The Mechanisms of Codon Reassignments in Mitochondrial Genetic Codes

Many cases of non-standard genetic codes are known in mitochondrial genomes. We carry out analysis of phylogeny and codon usage of organisms for which the complete mitochondrial genome is available, and we determine the most likely mechanism for codon reassignment in each case. Reassignment events can be classified according to the gain-loss framework. The gain represents the appearance of a new tRNA for the reassigned codon or the change of an existing tRNA such that it gains the ability to pair with the codon. The loss represents the deletion of a tRNA or the change in a tRNA so that it no longer translates the codon. One possible mechanism is Codon Disappearance, where the codon disappears from the genome prior to the gain and loss events. In the alternative mechanisms the codon does not disappear. In the Unassigned Codon mechanism, the loss occurs first, whereas in the Ambiguous Intermediate mechanism, the gain occurs first. Codon usage analysis gives clear evidence of cases where the codon disappeared at the point of the reassignment and also cases where it did not disappear. Codon disappearance is the probable explanation for stop to sense reassignments and a small number of reassignments of sense codons. However, the majority of sense to sense reassignments cannot be explained by codon disappearance. In the latter cases, by analysis of the presence or absence of tRNAs in the genome and of the changes in tRNA sequences, it is sometimes possible to distinguish between the Unassigned Codon and Ambiguous Intermediate mechanisms. We emphasize that not all reassignments follow the same scenario and that it is necessary to consider the details of each case carefully.Comment: 53 pages (45 pages, including 4 figures + 8 pages of supplementary information). To appear in J.Mol.Evo

Global Genome Responses to DNA-Repair Deficiency Modulate Aging and Stress Response Pathways

The genomes of all animals are constantly challenged by exogenous and endogenous sources of DNA damaging agents. UV radiation, chemicals, pollutants, and by-products of the cells’ own metabolism may damage the genetic material. Such damages are harmful to the animal as they may cause mutations or generate cytotoxic lesions, which in turn may lead to disease, cancer and aging. Protection of the genome is therefore of the utmost importance. To counteract such potential detrimental effects, all organisms have developed protective mechanisms such as antioxidants and DNA repair mechanisms. DNA excision repair proteins detect lesions in DNA, excise the damaged base and re-insert a correct base, thus maintaining the correct coding properties of the genome. Defects in DNA repair mechanisms may lead to cancer, neurodegeneration, other age-related pathologies or senescence. The nematode Caenorhabditis elegans (C. elegans) contains very few DNA glycosylases, which are the lesion-detecting proteins in DNA excision repair, compared to other animals and organisms. Analysis of all transcribed genes in DNA repair-deficient mutants in C. elegans revealed a global transcriptional response aimed at minimizing further damage to the genome. This involved a down-regulation of insulin-like signaling and an upregulation of antioxidants and stress response genes, similar to the response seen in both long-lived and old animals. This response seems to be conserved across different species as analysis of comparable mutants in the yeast Saccharomyces cerevisiae and mouse showed a similar response. Pathway reconstruction and literature mining suggests that this response is not elicited only by lack of repair per se, but rather from aberrant or attempted processing of lesions by other repair pathways than those normally repairing such lesions. This result in lesions that block the transcription of active genes and signal the transcription of other genes aimed at reducing further damage to DNA. Analysis of C. elegans mutants deficient in two different repair pathways revealed a completely different response with downregulation of Aurora-B and Polo-like kinase 1 signaling networks as well as downregulation of other DNA repair pathways. The mechanism and signaling origin of this response is yet unknown. Gene expression profiling is emerging as a powerful complementary tool to classical genetics and molecular analysis. By taking a systems biology approach, which takes into account the interplay between many pathways, gene expression profiling may aid in the interpretation of observed phenotypes and assist in the generation of new testable hypotheses

Gene duplications during experimental evolution of Caenorhabditis elegans : duplication rates and evolutionary responses

Copy-number variants (CNVs) are a ubiquitous form of genetic variation. How often this form of variation arises and its adaptive significance are active areas of contemporary research. This work presents evidence regarding both of these subjects. First, it demonstrates that gene duplications occur at a frequency two orders of magnitude greater than point mutations. Specifically, the gene duplication rate is estimated to be 1.2 x 10-7/gene/generation, compared to a point mutation rate on the order of ~10-9/site/ generation. Second, it was found that populations in a low state of fitness due to mutation accumulation could recover some or all of their fitness over short spans of generations concurrent with an increase in frequency of duplications and deletions that arose during the recovery process. The pattern of frequency increase among CNVs over generations during recovery was consistent with the signature of positive selection. The median size of duplications that were identified after selection for ~200 generations were significantly larger (191.5 kb) than both duplications that occurred spontaneously (2 kb) in the absence of selection and deletions identified after selection for ~200 generations (12.5 kb). The median number of genes contained in the duplications during recovery was 38, evincing the ability of these events to increase the genetic information available for selection to act on. These results clearly demonstrate that gene duplication and deletion processes contribute significantly to the adaptability of populations

Phenotypic and transcriptomic consequences of ribosomal DNA copy number variation in Caenorhabditis elegans and Saccharomyces cerevisiae

Ribosomal DNA (rDNA) forms the template for mature ribosomal RNA (rRNA) and shows considerable sequence conservation through evolution. In eukaryotic genomes rDNA is present in one or more tandem repeat arrays, with copy number variation (CNV) commonly observed in inter- and intra-species comparisons. rDNA CNV has been primarily studied in the budding yeast Saccharomyces cerevisiae, where an environmentally sensitive copy number amplification mechanism corrects for repeat loss from this inherently unstable locus. Furthermore, there is much evidence implicating rDNA instability and production of extrachromosomal rDNA circles (ERCs) as causative processes in yeast replicative ageing. By contrast, the functional consequences of rDNA CNV have been largely understudied in metazoans and as such rDNA CNV represents a source of poorly understood genomic variation. I undertook this project to investigate possible functional and transcriptomic consequences of rDNA CNV in an animal model, the nematode Caenorhabditis elegans. Additionally, I produced and analysed RNA-seq datasets studying the transcriptomic effects of rDNA CNV, either due to genomic CNV or ERC accumulation, in S. cerevisiae. After developing methods for assaying rDNA copy number in C. elegans by pulsed field gel electrophoresis (PFGE) and quantitative PCR, I determined that there was notable variation between and within laboratory strains. Using a genetic crossing strategy, I derived strains believed to be essentially isogenic with either wild type (WT) or approximately 2.5-fold amplified rDNA. A variety of phenotypic assays found generally small effect sizes for differences unambiguously attributed to rDNA CNV. Some experiments were, however, confounded by unexpected developmental differences and strain pathology, likely the consequence of unidentified background genetic variation. Transcriptomic analyses were consistent with these observations and revealed some genes differentially expressed between samples with different rDNA CN at either young or aged timepoints. Comparison of log-phase yeast samples with divergent rDNA CN similarly revealed few differentially expressed genes, although gene ontology enrichment analysis suggested effects on specific processes such as ribosome biogenesis. I also generated and analysed an ageing RNA-seq dataset which demonstrated that rDNA CNV in the form of ERC accumulation has significant effects on the ageing transcriptome. ERC accumulation influences the expression of hundreds of genes with strong enrichment for several previously identified age-related processes. Finally, using Northern blotting, I identified a common set of changes in rRNA processing intermediates, reminiscent of stress-related effects, which occur with ageing in variety of genetic backgrounds

Discovering circadian clocks in microbes

We humans experience the influence of our circadian clock every day. This clock mechanism causes, for example, a jet lag during transatlantic air travel. We now believe that almost all organisms have developed a circadian clock mechanism.In this thesis I describe the analysis techniques we developed and the newly discovered molecular components of a circadian mechanism in Saccharomyces cerevisiae and Bacillus subtilis. To identify these molecular components, I applied structured zeitgebers, i.e. light and temperature cycling, to yeast and bacillus cultures. All this in conjunction with bioinformatic in-silico approachesIn Bacillus biofilm populations, we found a free-running rhythm of ytvA and KinC activity of nearly 24 hours after entrainment and release to constant dark and temperature conditions. The free-running oscillations are temperature compensated. This is one of the most important features of a circadian clock mechanism, making it very likely that such a system exists in B. subtilis.We found in yeasts that temperature appears to mainly regulate metabolic processes. Light appears to act more indirectly via photo-oxidation of mitochondrial cytochromes.Finally, I present a hypothetical model for an integrated circadian clock mechanism in unicellular microbes with an emphasis on S. cerevisiae. This mechanism involves several metabolic pathways and the main regulator is the stress sensitive transcriptional activator Msn2p. The model shows that in the circadian clock mechanism in yeast, energy metabolism appears to be an important theme. Other processes that are relevant: metabolic process of nitrogen compounds, oxidation-reduction process and fatty acid metabolism. All could serve as a starting point for further research on the circadian clock in yeast

Genome-wide characterization of the Complex Trancriptome Architecture of S.cerevisiae with tiling arrays

Recent genome-wide transcriptome analysis in humans, Drosophila, Arabidopsis and yeast challenged the old notion of fundamental aspects of gene regulation, providing evidence that protein-encoding genes are not the only agents controlling cellular processes. Non-coding RNAs comprising untranslated regions of protein coding genes, antisense transcripts of annotated genes, micro RNAs and small interfering RNAs present another tier in gene regulation, enabling integration and networking of complex suites of gene activity. Sophisticated RNA signaling networks operate in higher eukaryotes, enabling gene to gene communication and regulation of chromatin structure, DNA methylation, transcription, translation, RNA silencing and stability, and coordinate multiple tasks of the whole cellular system. Fundamental mechanisms and structure of such control architecture remained largely obscure due to limitations of available approaches, such as noise in the data, strand–unspecific transcription analysis and difficulties in functional follow-up opportunities in higher eukaryotes. To address the complexity of transcriptome architecture we undertook the genome-wide transcriptome study in a simpler genome of S.cerevisiae with the help of a new tiling array. We have shown that 85% of the genome is expressed in rich media. Apart from expected transcripts, we found operon-like transcripts, transcripts from neighboring genes not separated by intergenic regions, and genes with complex transcriptional architecture where different parts of the same gene are expressed at different levels. We mapped the positions of 3' and 5' UTRs of coding genes and identified hundreds of RNA transcripts distinct from annotated genes. These non-annotated transcripts, on average, have lower sequence conservation and lower rates of deletion phenotype than protein coding genes. Many other transcripts overlap known genes in antisense orientation, and for these pairs global correlations were discovered: UTR lengths correlated with gene function, localization, and requirements for regulation; antisense transcripts overlapped 3' UTRs more than 5' UTRs; UTRs with overlapping antisense tended to be longer; and the presence of antisense associated with gene function. Overall our study revealed complexity of yeast transcriptional architecture calling for additional annotation of the genome and putting forward an important role for RNA-mediated regulation. An attractive model for the study of the genome-wide RNA-mediated regulation of gene activity in yeast is mitotic cell cycle, which has been extensively studied over past decade and is therefore a well characterized system. Mitosis is associated with important physiological changes in the cell and diverse biological events depend on this periodicity. To ensure the proper functioning of the mechanisms that maintain order during cell division about 800 genes of diverse GO categories are coordinately regulated in a periodic manner coincident with the cell cycle. This includes genes involved in DNA replication, budding, glycosylation, nuclear division, control of mRNA transcription, responsiveness to external stimuli and subcellular localization of proteins. Several genome-wide studies have been done to catalogue cell cycle-regulated genes with the help of early expression arrays. Given the high resolution of our technique, profiling genome-wide periodic expression with the tiling arrays allowed taking a step forward to prove the existence of RNA-mediated regulation of transcription. Using two methods of synchronization, I have monitored cell-cycle dependent transcription for more than 3 complete cell cycles. I have identified about ~600 periodic ORFs. In consent with previous studies on transcriptional regulation during specific mitotic phases, I have shown prevalence of periodic expression of annotated genes in three distinct periods of cell cycle progression: late G1/S transition, G2/M transition and exit of M phase of mitosis. Moreover, I have shown antisense transcription throughout the cell cycle phases. Out of ~260 antisense transcripts that we discovered, 37 display periodic patterns; half of them are expressed coincidentally with peak expression intensity of cell cycle-regulated ORFs, whereas the other half peaks at the periods of relaxation of the transcriptional machinery, which drives phase transition. Cycling antisense has been registered opposite several important cell cycle regulators. Additionally, periodic novel isolated transcripts were detected in the dataset, which may represent non-annotated ncRNAs involved in regulation of mitosis or regulated by cell cycle controlling genes

Meiosis

Meiosis, the process of forming gametes in preparation for sexual reproduction, has long been a focus of intense study. Meiosis has been studied at the cytological, genetic, molecular and cellular levels. Studies in model systems have revealed common underlying mechanisms while in parallel, studies in diverse organisms have revealed the incredible variation in meiotic mechanisms. This book brings together many of the diverse strands of investigation into this fascinating and challenging field of biology