Réjean Morais

Sequence and gene organization of the chicken mitochondrial genome: A novel gene order in higher vertebrates

The 16,775 base-pair mitochondrial genome of the white Leghorn chicken has been cloned and sequenced. The avian genome encodes the same set of genes (13 proteins, 2 rRNAs and 22 tRNAs) as do other vertebrate mitochondrial DNAs and is organized in a very similar economical fashion. There are very few intergenic nucleotides and several instances of overlaps between protein or tRNA genes. The protein genes are highly similar to their mammalian and amphibian counterparts and are translated according to the same variant genetic code. Despite these highly conserved features, the chicken mitochondrial genome displays two distinctive characteristics. First, it exhibits a novel gene order, the contiguous tRNA(Glu) and ND6 genes are located immediately adjacent to the displacement loop region of the molecule, just ahead of the contiguous tRNA(Pro), tRNA(Thr) and cytochrome b genes, which border the displacement loop region in other vertebrate mitochondrial genomes. This unusual gene order is conserved among the galliform birds. Second, a light-strand replication origin, equivalent to the conserved sequence found between the tRNA(Cys) and tRNA(Asn) genes in all vertebrate mitochondrial genomes sequenced thus far, is absent in the chicken genome. These observations indicate that galliform mitochondrial genomes departed from their mammalian and amphibian counterparts during the course of evolution of vertebrate species. These unexpected characteristics represent useful markers for investigating phylogenetic relationships at a higher taxonomic level.

Nucleotide sequence and evolution of coding and noncoding regions of a quail mitochondrial genome.

SummarySegments of the Japanese quail mito-chondrial genome encompassing many tRNA and protein genes, the small and part of the large rRNA genes, and the control region have been cloned and sequenced. Analysis of the relative position of these genes confirmed that the tRNAGlu and ND6 genes in galliform mitochondrial DNA are located immediately adjacent to the control region of the molecule instead of between the cytochrome b and ND5 genes as in other vertebrates. Japanese quail and chicken display another distinctive characteristic, that is, they both lack an equivalent to the light-strand replication origin found between the tRNACys and tRNAAsn genes in all vertebrate mitochondrial genomes sequenced thus far. Comparison of the protein-encoding genes revealed that a great proportion of the substitutions are silent and involve mainly transitions. This bias toward transitions also occurs in the tRNA and rRNA genes but is not observed in the control region where transversions account for many of the substitutions. Sequence alignment indicated that the two avian control regions evolve mainly through base substitutions but are also characterized by the occurrence of a 57-bp deletion/addition event at their 5′ end. The overall sequence divergence between the two gallinaceous birds suggests that avian mitochondrial genomes evolve at a similar rate to other vertebrate mitochondrial DNAs.

An established avian fibroblast cell line without mitochondrial DNA

AbstracttAn established avian fibroblast cell line (LSCC-H32) has been found to be inherently resistant to the growth-inhibitory effect of ethidium bromide, when supplied with exogenous urdine. After long-term exposure to ethidium bromide (90 days), the cell population has been transferred to drug-free medium for 60 days, and then seeded at low cell density. Three clones have been isolated and propagated in drug-free medium for 5, 6, and more than 12 months, respectively. It was found that none of these cell lines had detectable cytochrome c oxidase activity and that they were virtually devoid of cytochromes aa3 and b. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 300 copies of mitochondrial DNA per cell was found in LSCC-H32 cells. Analysis of DNA extracted from cell populations exposed to ethidium bromide for 90 days and then transferred to drug-free medium for long periods of time revealed no mitochondrial DNA molecules by reassociation kinetics or by Southern blot hybridization of HindIII- or Aval-digested total cellular DNA.

Molecular characterization and evolution of a duck mitochondrial genome

We sequenced 6,478 by of mitochondrial DNA from Peking duck (Anas platyrhyncos). Eight protein genes, 11 tRNAs, part of the small and large ribosomal subunits, and the control region sequences were compared to homologous chicken sequences. The gene organization in duck and chicken is identical but differs from other vertebrates in the juxtaposition of the tRNAGlu-ND6 genes next to the control region and in the lack of a hairpinlike structure between the genes for tRNAAsn and tRNACys used for light-strand replication. Protein, tRNA, and rRNA genes evolved mainly through base substitutions and small insertions and deletions. Transitions greatly outnumber transversions in the tRNA and rRNA genes, but this bias is not evident in protein genes; the control region has a higher proportion of transversions. The duck and chicken control regions show a high frequency of length mutations. Large A-T-rich nucleotide stretches dispersed across the region between the bidirectional transcription promoter and the heavy-strand replication origin in the chicken are absent in the duck. Sequence elements for heavystrand replication in mammals are conserved in the duck and chicken control regions. Estimates of divergence for ribosomal RNAs and proteins based on total substitutions, transversions, and amino acid replacements show that all the duck/chicken values are lower than the corresponding mammal/mammal (cow, human, mouse) values. If paleontological data suggesting that avian and eutherian ordinal radiation occurred at approximately the same time are correct, this suggests that at great evolutionary distance, rate of mitochondrial DNA evolution in birds is somewhat decelerated compared to mammals.

Development and characterization of continuous avian cell lines depleted of mitochondrial DNA

SummaryPopulations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.

Chick embryo cells rendered respiration-deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines

Summary The present results demonstrate that uridine confers on cultured chick embryo cells resistance to the growth inhibitory effect of chloramphenicol and ethidium bromide. Cellular cytochrome oxidase activity is lost suggesting that uridine does not prevent the inhibitory effect of the drugs on mitochondrial transcription and translation. Other than cytidine, none of the precursors and derivatives of uridine tested supports cell growth.

Up-regulation of nuclear genes in response to inhibition of mitochondrial DNA expression in chicken cells.

Vertebrate cells depleted of (rho0) mitochondrial DNA (mtDNA) exhibited phenotypic traits that differed from the parental (rho+) cells. To isolate genes whose expression is associated with mtDNA depletion, we constructed cDNA libraries from mRNAs isolated from chicken rho+ cells transformed by the MC29 (v-myc-containing) retrovirus and from rho0 cells developed by long-term exposure of the rho+ cells to ethidium bromide (EtdBr). Through subtractive hybridization procedures, three genes, elongation factor 1 alpha (EF- 1 alpha), beta-actin and v-myc were identified and found to be up-regulated in rho0 cells. In addition, Northern analysis demonstrated that the mRNA content for GAPDH was also elevated in rho0 cells. Run-on transcription assays and mRNA stability studies in the presence of actinomycin D indicated that elevated expression of these four genes depends, at least in part, upon increased rate of transcription. Other regulatory mechanisms contribute to the elevated expression of the transcripts in rho0 cells, as suggested by cycloheximide enhancement of the accumulation of the mRNAs for EF-1 alpha and beta-actin in rho0 cells, but not in parental rho+ cells. Moreover, inhibition of mtDNA replication and transcription by EtdBr and inhibition of translation on mitoribosomes by chloramphenicol also increased the expression of the four genes in parental rho+ cells, thus mimicking the situation in rho0 cells. These data suggest that information encoded within mtDNA participates in the regulation of nuclear genes in chicken cells.

Autodegradation of ribonucleic acid of rat-liver microsomes

Abstract The autodegradation of the endogenous ribonucleic acid of the microsomal fraction of rat liver was studied. The results indicate that ribonuclease (ribonucleate oligotido-nucleotidohydrolase) and a phosphodiesterase (orthophosphoric diester phosphohydrolase) are present in this fraction. The former is stimulated by phosphate, arsenate and EDTA, and is inhibited by Mg 2+ whereas the latter is activated by phosphate but inhibited by arsenate and EDTA. The autodegradation of RNA reaches a plateau in the presence of phosphate or arsenate and suggests the presence in the microsomal fraction of rat liver of another enzymatic system. Chromatographic analysis of the acid-soluble products of reaction shows that the main products of degradation are oligonucleotides, and nucleosides. The absence of nucleotides in the incubation mixture is due to the presence of nucleotidases.

Intracellular distribution of 5′-ribonuclease and 5′-phosphodiesterase in rat liver☆

Abstract The intracellular distribution of 5′-ribonuclease and 5′-phosphodiesterase was studied in rat liver. Glucose 6-phosphatase and acid phosphatase were also studied as reference enzymes. The results show that the 5′-ribonuclease is essentially a mitochondrial enzyme whereas 5′-phosphodiesterase is present in the nuclear and microsomal fractions. Analysis of the degradation products of the mitochondrial ribonuclease showed that this ribonuclease yields oligonucleotides terminated by 5′-phosphate end-group.

Studies of the Effect of Chloramphenicol, Ethidium Bromide and Camptothecin on the Reproduction of Rous Sarcoma virus in Infected Chick Embryo Cells

A function for mitochondria in the reproduction of Rous sarcoma virus (RSV) in chronically and newly infected chick embryo cells was studied by using chloramphenicol and ethidium bromide. Chloramphenicol (CAM) and ethidium bromide (EB) were both shown to decrease the rate of growth of infected chick embryo cells and to inhibit the synthesis of mitochondrial macromolecules. Both drugs however had little or no effect on the incorporation of labelled leucine, thymidine and uridine into total cellular macromolecules. Neither CAM (80 microgram/ml) nor EB (0.4 microgram/ml) inhibited the production of infectious virus. In contrast, camptothecin, an inhibitor of cellular but not mitochondrial macromolecular synthesis, was shown to depress the production of infectious virus. The results indicate that the mitochondrial macromolecular synthesis machinery of RSV-infected chick embryo cells does not contribute to virus production.