Report on a New Truffle Species,Tuber koreanum sp. nov., from Korea

The truffle and ectomycorrhizal roots formed by Tuber sp. were collected from the rhizosphere of Quercus aliena in Korea. The morphological characteristics of the ascoma, and molecular phylogenetic analysis using sequences from the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA, translation elongation factor 1-alpha (TEF), and RNA polymerase second largest subunit (RPB2) regions confirmed the distinct morphology of the truffle. This truffle belongs to a monophyletic clade among the other Tuber species in the phylogeny. This study describes the truffle, Tuber koreanum, as a new species reported from Korea.     

Comparative study of calf thymus and wheat germ RNA polymerase II: stability of initiation complexes and elongation rates.

Pure wheat germ RNA polymerase II but not calf thymus RNA polymerase II forms relatively stable binary complexes (half life time of 30 minutes at 0°C) with superhelical SV 40 DNA. On the contrary, the addition of a specific dinucleotide and a single ribotriphosphate permits the formation of highly stable complexes between both enzymes and SV 40 DNA. The elongation of RNA chains with preinitiated wheat germ enzyme only is stimulated by sarkosyl. These observations suggest that the wheat germ enzyme, as compared to that isolated from calf thymus, may contain a protein factor, a more native structure or both that permit efficient initiation and elongation of RNA chains on double stranded DNA.     

Structural insights into the dual activities of the two-barrel RNA polymerase QDE-1

Neurospora crassa protein QDE-1, a member of the two-barrel polymerase superfamily, possesses both DNA- and RNA-dependent RNA polymerase (DdRP and RdRP) activities. The dual activities are essential for the production of double-stranded RNAs (dsRNAs), the precursors of small interfering RNAs (siRNAs) in N. crassa. Here, we report five complex structures of N-terminal truncated QDE-1 (QDE-1ΔN), representing four different reaction states: DNA/RNA-templated elongation, the de novo initiation of RNA synthesis, the first step of nucleotide condensation during de novo initiation and initial NTP loading. The template strand is aligned by a bridge-helix and double-psi beta-barrels 2 (DPBB2), the RNA product is held by DPBB1 and the slab domain. The DNA template unpairs with the RNA product at position –7, but the RNA template remains paired. The NTP analog coordinates with cations and is precisely positioned at the addition site by a rigid trigger loop and a proline-containing loop in the active center. The unique C-terminal tail from the QDE-1 dimer partner inserts into the substrate-binding cleft and plays regulatory roles in RNA synthesis. Collectively, this work elucidates the conserved mechanisms for DNA/RNA-dependent dual activities by QDE-1 and other two-barrel polymerase superfamily members.     

HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor.

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.     

A novel Trichoderma species isolated from soil in Guizhou, T. guizhouense

A new species of Trichoderma, Trichoderma guizhouense, isolated from soil in Guizhou Province, is described based on morphology and phylogenetic analyses. This species exhibits characteristic Trichoderma morphology but is distinct from related species based on characters of phialides and conidia. Two DNA markers, the translation elongation factor 1 alpha (tef1) and RNA polymerase II subunit b (rpb2) were used for phylogenetic analyses. The correlation between morphological and molecular-based clustering demonstrated two studied isolates are a new species.     

Stachybotrys from soil in China, identified by morphology and molecular phylogeny

Four Stachybotrys strains were isolated from soil in China. One was identified as a novel species by morphological characters of phialides and conidia. It produced cylindrical conidia with irregular striations and smooth, hyaline conidiophores. Phylogenetic analysis of three DNA markers, the internal transcribed spacer region of rDNA (ITS1–5.8S–ITS2), the translation elongation factor 1 alpha (tef1) and RNA polymerase II subunit (rpb2), supported the morphological results. The correlation between morphological and molecular-based clustering demonstrated that the studied isolate was a new species. Two other isolates were identified as S. cf. elegans.     

Reinitiation of synthesis of small cytoplasmic RNA species K and L in isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.     

Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.

The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.     

Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition.

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.