Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

New versatile cloning and sequencing vectors based on bacteriophage M13

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.     

Interaction of exogenous benzoquinone with Photosystem II in chloroplasts. The semiquinone form acts as a dichlorophenyldimethylurea-insensitive secondary acceptor

Chloroplasts were submitted to a sequence of saturating short flashes and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor (B^?) present was estimated from the DCMU-induced increase in fluorescence in the dark caused by the reaction: QB^?

Q^?B. By varying the time interval between the preillumination and the mixing, the time course of B^? reoxidation by externally added benzoquinone was investigated. It was found that benzoquinone oxidizes B^? in a bimolecular reaction, and does not interact directly with Q^?. When a sufficient delay after the preillumination was allowed in order to let benzoquinone reoxidize B^? before the injection of DCMU, the fluorescence increase caused by one subsequent flash fired in the presence of DCMU was followed by a fast decay phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 100 μ}\text{s}$). The amplitude of this phase was proportional to the amount of B^? produced by the preillumination. This fast decay was observed only after the first flash in the presence of DCMU. These results are interpreted by assuming a binding of the singly reduced benzoquinone to Photosystem II where it acts as an efficient, DCMU-insensitive, secondary (exogenous) acceptor.     

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes

Incubation of human erythrocytes for 1–2 h at 37°C in a suspension of dipalmitoylphosphatidylcholine (DPPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45–55% and a depletion of cholesterol by 19–24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K⁺ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K⁺ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias: Detection of ligand-induced conformational changes

1. Modification of the Class II sulphydryl groups on the (Na⁺ + K⁺)-ATPase from rectal glands of Squalus acanthias with N-ethylmaleimide has been used to detect conformational changes in the protein. The rates of inactivation of the enzyme and the incorporation of N-ethylmaleimide depend on the ligands present in the incubation medium. With 150 mM K⁺ the rate of inactivation is largest (k₁ = 1.73 mM^?1 · min^?1) and four SH groups per α-subunit are modified. The rate of inactivation in the presence of 150 mM Na⁺ is smaller (k₁ = 1.08 mM^?1 · min^-1) but the incorporation of N-ethylmaleimide is the same as with K⁺. 2. ATP in micromolar concentrations protects the Class II groups in the presence of Na⁺ (k₁ = 0.08 mM^?1 · min^?1 at saturating ATP) and the incorporation id drastically reduced. ATP in millimolar concentrations protects the Class II groups partially in the presence of K⁺ (k₁ = 1.08 mM^?1 · min^?1) and three SH groups are labelled per α subunit. 3. The K⁺ -dependent phosphatase is inhibited in parallel to the (Na⁺ + K⁺)-ATPase under all conditions, and the ligand-dependent incorporation of N-ethylmaleimide was on the α-subunit only. 4. It is shown that the difference between the Na⁺ and K⁺ conformations sensed with N-ethylmaleimide depends on the pH of the incubation medium. At pH 6 there is a very small difference between the rates of inactivation in the presence of Na⁺ and K⁺, but at higher pH the difference increases. It is also shown that the rate of inactivation has a minimum at pH 6.9, which suggests that the conformation of the enzyme changes with pH. 5. Modification of the Class III groups with N-ethylmaleimide-whereby the enzyme activity is reduced from about 16% to zero-shows that these groups are also sensitive to conformational changes. As with the Class II groups, ATP in micromolar concentrations protects in the presence of Na⁺ relative to Na⁺ or K⁺ alone. ATP in millimolar concentrations with K⁺ present increases the rate of inactivation relative to K⁺ alone, in contrast to the effect on the Class II groups. 6. Modification of the Class II groups with a maleimide spin label shows a difference between Class II groups labelled in the presence of Na⁺ (or K⁺) and Class II groups labelled in the presence of K + ATP, in agreement with the difference in incorporation of N-ethylmaleimide. The spectra suggest that the SH group protected by ATP in the presence of K⁺ is buried in the protein. 7. The results suggest that at least four different conformations of the (Na⁺ + K⁺)-ATPase can be sensed with N-ethylmaleimide: (i) a Na⁺ form of the enzyme with ATP bound to a high-affinity site (E₁-Na-ATP); (ii) a Na⁺ form without ATP bound (E₁-Na); (iii) a K⁺ form without ATP bound (E₂-K); and (iv) an enzyme form with ATP bound to a low-affinity site in the presence of K⁺, probably and E₁-K-ATP form.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Forskolin as a novel lipolytic agent

Forskolin is a novel lipolytic agent which elevates cAMP and FFA release in rat adipocytes in a manner different from existing lipolytic factors. This effect of Forskolin is potentiated by all lipolytic hormones tested, i.e. epinephrine, ACTH, and glucagon and is also reversible. The same batch of adipocytes can be repeatedly stimulated after washing. The effective concentration of Forskolin is in the micromolar range. Its action is due to an activation of cAMP synthesis by adenylate cyclase. There is no effect on cAMP hydrolysis. In contrast to stimulation by lipolytic hormones, Forskolin-activated membrane adenylate cyclase was not further stimulated by GPP(NH)P. These results suggest that Forskolin may be a useful analytical agent in the study of adenylate cyclase mediated function in intact adipocytes.     

The effect of temperature on CL 218872 and propyl beta-carboline-3-carboxylate inhibition of [3H]-flunitrazepam binding in rat brain

The competitive inhibition of [³H]-flunitrazepam binding by CL 218872 and propyl beta-carboline-3-carboxylate (PCC), non-benzodiazepine compounds that show differential affinities for benzodiazepine (BZD) receptor subtypes, was studied in the rat cerebral cortex and hippocampus at different temperatures of incubation. The potency of both inhibitors was significantly greater at 0° than at 37°C. The magnitude of temperature induced enhancement of potency may correlate with the pharmacological efficacy of compounds that interact with BZD receptors. Hill slopes for CL 218872 shifted from 0.52 to 0.97 in the cerebral cortex when incubations were performed at 0° and 37°C, respectively. Hill values for PCC changed from 0.68 to 0.93 under similar temperature conditions. These observations suggest the presence of a homogenous population of benzodiazepine receptors at physiological temperatures or the inability of CL 218872 and PCC to distinguish between receptor subtypes at 37°C.