Intracellular fate of fibrinogen B beta chain expressed in COS cells

Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.     

Use of simulated data sets to evaluate the fidelity of metagenomic processing methods

Metagenomics is a rapidly emerging field of research for studying microbial communities. To evaluate methods presently used to process metagenomic sequences, we constructed three simulated data sets of varying complexity by combining sequencing reads randomly selected from 113 isolate genomes. These data sets were designed to model real metagenomes in terms of complexity and phylogenetic composition. We assembled sampled reads using three commonly used genome assemblers (Phrap, Arachne and JAZZ), and predicted genes using two popular gene-finding pipelines (fgenesb and CRITICA/GLIMMER). The phylogenetic origins of the assembled contigs were predicted using one sequence similarity-based (blast hit distribution) and two sequence composition-based (PhyloPythia, oligonucleotide frequencies) binning methods. We explored the effects of the simulated community structure and method combinations on the fidelity of each processing step by comparison to the corresponding isolate genomes. The simulated data sets are available online to facilitate standardized benchmarking of tools for metagenomic analysis.     

Dictionary-driven prokaryotic gene finding

Gene identification, also known as gene finding or gene recognition, is among the important problems of molecular biology that have been receiving increasing attention with the advent of large scale sequencing projects. Previous strategies for solving this problem can be categorized into essentially two schools of thought: one school employs sequence composition statistics, whereas the other relies on database similarity searches. In this paper, we propose a new gene identification scheme that combines the best characteristics from each of these two schools. In particular, our method determines gene candidates among the ORFs that can be identified in a given DNA strand through the use of the Bio-Dictionary, a database of patterns that covers essentially all of the currently available sample of the natural protein sequence space. Our approach relies entirely on the use of redundant patterns as the agents on which the presence or absence of genes is predicated and does not employ any additional evidence, e.g. ribosome-binding site signals. The Bio-Dictionary Gene Finder (BDGF), the algorithm’s implementation, is a single computational engine able to handle the gene identification task across distinct archaeal and bacterial genomes. The engine exhibits performance that is characterized by simultaneous very high values of sensitivity and specificity, and a high percentage of correctly predicted start sites. Using a collection of patterns derived from an old (June 2000) release of the Swiss-Prot/TrEMBL database that contained 451 602 proteins and fragments, we demonstrate our method’s generality and capabilities through an extensive analysis of 17 complete archaeal and bacterial genomes. Examples of previously unreported genes are also shown and discussed in detail.     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.     

Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase

Arginine-386, the active-site residue of Escherichia coli aspartate aminotransferase (EC 2.6.1.1) that binds the substrate alpha-carboxylate, was replaced with tyrosine and phenylalanine by site-directed mutagenesis. This experiment was undertaken to elucidate the roles of particular enzyme-substrate interactions in triggering the substrate-induced conformational change in the enzyme. The activity and crystal structure of the resulting mutants were examined. The apparent second-order rate constants of both of these mutants are reduced by more than 5 orders of magnitude as compared to that of wild-type enzyme, though R386Y is slightly more active than R386F. The 2.5-A resolution structure of R386F in its native state was determined by using difference Fourier methods. The overall structure is very similar to that of the wild-type enzyme in the open conformation. The position of the Phe-386 side chain, however, appears to shift with respect to that of Arg-386 in the wild-type enzyme and to form new contacts with neighboring residues.     

Induction of citrate synthase by aldosterone in the rat kidney

Summary The possible induction of renal citrate synthase (E.C. 4.1.3.7), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 g/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968.Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on theK _m s for acetyl-CoA and oxalacetate but augmentedV _max of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation.The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 g/100 g body wt) or the diluent, and simultaneously with³H or³⁵S methionine (500 Ci/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 g/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either³H-or³⁵S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70–80 g/100 g body wt) or spirolactone (SC-26304) (80 g/100 g body wt). An equimolar dose of dexamethasone (0.8 g/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.     

Studies on the assembly and secretion of fibrinogen.

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.     

The Mechanism of the Calorigenic Action of Thyroid Hormone : Stimulation of Na+ + K+-activated adenosinetriphosphatase activity

In an earlier study, we proposed that thyroid hormone stimulation of energy utilization by the Na⁺ pump mediates the calorigenic response. In this study, the effects of triiodothyronine (T₃) on total oxygen consumption (Q_OO2), the ouabain-sensitive oxygen consumption [Q_OO2(t)], and NaK-ATPase in liver, kidney, and cerebrum were measured. In liver, ~90% of the increase in Q_OO2 produced by T₃ in either thyroidectomized or euthyroid rats was attributable to the increase in Q_OO2(t). In kidney, the increase in Q_OO2(t) accounted for 29% of the increase in Q_OO2 in thyroidectomized and 46% of the increase in Q_OO2 in euthyroid rats. There was no demonstrable effect of T₃ in euthyroid rats on Q_OO2 or Q_OO2(t) of cerebral slices. The effects of T₃ on NaK-ATPase activity in homogenates were as follows: In liver +81% from euthyroid rats and +54% from hypothyroid rats. In kidney, +21% from euthyroid rats and +69% from hypothyroid rats. T₃ in euthyroid rats produced no significant changes in NaK-ATPase or Mg-ATPase activity of cerebral homogenates. Liver plasma membrane fractions showed a 69% increase in NaK-ATPase and no significant changes in either Mg-ATPase or 5'-nucleotidase activities after T₃ injection. These results indicate that thyroid hormones stimulate NaK-ATPase activity differentially. This effect may account, at least in part, for the calorigenic effects of these hormones.     

Effects of aldosterone on Na transport in the toad bladder II. The anaerobic response

The action of aldosterone on active Na⁺ transport was assessed under aerobic and anaerobic conditions in the isolated urinary bladder of the toad, BUfo marinus. Aldesterone augmented the short-circuit current (I_sc) under rigorous anaerobiosis. Four lines of evidence indicate that the increase in anaerobic I_sc does not represent an equivalent increase in active Na⁺ transport: 1. Net Na⁺ transport, determined by isotopic fluxes, was the same in the aldosterone-treated and control quarter-bladders, and significantly greater than the simultaneously measured I_sc. 2. Amiloride, an inhibitor of the apiral entry of Na⁺, did not reduce the steroid-dependent increase in the anaerobic I_sc. 3. Substitution of choline for Na⁺ in the mucosal medium reduced the magnitude of the anaerobic I_sc values did not eliminate the effect of aldosterone. 4. Addition of ouabain, a potent inhibitor of the Na⁺ pump, partially inhibited the effect of aldosterone on the anerobic I_sc but a significant hormonal increment remained. The source of the anaerobic I_sc was not identified; an effort was made, however, to determine the dependence of this current on glycolysis. During anaerobics, aldosterone increased the integral I_sc by 42% but did not alter lactate production. These results suggest that the steroid-dependent increase in the anaerobic I_sc may involve effects on permeability properties of the epithelium rather than on active tranport systems.