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Biomedical subjects

N W Gillham

Publications and source records attributed to N W Gillham.

At least 73 records · Page 4Linked to original sources
PubMed1977

Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine.

5-Fluorodeoxyuridine selectively decreases the rate of chloroplast DNA replication in Chlamydomonas resulting after several generations of growth in equilibrium levels as low as one-seventh of normal. When the maternal parent is treated prior to mating, the decrease of chloroplast DNA appears to perturb the normal maternal transmission of chloroplast genes, dramatically increasing the proportion of exceptional zygotes transmitting chloroplast genes from the paternal parent.

Journal Article↗
PubMed1977

Nuclear mutations affecting mitochondrial structure and function in Chlamydomonas.

Wild type cells of the green alga Chlamydomonas reinhardtii can grow in the in the dark by taking up and respiring exogenously supplied acetate. Obligate photoautotrophic (dark dier, dk) mutants of this alga have been selected which grow at near wild type rates in the light, but rapidly die when transferred to darkness because of defects in mitochondrial structure and function. In crosses of the dk mutants to wild type, the majority of the mutants are inherited in a mendelian fashion, although two have been isolated which are inherited in a clearly nonmendelian fashion. Nine mendelian dk mutants have been analyzed in detail, and belong to eight different complementation groups representing eight gene loci. These mutants have been tentatively grouped into three classes on the basis of the pleiotropic nature of their phenotypic defects. Mutants in Class I have gross alterations in the ultrastructure of their mitochondrial inner membranes together with deficiencies in cytochrome oxidase and antimycin/rotenone-sensitive NADH-cytochrome c reductase activities. Mutants in Class II have a variety of less severe alterations in mitochondrial ultrastructure and deficiencies in cytochrome oxidase activity. Mutants in Class III have normal or near normal mitochondrial ultrastructure and reduced cytochrome oxidase activity. Eight of the nine mutants show corresponding reductions in cyanide-sensitive respiration.

Alleles↗
PubMed1977

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii.

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.

Acetates↗
PubMed1975

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.

Chlamydomonas↗
PubMed1975

Genetic control of chlorophyll biosynthesis in chlamydomonas: analysis of a mutant affecting synthesis of delta-aminolevulinic acid.

A Mendelian mutation, r-1, in Chlamydomonas reinhardtii has been isolated which elevates protoporphyrin accumulation of the Mendelian protoporphyrin mutants brS-1 and brC-1 more than 20 fold. This increased protoporphyrin accumulation is shown to result from increased delta-aminolevulinic acid synthesis in the double mutants brS-1 r-1 and brC-1 r-1 over that of brS-1 and brC-1 alone. By itself, the r-1 mutation has no detectable protoporphyrin accumulation and has reduced levels of delta-aminolevulinic acid synthesizing activity, chlorophyll, protoheme, and cytochrome oxidase activity. The low levels of chlorophyll and protoheme in r-1 can be increased by feeding delta-aminolevulinic acid. We hypothesize that r-1 may be a mutation of the gene coding for the delta-aminolevulinic acid synthesizing enzyme which reduces the sensitivity of this enzyme to feedback inhibition by protoporphyrin or heme as well as reducing the overall activity of the enzyme. Evidence is also presented for a single delta-aminolevulinic acid synthesizing enzyme serving both chlorophyll and heme biosynthesis.

5-Aminolevulinate Synthetase↗
PubMed1974

Chloroplast ribosome biogenesis in Chlamydomonas. Selection and characterization of mutants blocked in ribosome formation.

Chloroplast protein synthesis in Chlamydomonas reinhardtii is dispensable when cells are provided acetate as a carbon source. Mutants defective in synthesis, assembly, or function of chloroplast ribosomes are therefore conditionally viable. Positive selection of nonphotosynthetic cells on arsenate has been combined with a simple screening procedure to isolate mutants with a broad spectrum of defects in chloroplast protein synthesis. Eight new mutants deficient in chloroplast ribosomes have been isolated. Three of these have been characterized genetically and phenotypically, and compared with two previously described ribosome mutants, ac-20 and cr-1. A working model of ribosome assembly is proposed which suggests possible biochemical roles for these five Mendelian gene loci.

Acetates↗
PubMed1974

Genetic control of chlorophyll biosynthesis in Chlamydomonas. Analysis of mutants at two loci mediating the conversion of protoporphyrin-IX to magnesium protoporphyrin.

In this report we describe two nonallelic Mendelian protoporphyrin accumulating mutants br(s)-1 and br(c)-1. Results of experiments with these mutants lead us to postulate that porphyrin biosynthesis branches into light and dark steps between protoporphyrin-IX and magnesium protoporphyrin. We hypothesize that the br(c) locus controls a dark step while the br(s) locus either controls a step in the main pathway before the branch or mediates the preparation of the magnesium ion for its insertion into protoporphyrin-IX. The br(s)-1 mutant is thought to be light sensitive because a block prior to the branch point in the porphyrin pathway prevents chlorophyll formation in either the light or the dark. The br(c)-1 mutant, which also accumulates protoporphyrin in the dark, forms chlorophyll and chloroplast lamellae when transferred to the light, showing that function of the porphyrin pathway is normal in the light.

Cell Count↗
PubMed1974

Segregation and recombination of non-Mendellan genes in Chlamydomonas.

Non-Mendelian genes in Chamydomonas reinhardtii are inherited in a uniparental (UP) fashion. Most zygotes and their progeny receive UP genes only from the mt(+) or maternal parent. However, a few exceptional zygotes are also found in which the mt(-) or paternal UP genome is transmitted. Most of the exceptional zygotes are biparental in that their progeny segregate UP genes transmitted by both parents. As a result, biparental zygotes have been extensively used to study the rules governing UP inheritance. The frequency of biparental zygotes can be greatly increased if the maternal parent is irradiated with ultraviolet light prior to mating. Based principally on studies with ultraviolet-induced biparental zygotes, Sager has argued that a vegetative cell contains two copies of the UP genome and that the progeny of a biparental zygote receive a copy derived from each parent. Results reported in this paper with spontaneous and ultraviolet-induced biparental zygotes do not support the two copy model, but argue for a mulitple copy model with most of the copies normally being transmitted by the maternal parent. A multiple copy model which accounts for both Sager's results and ours is presented.

Alleles↗
PubMed1973

Can a non-Mendelian mutation affect both chloroplast and mithchondrial ribosomes?

Chloroplast ribosomes isolated from a spectinomycin-resistant mutant (spr-1-27-3) of Chlamydomonas reinhardtii that displays non-Mendelian inheritance fail to bind labeled antibiotic, in contrast to ribosomes from wild-type cells. In vitro resistance of this mutant appears to result from the absence of a specific protein in the small subunit of the chloroplast ribosome. However, chloroplast protein synthesis in the mutant and wild type shows identical sensitivity to spectinomycin in short-term in vivo experiments where ribulosediphosphate carboxylase serves as the marker. Long-term experiments demonstrate that the mutant can grow in the presence of spectinomycin only when acetate is supplied as a carbon source. Mitochondrial structure and function of the mutant are not affected by the antibiotic, whereas chloroplast structure and function are. Apparently, the mitochondrion, rather than the chloroplast, of this mutant is resistant to spectinomycin in vivo. We hypothesize that the gene product of the spr locus is a protein common to both chloroplast and mitochondrial ribosomes. The mutant gene product, in vivo, confers resistance on mitochondrial, but not chloroplast, ribosomes. We suppose that the mutant spr protein loosely attaches to chloroplast ribosomes in vivo so that the antibiotic is bound and blocks protein synthesis, but it dissociates during isolation, resulting in loss of the binding site.

Binding Sites↗
PubMed1971

Organelle mutations and their expression in Chlamydomonas reinhardi.

A new method for classifying Mendelian and non-Mendelian antibiotic-resistant mutants in Chlamydomonas reinhardi is proposed. This classification takes into account the cellular site of expression of the resistant phenotype, the location of genetic information for resistance in the cell, and the pattern of inheritance of the different mutants. The classification is consistent with the idea that C. reinhardi contains three protein-synthesizing systems, two of which are sensitive to antibacterial antibiotics and located in the chloroplast and mitochondria, respectively. The information for resistance can be coded either in the chloroplast, the mitochondria, or the nucleus. A new experimental approach to the problem of cellular localization of mutations to antibiotic resistance is prescribed and used for the classification of known resistant mutations.

Anti-Bacterial Agents↗
PubMed1970

Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, II. A New Mendelian Mutation.

A new mutation of Chlamydomonas reinhardi, cr-1, is characterized. The mutation exhibits Mendelian inheritance and affects the sedimentation velocity and formation of intact chloroplast ribosomes. The mutant grows reasonably well when supplied with sodium acetate as a carbon source, but poorly when forced to grow photosynthetically using carbon dioxide. Since the mutant cr-1 accumulates large subunits of the chloroplast ribosome, we postulate that it is blocked in the formation of the small subunit. A tentative model explaining the behavior of the several mutants in Chlamydomonas now known to have altered chloroplast ribosomal phenotypes is presented.

Journal Article↗
PubMed1968

Recombination of nonchromosomal mutations: a 3-point cross in the green alga Chlamydomonas reinhardi.

The heterothallic, unicellular green alga Chlamydomonas reinhardi possesses both chromosomal and nonchromosomal systems of inheritance. Mutations belonging to the latter system are capable of recombination. The experiments reported show that double and triple recombinants can be obtained from crosses employing three phenotypically distinct mutations and that certain pairs of mutants recombine more freely than others.

Crosses, Genetic↗