The topoisomerase I gene from Ustilago maydis: sequence, disruption and mutant phenotype.

The Ustilago maydis genomic TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Necropsy findings and arbovirus surveillance in mourning doves from the southeastern United States

Mourning doves (Zenaida macroura) are the most abundant and widespread native member of the columbid family, as well as a major migratory game species, in the United States. However, there is little information on mortality factors in mourning doves. Records of necropsy accessions at the Southeastern Cooperative Wildlife Disease Study (SCWDS) from 15 southeastern states, from 1971 through 2005, were reviewed. One hundred thirty-five mourning doves were submitted from nine states during the 35-yr period. Trichomonosis constituted 40% (n = 54) of all diagnoses and was the most frequent diagnosis. Toxicoses and avian pox constituted 18.5% (n = 25) and 14.8% (n = 20) of all diagnoses, respectively. Remaining diagnoses included trauma, suspected toxicosis, Ascaridia columbae infection, suspected tick paralysis, and undetermined. Adults were observed more frequently with trichomonosis (94.1%) and toxicoses (68%) as compared to juveniles, but a gender predisposition was not apparent for either disease. Age and gender predilections were not apparent for cases of avian pox. The majority of the trichomonosis and avian pox cases were observed in the spring-summer, whereas the majority of the toxicosis cases were observed in the winter-spring. Additionally, the Georgia Department of Human Resources-Division of Public Health and West Virginia Department of Health and Human Resources submitted 809 mourning doves to SCWDS from 2001 through 2005 for West Nile virus surveillance efforts. West Nile virus was isolated from 2.1% (n = 17) and eastern equine encephalitis virus (EEEV) was isolated from 0.2% (n = 2) of the submitted birds.     

Androgens drive divergent responses to salt stress in male versus female rat kidneys

Dahl-Iwai (DI) salt-sensitive rats were studied using microarrays to identify sex-specific differences in the kidney, both basal differences and differences in responses to a high-salt diet. In DI rat kidneys, gene expression profiles demonstrated inflammatory and fibrotic responses selectively in females. Gonadectomy of DI rats abrogated sex differences in gene expression. Gonadectomized female and gonadectomized male DI rats both responded to high salt with the same spectrum of gene expression changes as intact female DI rats. Androgens dominated the sex-selective responses to salt. Several androgen-responsive genes with roles potentiating the differential responses to salt were identified, including increased male expression of angiotensin-vasopressin receptor and prolactin receptor, decreased 5 alpha-reductase, and mixed increases and decreases in expression of Cyp4a genes that can produce eicosanoid hormones. These sex differences potentiate sodium retention by males and increase kidney function during gestation in females.     

Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.