LmbJ and LmbIH protein levels correlate with lincomycin production in Streptomyces lincolnensis

AIMS: To demonstrate the expression of two overlapping genes lmbJ and lmbIH in Streptomyces lincolnensis and to document LmbJ and LmbIH protein levels during the lincomycin production phase. To analyse presumable function of the LmbIH protein. METHODS AND RESULTS: Lincomycin production was monitored by thin-layer chromatography, proteins LmbJ and LmbIH were assayed in the cell-free extracts of S. lincolnensis by immunodetection. LmbJ occurred at stable level (2-4 mg x g(-1) of total proteins) for a long time period (36-96 h of cultivation) covering the whole production phase. This fairly corresponds to the catalytic function of the protein in the antibiotic biosynthesis (N-demethyllincomycin methyltransferase). On the contrary, LmbIH reached the detectable level (0.1 and 0.7 mg x g(-1)) just for a short period at 60-72 h. CONCLUSIONS: The absence of LmbIH protein at a detectable level during the major part of the antibiotic production phase casts doubt on its possible catalytic function. Rather a different connection with the final biosynthetic steps, e.g. regulatory, can be envisaged. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of a newly found putative regulatory gene was demonstrated during production of industrial antibiotic, lincomycin.     

Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.     

Acid hydrolase activity of cultured bovine oligodendrocytes

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8–11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.     

Characterization and chromosomal localization of the chicken avidin gene family

Chicken avidin is a biotin-binding protein expressed under inflammation in several chicken tissues and in the oviduct after progesterone induction. The gene encoding avidin belongs to a family that has been shown to include multiple genes homologous to each other. The screening and chromosomal localization studies performed to reveal the structure and organization of the complete avidin gene family is described. The avidin gene family is arranged in a single cluster within a 27-kb genomic region. The cluster is located on the sex chromosome Z on band q21. The organization of the genes was determined and two novel avidin-related genes, AVR6 and AVR7, were cloned and sequenced.     

The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster

Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30–45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented. Received: 25 November 1999 / Revised: 10 January 2000     

Mining of assembled expressed sequence tag (EST) data for protein families: application to the G protein-coupled receptor superfamily

The availability of large expressed sequence tag (EST) databases has led to a revolution in the way new genes are identified. Mining of these databases using known protein sequences as queries is a powerful technique for discovering orthologous and paralogous genes. The scientist is often confronted, however, by an enormous amount of search output owing to the inherent redundancy of EST data. In addition, high search sensitivity often cannot be achieved using only a single member of a protein superfamily as a query. In this paper a technique for addressing both of these issues is described. Assembled EST databases are queried with every member of a protein superfamily, the results are integrated and false positives are pruned from the set. The result is a set of assemblies enriched in members of the protein superfamily under consideration. The technique is applied to the G protein-coupled receptor (GPCR) superfamily in the construction of a GPCR Resource. A novel full-length human GPCR identified from the GPCR Resource is presented, illustrating the utility of the method.     

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity.The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.     

A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism)

Tetracapsula bryosalmonae, formerly PKX organism, is a myxozoan parasite that causes proliferative kidney disease in salmonid fish. Its primary hosts, in which it undergoes a sexual phase, are phylactolaemate bryozoans. It develops in the bryozoan coelomic cavity as freely floating sacs which contain two types of cells, stellate cells and sporoplasmogenic cells, which become organised as spores. Eight stellate cells differentiate as four capsulogenic cells and four valve cells which surround a single sporoplasmogenic cell. The sporoplasmogenic cell undergoes meiosis and cytoplasmic fission to produce two sporoplasms with haploid nuclei. Sporoplasms contain secondary cells. The unusual development supports previously obtained data from 18S rDNA sequences, indicating that species of Tetracapsula form a clade. It diverged early in the evolution of the Myxozoa, before the radiation that gave rise to the better known genera belonging to the two orders in the single class Myxosporea. The genus Tetracapsula as seen in bryozoans shares some of the characters unique to the myxosporean phase and others typical of the actinosporean phase of genera belonging to the class Myxosporea. However, it exhibits other features which are not found in either phase. A new class Malacosporea and order Malacovalvulida are proposed to accommodate the family Saccosporidae and genus Tetracapsula. Special features of the new class are the sac-like proliferative body, valve cells not covering the exit point of the polar filament, lack of a stopper-like structure sealing the exit, maintenance of valve cell integrity even at spore maturity, absence of hardened spore walls and unique structure of sporoplasmosomes in the sporoplasms.