Origin and diversity of the Sox transcription factor gene family: genome-wide analysis in Fugu rubripes

The SOX family of transcription factors are found throughout the animal kingdom and are important in a variety of developmental contexts. Genome analysis has identified 20 Sox genes in human and mouse, which can be subdivided into 8 groups, based on sequence comparison and intron-exon structure. Most of the SOX groups identified in mammals are represented by a single SOX sequence in invertebrate model organisms, suggesting a duplication and divergence mechanism has operated during vertebrate evolution. We have now analysed the Sox gene complement in the pufferfish, Fugu rubripes, in order to shed further light on the diversity and origins of the Sox gene family. Major differences were found between the Sox family in Fugu and those in humans and mice. In particular, Fugu does not have orthologues of Sry, Sox,15 and Sox30, which appear to be specific to mammals, while Sox19, found in Fugu and zebrafish but absent in mammals, seems to be specific to fishes. Six mammalian Sox genes are represented by two copies each in Fugu, indicating a large-scale gene duplication in the fish lineage. These findings point to recent Sox gene loss, duplication and divergence occurring during the evolution of tetrapod and teleost lineages, and provide further evidence for large-scale segmental or a whole-genome duplication occurring early in the radiation of teleosts. (C) 2004 Elsevier B.V. All rights reserved.

Opsins: Evolution in waiting

Complete vertebrate genome sequencing has revealed a remarkable stability and uniformity in the protein-coding gene set, which at first glance might suggest that gene duplication events are relatively rare. This may be a red herring, or at least a red cichlid, as the Lake Malawi cichlid fishes show rapid and extensive duplication and diversification of their retinal cone photoreceptor opsin genes.

The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains similar to16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at similar to3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 mug/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Piecing together evolution of the vertebrate endocrine system

One of the great challenges in biology is to understand how particular complex morphological and physiological characters originated in specific evolutionary lineages. In this article, we address the origin of the vertebrate hypothalamic-pituitary-peripheral gland (H-P-PG) endocrine system, a complex network of specialized tissues, ligands and receptors. Analysis of metazoan nucleotide and protein sequences reveals a patchwork pattern of H-P-PG gene conservation between vertebrates and closely related invertebrates (ascidians). This is consistent with a model of how the vertebrate H-P-PG endocrine system could have emerged in relatively few steps by gene family expansion and by regulatory and structural modifications to genes that are present in a chordate ancestor. Some of these changes might have resulted in new connections between metabolic or signaling pathways, such as the bridging of 'synthesis islands' to form an efficient system for steroid hormone synthesis.

Determination of Wolbachia genome size by Pulsed-Field Gel Electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

Wolbachia: why these bacteria are important to genome research

Intracellular bacteria of the genus Wolbachia were first discovered in mosquitoes in the 1920s. Their superficial similarity to pathogenic rickettsia initially raised interest in them as potential human pathogens. However, injection experiments with mice showed that they were non-pathogenic, and they were subsequently classified as symbionts of insects. Until the 1970s, Wolbachia was considered to infect a limited number of species of mosquitoes. It is now clear that Wolbachia is an extremely common intracellular agent of invertebrates, infecting nearly all the major groups of arthropods and other terrestrial invertebrates. Its wide host range and abundance can be attributed partly to the unusual phenotypes it exerts on the host it infects. These include the induction of parthenogenesis (the production of female offspring from unmated mothers) in certain insects, the feminization of genetic male crustaceans to functional phenotypic females, and the failure of fertilization in hosts when males and females have a different infection status (cytoplasmic incompatibility). All of these phenotypes favor maternal transmission of the intracellular Wolbachia. In the last year, Wolbachia has also been shown to be a widespread symbiont of filarial nematodes. It appears that Wolbachia is needed by the adult worm for normal fertility, indicating that Wolbachia is behaving like a classic mutualist in this case. This discovery exemplifies that the extent of the host range of Wolbachia and its associated phenotypes is still far from fully understood.

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromosome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively. (C) 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-systern in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-systern open to the outside, the sealed t-systern of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres. (C) 2002 Elsevier Science Ltd. All rights reserved.