Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

L-pilin variants of Neisseria gonorrhoeae MS11

Phase- and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS) and the pilin expression locus (pilE). Similarly, the non-piliated L-variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilE locus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a pilE(L) locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L-variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non-revertible and non-piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pilE can be regarded as a third mechanism of pilin variation in gonococci.     

Discrepancies in lipolytic activities induced by beta-adrenoceptor agonists in human and rat adipocytes

A number of catecholamine and non-catecholamine beta-adrenoceptor agonists, including the lipolytically selective compound BRL 37344, were compared for lipolytic activity on human and rat adipocytes. On rat adipocytes, all compounds were full agonists, BRL 37344 being the most potent. On human adipocytes, only the catecholamines were full beta-adrenoceptor agonists. The other compounds were partial agonists, with intrinsic activities declining in the order fenoterol greater than salbutamol greater than clenbuterol greater than BRL 37344. This was the case with FFA- as well as with glycerol-production. Addition of 20 microM phentolamine did not enhance BRL 37344 activity. The isoprenaline- and BRL 37344-induced lipolysis on rat white adipocytes was stereoselectively antagonized by enantiomers of alprenolol, with atypical low potencies and stereoselectivity. It was concluded that (1) human and rat adipocyte beta-adrenoceptors mediating lipolysis are not essentially different, (2) partial agonism in human adipocytes is not explained by enhanced re-esterification and (3) BRL 37344 selectively stimulates rat adipocyte lipolysis.     

Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains

Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product. Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease. In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme. Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis. Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates. Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Competitive inhibition of lipolytic enzymes. IV. Structural details of acylamino phospholipid analogues important for the potent inhibitory effects on pancreatic phospholipase A2

1-Acyl-2(R)-acylamino phospholipids are effective competitive inhibitors of porcine pancreatic phospholipase A2 (EC 3.1.1.4, Bonsen et al. (1972) Biochim. Biophys. Acta 270, 364-382). By systematically varying the substituent at C-1 and the acyl chain length at C-2, a series of phospholipid analogues was obtained for which the inhibitory power was determined in a detergent-containing and occasionally also in a detergent-free micellar substrate system. The recently proposed kinetic model applicable to water-insoluble inhibitors (Ransac et al. (1990) Biochim. Biophys. Acta 1043, 57-66) allowed a quantitative comparison of the inhibitory power Z of the various substrate analogues. The most powerful inhibitors of the enzyme were found to possess the general 2-(R)-structure: (formula; see text) Using as substrate (R)-1,2-didodecanoylglycero-3-phosphocholine in mixed micelles with sodium taurodeoxycholate, the inhibitor molecule with m = 4 and n = 11 showed a Z-value of 15,000. This implies an affinity of the inhibitor for the active site of the enzyme higher than 4 orders of magnitude stronger as compared with the substrate molecule. Slightly higher and lower m-values resulted in a sharp drop of the inhibitory power, which suggests that the enzyme must possess a rather short, but well-defined hydrophobic binding pocket for the C-1 alkyl chain. Variation of n (keeping m = 2 constant) resulted in inhibitors with nearly equal Z-values for n = 11, 13 and 15. Most probably the binding cleft on the enzyme for the C-2 acylamino chain is longer, more losely constructed and contributing less to the overall binding energy. Several members of the 2-acylamino phospholipids are water-soluble and possess relatively high critical micelle concentrations. Their inhibitory power could be tested not only in micellar substrate dispersions but also in assay systems where both the inhibitor and substrate are molecularly dispersed. It appeared that these water-soluble phospholipid analogues are effective inhibitors of the enzyme only after incorporation into an organized substrate/water interface. In contrast, in molecularly dispersed substrate solutions the same molecules have completely lost their inhibitory power. These observations support our kinetic model of lipolysis and interfacial inhibition.     

Properties and fate of plasma fibronectin bound to the tissue culture substratum

Plasma fibronectin (pFn) is a serum protein which, when adsorbed to a glass or plastic substratum, mediates the adhesion of fibroblasts in culture. We have studied some of the details of its adsorption and subsequent fate. By using ¹²⁵l-labeled pFn, we show that a substratum incubated with pFn adsorbs approximately 0.4 μg/cm² pFn (a monomolecular layer), and one incubated with medium containing serum adsorbs approximately 7 ng/cm² pFn (a 12-fold enrichment relative to a random selection of the soluble proteins). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) suggests the bound serum proteins (eluted with SDS) are primarily BSA and β-globulins. The bound pFn adheres so tightly, though, that most resists elution, as assayed (1) with pFn radioiodinated before binding, (2) with pFn radioiodinated after binding, or (3) by the cell spreading activity of the bound pFn retained after SDS treatment. Under culture conditions, there is a continuous “turnover” of substratum-bound pFn: soluble pFn can bind to a serum-coated substratum, while bound pFn is gradually removed by incubation with serum proteins. The presence of fibroblasts increases the rate of this removal several-fold. By SDS-PAGE the material removed (as well as that eluted from the substratum with SDS after cell detachment) is intact pFn or large (possibly proteolytically generated) fragments. Thus, pFn binds preferentially to the tissue culture substratum, but can be removed subsequently by the combined action of cells and other serum proteins.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.