Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryza sativa L.) with molecular markers

The thermo-sensititve genic male-sterile (TGMS) gene in rice can alter fertility in response to temperature and is useful in the two-line system of hybrid rice production. However, little is known about the TGMS gene at the molecular level. The objective of this study was to identify molecular markers tightly linked with the TGMS gene and to map the gene onto a specific rice chromosome. Bulked segregant analysis of an F₂ population from 5460s (a TGMS mutant line) x Hong Wan 52 was used to identify RAPD markers linked to the rice TGMS gene. Four hundred RAPD primers were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants; of these, 4 primers produced polymorphic products. Most of the polymorphic fragments contained repetitive sequences. Only one singlecopy sequence fragment was found, a 1.2-kb fragment amplified by primer OPB-19 and subsequently named TGMS1.2. TGMS1.2 was mapped on chromosome 8 with a RIL population and confirmed by remapping with a DHL population. Segregation analysis using TGMS1.2 as a probe indicated that TGMS1.2 both consegregated and was lined with the TGMS gene in this population. It is located about 6.7 cM from the TGMS gene. As TGMS1.2 is linked to the TGMS gene, the TGMS gene must be located on chromosome 8.This research was supported by the Rockefeller Foundation and China National High-Tech Research and Development Program. The first author is a Rockefeller Career Fellow at Texas Tech University     

Regulation,purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg²⁺ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn²⁺ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.     

Caenorhabditis Elegans Mutants Resistant to Inhibitors of Acetylcholinesterase

We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions.     

Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides,calcium, protein kinases and phosphatases and depolarization

Protein phosphorylation was investigated in [³²P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both ₁- and ₂-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the ₂-AR only marginally increased while the stimulation of ₁-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M₂-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated G_i-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of ₁-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported.     

Identification of Trans-Acting Genes Necessary for Centromere Function in Drosophila Melanogaster Using Centromere-Defective Minichromosomes

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.     

Protein interactions during coronavirus assembly.

Coronaviruses assemble and obtain their envelope at membranes of the intermediate compartment between the endoplasmic reticulum and Golgi complex. Like other enveloped viruses, coronavirus assembly is presumably dependent on protein localization and protein-protein as well as protein-RNA interactions. We have used the bovine coronavirus (BCV) as a model to study interactions between the viral proteins in virus-infected cells that are important for coronavirus assembly. BCV is a prototype for the coronaviruses that express an additional major structural protein, the hemagglutinin esterase (HE), in addition to the spike (S) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. Complexes consisting of the M, S, and HE proteins were detected in virus-infected cells by coimmunoprecipitations. Kinetic analyses demonstrated that S protein and HE each quickly formed a complex with M protein after synthesis, whereas heterocomplexes consisting of all three proteins formed more slowly. The kinetics of HE biosynthesis revealed that the half-life of oligomerization was approximately 30 min, which correlated with the appearance of complexes consisting of M, HE, and S proteins, suggesting that oligomerization and/or conformational changes may be important for the S-M-HE protein complexes to form. Only HE dimers were found associated with the heterocomplexes consisting of all three proteins. S-M-HE protein complexes were detected prior to processing of the oligosaccharide chains on HE, indicating that these protein complexes formed in a premedial Golgi compartment before trimming of sugar chains. Transient coexpressions and double-labeling immunofluorescence demonstrated that HE and S proteins colocalized with M protein. This was further supported by coimmunoprecipitation of specific HE-M and S-M protein complexes from transfected cells, indicating that these proteins can form complexes in the absence of other viral proteins.     

The MUC5AC gene: RFLP analysis with the Jer58 probe

We have recently obtained evidence that the locus corresponding to three groups of partial tracheobronchial cDNAs (A=Jer47, B=Jer57, C=Jer58) which mapped to chromosome 11p15 and was given the symbol MUC5 corresponds to two distinct genes which we have provisionally called MUC5B and MUC5AC. Here we describe the detection, using the Jer58 probe, which contains a 24-bp tandem repeat, of polymorphism in the MUC5AC gene with seven different restriction enzymes.     

Influence of broth dilution on the disruption of Escherichia coli

Summary The effect of cell concentration (5 to 150 g/L wet wt after broth dilution) on homogenizer disruption efficiency and homogenate viscosity is reported for E. coli. Broth dilution increases homogenizer efficiency and decreases feed and homogenate viscosity. However, this increase in disruption efficiency is not sufficient to warrant dilution of the broth prior to homogenization. The optimal feed concentration is the maximum possible that does not lead to practical handling difficulties due to high viscosity.