Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Characterization of specific HLA-DQ alpha allospecificities by genomic, biochemical, and serologic analysis

Bgl II restriction endonuclease digestion of genomic DNA from lymphoblastoid cell lines homozygous for HLA DR and DQ serological specificities, followed by hybridization with a DQ alpha cDNA probe, identified a genomic polymorphism characterized by two reciprocal patterns, one associated with DR 3, 5 and 8 and the other with DR 1, 2, 4, 7, and 9. The former pattern corresponded precisely to the reactivity of monoclonal antibody SFR20-DQ alpha 5, shown by Western blotting to react with isolated alpha-chains, but not with beta-chains. Additional variants of the DQ alpha genes were identified by using a locus-specific oligonucleotide probe for the DQ alpha gene, indicating differences among the DQ alpha 5-negative set of alleles. This analysis defines a set of DQ alpha allelic markers that are distinct from the well-established DQ serologic specificities DQw1, 2, 3 or "blank." Although most DQ alpha 5+ cells carry the DRw52 specificity associated with the DR beta 2 gene, analysis of DQ alpha polymorphisms on DR5, DQw1; DR8, DQw1; and DRw13, DQw1 cells verified that this DQ alpha family of alleles was not invariably linked to the DR beta 2 locus.     

Internal anion binding site and membrane potential dominate the regulation of proton pumping by the chromaffin granule ATPase

Effects of anions and membrane potential on the reconstituted proton pump from chromaffin granules were investigated. When acetate was present inside of the vesicles, ATP-dependent proton uptake was absolutely dependent on external chloride. Without external chloride, however, substantial proton uptake was observed when chloride or sulfate was present inside of the vesicles. Inside negative membrane potential drove ATP-dependent proton uptake regardless of the anion species present inside or outside of the vesicles. It is concluded that the internal anion binding site and membrane potential regulate the proton pumping activity of the ATPase.     

Compatability of Metarhizium anisopliae var. anisopliae with chemical pesticides

The effects of various insecticides on the mycelial growth, sporulation and conidial germination of Metarhizium anisopliae var. anisopliae isolate E₉ were studied in the laboratory. Chlorpyrifos was the most toxic organophosphate to mycelial growth and sporulation at all concentrations. Temephos, malathion and leptophos were highly toxic to sporulation while malathion was the most inhibitory to germination. The carbamates, carbofuran, methomyl and oxamyl were moderately toxic to mycelial growth and sporulation while oxamyl had an adverse effect on germination. The pyrethroids (pyrethrin, permethrin and resmethrin) and the insect growth regulators (diflubenzuron and methoprene) were not inhibitory to the various developmental stages of isolate E₉. The chlorinated hydrocarbons (chlordane, lindane and toxaphene) were more deleterious than all other insecticide groups tested. Among the fungicides, benomyl and maneb produced the greatest inhibition.     

Chicken erythrocyte beta-globin chromatin: enhanced solubility is a direct consequence of induced histone hyperacetylation.

Chicken immature red blood cells were incubated for 1 hour in Swim's medium containing 3H-acetate and 10 mM n-butyrate. During the incubation period, the small percentage of dynamically acetylated and deacetylated histone is radiolabeled and hyperacetylated. A second effect of the n-butyrate incubation is to shift a small subset of nucleohistone into a soluble form. This chromatin is predominantly polynucleosome size (approximately dimer to pentamer) and can be separated from soluble mononucleosomes by 5-30% sucrose gradient centrifugation. The soluble polynucleosomes are 25-30 fold enriched for adult beta-globin (beta A) DNA and contain the hyperacetylated histones. We have tested whether histone hyperacetylation is responsible for the enhanced beta-globin chromatin solubility by in vitro deacetylation of the soluble chromatin histones. This procedure converts the beta-globin polynucleosomes to an insoluble form, demonstrating that histone hyperacetylation is in fact directly responsible for the increased solubility of the beta A chromatin.     

Restriction endonucleases for pulsed field mapping of bacterial genomes.

Fundamental to many bacterial genome mapping strategies currently under development is the need to cleave the genome into a few large DNA fragments that can be resolved by pulsed field gel electrophoresis. Identification of endonucleases that infrequently cut a genome is of key importance in this process. We show that the tetranucleotide CTAG is extremely rare in most bacterial genomes with G+C contents above 45%. As a consequence, most of the sixteen bacterial genomes we have tested are cleaved less than once every 100,000 base pairs by one or more endonucleases that have CTAG in their recognition sequences: Xba I (TCTAGA), Spe I (ACTAGT), Avr II (CCTAGG) and Nhe I (GCTAGC). Similarly, CCG and CGG are the rarest trinucleotides in many genomes with G+C content of less than 45%. Thus, Sma I (CCCGGG), Rsr II (CGGWCCG), Nae I (GCCGGC) and Sac II (CCGCGG) are often suitable endonucleases for producing fragments that average over 100,000 base pairs from such genomes. Pulsed field gel electrophoresis of the fragments that result from cleavage with endonucleases that cleave only a few times per genome should assist in the physical mapping of many prokaryotic genomes.     

Depression of cell-mediated immunity by tumour cell products: induction of resistance by immunotherapeutically active extracts of bovine ocular squamous cell carcinoma

Summary Tumours produce substances that inhibit the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity in mice. Phenol-saline extracts of bovine ocular squamous cell carcinoma (BOSCC) which have immunotherapeutic activity in cattle were able to immunize mice against this depressive effect. Such immunization was effective against products of BOSCC, a spontaneous rat tumour, three of four human tumour cell lines and (in other experiments) mouse tumours. Phenol-saline extracts of mouse tumour cell lines were immunogenic (protective against depression of delayed-type hypersensitivity) in mice. Fractions of BOSCC phenol-saline extracts which were immunotherapeutically active in cattle were generally also protective in mice. The protective activity was lost after treatment with proteinase K, and was present in the supernatant after precipitation with 55% ammonium sulphate. It was not affected by treatment with RNase or DNase or by heating to 50 °C for 2 h. It was present in gel filtration fractions with an apparent molecular weight of 10,000–37,000 daltons. The immunogenic factor in mice and the immunotherapeutic factor in cattle may be related to each other.