Cloning of the human glucocorticoid receptor cDNA.

We show that the human glucocorticoid receptor (GR), isolated from the breast cancer cell line MCF-7, has an apparent molecular weight identical to that of rat liver GR (94 kDa) and reacts with antibodies raised against the latter. These antibodies were used to clone cDNA sequences corresponding to the human GR from a lambda gt11 expression library constructed using MCF-7 poly(A)+ RNA. Three non-homologous cDNA clones with inserts of 125, 220 and 350 bp, which express epitopes recognised by the rat liver GR antibodies, were isolated. Rat liver GR antibodies, immunopurified using the immobilised purified beta-galactosidase fusion proteins, detect partially purified rat liver and human GRs on Western blots. In addition, these antibodies immuno-adsorb rat liver and human GRs affinity-labelled with [3H] triamcinolone acetonide. Northern blot analysis, using all three cDNA probes, reveals the presence of a major MCF-7 poly(A)+ RNA species of approximately 7 kb.     

Organization of Plasmodium falciparum genome: 1. Evidence for a highly repeated DNA sequence.

Plasmodium falciparum DNA, isolated from the merozoite stage, was cleaved with HindIII and cloned in pBR322 and lambda L47.1 vectors. Plasmid clones containing 13.4, 7.0, 4.3, 4.1 and 1.5 kb inserts were characterized in some detail. The inserts contain several repeating units of smaller size. Nucleic acid hybridization studies showed that the repeat element is present in the Plasmodium DNA at a very high copy number and appears to be distributed widely throughout the genome.     

Preparation of h and m antigens ofHistoplasma capsulatum free of heterologous antigens

Salt gradient elution of crude histoplasmin on CM-Sepharose CL6B at pH 3.0 was used in essentially a one-step procedure to isolate the h, m, and non-m antigens ofHistoplasma capsulatum and free them of any c antigen common to other pathogenic fungi. The h antigen fraction was pure; the isolated m and non-m antigens contained less than 0.1% of the c antigen present in the original preparation. The residual c antigen in these fractions was removed by affinity chromatography on Affigel-10 coupled to aBlastomyces dermatitidis fungal antiserum. The final preparations of h, m, and non-m were pure when tested by immunodiffusion (ID), sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), or electrofocusing. The antigens were serologically stable at neutral pH for at least three months at 10°C. The relative molecular weights, isoelectric points, and amino acid composition of the antigens are described.     

Role of quinones in the branch of the Escherichia coli respiratory chain that terminates in cytochrome o.

The role of quinones in the cytochrome o branch of the Escherichia coli respiratory chain was investigated by using mutant strains lacking the cytochrome d terminal oxidase complex. The only cytochromes present were cytochrome b556 and the cytochrome o complex, consisting of cytochrome b555-b562. Mutant strains missing ubiquinone, menaquinone, or both were constructed in the cytochrome d-minus (cyd) background. The steady-state levels of cytochrome b reduction were examined and compared in these strains to assess the effects of the quinone deficiencies. The data clearly show that a ubiquinone deficiency results in a lower level of cytochrome b reduction in the steady state. The data are consistent with a simple model in which ubiquinone is placed on the dehydrogenase side of all the cytochromes in this branch of the respiratory chain. There is no evidence from these experiments for a role of quinones in the respiratory chain at any site besides this one.     

Potentiometric analysis of the cytochromes of an Escherichia coli mutant strain lacking the cytochrome d terminal oxidase complex.

A combination of potentiometric analysis and electrochemically poised low-temperature difference spectroscopy was used to examine a mutant strain of Escherichia coli that was previously shown by immunological criteria to be lacking the cytochrome d terminal oxidase. It was shown that this strain is missing cytochromes d, a1, and b558 and that the cytochrome composition of the mutant is similar to that of the wild-type strain grown under conditions of high aeration. The data indicate that the high-aeration branch of the respiratory chain contains two cytochrome components, b556 (midpoint potential [Em] = +35 mV) and cytochrome o (Em = +165 mV). The latter component binds to CO and apparently has a reduced-minus-oxidized split-alpha band with peaks at 555 and 562 nm. When the wild-type strain was grown under conditions of low aeration, the components of the cytochrome d terminal oxidase complex were observed: cytochrome d (Em = +260 mV), cytochrome a1 (Em = +150 mV) and cytochrome b558 (Em = +180 mV). All cytochromes appeared to undergo simple one-electron oxidation-reduction reactions. In the absence of CO, cytochromes b558 and o have nearly the same Em values. In the presence of CO, the Em of cytochrome o is raised, thus allowing cytochromes b558 and o to be individually quantitated by potentiometric analysis when they are both present.     

Asymmetric reconstitution of homogeneous Escherichia coli sn-glycerol-3-phosphate acyltransferase into phospholipid vesicles

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside. Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50. Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000. These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules. Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle. The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1. The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid). Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles. Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles. Disruption of vesicles with Triton X-100 did not reveal significant latent activity. These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.     

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.     

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.     

Effect of the beta-gamma phosphate bond of ATP on synthesis of leader RNA and mRNAs of vesicular stomatitis virus.

RNA products synthesized in vitro by vesicular stomatitis virus with normal nucleotides or imido analogs were compared by polyacrylamide gel electrophoresis. When imido ATP, which has a nonhydrolyzable beta-gamma bond, was substituted for ATP, leader RNA and DI particle product were synthesized, but appreciable mRNA synthesis was not detected.     

Connective tissue activation. XXX: Isoelectric point microheterogeneity of CTAP-III, a human platelet-derived growth factor

Connective tissue activating peptide (CTAP-III) is one of the growth factors found in the alpha granules of human platelets. This small cationic platelet protein shows several apparent isoelectric point variants after the preparative isoelectric focusing step in large scale preparations from multiple donors. Analytical isoelectricfocusing techniques under denaturing conditions, coupled with Western blotting and immunodetection, now show that CTAP-III prepared rapidly from single donors also has multiple isoelectric point variants, suggesting that this finding is more likely related to post-translational modification than preparative artifact or genetic polymorphism.