Tsutomu Morinaga

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf‐SRP54) and the N‐terminal G‐domain and C‐terminal M‐domain (pf‐SRP54M) of pf‐SRP54 with an amino‐terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non‐conserved 213‐nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107‐nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine‐tagged pf‐SRP54M bound specifically to the conserved 107‐nucleotide RNA in the absence of pf‐SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

HSP70 and ribosomal protein L2: novel 5S rRNA binding proteins in Escherichia coli

A Northwestern analysis of Escherichia coli total proteins with radiolabeled 5S rRNA identified two novel 5S rRNA interacting proteins, a 70 kDa and a 37 kDa protein, and three ribosomal proteins reported on already. The N‐terminal sequencing of the 70 kDa protein separated by SDS–PAGE from the high‐salt‐washed fraction of crude ribosome led to the discovery of a polypeptide identical in its first 10 amino acid residues to E. coli heat shock protein 70. The N‐terminal eight amino acid sequence of the 37 kDa protein extracted from the high‐salt‐washed ribosome is identical to that of the ribosomal protein L2. In addition, the interaction of these proteins with 5S rRNA has been confirmed with gel mobility shift assays.

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix.

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.

Nucleotide sequences of genes for ribosomal protein L41 and tRNAThr(AGU) from Coprinus cinereus

The nucleotide sequences of genes for the homolog in Coprinus cinereus of the eukaryotic ribosomal protein L41 and for tRNAThr(AGU) are reported. The gene for tRNAThr(AGU) was located upstream of the gene for the L41 ribosomal protein, and these genes were adjacent to each other but in opposite orientations. The deduced amino acid sequence of ribosomal protein L41 exhibited strong homology to those of L41 proteins of several yeasts. The 56th amino acid of the deduced protein was proline, as it is in the L41 protein of a cycloheximide-sensitive strain of yeast. The putative secondary structure of the tRNA gene resembled the characteristic cloverleaf structure of tRNAs. Elements resembling an A-box and a B-box were found in the gene for tRNAThr(AGU). These boxes are known as internal promoter elements in genes for eukaryotic tRNAs.

Cloning and characterization of the gene for the iron-sulfur subunit of succinate dehydrogenase from the violet root rot fungus, Helicobasidium mompa

The sdhB gene, encoding the iron‐sulfur protein (Ip) subunit of succinate dehydrogenase (Sdh, EC 1.3.99.1), has been cloned from the violet root rot fungus, Helicobasidium mompa, and characterized. The promoter region contains a CCAAT box, TATA‐like box, and CT‐rich region. The gene is interrupted by eight introns and is predicted to encode a polypeptide of 291 amino acid residues. The putative amino acid sequence of the encoded product of sdhB gene from H. mompa shows high homology to the other known sdhB genes and is 79% identical to the Ip subunit of SdhB of Uromyces fabae. Three cysteine‐rich clusters associated with the iron‐sulfur centers involved in electron transport were particularly well conserved. One of these clusters contains a critical histidine residue implicated in carboxin sensitivity in the basidiomycetes. Only one copy of the gene was present in the genome of H. mompa, and reverse transcription (RT)‐PCR analysis of mRNA expression showed that the sdhB gene was transcribed in potato dextrose broth. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)