19.6 kb inserts of $\text{Streptomyces hygroscopicus}$ DNA, respectively, which express the modifying enzyme, have been isolated. A 3.1 kb PstI restriction fragment from pFM4 was inserted in the $\text{Streptomyces}$ vector pIJ350 and the resulting plasmids, pMZ11.1 and pMZ11.2, express the hygromycin B-resistance phenotype. The utility of this dominant marker for cloning experiments is discussed in the text.     

Expression of mitochondrial DNA in Saccharomyces cerevisiae: the construction of sets of isonuclear haploid strains containing different specified mitochondrial genomes.

In order to perform systematic biochemical studies of the expression of the mitochondrial genome in different strains of $\text{Saccharomyces}\text{cerevisiae}$, we have constructed a series of haploid strains in which a specified mitochondrial genome has been placed in cells containing a particular nuclear genome. The desired strains were obtained from crosses between $\text{rho}{}^{\mathrm{+}}$ and $\text{rho}{}^{\mathrm{o}}$ haploids, one of which carried the $\text{karl}$-l mutation which interferes with nuclear fusion. These newly constructed strains provide the basis for studying mitochondrial gene expression as a function of (i) the nucleotide sequence differences that are apparent in the mitochondrial DNAs from different strains, and (ii) the role played by different nuclear genomes in modulating the expression of mitochondrial genes.     

Generation of small plasmids from colicin E1 factor carrying genes for galactose utilization and ampicillin resistance,.

Ampicillin-resistant colonies that did not utilize galactose appeared sporadically in cultures of galactose genedeleted $\text{Escherichia coli}$ K-12 cells containing colicin E1 factor carrying genes for galactose utilization and ampicillin resistance. Most of these colonies contained small plasmid DNAs. These plasmids existed as monomer DNAs within $\text{E. coli}$ K-12 cells and formed a series of covalently closed circular DNA molecules ranging in size from 6.3 × 10⁶ to 15.1 × 10⁶ daltons. The use of these plasmid DNAs was discussed.     

The mitochondrial genome of wild-type yeast cells. VII. Recombination in crosses.

Two Saccharomyces cerevisiae wild-type strains were crossed, and 26 diploid clones were obtained from (1) mass mating; (2) individual buds in zygote lineages; (3) individual zygotes. The mitochondrial DNAs from these diploids were investigated in their recombination and segregation by analyzing their restriction fragment patterns.Recombinant mitochondrial genomes were present in 75% of the diploid clones. Such recombinant genomes had unit sizes different from, yet within ± 5% of, the parental ones and showed EcoRI and HindII + III fragment patterns of parental types, two strong indications that both the gene complement and the gene order were very largely preserved in the progeny.Fragment patterns produced by HpaII and HaeIII were characterized by (1) fragments originating from the DNAs of both parents; and (2) new fragments, namely fragments absent in either parent. The new fragments appear to arise from unequal crossing-over events occurring in the spacers of allelic parental genetic units and usually have preferential localizations in the genome.These results provide the first evidence for physical recombinations of mitochondrial DNA in crosses of wild-type yeast cells, indicate that recombination is very frequent in crosses, and shed some light on mitochondrial segregation. They also have interesting implications for recombination phenomena in interspersed systems of unique and repetitive nucleotide sequences.     

Detection of sequence heterology by use of the N. Crassa nucleases

We have used the single-strand specific nucleases of $\text{Neurospora crassa}$ to detect sequence divergencies between two similar DNA molecules: restriction endonuc lease $\text{Eco}$RI produced linears from Simian Virus 40 and a variant of human origin, DAR. Enzyme treatment of the heteroduplex DNA resulted in specific cleavage into two fragments of one-third and two-thirds genome length. These two viral DNAs therefore have at least one region of heterology located about 0.35 map units from the $\text{Eco}$RI site. Due to the known specificities of the $\text{N.crassa}$ nucleases, this technique is applicable to detect mutations in RNA or DNA genomes.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

The use of agar for gel electrophoresis of DNA

Agar can be used instead of agarose for electrophoresis of DNA. DNA restriction fragments migrate in proportion to the log of their molecular weights in the ranges studied. Bands of both restriction fragments and discrete small low molecular weight DNAs such as plasmids are sharp and clearly visible. The DNA can be Southern blotted with very low nonspecific background binding of radioactivity. Fragments can be removed from the gel and can be further restricted and ligated. Plasmid DNA retains its capacity to transform host bacterial cells. Agar is about $\text{1}\text{10}$ the cost of electrophoresis-grade agarose.     

Mechanism of feedback inhibition by leucine: Purification and properties of a feedback-resistant α-isopropylmalate synthase

A mutationally altered, l-leucine-resistant form of α-isopropylmalate synthase, the first committed enzyme in leucine biosynthesis, has been purified to near homogeneity. Comparison of the feedback-resistant enzyme with its wild-type parent shows the following: Both enzymes are very similar with respect to substrate specificity and maximal activity, but the feedback-resistant enzyme has a greater affinity for one of the substrates, α-ketoisovalerate. The feedback-resistant enzyme is about three orders of magnitude less sensitive to l-leucine than wild-type enzyme. By contrast, it is slightly more sensitive to l-isoleucine, the only other naturally occurring amino acid known to inhibit α-isopropylmalate synthase. Results of chemical densensitization experiments suggest that the leucine, isoleucine, and active sites are distinct. The kinetic pattern of leucine inhibition at pH 7.0 shows that leucine is a noncompetitive inhibitor with respect to both substrates with wild-type enzyme, whereas the weak inhibition by leucine of the feedback-resistant enzyme is of a competitive type. Intersubunit cross-linking of the feedback-resistant enzyme followed by gel electrophoresis in sodium dodecyl sulfate reveals the presence of monomers, dimers, and tetramers with molecular weights of approximately 52,000, 110,000, and 200,000, respectively. Very similar results had been obtained with wild-type enzyme. Sedimentation equilibrium analyses indicate that both enzymes exist as associating-dissociating systems that can be adequately described by either a monomer-tetramer or a monomer-dimer-tetramer equilibrium. With the feedback-resistant enzyme, the equilibrium constant for the monomer-tetramer equilibrium. $\text{K}{}_4\text{=}\text{[}\text{A}{}_4\text{]}\text{[A]}{}^4$, is 1 × 10¹⁹m^?3, compared with 9 × 10¹⁶m^?3 for wild-type enzyme. This suggests a stronger tendency of the subunits of the feedback-resistant enzyme to aggregate, a conclusion supported by gel filtration experiments. These results, together with previous observations that wild-type enzyme is dissociated by leucine whereas the feedback-resistant enzyme is not, suggest that efficient inhibition of α-isopropylmalate synthase by leucine may be coupled to a relatively loose arrangement of subunits within the oligomeric structure of the enzyme.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Ligation of highly modified bacteriophage DNA

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4′,5′-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, λ DNA to be ligated was as follows: $\text{λ}\text{DNA}\text{=}\text{XP}\text{12}\text{DNA}\text{>}\text{SP}\text{82}\text{DNA}\text{?}\text{nonglucosylated}\text{T}\text{4}\text{DNA}\text{>}\text{T}\text{4}\text{DNA}\text{=}\text{PBS}\text{1}\text{DNA}\text{?}\text{SP}\text{15}\text{DNA}$. TaqI-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and λ DNAs was equally efficient. We conclude that the poor ligation of TaqI fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.     

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

Glycosylation of rat liver cytochrome b5 on the ribosomal level

The distribution and site of synthesis of cytochrome $\text{b}{}_5$ was studied by antibody precipitation of the enzyme labeled $\text{in}\text{vivo}$. The enzyme is present in rough and smooth microsomes, Golgi and outer mitochondrial membranes. The cytochrome is synthesized only on bound ribosomes, where glucosamine and galactose moieties are also added. The enzyme seems to be devoid of mannose and sialic acid residues.     

Physical and genetic organization of Petite and Grande yeast mitochondrial DNA. III. High resolution melting and reassociation studies

Mitochondrial DNAs from 13 petite mutants have been analyzed by means of high-resolution melting and reassociation in solution, in an attempt to relate their physical chemical properties to the mitochondrial genotype, which displays various combinations of genetic marker deletions. The kinetic complexities of the petite mtDNAs were found to range from $\text{1}\text{3}$ down to $\text{1}\text{500}$ of that of the grande mtDNA; the loss in sequential complexity undergone by petite mtDNAs parallels the loss in mitochondrial genetic complexity. Melting profiles of petite mtDNAs can be resolved into well-defined peaks. Some of them are specific to the marker genes. The genotypic specificity increases as the sequential complexity of mtDNA decreases. The mtDNA region conferring resistance to erythromycin could in this way be shown to be characterized by two melting peaks, at 72 °C and 75 °C. The results are interpreted in terms of selective enrichment in gene-specific sequences.     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosusforma paromomycinus

The paromomycin producing organism $\text{Streptomyces}$$\text{rimosus}$$\text{forma paromomycinus}$ is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the $\text{Streptomyces}$ vector pIJ702 and then cloned in $\text{Streptomyces}$$\text{lividans}$, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for $\text{Streptomyces}$ cloning vectors with the versatility of insertional inactivation.