Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

In vivo regulation of hepatic LDL receptor mRNA in the baboon. Differential effects of saturated and unsaturated fat

The effects of diets enriched with cholesterol and different fats upon plasma lipoproteins and hepatic low density lipoprotein (LDL) receptor mRNA levels were studied in a group of 18 normal baboons. Animals were fed diets containing 1% cholesterol and 25% fat as either coconut oil, peanut oil, or olive oil for a period of 20 weeks. Plasma total cholesterol, high density lipoprotein (HDL) cholesterol, beta-lipoprotein (LDL + very low density lipoprotein) cholesterol, apolipoprotein B and apolipoprotein A-I were measured in samples obtained at 4-week intervals. All three diet groups demonstrated a statistically significant increase in plasma cholesterol as compared to base line throughout the experiment. Hepatic LDL receptor (LDL-R) mRNA levels were quantified by dot blot hybridization in serial liver biopsies. Animals fed saturated fat sustained a significant reduction in hepatic LDL-R mRNA as compared to those fed either monounsaturated or polyunsaturated fat. A strong negative correlation between LDL-R mRNA and plasma total cholesterol (r = -0.71), HDL cholesterol (r = -0.76), and plasma apo A-I (r = -0.77) was observed only in those animals fed coconut oil. Weak negative correlations between LDL-R mRNA and other plasma parameters did not achieve statistical significance. We conclude that saturated and unsaturated oils may influence plasma cholesterol levels in part through differential effects on LDL receptor biosynthesis in baboons.     

Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming bacilli and non-spore-forming relatives

The phylogenetic incoherency of the genus Bacillus as presently described is demonstrated by analysis of both published and new data from comparative 16S rRNA cataloguing of nine Bacillus species and a number of related non-Bacillus taxa, i.e. Caryophanon latum, Filibacter limicola and Planococcus citreus. While the ellipsoidal-spore-forming bacilli, e.g. B. subtilis and allied species, formed a coherent cluster, the round-spore-forming bacilli showed a higher degree of relationship to the non-spore-forming organisms than these bacilli show among each other. Thus B. sphaericus clustered with C. latum, B. globisporus grouped with F. limicola, B. pasteurii with Sporosarcina ureae, and 'B. aminovorans' with P. citreus, respectively. These organisms formed two related subclusters which, in their phylogenetic depth, are comparable to that of the B. subtilis subline. With the exception of 'B. aminovorans', the 16S rRNA phylogeny was entirely consistent with the distribution of murein types. Even more distantly related to and grouping outside the main Bacillus cluster was B. stearothermophilus, which displayed a moderate relationship to Thermoactinomyces vulgaris. Taxonomic problems arising from the new insights into the intrageneric relationships of Bacillus are discussed.     

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

Luteal morphology, atresia, and plasma progesterone concentrations during the reproductive cycle of two oviparous lizards, Crotaphytus collaris and Eumeces obsoletus

From ovulation to oviposition, the corpora lutea of the oviparous lizards Crotaphytus collaris and Eumeces obsoletus exhibit three stages of luteal development: 1) luteogenesis, 2) luteal maturity, and 3) luteal regression. Each stage exhibits distinct characteristics, involving changes in: 1) luteal volume, 2) nuclear diameter of cells within the luteal cell mass, and 3) thecal development. Plasma progesterone concentration is greatest during luteogenesis and is positively correlated with ovarian atresia, although atresia occurred throughout the period of gravidity. These data suggest that in these two species, the corpora lutea secrete high amounts of progesterone immediately following ovulation and exhibit morphologically distinct stages of growth and regression.     

Influence of Physical Disruption on Growth of Attached Bacteria

Attached bacterial populations cultured without an exogenous carbon source or grown in conjunction with attached diatoms incorporated [³H]thymidine at a rate between four and five times lower than that of replicate bacterial populations which were dispersed before being assayed.     

Evidence for the existence of an expressed minor variant tRNAPhe in yeast

Two expressed brewer's yeast tRNAsPhe, a major and a minor one, have been purified and sequenced. The major tRNAPhe corresponds to the already known tRNAPhe, whereas the minor one differs from the former in the substitution of T6-A67 by C6-G67 base pair in the "acceptor stem". The minor tRNAPhe contaminates all preparations of yeast tRNAPhe except those prepared by polyacrylamide gel electrophoresis.     

Nuclear Overhauser effect studies of the conformations of MgATP bound to the active and secondary sites of muscle pyruvate kinase

MgATP binds both at the active site (site 1) and at a secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (chi = 68 +/- 10 degrees) and a C3'-endo ribose pucker (delta = 90 +/- 10 degrees). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 +/- 10% low anti (chi = 30 degrees), C3'-endo; 20 +/- 8% high anti (chi = 55 degrees), O1'-endo; and 18 +/- 8% syn (chi = 217 degrees), C2'-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., & Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O'Brian, C., Kaiser, E. T., & Mildvan, A. S. (1983) Biochemistry 22, 3439].(ABSTRACT TRUNCATED AT 250 WORDS)     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.