[Evolutionary history of the SSX family of human C/T-antigens].

C/T-antigens are endogenous proteins expressed in normal testis, ovary and placenta, or in a variety of tumors. Such expression pattern sets the C/T antigens as promising targets for cancer vaccines. The SSX family comprises several C/T antigens. Here we applied comparative genomics techniques to study the evolution of the SSX genes. The human genomic locus includes 11 genes localized on the X chromosome in two separate regions 4 Mb apart. The recent pseudogenization of two SSX genes was demonstrated using the available expression data. The comparative analysis of the human, chimpanzee and mouse genomic loci allowed us to describe the phylogeny of the family and to reconstruct the evolutionary history of the locus in terms of elementary events.

Antibacterial non-glycosidase activity of invertebrate destabilase-lysozyme and of its helical amphipathic peptides.

BACKGROUND: Since bactericidal properties of some lysozymes are independent of their glycosidase activity, we have investigated this phenomenon for destabilase-lysozyme (DL) from medicinal leech (Hirudo medicinalis). METHODS: Glycosidase activity was determined on Micrococcus luteus, non-enzymatic antibacterial activity of heat-treated DL and of synthetic peptides alpha1, alpha2 and alpha3 (fragments of its primary structure) on M. luteus, Escherichia coli, Bacillus brevis and Streptomyces chrysomallus. RESULTS: Glycosidase activity disappeared after the heating of native DL at 100 degrees C for 40 min. Antibacterial activity of heat-treated DL for M. luteus MDMSU128 and MDMSU140 expressed as minimal inhibitory concentration was 9.8.10(-8) and 12.10(-8) M, respectively, and to E. coli MDMSU52 11.10(-8) M. Antibacterial activity of synthetic peptide alpha1 for M. luteus MDMSU128 and for E. coli MDMSU52 was 8.3.10(-5) and 4.9.10(-5) M, respectively. CONCLUSION: DL is the first invertebrate lysozyme with combined enzymatic and non-enzymatic antibacterial action.

Alternative splicing and protein function.

BACKGROUND: Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data. RESULTS: We have developed a method that generates possible mRNA isoforms for human genes contained in the EDAS database, taking into account the effects of nonsense-mediated decay and translation initiation rules, and a procedure for offsetting the effects of uneven EST coverage. Then we computed the number of mRNA isoforms for genes from different functional categories. Genes encoding ribosomal proteins and genes in the category "Small GTPase-mediated signal transduction" tend to have fewer isoforms than the average, whereas the genes in the category "DNA replication and chromosome cycle" have more isoforms than the average. Genes encoding proteins involved in protein-protein interactions tend to be alternatively spliced more often than genes encoding non-interacting proteins, although there is no significant difference in the number of isoforms of alternatively spliced genes. CONCLUSION: Filtering for functional isoforms satisfying biological constraints and accounting for uneven EST coverage allowed us to describe differences in alternative splicing of genes from different functional categories. The observations seem to be consistent with expectations based on current biological knowledge: less isoforms for ribosomal and signal transduction proteins, and more alternative splicing of interacting and cell cycle proteins.

Multiple forms of medicinal leech destabilase-lysozyme.

Earlier, three genes Ds1, Ds2, and Ds3 encoding corresponding destabilase-lysozyme isoforms were identified. However only one form of the enzyme encoded by Ds3 gene coincided with the protein CNBr fragments [Mol. Gen. Genet. 253 (1996) 20]. In this work we found by ESI-TOF mass spectrometry that the enzyme preparation consists of at least three forms with molecular masses of 12677.6, 12839.7, and 12938.2Da, each of which contains seven disulfide bridges. Only one mass (12839.7Da) fits to the calculated mass for the protein encoded by Ds3 gene. Further analysis of the CNBr fragments of the enzyme showed the heterogeneity of large 5.5 kDa peptide at positions 64 (threonine or arginine) and 67 (histidine or arginine) in the wild-type amino acid sequence. One CNBr peptide, with Arg and His at positions 64 and 67, respectively, correlates in the molecular mass with the protein encoded by Ds3. In addition, we have found a new acid form of destabilase-lysozyme, P-Ac, which differs from all known destabilase-lysozyme structures by its N-terminal amino acid sequence.

Full-sized HERV-K (HML-2) human endogenous retroviral LTR sequences on human chromosome 21: map locations and evolutionary history.

One of the evolutionary mechanisms for acquisition of novel functional sequences can be domestication of exogenous retroviruses that have been integrated into the germ line. The whole genome mapping of such elements in various species could reveal differences in positions of the retroviral integration and suggest possible roles of these differences in speciation. Here, we describe the number, locations and sequence features of the human endogenous retrovirus HERV-K (HML-2) long terminal repeat (LTR) sequences on human chromosome 21. We show that their distribution along the chromosome is not only non-random but also roughly correlated with the gene density. Amplification of orthologous LTR sites from a number of primate genomes produced patterns of presence and absence for each LTR sequence and allowed determination of the phylogenetic ages and evolutionary order of appearance of individual LTRs. The identity level and phylogenetic age of the LTRs did not correlate with their map locations. Thus, despite the non-random distribution of LTRs, they have apparently been inserted randomly into the chromosome relative to each other. As evidenced in previous studies of chromosomes 19 and 22, this is a characteristic of HERV-K integration.

Destabilase from the medicinal leech is a representative of a novel family of lysozymes.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

[The stimulating effect of destabilase, a component of Hirudo medicinalis salivary gland secretion, on sensory neuron neurite growth in organotypic culture]. / Stimuliruiushchee vliianie destabilazy, komponenta sekreta, sliunnykh zhelez meditsinskoi piiavki, na rost neiritov chuvstvitel'nykh neironov v organotipicheskoi kul'ture.

The effect of destabilase, a component of Hirudo medicinalis salivary gland secret, was investigated in organotypic tissue culture of dorsal root ganglia (DRG) of 10-11-day old chick embryos. Native destabilase in concentrations 0.01 and 0.05 ng/ml was active, inducing a more intensive neurite growth in DRG that in the control. The stabilizing activity of destabilase was lost following reverse-phase chromatography. Neurite-stimulating effects of the drug "pyjavit" is due presumably to neurite-stimulating activity of destabilase.