Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

INTRODUCTIONAsearlyasin1948wehavefr8CtionatedisolatednucleifromnormalandtumorcellsbyextractionwithiMNaCIanddilutealkali[1].Thenuclearresiduewasthenstudiedmorethoroughly[2,3].Lateron,sillillarproteinousnuclearresidueswereisolatedbyotherworkers[46]andasstud…     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

PHB synthase from Streptomyces aureofaciens NRRL 2209

An approximately 4.9 kb Sau3A I genomic DNA fragment from the Streptomyces aureofaciens NRRL 2209 aiding in the biosynthesis of PHB in recombinant Escherichia coli has been sequenced and analysed for phaC gene. The putative phaC(Sa) gene of 2 kb is 79.1% GC rich and encodes a 63.5 kDa protein. It expressed under its own promoter and significant PHA synthase activity was detected in the recombinant E. coli. This is the first putative PHA synthase gene reported from a Streptomyces sp. with serine as the active nucleophile in the conserved lipase box. The phaC(Sa) was found in close proximity to a regulatory gene, which apparently regulated the phaC expression.     

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediaesp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.     

Construction of a Streptomyces sp.–Escherichia coli conjugative shuttle vector and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acid)

pGTR760 and pGTR761, two new shuttle vectors, withmultiple cloning sites and capable of conjugal transfer from E. coli to Streptomyces sp. were constructed. The poly-3-hydroxybutyrate (PHB) biosynthetic polycistron from Ralstonia eutropha was cloned into the pGTR760 vector to derive the pCAB_Re plasmid. The pCAB_Re plasmid was conjugally transferred from E. coli S17-1 to Streptomyces lividans TK64. Fluorescence microscopy of the recombinant and the untransformed S. lividans TK64 revealed presence of polyhydroxyalkanoates (PHAs) in both cell types. GC/GC-MS analysis revealed the accumulated polymer to be polyhydroxyoctanoate (PHO). While the untransformed S. lividans cells accumulate 3.5% PHO of cell dry wt, the recombinant cells accumulate 8% PHO of the cell dry wt. The transformation of S. lividans, however, resulted in slower growth rate, delayed sporulation and impaired pigment formation. Scanning electron microscope analysis revealed broken mycelia probably due to release of accumulated PHO granules from the cells.