Isolation and Genetic Analysis of Mutant Strains of CHLAMYDOMONAS REINHARDI Defective in Gametic Differentiation

Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-l, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt- or mt+ gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt- gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt+ locus, and fine-structural studies reveal that imp-1 gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.     

Mutants of the Biological Clock in CHLAMYDOMONAS REINHARDI

A genetic analysis of the biological clock in Chlamydomonas reinhardi has been initiated. Of six wild-type strains tested (3 mt(+) and 3 mt(-)), five had periods close to 24 hr whereas one had a 21-hr period. Mutants with altered clock period have been isolated. The periods of 3 of these variant strains are temperature compensated. Genetic crosses involving a long-period mutant suggest that a single gene confers the long-period character, and in general clock-period length seems to be a useful phenotypic measure of alterations in the clock due to genetic differences. One phase mutant was found but its behavior was variable and the phase of the rhythm, relative to a light-dark transition which initiates the rhythm, does not seem to be reliable as a parameter of clock differences. No markers have yet been mapped.     

Genetic hitchhiking,mitonuclear coadaptation,and the origins of mt DNA barcode gaps

DNA barcoding based on mitochondrial (mt) nucleotide sequences is an enigma. Neutral models of mt evolution predict DNA barcoding cannot work for recently diverged taxa, and yet, mt DNA barcoding accurately delimits species for many bilaterian animals. Meanwhile, mt DNA barcoding often fails for plants and fungi. I propose that because mt gene products must cofunction with nuclear gene products, the evolution of mt genomes is best understood with full consideration of the two environments that impose selective pressure on mt genes: the external environment and the internal genomic environment. Moreover, it is critical to fully consider the potential for adaptive evolution of not just protein products of mt genes but also of mt transfer RNAs and mt ribosomal RNAs. The tight linkage of genes on mt genomes that do not engage in recombination could facilitate selective sweeps whenever there is positive selection on any element in the mt genome, leading to the purging of mt genetic diversity within a population and to the rapid fixation of novel mt DNA sequences. Accordingly, the most important factor determining whether or not mt DNA sequences diagnose species boundaries may be the extent to which the mt chromosomes engage in recombination.     

Translocation within the acceptor helix of a major tRNA identity determinant

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl-tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific-a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNA(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.     

Genetic control of amino acid permeability in Neurospora crassa

Lester, Gabriel (Reed College, Portland, Ore.). Genetic control of amino acid permeability in Neurospora crassa. J. Bacteriol. 91:677-684. 1966.-Strains of Neurospora crassa resistant to 4-methyltryptophan (4-MT) were isolated from populations of conidia exposed to ultraviolet light. In genetic crosses, 4-MT resistance behaved as a single-gene difference. Resistance to 4-MT could not be attributed to a relaxation of control of the formation or the activity of the enzymes of tryptophan biosynthesis. Growth studies involving tryptophan auxotrophs carrying the aberrant mt gene and uptake studies with normal and 4-MT-resistant strains showed that 4-MT resistance could be attributed to an inability of 4-MT-resistant strains to take up tryptophan and its methyl analogues. The mt gene is not specific for tryptophan; strains resistant to 4-MT are also resistant to ethionine, and they have a markedly reduced ability to take up serine, leucine, and alpha-aminoisobutyric acid. No difference was observed between strains carrying either mt allele in their ability to take up glucose; also, the uptake of anthranilic acid or of indole was not sufficiently impaired by the aberrant mt gene to prevent these tryptophan precursors from satisfying the nutritional requirement of certain tryptophan auxotrophs. The role of the mt gene in determining the permeability of N. crassa to amino acids is discussed.     

Polymorphisms in the Mitochondrial DNA Control Region and Frailty in Older Adults

Background

Mitochondria contribute to the dynamics of cellular metabolism, the production of reactive oxygen species, and apoptotic pathways. Consequently, mitochondrial function has been hypothesized to influence functional decline and vulnerability to disease in later life. Mitochondrial genetic variation may contribute to altered susceptibility to the frailty syndrome in older adults.

Methodology/Principal Findings

To assess potential mitochondrial genetic contributions to the likelihood of frailty, mitochondrial DNA (mtDNA) variation was compared in frail and non-frail older adults. Associations of selected SNPs with a muscle strength phenotype were also explored. Participants were selected from the Cardiovascular Health Study (CHS), a population-based observational study (1989–1990, 1992–1993). At baseline, frailty was identified as the presence of three or more of five indicators (weakness, slowness, shrinking, low physical activity, and exhaustion). mtDNA variation was assessed in a pilot study, including 315 individuals selected as extremes of the frailty phenotype, using an oligonucleotide sequencing microarray based on the Revised Cambridge Reference Sequence. Three mtDNA SNPs were statistically significantly associated with frailty across all pilot participants or in sex-stratified comparisons: mt146, mt204, and mt228. In addition to pilot participants, 4,459 additional men and women with frailty classifications, and an overlapping subset of 4,453 individuals with grip strength measurements, were included in the study population genotyped at mt204 and mt228. In the study population, the mt204 C allele was associated with greater likelihood of frailty (adjusted odds ratio = 2.04, 95% CI = 1.07–3.60, p = 0.020) and lower grip strength (adjusted coefficient = −2.04, 95% CI = −3.33– −0.74, p = 0.002).

Conclusions

This study supports a role for mitochondrial genetic variation in the frailty syndrome and later life muscle strength, demonstrating the importance of the mitochondrial genome in complex geriatric phenotypes.     

The complete mitochondrial genome of yarrowia lipolytica

We here report the complete nucleotide sequence of the 47.9 kb mitochondrial (mt) genome from the obligate aerobic yeast Yarrowia lipolytica. It encodes, all on the same strand, seven subunits of NADH: ubiquinone oxidoreductase (ND1-6, ND4L), apocytochrome b (COB), three subunits of cytochrome oxidase (COX1, 2, 3), three subunits of ATP synthetase (ATP6, 8 and 9), small and large ribosomal RNAs and an incomplete set of tRNAs. The Y. lipolytica mt genome is very similar to the Hansenula wingei mt genome, as judged from blocks of conserved gene order and from sequence homology. The extra DNA in the Y. lipolytica mt genome consists of 17 group 1 introns and stretches of A+Trich sequence, interspersed with potentially transposable GC clusters. The usual mould mt genetic code is used. Interestingly, there is no tRNA able to read CGN (arginine) codons. CGN codons could not be found in exonic open reading frames, whereas they do occur in intronic open reading frames. However, several of the intronic open reading frames have accumulated mutations and must be regarded as pseudogenes. We propose that this may have been triggered by the presence of untranslatable CGN codons. This sequence is available under EMBL Accession No. AJ307410.     

Phylogenetic and taxonomic incongruence involving nuclear and mitochondrial markers in Korean populations of the freshwater snail genus Semisulcospira (Cerithioidea: Pleuroceridae)

We investigated the population genetic structure and phylogenetic relationships of four morphospecies of Semisulcospira sampled from multiple South Korean drainages. One, S. extensa, displayed modest levels of genetic diversity and formed a species-specific clade for both mitochondrial (mt) and nuclear markers. In contrast, the other three, S. coreana, S. gottschei, and S. libertina, were polyphyletic for both mt and nuclear markers. They formed, together with other nominal congeners (S. forticosta, S. multicincta, S. nodiperda, and S. tegulata), a taxonomically heterogeneous species complex containing population-level admixtures of genotypes from numerically predominant modal clades in addition to rare, phylogenetically divergent, mt and nuclear genotypes. The modal mt haplotypes exhibited far more geographic than taxonomic congruence and typically co-clustered into drainage-specific tip clades, irrespective of nominal taxonomic status. The evolutionary origins of the observed non-extensa phylogenetic heterogeneity are unclear at present although the available data do not support paralogous mt marker phenomena or the presence of cryptic species. We cannot distinguish among retention of ancestral polymorphisms or recticulate evolutionary origins as explanatory mechanisms and it may require the genetic characterization of Semisulcospira lineages throughout much of their collective east Asian range to address this issue. Based on the available data, we consider it best to view all of our non-extensa Korean study taxa as a single polymorphic species complex of the type species S. libertina. There is considerable evidence that similarly complex genetic structuring, at least for mt, may be typical of many other freshwater cerithioidean snail taxa. In light of our Semisulcospira results, we suggest that meaningful molecular phylogenetic characterization of freshwater cerithioidean lineages may require the use of both mt and nuclear markers together with population level sampling of all nominal taxa within regional drainages.     

Down-regulation of the mitochondrial translation system during terminal differentiation of HL-60 cells by 12-O-tetradecanoyl-1-phorbol-13-acetate: comparison with the cytoplasmic translation system

Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly down-regulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPA-induced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation (in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.     

A mitochondrial phosphatase required for cardiolipin biosynthesis: the PGP phosphatase Gep4

Cardiolipin (CL), a unique dimeric phosphoglycerolipid predominantly present in mitochondrial membranes, has pivotal functions for the cellular energy metabolism, mitochondrial dynamics and the initiation of apoptotic pathways. Perturbations in the mitochondrial CL metabolism cause cardiomyopathy in Barth syndrome. Here, we identify a novel phosphatase in the mitochondrial matrix space, Gep4, and demonstrate that it dephosphorylates phosphatidylglycerolphosphate to generate phosphatidylglycerol, an essential step during CL biosynthesis. Expression of a mitochondrially targeted variant of Escherichia coli phosphatase PgpA restores CL levels in Gep4‐deficient cells, indicating functional conservation. A genetic epistasis analysis combined with the identification of intermediates of CL biosynthesis allowed us to integrate Gep4 in the CL‐biosynthetic pathway and assign an essential function during early steps of CL synthesis to Tam41, which has previously been shown to be essential for the maintenance of normal CL levels. Our experiments provide the framework for the further dissection of mechanisms that are required for accumulation and maintenance of CL levels in mitochondria.     

Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.     

Evolution of multipartite mitochondrial genomes in the booklice of the genus Liposcelis (Psocoptera)

Background

The genus Liposcelis (Psocoptera: Troctomorpha) has more than 120 species with a worldwide distribution and they pose a risk for global food security. The organization of mitochondrial (mt) genomes varies between the two species of booklice investigated in the genus Liposcelis. Liposcelis decolor has its mt genes on a single chromosome, like most other insects; L. bostrychophila, however, has a multipartite mt genome with genes on two chromosomes.

Results

To understand how multipartite mt genome organization evolved in the genus Liposcelis, we sequenced the mt genomes of L. entomophila and L. paeta in this study. We found that these two species of booklice also have multipartite mt genomes, like L. bostrychophila, with the mt genes we identified on two chromosomes. Numerous pseudo mt genes and non-coding regions were found in the mt genomes of these two booklice, and account for 30% and 10% respectively of the entire length we sequenced. In L. bostrychophila, the mt genes are distributed approximately equally between the two chromosomes. In L. entomophila and L. paeta, however, one mt chromosome has most of the genes we identified whereas the other chromosome has largely pseudogenes and non-coding regions. L. entomophila and L. paeta differ substantially from each other and from L. bostrychophila in gene content and gene arrangement in their mt chromosomes.

Conclusions

Our results indicate unusually fast evolution in mt genome organization in the booklice of the genus Liposcelis, and reveal different patterns of mt genome fragmentation among L. bostrychophila, L. entomophila and L. paeta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-861) contains supplementary material, which is available to authorized users.     

A digenic cause of cleft lip in A-strain mice and definition of candidate genes for the two loci

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate, CL(P), is a common human birth defect with a complex unknown genetic cause. The mouse model is the "A/-" strains. Our previous studies mapped two loci: clf1 on Chr11 and clf2 on Chr13--with a strong genetic maternal effect on the level of risk. Here we test the hypothesis that CL(P) is digenic and identify candidate genes for clf1 and clf2. METHODS: We observed E14 CL(P) frequencies in backcross (BC1) embryos from a new cross of A/WySn to AXB-4/Pgn and from test crosses of three new "congenic RI" lines. Using new polymorphic markers from genes and our mapping panels of segregants and RI strains, we identified the candidate genes for clf1 and clf2. We sequenced the coding region of Ptch in A/WySn cDNA. RESULTS: Seventy new BC1 CL(P) segregants (4%) were obtained, as predicted. All three new congenic RI lines homozygous for both clf1 and clf2 had A/WySn-level CL(P) frequencies (10-30%) in test crosses. The clf1 region contains 10 known genes (Arf2, Cdc27, Crhr1, Gosr2, Itgb3, Mapt, Myl4, Nsf, Wnt3, and Wnt9b). The clf2 region contains 17 known genes with human orthologs. Both regions contain additional potential genes. No causal mutation in Ptch coding sequence was found. CONCLUSIONS: In A-strain mice, nonsyndromic CL(P) is digenic, suggesting that nonsyndromic human CL(P) may also be digenic. The orthologous human genes are on 17q (clf1) and 9q, 8q and 5p (clf2), and good candidate genes are WNT3 or WNT9B (17q), and PTCH (9q) or MTRR (5p).     

Mitochondrial DNA polymorphisms are associated with the longevity in the Guangxi Bama population of China

Human longevity is an interesting and complicated subject, with many associated variations, geographic and genetic, including some known mitochondrial variations. The population of the Bama County of Guangxi Province of China is well known for its longevity and serves as a good model for studying a potential molecular mechanism. In this study, a full sequence analysis of mitochondrial DNA (mtDNA) has been done in ten Bama centenarians using direct sequencing. Polymorphisms of the displacement loop (D-loop) region of mtDNA and several serum parameters were analyzed for a total of 313 Bama individuals with ages between 10 and 110 years. The results showed that there were seven mitochondrial variations, A73G, A263G, A2076G, A8860G, G11719A, C14766T, and A15326G, and four haplogroups, M(*), F1, D* and D(4) in 10 Bama centenarians. In the D-loop region of mtDNA, the mt146T occurred at a significantly lower frequency in those is the older age group (90-110 years) than in the middle (80-89 years) and in the younger (10-79 years) groups (P < 0.05). The mt146T also had lower systolic blood pressure and serum markers such as total cholesterol, triglyceride and low density lipoprotein than did mt146C in the older age group (P < 0.05). No significant differences were observed between the mt146C and the mt146T individuals in the middle and the younger groups (P > 0.05). The mt5178C/A polymorphisms did not show any significant differences among the three age-groups (P > 0.05), but different nationalities in the Bama County did show a significant difference in the mt5178C/A polymorphisms (P < 0.05). These results suggest that the mt146T/C polymorphisms in Guangxi Bama individuals may partly account for the Bama longevity whereas the mt5178C/A polymorphisms are strongly associated with the nationalities in the Guangxi Bama population.     

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses

Background

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change.

Methodology/Principal Findings

We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses.

Conclusions/Significance

Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.     

Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.