The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.
Crystallography, X-Ray ; GTP Phosphohydrolases ; chemistry ; metabolism ; Humans ; Protein Domains ; Ribosome Subunits, Large, Eukaryotic ; chemistry ; metabolism ; Saccharomyces cerevisiae ; chemistry ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; metabolism

A seed-borne fungus, Curvularia sp. EML-KWD01, was isolated from an indigenous wheat seed by standard blotter method. This fungus was characterized based on the morphological characteristics and molecular phylogenetic analysis. Phylogenetic status of the fungus was determined using sequences of three loci: rDNA internal transcribed spacer, large ribosomal subunit, and glyceraldehyde 3-phosphate dehydrogenase gene. Multi loci sequencing analysis revealed that this fungus was Curvularia spicifera within Curvularia group 2 of family Pleosporaceae.
Classification* ; DNA, Ribosomal ; Fungi ; Glyceraldehyde 3-Phosphate ; Humans ; Oxidoreductases ; Ribosome Subunits, Large ; Triticum

Six genes for nucleoprotein, phosphoprotein, matrix protein, hemagglutinin neuramindase protein, fusion protein and large protein were obtained by reverse transcription and PCR methods based on our previous work of sequencing full length genome of sendai virus BB1 strain (DQ219803 in GenBank). Sequencing showed the six genes were completely identical to that we reported. In order to supply the function necessary for rescuing and packaging of sendai virus vector in trans, the N, P, M, F, HN and L genes were separately cloned into an adenoviral shuttle expression vector pDC316 resulting in six recombinant adenoviral plasimds. Six replicating defective recombinant adenoviruses Ad5-N, Ad5-P, Ad5-M, Ad5-F, Ad5-HN and Ad5-L were obtained by separately cotransfection of pDC316 carrying N, P, M, F, HN and L genes with the adenoviral genomic plasmid pBHGloxdeltaE1, 3Cre into HEK293cells. Restrictive enzymatic results indicated that the six recombinant plasmids were correctly constructed. PCR results showed the recombinant adenoviruses contained the respective SeV genes . Western blotting as well as immunofluorescence assay indicated the expression of the corresponding proteins of sendai virus. These work laid the basis for the construction of the full length genome plasmid of sendai virus BB1 strain and the setup of SeV virus vector system based on SeV BB1 strain.
Adenoviridae ; genetics ; Animals ; Cell Line ; Cloning, Molecular ; Gene Expression Regulation, Viral ; Genetic Vectors ; genetics ; HN Protein ; genetics ; metabolism ; Humans ; Macaca mulatta ; Nucleoproteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosome Subunits, Large ; genetics ; metabolism ; Sendai virus ; genetics ; metabolism ; Viral Fusion Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism