Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by <2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of >99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.
Heterotrimeric G proteins couple various receptors to intracellular effector molecules. Although the role of the G alpha subunit in effector activation, guanine nucleotide exchange and GTP hydrolysis has been well studied, the cellular functions of the G beta subunits are less well understood. G beta gamma dimers bind G alpha subunits and anchor them to the membrane for presentation to the receptor. In specific systems, the G beta subunits have also been implicated in direct coupling to ion channels and to effector molecules. We have isolated Drosophila melanogaster mutants defective in an eye-specific G-protein beta-subunit (G beta e), and show here that the beta-subunit is essential for G-protein-receptor coupling in vivo. Remarkably, G beta mutants are also severely defective in the deactivation of the light response, demonstrating an essential role for the G beta subunit in terminating the active state of this signalling cascade.
We have isolated a gram-positive, weakly acid-alcohol-fast, irregular rod-shaped bacterium from cultures of blood from a 5-year-old girl with acute myelogenous leukemia. This isolate was compared with 14 other strains including reference strains of Tsukamurella species by a polyphasic approach based on physiological and biochemical properties, whole-cell short-chain fatty acid and mycolic acid analyses, DNA-DNA hybridization, and sequencing of the 16S rRNA gene. This isolate represents a new taxon within the genus Tsukamurella for which we propose the name Tsukamurella strandjordae sp. nov. Our study also revealed that Tsukamurella paurometabola ATCC 25938 represents a misnamed Tsukamurella inchonensis isolate and confirms that Tsukamurella wratislaviensis belongs to the genus Rhodococcus.Tsukamurellae are members of the mycolic acid-containing aerobic actinomycetes. The genus was created in 1988 to accommodate a group of chemically unique organisms characterized by a series of very long chain (68 to 76 carbons) highly unsaturated (two to six double bonds) mycolic acids, in addition to possessing meso-diaminopimelic acid and arabinogalactan, common to the genus Corynebacterium (6). The type species, Tsukamurella paurometabola, described by Steinhaus as Corynebacterium paurometabolum in 1941, was originally isolated from the mycetomes and ovaries of bed bugs (27). The first human isolate of Tsukamurella was reported in 1971 as Gordona aurantiaca (33). Four additional species were proposed in the 1990s. Tsukamurella wratislaviensis was isolated from soil (10). Strains of Tsukamurella inchonensis, Tsukamurella pulmonis, and Tsukamurella tyrosinosolvens have been isolated only from human specimens, and all were associated with clinical disease (34-36). We have encountered an unusual isolate in multiple cultures of blood from a 5-year-old girl with acute myelogenous leukemia who presented with sepsis. On the basis of physiological and biochemical characteristics, analysis of cell components, DNA-DNA hybridizations, and the 16S rRNA gene sequence, we propose that this strain represents a new taxon within the genus Tsukamurella to which we assign the name Tsukamurella strandjordae sp. nov. Our study compared this isolate to reference and clinical strains of the other Tsukamurella species and found that T. paurometabola ATCC 25938 is a misnamed T. inchonensis isolate. MATERIAL AND METHODSStrains. The study included type strains of all proposed Tsukamurella species. T. paurometabola ATCC 8368 and T. wratislaviensis ATCC 51786 were purchased from the American Type Culture Collection (ATCC). A. F. Yassin generously provided the type strains T. pulmonis ATCC 700081 and T. inchonesis ATCC 700082. Type strain T. tyrosinosolvens DSMZ 44234 was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany). Other purchased reference strains included strains T. paurometabola ATCC 25938 and T. tyrosinosolvens DSMZ 44316. The strain of T. strandjordae, the subject of this study, was is...
A G protein beta subunit gene (Gbe) is expressed only in the eyes of adult D. melanogaster. This gene was identified by probing a Drosophila head cDNA expression library with monoclonal antibodies to a previously characterized Drosophila G protein beta subunit (Gbb). Immunoblot and Northern analyses demonstrate that Gbe protein and mRNA is not present in Drosophila mutants that lack eyes. Immunocytochemical and in situ hybridization analyses further demonstrate that Gbe is expressed in the eyes but not in the brain, whereas Gbb is abundantly expressed in the brain. The Gbe product is approximately 45% identical to previously identified G beta subunits and defines a new G beta class. Its localization suggests a possible role in phototransduction.
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