Regulation of cell separation in the dimorphic fungus Ustilago maydis

During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree-like clusters in liquid culture and grow as ring-like (donut-shaped) colonies on solid medium. In wild-type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal-centre-kinase (GC) subfamily of Ste20-like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two-hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.     

The Epstein-Barr virus lytic program is controlled by the co-operative functions of two transactivators

The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of specific viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein-Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective influence of these molecules on the expression of various viral target genes varied greatly. In replication-defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that influence all stages of viral production.     

Villin-like actin-binding proteins are expressed ubiquitously in Arabidopsis

In an attempt to elucidate the biological function of villin-like actin-binding proteins in plants we have cloned several genes encoding Arabidopsis proteins with high homology to animal villin. We found that Arabidopsis contains at least four villin-like genes (AtVLNs) encoding four different VLN isoforms. Two AtVLN isoforms are more closely related to mammalian villin in their primary structure and are also antigenically related, whereas the other two contain significant changes in the C-terminal headpiece domain. RNA and promoter/beta-glucuronidase expression studies demonstrated that AtVLN genes are expressed in all organs, with elevated expression levels in certain types of cells. These results suggest that AtVLNs have less-specialized functions than mammalian villin, which is found only in the microvilli of brush border cells. Immunoblot experiments using a monoclonal antibody against pig villin showed that AtVLNs are widely distributed in a variety of plant tissues. Green fluorescent protein fused to full-length AtVLN and individual AtVLN headpiece domains can bind to both animal and plant actin filaments in vivo.     

LESS IS MORE: SELECTIVE ADVANTAGES CAN EXPLAIN THE PREVALENT LOSS OF BIOSYNTHETIC GENES IN BACTERIA

Bacteria that have adapted to nutrient‐rich, stable environments are typically characterized by reduced genomes. The loss of biosynthetic genes frequently renders these lineages auxotroph, hinging their survival on an environmental uptake of certain metabolites. The evolutionary forces that drive this genome degradation, however, remain elusive. Our analysis of 949 metabolic networks revealed auxotrophies are likely highly prevalent in both symbiotic and free‐living bacteria. To unravel whether selective advantages can account for the rampant loss of anabolic genes, we systematically determined the fitness consequences that result from deleting conditionally essential biosynthetic genes from the genomes of Escherichia coli and Acinetobacter baylyi in the presence of the focal nutrient. Pairwise competition experiments with each of 20 mutants auxotrophic for different amino acids, vitamins, and nucleobases against the prototrophic wild type unveiled a pronounced, concentration‐dependent growth advantage of around 13% for virtually all mutants tested. Individually deleting different genes from the same biosynthesis pathway entailed gene‐specific fitness consequences and loss of the same biosynthetic genes from the genomes of E. coli and A. baylyi differentially affected the fitness of the resulting auxotrophic mutants. Taken together, our findings suggest adaptive benefits could drive the loss of conditionally essential biosynthetic genes.     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.     

Influence of human B-casomorphin-7 on specific binding of 3H-spiperone to the 5-HT2-receptors of rat brain frontal cortex

Using radio-receptor analysis, it has been demonstrated that human beta-casomorphin-7 (Tyr-Pro-Phe-Val-Glu-Pro-Ile) displaces 3H-spiperone from 5-HT2-receptors of rat brain frontal cortex. IC50 of human beta-casomorphin-7 was 8 microM. These data suggest that one of the mechanisms of neurotropic action of beta-casomorphin-7 is might be associated with its influence on the serotoninergic system.     

Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.