Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family.

Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N-glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome.     

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion

Abstract Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells. HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.     

Effect of agitation on growth and enzyme production of Trichoderma reesei in batch fermentation

Growth, enzyme-producing activity and respiratory properties of Trichoderma reesei QM 9414 were examined under various agitation intensities. Two substrates were compared: lactose and Avicel. Pellet formation occurred at all agitation intensities for both substrates. Oxygen dependence at the lower agitation rate varied with the substrate type. With lactose as the carbon source, linear growth was observed, despite a regulation of the dissolved oxygen concentration at 30% saturation. The enzyme production was strongly affected by the agitation. At the higher agitation rates the enzyme production dropped. With Avicel as the carbon source, the production of enzymes surged as soon as the growth was limited by the hydrolysis of Avicel.Growth on Avicel, in the conditions we used, was limited by Avicel hydrolysis. Cubic growth was observed when lactose was the carbon source. A new derivation for a model of the observed cubic growth is proposed and is used to correlate growth, CO₂ production and oxygen consumption in a consistent way, impossible with exponential growth models.     

A broadly active viral inhibitor in human and animal organ extracts and body fluids

To help assess the possibility that a newly described viral inhibitor from cell cultures might play a natural defensive role in vivo, its distribution and concentration in human and animal organ extracts and body fluids were investigated. The concentration of the inhibitor was high in human liver, heart muscle, splenic extracts, and human serum and milk. The inhibitor in the body was indistinguishable from a previously described inhibitor produced in cell cultures that was characterized by broad antiviral activity, lack of target cell species specificity, lack of induction of stable antiviral activity in cells, rapid reversibility of antiviral action, prevention of virus attachment, and stability at 100 degrees C. Sixteen virus plaque reduction units of the inhibitor diminished the yield of poliovirus in vitro by more than 1000-fold. Additional evidence that contact-blocking viral inhibitor (CVI) inhibits vaccinia virus attachment to cells is presented. A role for the inhibitor in natural defense against viral infections is possible.     

Fatty acid incorporation increases the affinity of muscarinic cholinergic receptors for agonists

Incorporation of unsaturated fatty acids into membrane fragments from rat brain cortex and medulla pons selectively increased the affinity of the muscarinic agonist, carbamylcholine. The affinity and number of binding sites for the labeled antagonist, N-[3H]methyl-4-piperidyl benzilate was unchanged. The effect on agonist binding was most prominent in the cortex, in which carbamylcholine IC50 values were decreased up to 5-fold. Selectivity of the effect was observed with fatty acids of chain length 18-20 carbons, unsaturation in position 11-12, and a cis conformation of the double bond being most effective. The effects of fatty acids on agonist binding were due primarily to alterations in the affinity constants for the binding reaction, with minor increases in the proportion of high-affinity sites. Transition metals selectively increased the percentage of high-affinity sites in the cortex, but in cis-vaccenic-acid-treated membranes more than additive effects of the metal were observed; both were reversed by GTP. GTP also reversed binding parameters in cis-vaccenic-acid-treated medulla membranes to control level. We conclude that the primary effect of the active fatty acids is to alter the thermodynamic properties of muscarinic agonist binding without markedly inducing interconversion.     

Segregation analysis of schizophrenia and related disorders

Segregation analysis was applied to 79 nuclear families ascertained through chronic schizophrenic probands. Analysis was performed on the diagnosis of schizophrenia alone and on schizophrenia and schizotypal personality disorder (milder phenotype) combined. The models used were the transmission probability model and the mixed model. Because the disease is associated with reduced fertility, all likelihoods were calculated conditional on parental phenotypes. However, compatibility of the mating-type distribution predicted by each model with the observed was also examined. In all analyses, results suggested consistency with genetic transmission. In the analysis of schizophrenia alone, discrimination among models was difficult. In the analysis including the milder phenotypes, all single-locus models without polygenic background were excluded, while pure polygenic inheritance could not be eliminated. The polygenic model also gave good agreement with supplementary observations (lifetime disease incidences, mating-type distribution, and monozygotic twin concordance). The estimated components of variance for the polygenic model were: polygenes (H) 81.9%; common sib environment (B) 6.9%; random environment (R) 11.2%. Although the polygenic model was parsimonious, segregation analysis and the supplementary observations were also consistent with a mixed model, with a single major locus making a large contribution to genetic liability. Such a locus is more likely to be recessive than dominant, with a high gene frequency and low penetrance. The most likely recessive mixed model gave the following partition of liability variance: major locus, 62.9%; polygenes, 19.5%; common sib environment, 6.6%; and random environment, 11.0%.     

Genetic Basis of Vi Antigen Expression in Salmonella paratyphi C

Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.