Molecular phylogeny and biogeography of the dung beetle genus Temnoplectron Westwood (Scarabaeidae: Scarabaeinae) from Australia’s wet tropics

The landscape of the Australian Wet Tropics can be described as islands of montane rainforest Surrounded by warmer or more xeric habitats. Historical glaciation cycles have caused expansion and contraction of these rainforest islands leading to consistent patterns of genetic divergence within species of vertebrates. To explore whether this dynamic history has promoted speciation in endemic and diverse groups Of insects, we used a combination of mtDNA sequencing and morphological characters to estimate relationships and the tempo of divergence among Australian representatives of the dung beetle genus Temnoplectron. This phylogenetic hypothesis shares a number of well-supported clades with a previously published phylogenetic hypothesis based on morphological data. though statistical support for several nodes is weak. Sister species relationships well-supported in both tree topologies. and a tree obtained by combining the two data sets. suggest that speciation has mostly been allopatric. We identify a number of speciation barriers, which coincide with phylogeographic breaks found in vertebrate species. Large sequence divergences between species emphasize that speciation events are ancient (pre-Pleistocene). The flightless, rainforest species appear to have speciated rapidly. but also in the distant past. (C) 2003 Elsevier Inc. All rights reserved.

Sociality and the rate of molecular evolution

The molecular clock does not tick at a uniform rate in all taxa but maybe influenced by species characteristics. Eusocial species (those with reproductive division of labor) have been predicted to have faster rates of molecular evolution than their nonsocial relatives because of greatly reduced effective population size; if most individuals in a population are nonreproductive and only one or few queens produce all the offspring, then eusocial animals could have much lower effective population sizes than their solitary relatives, which should increase the rate of substitution of nearly neutral mutations. An earlier study reported faster rates in eusocial honeybees and vespid wasps but failed to correct for phylogenetic nonindependence or to distinguish between potential causes of rate variation. Because sociality has evolved independently in many different lineages, it is possible to conduct a more wide-ranging study to test the generality of the relationship. We have conducted a comparative analysis of 25 phylogenetically independent pairs of social lineages and their nonsocial relatives, including bees, wasps, ants, termites, shrimps, and mole rats, using a range of available DNA sequences (mitochondrial and nuclear DNA coding for proteins and RNAs, and nontranslated sequences). By including a wide range of social taxa, we were able to test whether there is a general influence of sociality on rates of molecular evolution and to test specific predictions of the hypothesis: (1) that social species have faster rates because they have reduced effective population sizes; (2) that mitochondrial genes would show a greater effect of sociality than nuclear genes; and (3) that rates of molecular evolution should be correlated with the degree of sociality. We find no consistent pattern in rates of molecular evolution between social and nonsocial lineages and no evidence that mitochondrial genes show faster rates in social taxa. However, we show that the most highly eusocial Hymenoptera do have faster rates than their nonsocial relatives. We also find that social parasites (that utilize the workers from related species to produce their own offspring) have faster rates than their social relatives, which is consistent with an effect of lower effective population size on rate of molecular evolution. Our results illustrate the importance of allowing for phylogenetic nonindependence when conducting investigations of determinants of variation in rate of molecular evolution.

Molecular crowding effects of linear polymers in protein solutions

Measurement of protein-polymer second virial coefficients (B-AP) by sedimentation equilibrium studies of carbonic anhydrase and cytochrome c in the presence of dextrans (T10-T80) has revealed an inverse dependence of B-AP upon dextran molecular mass that conforms well with the behaviour predicted for the excluded-volume interaction between a spherical protein solute A and a random-flight representation of the polymeric cosolute P. That model of the protein-polymer interaction is also shown to provide a reasonable description of published gel chromatographic and equilibrium dialysis data on the effect of polymer molecular mass on BAP for human serum albumin in the presence of polyethylene glycols, a contrary finding from analysis of albumin solubility measurements being rejected on theoretical grounds. Inverse dependence upon polymer chainlength is also the predicted excluded-volume effect on the strength of several types of macromolecular equilibria-protein isomerization, protein dimerization, and 1 : 1 complex formation between dissimilar protein reactants. It is therefore concluded that published experimental observations of the reverse dependence, preferential reaction enhancement within DNA replication complexes by larger polyethylene glycols, must reflect the consequences of cosolute chemical interactions that outweigh those of thermodynamic nonideality arising from excluded-volume effects. (c) 2005 Elsevier B.V. All rights reserved.

Molecular mechanisms for the variation of mitochondrial gene content and gene arrangement among chigger mites of the genus Leptotrombidium (Acari : Acariformes)

The gene content of a mitochondrial (mt) genome, i.e., 37 genes and a large noncoding region (LNR), is usually conserved in Metazoa. The arrangement of these genes and the LNR is generally conserved at low taxonomic levels but varies substantially at high levels. We report here a variation in mt gene content and gene arrangement among chigger mites of the genus Leptotrombidium. We found previously that the mt genome of Leptotrombidium pallidum has an extra gene for large-subunit rRNA (rrnL), a pseudo-gene for small-subunit rRNA (PrrnS), and three extra LNRs, additional to the 37 genes and an LNR typical of Metazoa. Further, the arrangement of mt genes of L. pallidum differs drastically from that of the hypothetical ancestor of the arthropods. To find to what extent the novel gene content and gene arrangement occurred in Leptotrombidium, we sequenced the entire or partial mt genomes of three other species, L. akamushi, L. deliense, and L. fletcheri. These three species share the arrangement of all genes with L. pallidum, except trnQ (for tRNA-glutamine). Unlike L. pallidum, however, these three species do not have extra rrnL or PrrnS and have only one extra LNR. By comparison between Leptotrombidium species and the ancestor of the arthropods, we propose that (1) the type of mt genome present in L. pallidum evolved from the type present in the other three Leptotrombidium species, and (2) three molecular mechanisms were involved in the evolution of mt gene content and gene arrangement in Leptotrombidium species.

Characterization of the human sulfate anion transporter (hsat-1) protein and gene (SAT1; SLC26A1)

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

Multivariate QTL linkage analysis suggests a QTL for platelet count on chromosome 19q

Platelet count is a highly heritable trait with genetic factors responsible for around 80% of the phenotypic variance. We measured platelet count longitudinally in 327 monozygotic and 418 dizygotic twin pairs at 12, 14 and 16 years of age. We also performed a genome-wide linkage scan of these twins and their families in an attempt to localize QTLs that influenced variation in platelet concentrations. Suggestive linkage was observed on chromosome 19q13.13-19q13.31 at 12 (LOD=2.12, P=0.0009), 14 (LOD=2.23, P=0.0007) and 16 (LOD=1.01, P=0.016) years of age and multivariate analysis of counts at all three ages increased the LOD to 2.59 (P=0.0003). A possible candidate in this region is the gene for glycoprotein VI, a receptor involved in platelet aggregation. Smaller linkage peaks were also seen at 2p, 5p, 5q, 10p and 15q. There was little evidence for linkage to the chromosomal regions containing the genes for thrombopoietin (3q27) and the thrombopoietin receptor (1q34), suggesting that polymorphisms in these genes do not contribute substantially to variation in platelet count between healthy individuals.

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described.

Gene-expression profiling reveals distinct expression patterns for classic versus variant Merkel cell phenotypes and new classifier genes to distinguish Merkel cell from small-cell lung carcinoma

Merkel cell carcinoma (MCC) is a rare aggressive skin tumor which shares histopathological and genetic features with small-cell lung carcinoma (SCLC), both are of neuroendocrine origin. Comparable to SCLC, MCC cell lines are classified into two different biochemical subgroups designated as 'Classic' and 'Variant'. With the aim to identify typical gene-expression signatures associated with these phenotypically different MCC cell lines subgroups and to search for differentially expressed genes between MCC and SCLC, we used cDNA arrays to pro. le 10 MCC cell lines and four SCLC cell lines. Using significance analysis of microarrays, we defined a set of 76 differentially expressed genes that allowed unequivocal identification of Classic and Variant MCC subgroups. We assume that the differential expression levels of some of these genes reflect, analogous to SCLC, the different biological and clinical properties of Classic and Variant MCC phenotypes. Therefore, they may serve as useful prognostic markers and potential targets for the development of new therapeutic interventions specific for each subgroup. Moreover, our analysis identified 17 powerful classifier genes capable of discriminating MCC from SCLC. Real-time quantitative RT-PCR analysis of these genes on 26 additional MCC and SCLC samples confirmed their diagnostic classification potential, opening opportunities for new investigations into these aggressive cancers.

A mitochondrial phylogeny of the rainforest skink genus Saproscincus, Wells and Wellington (1984)

The phylogenetic relationships and historical biogeography of 10 currently described rainforest skinks in the genus Saproscincus were investigated using mitochondrial protein-coding ND4 and ribosomal RNA 16S genes. A robust phylogeny is inferred using both maximum likelihood and Bayesian analysis, with all inter-specific nodes strongly supported when datasets are combined. The phylogeny supports the recognition of two major lineages (northern and southern), each of which comprises two divergent clades. Both northern and southern lineages have comparably divergent representatives in mid-east Queensland (MEQ), providing further molecular evidence for the importance of two major biogeographic breaks, the St. Lawrence gap and Burdekin gap separating MEQ from southern and northern counterparts respectively. Vicariance associated with the fragmentation and contraction of temperate rainforest during the mid-late Miocene epoch underpins the deep divergence between morphologically conservative lineages in at least three instances. In contrast, one species, Saproseincus oriarus, shows very low sequence divergence but distinct morphological and ecological differentiation from its allopatric sister clade within Saproseincus mustelinus. These results suggest that while vicariance has played a prominent role in diversification and historical biogeography of Saproscincus, divergent selection may also be important. (C) 2004 Elsevier Inc. All rights reserved.

Disruption of the BLM gene in ATM-null DT40 cells does not exacerbate either phenotype

Bloom syndrome and ataxia-telangiectasia are autosomal recessive human disorders characterized by immunodeficiency, genome instability and predisposition to develop cancer. Recent data reveal that the products of these two genes, BLM and ATM, interact and function together in recognizing abnormal DNA structures. To investigate the function of these two molecules in DNA damage recognition, we generated double knockouts of ATM(-/-) BLM-/- in the DT40 chicken B-lymphocyte cell line. The double mutant cells were viable and exhibited a variety of characteristics of both ATM(-/-) and BLM-/- cells. There was no evidence for exacerbation of either phenotype; however, the more extreme radiosensitivity seen in ATM(-/-) and the elevated sister chromatid exchange seen in BLM-/- cells were retained in the double mutants. These results suggest that ATM and BLM have largely distinct roles in recognizing different forms of damage in DNA, but are also compatible with partially overlapping functions in recognizing breaks in radiation-damaged DNA.

Aprataxin, a novel protein that protects against genotoxic stress

Ataxia-oculomotor apraxia (AOA1) is a neurological disorder with symptoms that overlap those of ataxia-telangiectasia, a syndrome characterized by abnormal responses to double-strand DNA breaks and genome instability. The gene mutated in AOA1, APTX, is predicted to code for a protein called aprataxin that contains domains of homology with proteins involved in DNA damage signalling and repair. We demonstrate that aprataxin is a nuclear protein, present in both the nucleoplasm and the nucleolus. Mutations in the APTX gene destabilize the aprataxin protein, and fusion constructs of enhanced green fluorescent protein and aprataxin, representing deletions of putative functional domains, generate highly unstable products. Cells from AOA1 patients are characterized by enhanced sensitivity to agents that cause single-strand breaks in DNA but there is no evidence for a gross defect in single-strand break repair. Sensitivity to hydrogen peroxide and the resulting genome instability are corrected by transfection with full-length aprataxin cDNA. We also demonstrate that aprataxin interacts with the repair proteins XRCC1, PARP-1 and p53 and that it co-localizes with XRCC1 along charged particle tracks on chromatin. These results demonstrate that aprataxin influences the cellular response to genotoxic stress very likely by its capacity to interact with a number of proteins involved in DNA repair.

Sequence variation in the proximity of IDE may impact age at onset of both Parkinson disease and Alzheimer disease

We recently reported that a linkage disequilibrium (LD) block on chromosome 10q encompassing the gene encoding insulin-degrading enzyme (IDE) harbors sequence variants that associate with Alzheimer disease (AD). Evidence also indicated effects upon a number of quantitative indices of AD severity, including age-at-onset (AAO). Since linkage of this immediate region to AAO has been shown in both AD and Parkinson disease (PD), we have explored the possibility that polymorphism within this LD block might also influence PD. Utilizing single nucleotide polymorphisms that delineate common haplotypes from this region, we observed significant evidence of association with AAO in an Australian PD case-control sample. Analyses were complemented with AAO data from two independent Swedish AD case samples, for which previously reported findings were replicated. Results were consistent between AD and PD, suggesting the presence of equivalent detrimental and protective alleles. These data highlight a genomic region in the proximity of IDE that may contribute to AD and PD in a similar manner.

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.