Stephen J. Aves

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cdc10 'start' gene.

The cdc10 ‘start’ gene from the fission yeast Schizosaccharomyces pombe has been cloned by rescue of mutant function. It is present as a single copy in the haploid genome. Hybridisation of the gene to Northern blots has identified a low abundance 2.7‐kb polyadenylated RNA. Study of RNA extracted from cells both entering stationary phase and undergoing synchronous cell divisions suggests that commitment to the cell cycle is not controlled by regulation of cdc10 transcript level. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of mol. wt. 85 400. The putative cdc10 gene product shows no significant primary structure similarity with products of other fission and budding yeast cell cycle genes, or with other protein sequences in several databases.

suc1 is an essential gene involved in both the cell cycle and growth in fission yeast

The gene suc1 encodes a product which suppresses certain temperature sensitive mutants of the cell cycle control gene cdc2 of Schizosaccharomyces pombe. Mutants in the suc1 gene or over‐expression of its product leads to delays in mitotic and meiotic nuclear division. Deletion of the suc1 gene is lethal and generates some cells blocked in the cell cycle and others impaired in cellular growth. It is likely that the suc1 gene product binds and forms unstable complexes with the cdc2 protein kinase and with other proteins necessary for the cell cycle and cellular growth. suc1 may have a regulatory role in these processes.

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.

Cell Cycle-Mediated Regulation of Plant Infection by the Rice Blast Fungus

This study uses a combination of live-cell imaging and gene functional analysis to explore the relationship between the cell cycle and appressorium-mediated plant infection. It finds that there are as many as three distinct cell cycle checkpoints in the establishment of plant disease by M. oryzae. To gain entry to plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we demonstrate that appressorium morphogenesis in the rice blast fungus Magnaporthe oryzae is tightly regulated by the cell cycle. Shortly after a fungus spore lands on the rice (Oryza sativa) leaf surface, a single round of mitosis always occurs in the germ tube. We found that initiation of infection structure development is regulated by a DNA replication-dependent checkpoint. Genetic intervention in DNA synthesis, by conditional mutation of the Never-in-Mitosis 1 gene, prevented germ tubes from developing nascent infection structures. Cellular differentiation of appressoria, however, required entry into mitosis because nimA temperature-sensitive mutants, blocked at mitotic entry, were unable to develop functional appressoria. Arresting the cell cycle after mitotic entry, by conditional inactivation of the Blocked-in-Mitosis 1 gene or expression of stabilized cyclinB-encoding alleles, did not impair appressorium differentiation, but instead prevented these cells from invading plant tissue. When considered together, these data suggest that appressorium-mediated plant infection is coordinated by three distinct cell cycle checkpoints that are necessary for establishment of plant disease.

Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.

Ancient diversification of eukaryotic MCM DNA replication proteins

BackgroundYeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear.ResultsWe used comparative genomics and phylogenetics to reconstruct the diversification of the eukaryotic Mcm2-9 gene family, demonstrating that Mcm2-9 were formed by seven gene duplication events before the last common ancestor of the eukaryotes. Mcm2-7 protein paralogues were present in all eukaryote genomes studied suggesting that no gene loss or functional replacements have been tolerated during the evolutionary diversification of eukaryotes. Mcm8 and 9 are widely distributed in eukaryotes and group together on the MCM phylogenetic tree to the exclusion of all other MCM paralogues suggesting co-ancestry. Mcm8 and Mcm9 are absent in some taxa, including Trichomonas and Giardia, and appear to have been secondarily lost in some fungi and some animals. The presence and absence of Mcm8 and 9 is concordant in all taxa sampled with the exception of Drosophila species. Mcm10 is present in most eukaryotes sampled but shows no concordant pattern of presence or absence with Mcm8 or 9.ConclusionA multifaceted and heterogeneous Mcm2-7 hexamer evolved during the early evolution of the eukaryote cell in parallel with numerous other acquisitions in cell complexity and prior to the diversification of extant eukaryotes. The conservation of all six paralogues throughout the eukaryotes suggests that each Mcm2-7 hexamer component has an exclusive functional role, either by a combination of unique lock and key interactions between MCM hexamer subunits and/or by a range of novel side interactions. Mcm8 and 9 evolved early in eukaryote cell evolution and their pattern of presence or absence suggests that they may have linked functions. Mcm8 is highly divergent in all Drosophila species and may not provide a good model for Mcm8 in other eukaryotes.

TRbase: a database relating tandem repeats to disease genes for the human genome

MOTIVATION Tandem repeats are associated with disease genes, play an important role in evolution and are important in genomic organization and function. Although much research has been done on short perfect patterns of repeats, there has been less focus on imperfect repeats. Thus, there is an acute need for a tandem repeats database that provides reliable and up to date information on both perfect and imperfect tandem repeats in the human genome and relates these to disease genes. RESULTS This paper presents a web-accessible relational tandem repeats database that relates tandem repeats to gene locations and disease genes of the human genome. In contrast to other available databases, this database identifies both perfect and imperfect repeats of 1-2000 bp unit lengths. The utility of this database has been illustrated by analysing these repeats for their distribution and frequencies across chromosomes and genomic locations and between protein-coding and non-coding regions. The applicability of this database to identify diseases associated with previously uncharacterized tandem repeats is demonstrated.

The essential Schizosaccharomyces pombe cdc23 DNA replication gene shares structural and functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene

Abstract The fission yeast cdc23 gene is required for correct DNA replication: cdc23 mutants show reduced rates of DNA synthesis and become elongated after cell-cycle arrest. We have cloned the Schizosaccharomyces pombe cdc23 gene by complementation of the temperature-sensitive phenotype of cdc23-M36 and confirmed the identity of the gene by integrative mapping. Analysis of the DNA sequence reveals that cdc23 can encode a protein of 593 amino acids (Mr=67 kDa) with 22% overall identity and many structural homologies with the product of the Saccharomyces cerevisiae DNA43 (MCM10) gene which is required for correct initiation of DNA synthesis at chromosomal origins of replication. Construction of a cdc23 null allele has established that the cdc23 gene is essential for viability, with cdc23 deletion mutant spores germinating but undergoing arrest with undivided nuclei in the first or second cell cycle. The S. pombe cdc23 gene on an expression plasmid is able to complement the S. cerevisiae dna43-1 mutant. These structural and functional homologies between two distantly related species suggest that cdc23 and DNA43 may represent genes for a conserved essential eukaryotic DNA replication function.

Fission yeast Cdc23 interactions with DNA replication initiation proteins.

Abstract.Schizosaccharomyces pombe Cdc23 is an essential DNA replication protein, conserved in eukaryotes and functionally homologous with Saccharomyces cerevisiae Dna43 (Mcm10). We sought evidence for interactions between Cdc23 and the MCM2–7 complex, a component of both the pre-replicative complex and the replication fork. Cdc23 shows genetic interactions with four MCM subunits: cdc23-M36 and cdc23-1E2 alleles both show synthetic phenotypes with mcm2 (cdc19-P1) and mcm6 (mis5-268), and cdc23-M36 is synthetically lethal with mcm4 (cdc21-K46) and with mcm5 (nda4-108). The wild-type cdc23 gene on multicopy plasmids can partially suppress temperature-dependent defects in mcm5 (nda4-108). Two-hybrid analysis demonstrates interactions at the protein–protein level between Cdc23 and Mcm4, Mcm5 and Mcm6. Cdc23 also interacts with four subunits of the Schizosaccharomyces pombe origin recognition complex (ORC) in yeast two-hybrid assay: Orc1, Orc2, Orc5 and Orc6. We found no evidence for interaction between Cdc23 and the MCM recruitment factor Cdc18 (the homologue of Saccharomyces cerevisiae Cdc6). Unlike Cdc18, Cdc23 mRNA shows no significant fluctuation in level through the cell cycle. These data suggest that fission yeast Cdc23 is an MCM-associated factor which has a role in the initiation of DNA replication.