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PubMed2006

DNA-protein interactions and bacterial chromosome architecture.

Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

Bacterial Proteins↗
PubMed1999

Classical and molecular cytogenetics of the pufferfish Tetraodon nigroviridis.

Because of its highly compact genome, the pufferfish has become an important animal model in genome research. Although the small chromosome size renders chromosome analysis difficult, we have established both classical and molecular cytogenetics in the freshwater pufferfish Tetraodon nigroviridis (TNI). The karyotype of T. nigroviridis consists of 2n = 42 biarmed chromosomes, in contrast to the known 2n = 44 chromosomes of the Japanese pufferfish Fugu rubripes (FRU). RBA banding can identify homologous chromosomes in both species. TNI 1 corresponds to two smaller FRU chromosomes, explaining the difference in chromosome number. TNI 2 is homologous to FRU 1. Fluorescence in-situ hybridization (FISH) allows one to map single-copy sequences, i.e. the Huntingtin gene, on chromosomes of the species of origin and also on chromosomes of the heterologous pufferfish species. Hybridization of total genomic DNA shows large blocks of (species-specific) repetitive sequences in the pericentromeric region of all TNI and FRU chromosomes. Hybridization with cloned human rDNA and classical silver staining reveal two large and actively transcribed rRNA gene clusters. Similar to the situation in mammals, the highly compact pufferfish genome is endowed with considerable amounts of localized repeat DNAs.

Animals↗
PubMed1998

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu.

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.

Alternative Splicing↗
PubMed1997

Close phylogenetic relationship between egg drop syndrome virus, bovine adenovirus serotype 7, and ovine adenovirus strain 287.

A cloned egg drop syndrome (EDS) virus genomic DNA fragment containing the protease gene has been identified and the complete nucleotide sequence of the protease and partial nucleotide sequence of the hexon genes has been determined. Phylogenetic analysis of the protease gene has revealed EDS virus to be genetically more closely related to bovine adenovirus type 7 (BAV-7) and ovine adenovirus isolate 287 (OAV287) than either of these two viruses are to other members of the genus Mastadenovirus or EDS virus is to an other member of the Aviadenovirus genus. The three viruses share further similarities in that they have a high percentage AT content in their genome and are characterized by having more compact genomes than other adenoviruses. The protease gene from all three viruses contained the active site residues (H55-D72-C122 triad) and C104 (providing a disulfide bond to cofactor pVIc). However, P137, found in all other members of the Mastadenovirus genus, and thought to be involved in trafficking, was missing from the protease of the EDS virus, BAV-7, and OAV287. These results suggest that EDS virus should be classified along with BAV-7 and OAV287 in a separate taxon.

Adenoviridae↗
PubMed1985

The complete nucleotide sequence of the Xenopus laevis mitochondrial genome.

The complete sequence of the 17,553-nucleotide Xenopus laevis mitochondrial genome has been determined. A comparison of this amphibian mitochondrial genomic sequence with those of the mammalian mitochondrial genomes reveals a similar gene order and compact genomic organization. The encoded genes for 22 tRNAs, two ribosomal RNAs, and 13 proteins (COI, COII, COIII, ATPase 6, cytochrome b, and eight additional unidentified reading frames) in the amphibian mitochondria are highly homologous to their mammalian counterparts. Although the amphibian mitochondrial genome contains a significantly larger displacement loop region than the mammalian mitochondrial genomes, there are several regions of sequence homology near the putative sites for heavy and light strand transcription initiation and heavy strand replication. The unique mitochondrial genetic code observed in the mammalian mitochondrial systems is similar to that of the X. laevis mitochondrial genome because of the presence of only 22 encoded tRNAs and the high degree of homology between the predicted protein sequences. However, the amphibian system exclusively utilizes AUG as the start codon in all 13 open reading frames and shows a preference for codons ending in U rather than ending in C. In addition, the X. laevis mitochondrial genome employs the encoded AGA stop codon once and the UAA stop codon three times and requires polyadenylation to provide the nine other UAA stop codons. These observations suggest that the mechanisms of replication, transcription, processing, and translation in mitochondria are highly conserved throughout higher vertebrates.

Animals↗
PubMed2004

Insertion bias and purifying selection of retrotransposons in the Arabidopsis thaliana genome.

BACKGROUND: Genome evolution and size variation in multicellular organisms are profoundly influenced by the activity of retrotransposons. In higher eukaryotes with compact genomes retrotransposons are found in lower copy numbers than in larger genomes, which could be due to either suppression of transposition or to elimination of insertions, and are non-randomly distributed along the chromosomes. The evolutionary mechanisms constraining retrotransposon copy number and chromosomal distribution are still poorly understood. RESULTS: I investigated the evolutionary dynamics of long terminal repeat (LTR)-retrotransposons in the compact Arabidopsis thaliana genome, using an automated method for obtaining genome-wide, age and physical distribution profiles for different groups of elements, and then comparing the distributions of young and old insertions. Elements of the Pseudoviridae family insert randomly along the chromosomes and have been recently active, but insertions tend to be lost from euchromatic regions where they are less likely to fix, with a half-life estimated at approximately 470,000 years. In contrast, members of the Metaviridae (particularly Athila) preferentially target heterochromatin, and were more active in the past. CONCLUSION: Diverse evolutionary mechanisms have constrained both the copy number and chromosomal distribution of retrotransposons within a single genome. In A. thaliana, their non-random genomic distribution is due to both selection against insertions in euchromatin and preferential targeting of heterochromatin. Constant turnover of euchromatic insertions and a decline in activity for the elements that target heterochromatin have both limited the contribution of retrotransposon DNA to genome size expansion in A. thaliana.

Arabidopsis↗
PubMed2025

The evolution of the plastid genomes in the holoparasitic Balanophoraceae.

The independent transition to a heterotrophic lifestyle in plants drove remarkably convergent evolutionary trajectories, characterized by morphological modifications and reductions in their plastomes. The characteristics of the minimum plastome required for survival, if they exist, remain a topic of debate. The holoparasitic family Balanophoraceae was initially presumed to have entirely lost their plastids, however, recent reports revealed the presence of reduced and aberrant plastids with odd genomes. Among the outstanding features of these genomes are the highest nucleotide composition bias across the tree of life and the only two genetic code changes ever recorded among plants. In this study, we assembled the plastomes from five genera, four of which had never been studied. Major common features include extremely high AT content, the lack of a typical quadripartite structure and extensive size reduction due to gene elimination and genome compaction. The family exhibits multiple gene and intron losses, and a broad range of scenarios regarding the evolution of the plastid trnE, a gene considered essential because of its dual function in tetrapyrrole biosynthesis and translation within the plastid. In addition, phylogenetic analyses suggest that the genus Scybalium is not monophyletic. An evolutionary model for the plastomes of the Balanophoraceae is proposed.

Genome, Plastid↗
PubMed2000

Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans.

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.

Amino Acid Sequence↗
PubMed2004

The contribution of epistasis to the architecture of fitness in an RNA virus.

The tendency for genetic architectures to exhibit epistasis among mutations plays a central role in the modern synthesis of evolutionary biology and in theoretical descriptions of many evolutionary processes. Nevertheless, few studies unquestionably show whether, and how, mutations typically interact. Beneficial mutations are especially difficult to identify because of their scarcity. Consequently, epistasis among pairs of this important class of mutations has, to our knowledge, never before been explored. Interactions among genome components should be of special relevance in compacted genomes such as those of RNA viruses. To tackle these issues, we first generated 47 genotypes of vesicular stomatitis virus carrying pairs of nucleotide substitution mutations whose separated and combined deleterious effects on fitness were determined. Several pairs exhibited significant interactions for fitness, including antagonistic and synergistic epistasis. Synthetic lethals represented 50% of the latter. In a second set of experiments, 15 genotypes carrying pairs of beneficial mutations were also created. In this case, all significant interactions were antagonistic. Our results show that the architecture of the fitness depends on complex interactions among genome components.

Cloning, Molecular↗
PubMed2004

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D.

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Actins↗
PubMed2006

Detecting overlapping coding sequences in virus genomes.

BACKGROUND: Detecting new coding sequences (CDSs) in viral genomes can be difficult for several reasons. The typically compact genomes often contain a number of overlapping coding and non-coding functional elements, which can result in unusual patterns of codon usage; conservation between related sequences can be difficult to interpret--especially within overlapping genes; and viruses often employ non-canonical translational mechanisms--e.g. frameshifting, stop codon read-through, leaky-scanning and internal ribosome entry sites--which can conceal potentially coding open reading frames (ORFs). RESULTS: In a previous paper we introduced a new statistic--MLOGD (Maximum Likelihood Overlapping Gene Detector)--for detecting and analysing overlapping CDSs. Here we present (a) an improved MLOGD statistic, (b) a greatly extended suite of software using MLOGD, (c) a database of results for 640 virus sequence alignments, and (d) a web-interface to the software and database. Tests show that, from an alignment with just 20 mutations, MLOGD can discriminate non-overlapping CDSs from non-coding ORFs with a typical accuracy of up to 98%, and can detect CDSs overlapping known CDSs with a typical accuracy of 90%. In addition, the software produces a variety of statistics and graphics, useful for analysing an input multiple sequence alignment. CONCLUSION: MLOGD is an easy-to-use tool for virus genome annotation, detecting new CDSs--in particular overlapping or short CDSs--and for analysing overlapping CDSs following frameshift sites. The software, web-server, database and supplementary material are available at http://guinevere.otago.ac.nz/mlogd.html.

Algorithms↗
PubMed2001

Growth hormone secretagogue receptor family members and ligands.

We have previously reported the cloning and characterization of a new orphan G-protein-coupled receptor (GPC-R), the growth hormone secretagogue receptor (GHS-R), and shown that this receptor mediates the activity of the growth hormone-releasing peptides (GHRPs) and nonpeptide ligands such as L-692,429 and MK-0677. Because the GHS-R obviously does not belong to any of the known GPC-R subfamilies, we searched for GHS-R family members by screening a human genomic library using low-stringency hybridization and screening a Pufferfish genomic library. The Pufferfish was selected because of its compact genome. From the human genomic library, a homolog, GPR38, with 52% identity to the GHS-R was isolated. From the Pufferfish library, three family members were isolated. The Pufferfish gene having 58% identity to the GHS-R, on expression in HEK293 cells, was activated with GHRP-6 and MK-0677. These results indicate that the GHS-R has been conserved for at least 400 million years and that the Pufferfish genome is appropriate for isolation of GHS-R family members. In our search for endogenous ligands for the orphan receptors GHS-R and GPR38, we showed that adenosine is a partial agonist of the GHS-R and that motilin is the endogenous ligand for GPR38. We also confirmed that the endogenous ligand ghrelin is a full agonist of the GHS-R.

Adenosine↗
PubMed1997

Regions of human chromosome 2 (2q32-q35) and mouse chromosome 1 show synteny with the pufferfish genome (Fugu rubripes).

We have isolated and sequenced a cosmid clone from the compact genome of the Japanese pufferfish (Fugu rubripes) containing portions of three genes that have the same order as in human. The gene order is microtubule-associated protein (MAP-2), myosin light chain (MYL-1), and carbamoyl phosphate synthetase (CPS III). The intron-exon organization of Fugu CPS III is identical with that of rat CPS I, although the equivalent genomic fragments of rat and Fugu CPS span 87.9 and 21 kb, respectively. This is the first report of a piscine CPS III genomic structure and predicts a close evolutionary link between CPS III and CPS I. The 8-kb intergenic region between MYL-1 and CPS gave no clear areas of transcription factor-binding sites by pairwise comparison with shark or rat CPS promoter regions. However, there was a match with the rat myosin light chain 2 (MLC-2) gene promoter and a MyoD transcription factor-binding site 874 bp upstream of the MYL-1 gene.

Amino Acid Sequence↗
PubMed2005

Transfer RNA gene-targeted integration: an adaptation of retrotransposable elements to survive in the compact Dictyostelium discoideum genome.

Almost every organism carries along a multitude of molecular parasites known as transposable elements (TEs). TEs influence their host genomes in many ways by expanding genome size and complexity, rearranging genomic DNA, mutagenizing host genes, and altering transcription levels of nearby genes. The eukaryotic microorganism Dictyostelium discoideum is attractive for the study of fundamental biological phenomena such as intercellular communication, formation of multicellularity, cell differentiation, and morphogenesis. D. discoideum has a highly compacted, haploid genome with less than 1 kb of genomic DNA separating coding regions. Nevertheless, the D. discoideum genome is loaded with 10% of TEs that managed to settle and survive in this inhospitable environment. In depth analysis of D. discoideum genome project data has provided intriguing insights into the evolutionary challenges that mobile elements face when they invade compact genomes. Two different mechanisms are used by D. discoideum TEs to avoid disruption of host genes upon retrotransposition. Several TEs have invented the specific targeting of tRNA gene-flanking regions as a means to avoid integration into coding regions. These elements have been dispersed on all chromosomes, closely following the distribution of tRNA genes. By contrast, TEs that lack bona fide integration specificities show a strong bias to nested integration, thus forming large TE clusters at certain chromosomal loci that are hardly resolved by bioinformatics approaches. We summarize our current view of D. discoideum TEs and present new data from the analysis of the complete sequences of D. discoideum chromosomes 1 and 2, which comprise more than one third of the total genome.

Animals↗
PubMed2005

Comparative genomics of the human and Fugu voltage-gated calcium channel alpha1-subunit gene family reveals greater diversity in Fugu.

Extensive search for the orthologs of 10 human voltage-gated calcium channel (VGCC) alpha(1)-subunit genes in the Fugu genome sequence revealed 21 alpha(1)-subunit genes in the compact genome of Fugu. Subtype classification of the identified Fugu alpha(1) orthologs based on phylogenetic analysis, genomic organization and sequence comparison of the most divergent II/III loop and the C-terminal regions of the alpha(1)-subunits indicated extra copies of alpha(1S)-, alpha(1D)-, alpha(1F)-, alpha(1A)-, alpha(1E)-, alpha(1H)- and alpha(1G)-subunit genes. Phylogenetic analysis reveals that this is likely due to fish lineage specific alpha(1)-subunit subtype duplication. Sequence comparison shows that many of the structural features characteristic of VGCC and specific channel subtypes are also present in the Fugu alpha(1)-subunits. All the Fugu alpha(1)-subunits showed similar expression profile to that of the mammalian alpha(1)-subunits except for Fugu alpha(1S), alpha(1A), alpha(1B) and alpha(1H) which have a more widespread tissue distribution. These results indicate that Fugu, a lower vertebrate, has more extensive channel heterogeneity compared to human.

Animals↗
PubMed2004

Genome evolution in yeasts.

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

Chromosomes, Fungal↗
PubMed2006

Genolevures complete genomes provide data and tools for comparative genomics of hemiascomycetous yeasts.

The Génolevures online database (http://cbi.labri.fr/Genolevures/) provides tools and data relative to 4 complete and 10 partial genome sequences determined and manually annotated by the Génolevures Consortium, to facilitate comparative genomic studies of hemiascomycetous yeasts. With their relatively small and compact genomes, yeasts offer a unique opportunity for exploring eukaryotic genome evolution. The new version of the Génolevures database provides truly complete (subtelomere to subtelomere) chromosome sequences, 25 000 protein-coding and tRNA genes, and in silico analyses for each gene element. A new feature of the database is a novel collection of conserved multi-species protein families and their mapping to metabolic pathways, coupled with an advanced search feature. Data are presented with a focus on relations between genes and genomes: conservation of genes and gene families, speciation, chromosomal reorganization and synteny. The Génolevures site includes an area for specific studies by members of its international community.

Databases, Genetic↗
PubMed2004

Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers.

We present an analysis of chromatin fiber structure across the human genome. Compact and open chromatin fiber structures were separated by sucrose sedimentation and their distributions analyzed by hybridization to metaphase chromosomes and genomic microarrays. We show that compact chromatin fibers originate from some sites of heterochromatin (C-bands), and G-bands (euchromatin). Open chromatin fibers correlate with regions of highest gene density, but not with gene expression since inactive genes can be in domains of open chromatin, and active genes in regions of low gene density can be embedded in compact chromatin fibers. Moreover, we show that chromatin fiber structure impacts on further levels of chromatin condensation. Regions of open chromatin fibers are cytologically decondensed and have a distinctive nuclear organization. We suggest that domains of open chromatin may create an environment that facilitates transcriptional activation and could provide an evolutionary constraint to maintain clusters of genes together along chromosomes.

Cell Line↗