Nucleotide sequence and organization of full length human U4 RNA pseudogenes

Two loci encoding human U4 RNA, designated U4/7 and U4/14, have been isolated and sequenced. Both are pseudogenes in that their sequences do not match any identified human U4 RNA species perfectly. The U4/7 locus harbours a full-length pseudogene of 144 bp with eight base substitutions in the structural region. This pseudogene might be derived from a hitherto unidentified human U4 RNA gene. The second locus, U4/14, has a complex structure; the structural sequence of a U4 gene has apparently been integrated into an Alu sequence.     

Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. isolated from fast ice from Adelie Land, Antarctica

Nine psychrotolerant bacteria were isolated from fast ice in the middle of Geologie Archipelago, Adelie Land, Antarctica and were categorized into two groups, based on their SDS-PAGE profiles. Representatives from each of the two groups, namely strains DD 48^T and SJ 14^T exhibited phenotypic and chemotaxonomic characteristics confirming to the genus Psychrobacter. The 16S rRNA gene sequence indicated that the two isolates are closely related to each other and to the already reported fifteen species of Psychrobacter. Detailed studies on the phenotypic characteristics, chemotaxonomic properties and phylogenetic analysis of strains DD 48^T and SJ 14^T indicated that they are distinctly different from each other and the reported species of Psychrobacter. At the DNA-DNA hybridisation level, the two species exhibit less than 70% similarity. Thus, strains DD 48^T and SJ 14^T are identified as new species of the genus Psychrobacter for which the names Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. respectively are proposed.     

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.     

Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant

Mycobacterium strain FA4^T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4^T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4^T is able to degrade aflatoxin B₁. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4^T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4^T (DSM 44556^T = CIP 108203^T).     

Cloning and sequencing of a full-length rat sucrase-isomaltase-encoding

Sucrase-isomaltase (SI) has been widely used as a marker enzyme to study cellular differentiation in the small intestine. We isolated a 6.1-kb SI cDNA clone (GC1.4) from a size-fractionated cDNA library from rat intestine. Sequencing of this cDNA clone showed 6066 nucleotides (nt) with an open reading frame (ORF) of 1841 amino acids (aa). The nt sequence correctly predicts several known aa stretches in the protein. The deduced aa sequence showed 78 and 75% overall identity with the rabbit and human SI, respectively. At the active sites of both S and I, the rat nt sequence encodes stretches of 14 and 16 aa, respectively, which show 100% identity to rabbit and human SI. In the region immediately beyond the transmembrane domain, the rat sequence encodes an extra 10 aa, as compared to rabbit and human. This 10-aa insertion consists almost entirely of Pro, Ser and Thr, and may be responsible for additional 0-glycosylations of rat SI. The cDNA contains a 3'-UTR (untranslated region) of 499 nt with polyadenylation signal sequence and a poly(A) tract. The ATG start codon was found 41 nt downstream from the 5' end of the cDNA. Primer extension experiments showed the cap site to be 61 nt upstream from the start codon. The results indicate that our cDNA clone lacks only 20 nt in the 5'-UTR. Given that this cDNA encodes the entire coding region of SI, it should be useful in elucidating the regulatory mechanisms of SI biosynthesis, localization and targeting during rat intestinal development and differentiation.     

Subtypes of genotype A Candida albicans isolates determined by restriction endonuclease and sequence analyses

Systemic yeast infections are the leading cause of mortality and morbidity in immunocompromized patients. Candida albicans, being the most frequently isolated fungal pathogen in these patients, can be divided into three genotypes (genotypes A, B and C) by 25S intron analysis. In our study, we found that molecular sizes of genotype A C. albicans isolates were heterogeneous. In order to determine the molecular basis of this difference, HaeIII digestion was applied, and strains forming different band patterns were analyzed by automated sequence analysis. As a result of sequence analysis, eight different subtypes (a→h) were found among genotype A C. albicans strains and an easy differentiation scheme consisting of HaeIII and MspI digestions was constructed.     

Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12

A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus.     

Geminicoccus roseus gen. nov., sp. nov., an aerobic phototrophic Alphaproteobacterium isolated from a marine aquaculture biofilter

A Gram-negative, strictly aerobic, diplococcoid bacterium (strain D2-3^T) was isolated from the biofilter of a recirculating marine aquaculture system. Phylogenetic analysis of the 16S rRNA gene sequence of D2-3^T indicated that the new organism occupied a novel lineage within the -1 subclass of Proteobacteria and was related to the genera Rhodothalassium, Azospirillum, Craurococcus, Acidiphilium, and Tistrella. The highest sequence similarity (90.8%) of the 16S rRNA gene sequence of D2-3^T was to that of Candidatus “Alysiosphaera europaea”. D2-3^T was mesophilic, heterotrophic, required sea salt, and had a pH optimum of 8.0. Growth in the presence of light resulted in the formation of pink colonies, a 25% increased cell yield, and a slightly increased growth rate. D2-3^T contained carotenoids and low amounts of bacteriochlorophyll a. Membranes of D2-3^T contained b-type cytochromes. The G+C content of the DNA was 60.3±0.1 mol%. Phylogenetic, morphological, physiological, and biochemical analyses demonstrated that D2-3^T represented a new aerobic phototrophic genus, for which the name Geminicoccus roseus gen. nov., sp. nov. is proposed for the type species (D2-3^T=DSM 18922^T=ATCC BAA-1445^T).     

Stereocontrol in diels—alder cycloaddition to unsaturated sugars: reactivities of cis-dienophiles with cyclopentadiene

Cycloaddition of cyclopentadiene with a -arabinose-derived cis-dienophile, methyl (Z)-4,5,6,7-tetra-O-acetyl-2,32-dideoxy- -arabino-hept-2-enonate (2), under thermal conditions gave essentially a single norbornene aduct, isolated crystaline in 81% yield and identified by NMR spectroscopy and X-ray crystallography as methyl (5R,6S)-6-endo-(1,2,3,4-tetra-O-acetyl- -arabino-tetritol-1-yl) bicyclo[2.2.1]-hept-2-ene-5-endo-carboxylate (3). The diene adds exclusively from the si-face of the dienophile and give only the endo product. The same sequence starting from -arabinose gave the enantiomer (7) of 3. In contrast, a related cis-dienophile (9) having a butenolide ring with cyclopentadiene from the opposite (re) face giving mainly the endo adduct (5S,6R)-6-endo-(2,3,4-tri-O-acetyl)- -arabino-tetritol-1-yl) bicyclo[2.2.1]hept-2-ene-5-endo-carboxylic acid 1,4-lactone (10), isolated crystalline in 70% yield, whose structure was again established by NMR spectroscopy, and firmly consolidated by X-ray crystallography. The minor (11%) product was the exo(5S,6R)isomer 11. A cis-enonate 14, analogous to 2 but deoxygenated at the allylic position, showed negligible diastereofacial selectivity ans reacted with cyclopentadiene to give a mixture of all four possible adducts. A 6-membered ring dienophile 16 was also subjected to the same cycloaddition for comparison with the butenolide 9; it gave principally the two endo products 17 and 19 in 31 and 38% yields, respectively, accompanied by 12% of a mixture of the two exo products (18 and 20). The quantitative distribution of cycloaddition products as a function of dienophile stereochemistry is discussed. The high degree of asymmetric induction observed, especially with the readily accesible dienophiles 2 and 7, providesa valuable route of access to enantiomerically pure tetra-C-substituted cycloalkanes.     

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.     

Isolation of a new vahlkampfiid amoeba from soil: Paravahlkampfia lenta n. sp.

When the genus Paravahlkampfia was distinguished from the genus Vahlkampfia on the basis of significant differences in small subunit ribosomal DNA sequences, Paravahlkampfia ustiana was the only described species of this new genus. More recently, a vahlkampfiid strain has been isolated (from soil from an upland farm in Scotland) which has trophozoite and cyst morphology more similar to P. ustiana than to other non-flagellating vahlkampfiid species. Also, it clusters with P. ustiana in phylogenetic trees derived from 5.8S ribosomal DNA sequences. However, it is significantly different from P. ustiana in both phenotype and internal transcribed spacer sequence. Consequently, a new species, Paravahlkampfia lenta, is proposed.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Molecular analysis of the responder satellite DNA in Drosophila melanogaster: DNA bending, nucleosome structure, and Rsp-binding proteins.

The Responder (Rsp) locus of Drosophila melanogaster, the target locus of segregation distortion, is a satellite DNA array. This repeat array imparts some fitness advantage to the chromosomes bearing it. In this paper, we report the following three related molecular properties of this satellite repeat: (1) Sequence-directed curvature--On a polyacrylamide gel, Rsp-containing fragments migrate slower than would be predicted on the basis of their physical sizes. The extent of migration retardation correlates with the size and position of the Rsp sequence in a DNA fragment, suggesting that Rsp DNA is bent. The bending is shown to be affected by a DNA-binding drug (Hoechst 33258). (2) Nucleosome structure--Nucleosomes associated with Rsp repeats have an unusual spacing pattern. Instead of being spaced at approximately 190-bp intervals as is the bulk chromatin, they are separated at approximately 240-bp intervals, roughly the size of a dimeric Rsp repeat. The nucleosomal structure in the Rsp region is preferentially disrupted by Hoechst 33258, whereas the bulk chromatin appears to be insensitive to the drug. (3) Rsp-DNA binding proteins--Gel mobility-shift assays using nuclear extracts from pupae and end-labeled Rsp repeat demonstrate the presence of three distinct DNA-protein complexes. Competition assays suggest that these complexes are specific to the Rsp sequence, and two of these nucleoprotein complexes seem to be influenced by the presence of Hoechst 33258. The observed complexes are formed by nonhistone proteins of somatic origin and may be related to the normal functions of Rsp, rather than to the germ-line segregation distortion activities.     

珠江口沉积物好氧反硝化细菌的筛选及鉴定

研究首次于珠江口沉积物中分离出多株好氧反硝化细菌,从中筛选出一株反硝化性能最强的菌株A14-1。综合其生理生化及分子生物学鉴定的结果确定此菌株为红球菌属Rhodococcus aetherivorar。此菌株可在48 h内将培养基中的硝酸盐含量从157.91mg·L^-1降低至32.07mg·L^-1,反硝化效率高达26.20 mg·L^-1·h^-1,且不会产生亚硝酸盐的明显积累。以细菌总基因组DNA为模板成功扩增出亚硝酸还原酶基因nirS,说明亚硝酸还原酶可能参与了此菌株的好氧反硝化过程,将亚硝酸盐进一步还原,从而不会造成水体亚硝酸盐的积累。菌株A14-1在珠江口多个站点均有分布,环境适应能力强,且不会对环境造成危害,因此有望应用于污水的生物脱氮处理中。     

Taxonomy and chemical characterization of antibiotics of Streptosporangium Sg 10 isolated from a Saharan soil

A new actinomycete strain designated Sg 10, producing antimicrobial substances was isolated from an Algerian soil. Morphological and chemical studies indicated that strain Sg 10 belonged to the genus Streptosporangium. The comparison of its physiological characteristics with those of known species of Streptosporangium showed significant differences with the nearest species Streptosporangium carneum. Analysis of the 16S rDNA sequence of strain Sg 10 showed a similarity level ranging between 96.3% and 97.8% within Streptosporangium species, with S. carneum the most closely related. However, the phylogenetic analysis indicated that strain Sg 10 represent a distinct phyletic line suggesting a new genomic species. The antimicrobial activity of strain Sg 10 showed an antibacterial activity against Gram-positive bacteria as well as an antifungal one. Four active products were isolated from the culture broth using various separation procedures. On the basis of UV-VIS spectrometry, infrared spectroscopy and chemical revelations, the antibiotics were classified in the group of glycosylated aromatics.     

Nocardioides halotolerans sp. nov., isolated from soil on Bigeum Island, Korea

A strictly aerobic, Gram-positive, motile, coccoid-shaped, halotolerant actinobacterium (10% NaCl, w/v), designated MSL-23^T, was isolated from a soil sample on Bigeum Island, Korea. Results of 16S rRNA gene sequence analysis indicated that the isolate belonged to the genus Nocardioides, with the highest sequence similarity (95.63%) being to Nocardioides kribbensis KCTC 19038^T. The major menaquinone was MK-8(H₄), and the predominant cellular fatty acids were i-C_16:0, ai-C_17:0, C_18:1 ω9c and 10-methyl-C_16:0. The DNA G+C content was 69.7 mol%. The 16S rRNA gene sequence of strain MSL 23^T and its chemotaxonomic properties showed it to be unique in the genus Nocardioides. Phenotypic characteristics distinguished strain MSL-23^T from other Nocardioides species. On the basis of the phenotypic, chemotaxonomic and phylogenetic data strain MSL-23^T represents a novel species, for which the name Nocardioides halotolerans sp. nov. is proposed, with MSL-23^T (=KCTC 19274^T=DSM 19273^T) as the type strain.     

Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.