The origins of replication of the mitochondrial genome of yeast

Recent investigations have provided information on the origin of replication of the mitochondrial genome of yeast and an explanation for the phenomenon of the suppressivity.     

The orir to ori+ mutation in spontaneous yeast petites is accompanied by a drastic change in mitochondrial genome replication

The orir petite mutants of Saccharomyces cerevisiae show a very low level of suppressivity (5-12%; suppressivity is the percentage of diploid petites issued from a cross of the parental haploid petite with a wild-type cell), indicating a poor replication efficiency of their mitochondrial genome. The latter is made up of repeat units containing two inverted ori sequences and arranged as tandem pairs in inverted orientation relative to their nearest neighbors. After subcloning orir petites or crossing with wild-type cells a large number of ori+ petites are found in the progeny. In contrast to the orir petites, from which they are derived, these ori+ petites are characterized by high suppressivity levels (approx. 90%) and contain mitochondrial genomes made up of tandem repeat units containing single ori sequences. The structural changes underlying the orir to ori+ mutation are therefore accompanied by a dramatic increase in suppressivity, indicating that the elimination of inverted ori sequences causes a drastic change from very poor to very good replicative efficiency in the mitochondrial genome. Finally, crosses of ori0 petites with wild-type cells were also studied; the results obtained have clarified the reasons for the high frequency of petites having genomes similar to those of orir petites after mutagenesis with ethidium bromide.     

The nucleoprotein is required for efficient coronavirus genome replication

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.     

Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast

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Whole genome sequences reveal the Xanthomonas perforans population is shaped by the tomato production system

Modern agricultural practices increase the potential for plant pathogen spread, while the advent of affordable whole genome sequencing enables in-depth studies of pathogen movement. Population genomic studies may decipher pathogen movement and population structure as a result of complex agricultural production systems. We used whole genome sequences of 281 Xanthomonas perforans strains collected within one tomato production season across Florida and southern Georgia fields to test for population genetic structure associated with tomato production system variables. We identified six clusters of X. perforans from core gene SNPs that corresponded with phylogenetic lineages. Using whole genome SNPs, we found genetic structure among farms, transplant facilities, cultivars, seed producers, grower operations, regions, and counties. Overall, grower operations that produced their own transplants were associated with genetically distinct and less diverse populations of strains compared to grower operations that received transplants from multiple sources. The degree of genetic differentiation among components of Florida’s tomato production system varied between clusters, suggesting differential dispersal of the strains, such as through seed or contaminated transplants versus local movement within farms. Overall, we showed that the genetic variation of a bacterial plant pathogen is shaped by the structure of the plant production system.Subject terms: Applied microbiology, Population genetics, Microbial ecology, Microbial ecology     

The role of mutational dynamics in genome shrinkage

Genome shrinkage occurs after whole genome duplications (WGDs) and in the evolution of parasitic or symbiotic species. The dynamics of this process, whether it occurs by single gene deletions or also by larger deletions are however unknown. In yeast, genome shrinkage has occurred after a WGD. Using a computational model of genome evolution, we show that in a random genome single gene deletions cannot explain the observed pattern of gene loss in yeast. The distribution of genes deleted per event can be very well described by a geometric distribution, with a mean of 1.1 genes per event. In terms of deletions of a stretch of base pairs, we find that a geometric distribution with an average of 500-600 base pairs per event describes the data very well. Moreover, in the model, as in the data, gene pairs that have a small intergenic distance are more likely to be both deleted. This proves that simultaneous deletion of multiple genes causes the observed pattern of gene deletions, rather than deletion of functionally clustered genes by selection. Furthermore, we found that in the bacterium Buchnera aphidicola larger deletions than in yeast are necessary to explain the clustering of deleted genes. We show that the excess clustering of deleted genes in B. aphidicola can be explained by the clustering of genes in operons. Therefore, we show that selection has little effect on the clustering of deleted genes after the WGD in yeast, while it has during genome shrinkage in B. aphidicola.     

The genome maintenance factor Mgs1 is targeted to sites of replication stress by ubiquitylated PCNA

Mgs1, the budding yeast homolog of mammalian Werner helicase-interacting protein 1 (WRNIP1/WHIP), contributes to genome stability during undisturbed replication and in response to DNA damage. A ubiquitin-binding zinc finger (UBZ) domain directs human WRNIP1 to nuclear foci, but the functional significance of its presence and the relevant ubiquitylation targets that this domain recognizes have remained unknown. Here, we provide a mechanistic basis for the ubiquitin-binding properties of the protein. We show that in yeast an analogous domain exclusively mediates the damage-related activities of Mgs1. By means of preferential physical interactions with the ubiquitylated forms of the replicative sliding clamp, proliferating cell nuclear antigen (PCNA), the UBZ domain facilitates recruitment of Mgs1 to sites of replication stress. Mgs1 appears to interfere with the function of polymerase δ, consistent with our observation that Mgs1 inhibits the interaction between the polymerase and PCNA. Our identification of Mgs1 as a UBZ-dependent downstream effector of ubiquitylated PCNA suggests an explanation for the ambivalent role of the protein in damage processing.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.     

The human SDF1 gene polymorphism is located on a mutational hot spot that was identified by the hominoid genome study.

Nucleotide sequences of a part of the stromal cell-derived factor-1 (SDF-1) gene 3' untranslated region were studied among hominoids (chimpanzees, gorillas, orangutans and gibbons). An identical sequence to the human SDF1-3'G allele was found in chimpanzees and gibbons, whereas that to the 3'A allele was found in gorillas. Based on the sequence data and the hominoid phylogenetic relation, it was suggested that an adenine nucleotide at nucleotide position (np) 801 in humans and gorillas was independently introduced into each lineage after the specific divergence and an ancestral hominoid sequence of this site (np 799-802) was deduced as CCGG. The present data showing a mutational hot spot on this site suggest the possible presence of multiple origins of the worldwide distribution of the SDF1-3'A allele in humans.