The role of cAMP in flagellation of Salmonella typhimurium

Summary A mutational alteration either in adenylate cyclase (cya ^-) or in cyclic-35-AMP (cAMP) receptor protein (crp ^-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation, cfs, partially suppressing the cya ^- mutation, was identified among the revertants of cya ^-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.     

Isolation of cDNA for Pea Phytochrome Using an Expression Vector

Partially purified phytochrome mRNA was obtained from etiolatedpea epicotyls by polyribosome immunoprecipitation or by sizefractionation of total poly(A)⁺RNA, and used for the synthesisof double-stranded complementary DNA (cDNA). cDNA librarieswere constructed using an Escherichia coli expression vector,pUC9, and screened for phytochrome cDNA by colony immunologicalassay. Nine colonies were found to produce a 27 kDa polypeptidethat was reactive to both polyclonal and monoclonal antipeaphytochrome antibodies. The plasmids from these colonies containedcDNA inserts of 1.2 or 2.0 kbp. Hybridization-arrest translationassay verified that the cDNA clones contained a sequence codingfor phytochrome polypeptide. RNA blot hybridization analysisindicated that the cDNA hybridized to a 4.1 kb poly(A)⁺RNA indark-grown pea. (Received March 22, 1986; Accepted June 13, 1986)     

Sugar-Dependent Expression of the CHS-A Gene for Chalcone Synthase from Petunia in Transgenic Arabidopsis

Transgenic Arabidopsis thaliana plants were constructed by introduction of a fusion of the gene for β-glucuronidase (GUS) to the CHS-A gene, which is one of the two genes for chalcone synthase that are actively expressed in the floral organs of petunia. The expression of the fusion gene CHS-A::GUS was low in transgenic Arabidopsis plantlets, but it was enhanced when plantlets or detached leaves were transferred to a medium that contained 0.3 molar sucrose, glucose, or fructose. No enhancement was observed when plantlets were transferred to a medium that contained 0.3 molar mannitol. Measurements of cellular levels of sugars revealed a tight linkage between the level of expression of the CHS-A::GUS gene and the level of accumulation of exogenously supplied sugars, in particular sucrose. The parallelism between the organ-specific accumulation of sugar and the organ-specific expression of the CHS-A::GUS gene was also observed in petunia and A. thaliana plants grown under normal conditions in soil. The consensus sequences for sugar responses, such as boxes II and III in members of the family of sporamin genes from the sweet potato, were found in the promoter region of the CHS-A gene that was used for fusion to the GUS gene. It is suggested that the expression of the CHS-A gene is regulated by sugars, as is the expression of other sugar-responsive genes, such as the genes for sporamin. A putative common mechanism for the control of expression of “sugar-related” genes, including the CHS-A gene, is discussed.     

Transient expression of β-glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun

Successful transient expression of -glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm²) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm² of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm² of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.     

Electrophoretic Analysis of Florally-Evoked Meristems of Pharbitis nil Choisy cv. Violet

Seedlings of Pharbitis nil Choisy cv. Violet were subjectedto a single SD (floral induction) or NB (inhibition of induction)treatment. Soluble proteins were extracted from the apical regionof seedlings just before the treatment or at various times afterthe treatment and were analyzed by two-dimensional polyacrylamidegel electrophoresis (2-D PAGE) to identify the changes specificallyinvolved in floral induction. The patterns of polypeptides werealmost identical between apices of short day-treated plants(SD-plants) and night break-treated plants (NB-plants), andquantitative differences were found in five polypeptide spots.Intensities of three of these polypeptide spots increased graduallyor transiently after SD treatment. The remaining two spots increasedin intensities after NB treatment. A fairly consistent resultswere obtained by a similar analysis of proteins labelled invivo. These differences were detected at relatively early stagesof flower formation, and some of them were retained or evenmagnified at a later stage. These findings suggest that thereare remarkably subtle differences between the complements ofproteins in the evoked and non-evoked apical meristems, andthat a few consistent quantitative differences are clearly apparentat the relatively early stages of formation of flowers. (Received September 20, 1989; Accepted November 16, 1989)     

Antibody expression in protease-deficient strains of the methylotrophic yeast Ogataea minuta

When human antibody genes were expressed in the methylotrophic yeast Ogataea minuta, the secreted antibody became partially degraded. To suppress the degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the vacuolar protease-deficient strain, the secreted antibody still became partially degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains.     

Chronological characterization of diabetes development in male Spontaneously Diabetic Torii rats

To characterize the underlying mechanisms of diabetes development in males of the Spontaneously Diabetic Torii (SDT) rat, a novel spontaneous model for diabetes, we chronologically examined them, focusing on their diabetic features and the pathological changes in the pancreatic islets. Male SDT rats exhibited glucose intolerance with impaired insulin secretion after 14 weeks and developed diabetes with remarkable hyperglycemia and marked hypoinsulinemia after 20 weeks. At prediabetic stage (10-20 weeks), they were normoglycemic, but had significantly lower insulin levels of plasma and pancreas than the normal rats. Their beta-cell volume was already smaller significantly at 10 weeks than that of normal rats. The primary changes of the pancreatic islets were microvascular events such as congestion and hemorrhage at 8-10 weeks. Thereafter, the SDT rat islets were affected with inflammation and progressive fibrosis (at 10-20 weeks), and eventually atrophied with a loss of beta-cells (at 38 weeks). These results indicate that the male SDT rats develop spontaneous diabetes with an absolute decrease in the insulin secretory capacity of the islets.