The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.     

Mismatch uracil glycosylase from Escherichia coli: a general mismatch or a specific DNA glycosylase?

The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U.G mismatches. Subsequently, 3,N4-ethenocytosine (epsilonC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme. The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme. Our results demonstrate that product release by the enzyme is very slow and hence comparing the "steady-state" parameters of the enzyme for different substrates is of limited use. Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions. Although the enzyme excised both epsilonC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis. Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate. This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism.     

Rush and grab strategies in foraging marine endotherms: the case for haste in penguins

The speed at which air-breathing marine predators that forage by diving should swim is likely to depend on a variety of factors that differ substantially from those relevant in animals for which access to oxygen is unlimited. We used loggers attached to free-living penguins to examine the speed at which three species swam during periods searching for prey and compared this to their speeds during actual prey pursuit. All penguin species appeared to travel at similar speeds around 2 m/s during normal commuting between the surface and feeding depths, which accords closely with minimum costs of transport. However, Adélie penguins, Pygoscelis adeliae, slowed down to feed, Magellanic penguins, Spheniscus magellanicus, speeded up and king penguins, Aptenodytes patagonicus, travelled at a variety of speeds, although mean speed did not change from normal commuting. Since energy expenditure, and therefore oxygen usage, in swimming animals increases with the cube of the speed, we hypothesized that prey escape speed (a function of prey size) and prey density would prove critical in determining optimum pursuit speeds in predators. Simple models of this type help explain why it is that some penguin species apparently benefit by increasing speed to capture prey while others benefit by decreasing speed.     

cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.     

Expression of three <Emphasis Type="Italic">spalt </Emphasis>(<Emphasis Type="Italic">sal</Emphasis>) gene homologues in zebrafish embryos

Three homologues of the Drosophilaregion-specific homeotic gene spalt (sal) have been isolated in zebrafish, sall1a, sall1b and sall3. Phylogenetic analysis of these genes against known salDNA sequences showed zebrafish sall1aand sall1b to be orthologous to other vertebrate sal-1 genes and zebrafish sall3to be orthologous to other vertebrate sal-3 genes, except Xenopus sall3. Phylogenetic reconstruction suggests that zebrafish sall1a and sall1bresulted from a gene duplication event occurring prior to the divergence of the ray-finned and lobe-finned fish lineages. Analysis of the expression pattern of the zebrafish sal genes shows that sall1a and sall3 share expression domains with both orthologous and non-orthologous vertebrate sal genes. Both are expressed in various regions of the CNS, including in primary motor neurons. Outside of the CNS, sall1a expression is observed in the otic vesicle (ear), heart and in a discrete region of the pronephric ducts. These analyses indicate that orthologies between zebrafish sal genes and other vertebrate sal genes do not imply equivalence of expression pattern and, therefore, that biological functions are not entirely conserved. However we suggest that, like other vertebrate sal genes, zebrafish sal genes have a role in neural development. Also, expression of zebrafish sall1a in the otic vesicle, heart sac and the pronephric ducts of zebrafish embryos is possibly consistent with some of the abnormalities seen in Sall1-deficient mice and in Townes-Brocks Syndrome, a human disorder which is caused by mutations in the human spalt gene SALL1.     

A random model for mapping imprinted quantitative trait loci in a structured pedigree: an implication for mapping canine hip dysplasia

Genetic imprinting may have played a more notable role in shaping embryonic development of plants, animals, and humans than previously appreciated. Quantitative trait loci that are imprinted (iQTL) exert monoallelic effects, depending on the parent of origin, which is an exception to the laws of Mendelian genetics. In this article, we present a modified random effect-based mapping model to use in a genome-wide scan for the distribution of iQTL that contribute to genetic variance for a complex trait in a structured pedigree. This model, implemented with the maximum likelihood method, capitalizes on a network of relatedness for maternally and paternally derived alleles through identical-by-descent sharing, thus allowing for the discrimination of the genetic variances due to alleles derived from maternal and paternal parents. The model was employed to map iQTL responsible for canine hip dysplasia in a multihierarchical canine pedigree, founded with seven greyhounds and six Labrador retrievers. Of eight significant QTL detected, three, located on CFA1, CFA8, and CF28, were found to trigger significant parent-of-origin effects on the age of femoral capital ossification measured at the left and right hips of a canine. The detected iQTL provide important candidate regions for fine-mapping of imprinted genes and for studying their structure and function in the control of complex traits.     

Comparison of microsatellites versus single-nucleotide polymorphisms in a genome linkage screen for prostate cancer-susceptibility Loci

Prostate cancer is one of the most common cancers among men and has long been recognized to occur in familial clusters. Brothers and sons of affected men have a 2-3-fold increased risk of developing prostate cancer. However, identification of genetic susceptibility loci for prostate cancer has been extremely difficult. Although the suggestion of linkage has been reported for many chromosomes, the most promising regions have been difficult to replicate. In this study, we compare genome linkage scans using microsatellites with those using single-nucleotide polymorphisms (SNPs), performed in 467 men with prostate cancer from 167 families. For the microsatellites, the ABI Prism Linkage Mapping Set version 2, with 402 microsatellite markers, was used, and, for the SNPs, the Early Access Affymetrix Mapping 10K array was used. Our results show that the presence of linkage disequilibrium (LD) among SNPs can lead to inflated LOD scores, and this seems to be an artifact due to the assumption of linkage equilibrium that is required by the current genetic-linkage software. After excluding SNPs with high LD, we found a number of new LOD-score peaks with values of at least 2.0 that were not found by the microsatellite markers: chromosome 8, with a maximum model-free LOD score of 2.2; chromosome 2, with a LOD score of 2.1; chromosome 6, with a LOD score of 4.2; and chromosome 12, with a LOD score of 3.9. The LOD scores for chromosomes 6 and 12 are difficult to interpret, because they occurred only at the extreme ends of the chromosomes. The greatest gain provided by the SNP markers was a large increase in the linkage information content, with an average information content of 61% for the SNPs, versus an average of 41% for the microsatellite markers. The strengths and weaknesses of microsatellite versus SNP markers are illustrated by the results of our genome linkage scans.