Isolation and Genetic Analysis of a Chloramphenicol-resistant Mutant of Pleurotus ostreatus

A chloramphenicol-resistant mutant was isolated by mutagenesis with manganese to make a study of the genetics and function of mitochondrial genes in the higher basidiomycete, Pleurotus ostreatus. The resistant mutant obtained was shown to grow well on media containing up to 4mg/ml chloramphenicol. The result of genetic analysis suggested that the origin of the factor for chloramphenicol resistance of the mutant might be mitochondrial.     

HISTOCHEMICAL LOCALIZATION OF ENZYME ACTIVITIES IN ROOT MERISTEM CELLS

Avers , Charlotte J. (Douglass Coll., Rutgers—The State University, New Brunswick, N. J.) Histochemical localization of enzyme activities in root meristem cells. Amer. Jour. Bot. 48(2): 137–143. Illus. 1961,—Particle counts were made in epidermal cells of the root meristem of 2 grasses after exposure of living seedlings to various substrates involved in dehydrogenation reactions. Hydrolytic enzyme activities also were recorded for 1 of these species. The mean number of particles stained with Janus green B was about 90 for each species, but significantly lower counts were obtained in all the dehydrogenase tests. With lactate, pyruvate, glutamate, citrate, and isocitrate as substrates, Phleum cells showed about 25% of the Janus green count, while Panicum cells were about 33% active. These substrates are known to be oxidized by DPN-linked enzymes. The succinic dehydrogenase counts were about 50% of the Janus green total, and 60–70% particle activity was recorded with hexose-phosphate substrates which are probably oxidized in TPN-mediated reactions. In Phleum, particulate activity occurred in the adenosine triphosphatase and aryl sulfatase tests, but a non-particulate distribution characterized 5-nucleotidase activity. The particle counts in the ATPase tests were not significantly different from the Janus green counts, but the 85% particle activity in the aryl sulfatase tests was significantly lower than the Janus green results. These intracellular distributions were compared with those obtained by various authors using biochemical and histochemical techniques and were found to be in close agreement. It was suggested that the evidence indicated intracellular differentiation of at least one kind of cellular organelle, which in all probability was mitochondria.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Studies on the induction of petite mutants in yeast by analogues of berenil

Summary Compound Hoe 15 030 is an analogue of berenil which is as effective as berenil in inducing petite mutants in Saccharomyces cerevisiae. Hoe 15 030 has greater stability than berenil in aqueous solution, and is less toxic to yeast at high drug concentrations. Mutants of S. cerevisia strain J69-1B have been isolated which are resistant to the petite inducing effects of Hoe 15 030. Three mutant strains (HR7, HR8 and HR10) were characterized and each was shown to carry a recessive nuclear mutation determining resistance to Hoe 15 030. The degree of resistance to Hoe 15 030 is different for each mutant, and each was found to be co-ordinately cross-resistant both to berenil and to another analogue of berenil, Hoe 13 548. However, the three mutants show no cross-resistance to other unrelated petite inducing drugs, including ethidium bromide, euflavine and 1-methyl phenyl neutral red.Further studies on the mutants revealed that each strain exhibits characteristic new properties indicative of changes in mitochondrial membrane functions concerned with the replication (and probably also repair) of mitochondrial DNA. Thus, mutant HR7 is hypersensitive to petite induction by the detergent sodium dodecyl sulphate under conditions where the parent J69-1B is unaffected by this agent. Mutant HR8 is even more sensitive to sodium dodecyl sulphate than is HR7, and additionally shows a markedly elevated spontaneous petite frequency. Isolated mitochondria from strains HR8 and HR10 (but not HR7) show resistance to the inhibitory effects of Hoe 15 030 on the replication of mitochondrial DNA in vitro.     

Evolution of fragmented mitochondrial ribosomal RNA genes inChlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences ofC. eugametos andC. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga,Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genusChlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the twoChlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between theChlamydomonas sequences andP. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed forC. eugametos andC. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor ofChlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

玉米黄绿叶突变体表型鉴定及基因初步定位

叶色突变体是研究光合作用及叶绿体发育的重要材料。开展玉米叶色突变体的相关研究,对光形态建成、光合作用、基因功能注释、蛋白质功能及抗逆性机制的阐述具有重要的理论意义。本研究以黄绿叶突变体ygl-F17138为材料,与玉米自交系B73进行杂交,构建F2分离群体,进行遗传效应分析和基因初步定位。遗传分析表明,该突变性状由单个隐性核基因控制,且能稳定遗传。利用BSR-seq结合连锁分析的方法将该基因初步定位在第3条染色体上一个约9.2 Mb的区间内(chr.3:173087201~182203992),查询该区间内已知基因功能注释,未发现类似前人报道的调控黄绿叶性状基因,说明YGL-F17138基因可能是一个控制玉米黄绿叶发育未被挖掘的候选基因。     

Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1–10μm) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under `nuclear' conditions and in the presence of Janus Green B and Ca²⁺ is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.     

Extrachromosomal inheritance in Schizosaccharomyces pombe. II. Evidence for extrakaryotically inherited respiratory deficient mutants.

Summary In contrast to the wild-type, mutant [ANT ^R8] is able spontaneously to throw off stable respiratory deficient mutants. The frequency of these mutants is considerably enhanced by treatment with ethidium bromide (EB) or the azo-dye Janus green (JG).An unstable cell state with a petite-like phenotype is found in both mutant [ANT ^R8] and wild-type after EB-treatment. However, only in the mutant is this unstable cell state followed by the appearance of stable respiratory deficient (RD) mutants. Formation of microcolonies is observed both in [ANT ^R8] and wild-type.RD mutants were isolated after EB treatment. Three of them (mit-12, mit-25, and mit-30) were analyzed and mit-25 characterized in more detail.Mutant mit-25 shows mitotic segregation in the diploid state, indicating non-Mendelian mode of inheritance.The results of haploidization experiments also indicate extrachromosomal inheritance. Mit-25 shows a spontaneous rate of reversion of 10^-6, which can considerably increased by EB. Mit-25 possesses enzymatically active complexes I, II, and III of the respiratory chain (the latter without cytochrome b-566), lacks complex IV and binds antimycin, showing that mitochondrial protein synthesis is functional. Several lines of evidence presented in this paper make it very likely that the lesion in the mutant mit-25 is a point mutation in mitochondrial DNA.     

Arabidopsis ELONGATED MITOCHONDRIA1 is required for localization of DYNAMIN-RELATED PROTEIN3A to mitochondrial fission sites

Mitochondrial fission is achieved partially by the activity of self-assembling dynamin-related proteins (DRPs) in diverse organisms. Mitochondrial fission in Arabidopsis thaliana is mediated by DRP3A and DRP3B, but the other genes and molecular mechanisms involved have yet to be elucidated. To identify these genes, we screened and analyzed Arabidopsis mutants with longer and fewer mitochondria than those of the wild type. ELM1 was found to be responsible for the phenotype of elongated mitochondria. This phenotype was also observed in drp3a plants. EST and genomic sequences similar to ELM1 were found in seed plants but not in other eukaryotes. ELM1:green fluorescent protein (GFP) was found to surround mitochondria, and ELM1 interacts with both DPR3A and DRP3B. In the elm1 mutant, DRP3A:GFP was observed in the cytosol, whereas in wild-type Arabidopsis, DRP3A:GFP localized to the ends and constricted sites of mitochondria. These results collectively suggest that mitochondrial fission in Arabidopsis is mediated by the plant-specific factor ELM1, which is required for the relocalization of DRP3A (and possibly also DRP3B) from the cytosol to mitochondrial fission sites.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710     

Mitochondrial genetics

Summary Cytoplasmic petite mutants of Saccharomyces cerevisiae carrying the gene conferring the resistance to chloramphenicol on one hand and the gene conferring the resistance to erythromycin on the other, have been crossed with each other. The two types of petites differed in the buoyant densities of their mitochondrial DNA. A novel type of evidence has been adduced, that the two genes are indeed located on mitochondrial DNA. Diploid petite recombinants were found, carrying both genes and containing not a mixture of the two parental DNAs but a new species of mitochondrial DNA of intermediate buoyant density. Recombination of mitochondrial genes involves therefore breakage and reunion of DNA molecules. New suppressiveness, different from the two parental ones, can result from the recombination of mitochondrial DNA. Recombination between petite mutants implies that the mitochondrial recombination enzymes have to be synthesized on cytosol ribosomes.     

Biogenesis of mitochondria 23. The biochemical and genetic characteristics of two different oligomycin resistant mutants ofSaccharomyces cerevisiae under the influence of cytoplasmic genetic modification

Two classes ofSaccharomyces cerevisiae mutants resistant to oligomycin, an inhibitor of mitochondrial membrane bound ATPase are described. Biochemical analysis shows thatin vitro the mitochondrial ATPase of both types of mutant are sensitive to oligomycin.In vivo sensitivity of the mutants to oligomycin can be demonstrated following anaerobic growth of the cells, which grossly alters the mitochondrial membrane and renders the ATPase of the mutants sensitive to oligomycin. It is concluded that the mutation to oligomycin resistance in both mutant types is phenotypically expressed as a change in the mitochondrial membrane. The intact mitochondrial membrane in the wild type cell is freely permeable to oligomycin, whereas the resistant mutant is impermeable to oligomycin; alteration of the mitochondrial membrane during isolation of the organelle or physiological modification of the membranes of the mitochondria by anaerobic growth renders the membranes permeable.These mitochondrial membrane mutants differ in their cross-reference patterns and their genetics. One is resistant to oligomycin only, and behaves like previously reported cytoplasmic mutants. The other shows cross-resistance to inhibitors of mitochondrial protein synthesis as well as to oligomycin; although the mutant appears to arise from a single step mutation its genetic properties are complex and show part-nuclear and part-cytoplasmic characteristics. The implications of the observations are discussed.     

Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and microarray analysis

To gain a better understanding of gene expression in bamboo (Bambusa edulis Murno), we have used a combination of suppressive subtractive hybridization (SSH), microarray hybridization analysis, sequencing, and bioinformatics to identify bamboo genes differentially expressed in a bamboo albino mutant. Ten expressed sequence tags (ESTs) were found to be differentially expressed; these were isolated and sequenced. RT-PCR analysis of these ESTs supported the results of the microarray analysis. Six ESTs that were nucleus-encoded exhibited differential expression patterns in the green wild-type bamboo relative to the albino mutant. These genes (exception being the Rubisco small subunit) were non-photosynthesis-related genes. The development of a specific SSH cDNA library in which most of the chloroplast-encoded or photosynthesis-related genes had been subtracted proved to be useful for studying the function of non-photosynthesis-related genes in the albino bamboo mutants with aberrant chloroplast genome. The combined use of this SSH library with microarray analysis will provide a powerful analytical tool for future studies of the bamboo genome.     

Genetic instability of an oligomycin resistance mutation in yeast is associated with an amplification of a mitochondrial DNA segment.

In the yeast Kluyveromyces lactis, mutations affecting mitochondrial functions are often highly unstable. In order to understand the basis of this genetic instability, we examined the case of an oligomycin resistant mutant. When the mutant was grown in the absence of the drug, the resistance was rapidly lost. This character showed a typical cytoplasmic inheritance. The unstable resistance was found to be associated with the presence of a repetitive DNA in which the repeating unit was a specific segment of the mitochondrial DNA. The amplified molecules were co-replicating with the wild type genome in the mutant cells. The spontaneous loss of the drug resistance was accompanied by the disappearance of the amplified DNA. The repetitive sequence came from a 405 base-pair segment immediately downstream of a cluster of two transfer RNA genes (threonyl 2 and glutamyl). Modified processing of these tRNAs was detected in the mutant. A possible mechanism by which these events could lead to drug resistance is discussed.     

Uth1p is involved in the autophagic degradation of mitochondria

The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation, two conditions that induce the autophagic process. Biochemical studies showed the onset of a fully active autophagic activity both in wild-type and Deltauth1 strains. On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the Deltauth1 strain. Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying null mutations of the vacuolar protease Pep4p, the autophagy-specific protein Atg5p, and Uth1p. These data show that, although the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation of mitochondria.     

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of M?ule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.     

A Method of Determination of Penta-, Tetra-, Tri-, and Disaccharides from Xylan by Ion-exchange Chromatography

Bacillus subtilis B7, a tmrA mutant, shows both tunicamycin resistance and a-amylase hyperproductivity. The tmrA characters can be transferred simultaneously to recipient cells by DNA-mediated transformation. We found a typical gene amplification phenomenon in the tmrA transformants and B7 strain. The amplified unit, 16.3kb in size, covers a-amylase structural gene amyE to another tunicamycin resistance gene tmrB, which is located 9kb downstream of the amyE gene. About 10 repeating units are supposed to be tandemly repeated in the transformants. Amplification of the wild amyE and tmrB genes could be the cause of the α-amylase hyperproductivity and tunicamycin resistance of the tmrA transformants and B7 strain.