Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice

Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

Distinct SNP Combinations Confer Susceptibility to Urinary Bladder Cancer in Smokers and Non-Smokers

Recently, genome-wide association studies have identified and validated genetic variations associated with urinary bladder cancer (UBC). However, it is still unknown whether the high-risk alleles of several SNPs interact with one another, leading to an even higher disease risk. Additionally, there is no information available on how the UBC risk due to these SNPs compare to the risk of cigarette smoking and to occupational exposure to urinary bladder carcinogens, and whether the same or different SNP combinations are relevant in smokers and non-smokers. To address these questions, we analyzed the genotypes of six SNPs, previously found to be associated with UBC, together with the GSTM1 deletion, in 1,595 UBC cases and 1,760 controls, stratified for smoking habits. We identified the strongest interactions of different orders and tested the stability of their effect by bootstrapping. We found that different SNP combinations were relevant in smokers and non-smokers. In smokers, polymorphisms involved in detoxification of cigarette smoke carcinogens were most relevant (GSTM1, rs11892031), in contrast to those in non-smokers with MYC and APOBEC3A near polymorphisms (rs9642880, rs1014971) being the most influential. Stable combinations of up to three high-risk alleles resulted in higher odds ratios (OR) than the individual SNPs, although the interaction effect was less than additive. The highest stable combination effects resulted in an OR of about 2.0, which is still lower than the ORs of cigarette smoking (here, current smokers'' OR: 3.28) and comparable to occupational carcinogen exposure risks which, depending on the workplace, show mostly ORs up to 2.0.     

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.