Expression of zinc transporter family genes in Dictyostelium

Regulation of the zinc ion concentration is physiologically important to control the activities of a variety of cellular molecules. A BLAST search against a conserved domain of known zinc transporters identified twelve putative zinc transporter family genes in the Dictyostelium genome. Phylogenetic analysis revealed the presence of three zinc transporter subfamilies in Dictyostelium. One subfamily of proteins, consisting of the ZntA-D proteins, has weak homology to the STAT3-inducible LIV-1 protein. In addition, in situ hybridization revealed that the zntA-D genes are expressed in the pstAB cells, this expression being absent in the Dd-STATa null mutant. Thus, Dd-STATa may control stalk cell differentiation through some members of the zinc transporter family genes during Dictyostelium development.     

<Emphasis Type="Italic">Drosophila</Emphasis> Bys is nuclear and shows dynamic tissue-specific expression during development

Although the bys-like family of genes has been conserved from yeast to humans, it is not apparent to what extent the function of Bys-like proteins has been conserved across phylogenetic groups. Human Bystin is thought to function in a novel cell adhesion complex involved in embryo implantation. The product of the yeast bys-like gene, Enp1, is nuclear and has a role in pre-ribosomal RNA (pre-rRNA) splicing and ribosome biogenesis. To gain insight into the function of the Drosophila melanogaster bys-like family member, termed bys, we examined bys mRNA expression and the localization of Bys protein. In embryos, bys mRNA is expressed in a tissue-specific pattern during gastrulation. In the larval wing imaginal disc, bys mRNA is expressed in the ventral and dorsal regions of the wing pouch, regions that give rise to epithelia that adhere to one another after the wing disc everts. The bys mRNA expression patterns could be interpreted as being consistent with a role for Bys in events requiring cell-cell interactions. However, embryonic bys mRNA expression patterns mirror those of genes that are potential targets of the growth regulator Myc and encode nucleolar proteins implicated in cell growth. Additionally, in Schneider line 2 (S2) cells, an epitope-tagged Bys protein is localized to the nucleus, suggesting that Drosophila Bys function may be conserved with that of yeast Enp1.Edited by D.A. Weisblat     

Brachyury homolog (HpTa) is involved in the formation of archenteron and secondary mesenchyme cell differentiation in the sea urchin embryo

Sea urchin Brachyury homolog (HpTa) is expressed exclusively in the vegetal plate and secondary mesenchyme cells in the embryos of sea urchin Hemicentrotus pulcherrimus. In order to gain insights into the role of HpTa during sea urchin development, we designed experiments to perturb the embryo by inducing ectopic overexpression of HpTa by injecting fertilized eggs with HpTa mRNA. The overexpression of HpTa resulted in suppression of the formation of vegetal plate and secondary mesenchyme cells. We assume that the interaction of HpTa with unknown factors is required for the activation of the HpTa target genes, and that the excess amount of HpTa proteins produced from injected HpTa mRNA depletes the co-factors. In consequence, the target genes of HpTa would be repressed by the overexpression of HpTa. We suggest that HpTa is involved in the formation of the vegetal plate and the differentiation of secondary mesenchyme cells.     

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.     

The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.     

Functional constraint and divergence in the G protein family in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis>

Part of the challenge of the post-genomic world is to identify functional elements within the wide array of information generated by genome sequencing. Although cross-species comparisons and investigation of rates of sequence divergence are an efficient approach, the relationship between sequence divergence and functional conservation is not clear. Here, we use a comparative approach to examine questions of evolutionary rates and conserved function within the guanine nucleotide-binding protein (G protein) gene family in nematodes of the genus Caenorhabditis. In particular, we show that, in cases where the Caenorhabditis elegans ortholog shows a loss-of-function phenotype, G protein genes of C. elegans and Caenorhabditis briggsae diverge on average three times more slowly than G protein genes that do not exhibit any phenotype when mutated in C. elegans, suggesting that genes with loss of function phenotypes are subject to stronger selective constraints in relation to their function in both species. Our results also indicate that selection is as strong on G proteins involved in environmental perception as it is on those controlling other important processes. Finally, using phylogenetic footprinting, we identify a conserved non-coding motif present in multiple copies in the genomes of four species of Caenorhabditis. The presence of this motif in the same intron in the gpa-1 genes of C. elegans, C. briggsae and Caenorhabditis remanei suggests that it plays a role in the regulation of gpa-1, as well as other loci.Electronic Supplementary Material Supplementary material is available for this article at     

Gene overexpression/suppression analysis of candidate virulence factors of Candida albicans

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors.