Primary structure of the herpesvirus saimiri genome.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.     

Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.

Cobyrinic acid a,c-diamide synthase, which catalyzes the conversion of cobyrinic acid to cobyrinic acid a,c-diamide via the intermediate formation of cobyrinic acid c-monoamide, was purified 155-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by high-performance liquid chromatography. The enzyme has an apparent molecular weight of 86,000 and consists of two identical subunits of Mr 45,000, as estimated by gel electrophoresis under denaturing conditions. Stepwise Edman degradation provided the N-terminal sequence of the first 15 amino acids. Glutamine was shown to be the preferred amino group donor (Km = 20.3 microM), but it could be replaced by ammonia (Km = 12 mM). The reaction was ATP dependent and exhibited a broad optimum pH around 7.3. Km values for (CN,aq)cobyrinic acid, (aq)2cobyrinic acid, and (CN,aq)cobyrinic acid c-monoamide were 160, greater than or equal to 250, and 71 microM, respectively. Hydrogenobyrinic acid and hydrogenobyrinic acid c-monoamide were shown to be much better substrates, with Km values of 0.41 and 0.21 microM, respectively.     

Cholesterol: free radical peroxidation and transfer into phospholipid membranes

Cholesterol, when sequestered in saturated liposomes of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC), undergoes peroxidation thermally initiated either by a lipid-soluble or a water-soluble azo initiator and in both cases the reaction is inhibited effectively by the water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox). Quantitative kinetic methods of autoxidation show that the oxidizability, kp/(2kt)1/2 (where kp and 2kt are the rate constants of radical chain propagation and termination, respectively) of cholesterol in DMPC or DPPC multilamellar liposomes, where kp/(2kt)1/2 is 3.0.10(-3) to 4.3.10(-3) M-1/2 s-1/2 at 37-45 degrees C, is similar to that measured in homogeneous solution in chlorobenzene, where kp/(2kt)1/2 is 3.32.10(-3). However, its oxidizability in smaller unilamellar vesicles of DMPC or DPPC increases by at least 3-times that measured in multilamellar systems. Autoxidation/antioxidant methods show that cholesterol partitions directly from the solid state into DMPC or DPPC liposomes by shaking and this is confirmed by 31P and 2H quadrupole NMR spectra of deuterated cholesterol when membrane bound. Analytical studies indicate that up to 21 mol% cholesterol will partition into the membranes by shaking.     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

Chemical and functional homology of myotoxin a from prairie rattlesnake venom and crotamine from South American rattlesnake venom

Myonecrosis is a serious result of rattlesnake bite and constitutes a persistent clinical problem. In the current study we have isolated crotamine from the venom of Crotalus durissus terrificus to test its ability to cause structural damage to skeletal muscle, and to make direct chemical comparisons with Myotoxin a, a myotoxic polypeptide we recently isolated from prairie rattlesnake (Crotalus viridis viridis) venom. Disc gel electrophoresis, isoelectric focusing, circular dichroic spectroscopy, and amino acid analysis, all indicated a high degree of chemical similarity. Light microscope histology revealed that crotamine caused vacuolizationof skeletal muscle fibers, qualitatively the same as the vacuolization caused by Myotoxin a. The ability of these two basic snake venom polypeptides to cause structural damage to skeletal muscle fibers has significant implications toward more complete understanding of the cause of snake venom-induced myonecrosis.     

EVOLUTION IN THE GENUS RUELLIA (ACANTHACEAE): A DISCUSSION BASED ON FLORAL FLAVONOIDS

A clear dichotomy exists in the genus Ruellia, separating the blue from the red flowered species. Flavonoids differ in a qualitative rather than a quantitative way. Apigenin 7-glucuronide and malvidin 3,5-diglucoside are common to all the blue flowered species, whereas chalcononaringenin 2'-glucoside (isosalipurposide) and pelargonidin 3,5-diglucoside are shared by the red flowered ones. The blue flowered species are linked with the red via apigenin 7-glucuronide and 3,5-diglucosylation of their respective anthocyanins. Both groups are involved in flavonoid race formation. All examined species (and some populations within species) differ in flavonoid content. The patterns of variability displayed provide a basis upon which an evolutionary scheme is constructed. Genetic drift is hypothesized as the effector of race formation in the blue flowered group.     

Nurse specialist in family planning.

In an experimental clinic, run by nurse specialists in family planning, a total of 768 patients were seen in the first year. Oral contraception was dispensed for 377 patients and 187 intrauterine devices (IUCDs) were inserted; a further 204 IUCD patients attended only for follow-up visits. All side effects were adequately diagnosed by the nurse specialist.     

Inhibition of cell division in amoebae: the incorporation of tritiated precursors into Amoeba proteus after the injection of non-homologous cytoplasm.

The injection of non-homologous cytoplasm into any strain of large free-living amoebae leads to a 60% inhibition of division amongst recipient cells. When the post-microsomal supernatant fraction of Amoeba discoides was injected into A. proteus, this inhibition of division was as high as 95%. The incorporation of tritiated precursors, either [3H]uridine or 3H-amino acids, into these inhibited amoebae was studied at various times after the injection of the inhibitory material using autoradiography. When cells were grown in [3H]uridine, autoradiographs indicated that RNA synthesis had ceased 2 days after the injection of non-homologous material. However, if [3H]uridine was injected into the inhibited cells, some synthesis of RNA could be detected up to 4 days after the injection of inhibitor. These results suggested that uptake of [3H]uridine was impaired and that one site of action of the inhibitory molecules was RNA synthesis for membrane components. Experiments with a variety of 3H-amino acids suggested that protein synthesis continued for at least 9 days after the injection of non-homologous cytoplasm, and that in these cells some informational RNA molecules were long-lived. There seemed to be accumulation of material containing [3H]lysine in the nuclei of control cells taken at random from cultures, and this was seen in the nuclei of inhibited cells 1 day after injection. However, 2 days after the injection of inhibitor, no accumulation of [3H]lysine-containing material was found in the nuclei.