Mediator structure and conformation change

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Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Cytological and mapping analysis of a novel male sterile type resulting from spontaneous floral organ homeotic conversion in marigold (<Emphasis Type="Italic">Tagetes erecta</Emphasis> L.)

A male sterile line was isolated in marigold (Tagetes erecta L.) and cytological analysis determined this to be a novel genic male sterility trait (Tems). Through the use of amplified fragment length polymorphisms (AFLPs) and bulked segregant analysis (BSA), tightly linked markers of Tems were identified with a view towards a map-based cloning strategy. It was found that spontaneous homeotic conversion of floral organs was the underlying cause of the male sterility in this marigold line. Thus, petals of male sterile plants resembled sepal-like structures and the stamens were partially converted to styles, although without the full characteristics or function of the true style organs. We have constructed a fine marker-based map for the Tems gene. This is intended to provide a tool for marker assisted selection (MAS) strategies in hybrid breeding and map-based cloning strategies for the male sterility locus. We discuss the significance of this spontaneously derived genic male sterility trait relating to the homeotic conversion of floral organs in marigold.     

A NEW RHEOCRICOTOPUS THIENEMANN & HARNISCHIA (DIPTERA: CHIRONOMIDAE) FROM TAIWAN PROVINCE CHINA

Abstract  Rheocricotopus (Psilocricotopus) taiwanensis sp. n. from Taiwan, China, is described as male imagines. The species is allied to R. (P.) chalybeatus (Edwards) but it is easily separated from the latter in lacking setae in all reins, much lower AR and more pronounced crista dorsalis in gonostylus. The genus is new to Taiwan Province, China. The specimen is deposited at the Department of Biology, Nankai University, Tianjin.