Linkage map of the fragments of herpesvirus papio DNA.

Herpesvirus papio (HVP), an Epstein-Barr-like virus, causes lymphoblastoid disease in baboons. The physical map of HVP DNA was constructed for the fragments produced by cleavage of HVP DNA with restriction endonucleases EcoRI, HindIII, SalI, and PvuI, which produced 12, 12, 10, and 4 fragments, respectively. The total molecular size of HVP DNA was calculated as close to 110 megadaltons. The following methods were used for construction of the map; (i) fragments near the ends of HVP DNA were identified by treating viral DNA with lambda exonuclease before restriction enzyme digestion; (ii) fragments containing nucleotide sequences in common with fragments from the second enzyme digest of HVP DNA were examined by Southern blot hybridization; and (iii) the location of some fragments was determined by isolating individual fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. Terminal heterogeneity and internal repeats were found to be unique features of HVP DNA molecule. One to five repeats of 0.8 megadaltons were found at both terminal ends. Although the repeats of both ends shared a certain degree of homology, it was not determined whether they were identical repeats. The internal repeat sequence of HVP DNA was found in the EcoRI-C region, which extended from 8.4 to 23 megadaltons from the left end of the molecule. The average number of the repeats was calculated to be seven, and the molecular size was determined to be 1.8 megadaltons. Similar unique features have been reported in EBV DNA (D. Given and E. Kieff, J. Virol. 28:524-542, 1978).     

Study of the thermal denaturation of ribonuclease A by differential thermal analysis and susceptibility to proteolysis

1. The thermally induced change in conformation of ribonuclease A in solution was investigated by differential thermal analysis and the susceptibility of the enzyme to proteolytic digestion by ficin. 2. A transition with a mid-point of 60.5°C at pH4.2 was observed directly by differential thermal analysis and shown to be a property of the native structure. 3. At pH4.2 ribonuclease A is susceptible to ficin digestion at 60°C but not at 18°C. 4. Chromatographic analysis of the digestion products reveals that transient active intermediates are produced during the digestion. 5. Three of these intermediates were purified and partially characterized. 6. The nature of those sections of the ribonuclease molecule that are involved in the thermal transition is discussed.     

MORPHOLOGICAL AND BIOCHEMICAL STUDIES OF ISOLATED MITOCHONDRIA FROM FETAL, NEONATAL, AND ADULT LIVER AND FROM NEOPLASTIC TISSUES

A combined morphological and biochemical investigation of mitochondria from developing and rapidly growing tissues ( tumors, fetal, and very early neonatal rat liver) revealed mitochondria which were deficient in respiratory control, showed no valinomycin induced K⁺ accumulation or spontaneous Ca⁺⁺ uptake, and were unable to undergo a swelling-contraction cycle. Electron microscopic examination of fetal and neonatal livers and a mammary tumor revealed mitochondria which differed from controls with respect to matrix density and ability to undergo reversible structural changes. The importance of isolation and assay media in interpretation of results is emphasized.     

Changes in Cerebral Acyl-CoA Concentrations Following Ischemia-Reperfusion in Awake Gerbils

Abstract: Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl-CoAs via acyl-CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia-reperfusion on brain fatty acid and acyl-CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia-reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4-fold in the ischemic-reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl-CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl- and arachidonoyl-CoA and a marked decrease in palmitoyl- and docosahexaenoyl-CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl-CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.     

Correlation Between Epstein-Barr Virus Membrane Antigen and Three Large Cell Surface Glycoproteins

A correlation between Epstein-Barr virus membrane antigen (MA) and three surface glycoproteins has been established on the basis of radio-immunoprecipitation and immunoabsorption experiments. For radio-immunoprecipitation, Epstein-Barr virus-infected cells were radiolabeled either with neuraminidase-galactose oxidase tritiated borohydride, a procedure highly specific for surface glycoproteins, or with a general tritiated amino acid mixture. Intact cells were incubated with MA(-) or MA(+) human sera, washed free of unbound immunoglobulins, and then lysed with Nonidet P-40. The antigen-antibody complexes were bound to protein A-Sepharose and after elution with sodium dodecyl sulfate were analyzed by acrylamide gel electrophoresis in sodium dodecyl sulfate. MA(+) sera specifically precipitated three glycoproteins with molecular weights of 236,000, 212,000, and 141,000 from B95-8 cells induced with 12-O-tetradecanoylphorbal-13-acetate (TPA) and from Raji cells superinfected with P3HR-1 virus. These glycoproteins were not detected on Epstein-Barr virus-negative Ramos cells treated with TPA or on B95-8 cells treated simultaneously with TPA and phosphonoacetic acid. Soybean lectin-Sepharose bound all three glycoproteins, and lectin-Sepharose-bound glycoproteins from TPA-induced P95-8 cells absorbed MA-specific antibody from MA(+) human sera. The data strongly suggest that either all three glycoproteins have MA determinants or they are part of a complex in which one or more of the components constitute the reactive antigen.     

High frequency of mutation to tubercidin resistance in CHO cells.

The acquisition of high-level resistance to tubercidin (an adenosine analog) in CHO cells occurs in a single step at high frequency (10(-3) to 10(-4)) without mutagenesis. Analysis of a large number of independent mutants by a fluctuation test (Luria and Delbruk, 1943) indicates that they arise independently of the selection medium and all fall into the same complementation group. All mutants tested lack detectable adenosine kinase activity. An analysis of hybrids between mutant and wild-type cells indicates that resistance to tubercidin is a recessive marker which segregates as would be expected if it were a haploid locus in the parental CHO cell. Resistance to tubercidin is not linked to the X chromosome in CHO cells and appears to occur at much lower frequency in primary Chinese hamster cells and other cultured cell lines.