Effect of base-pair stability of nearest-neighbor nucleotides on the fidelity of deoxyribonucleic acid synthesis

The influence of the stability of base pairs formed by nearest-neighbor nucleotides on misincorporation frequency has been studied with the large fragment of DNA polymerase I, the alternating DNA copolymers, poly(dI-dC) and poly-(dG-dC), as template-primers, and dGTP, dITP, and dCTP as substrates. We have utilized the difference in thermodynamic stability between the doubly H-bonded I X C base pair and triply H-bonded G X C base pair to examine the effects of base-pair stability of both the "preceding" and the "following" nucleotides on the frequency of insertion of a mismatched nucleotide, as well as on its stable incorporation into polynucleotide. The present studies demonstrate that the stability of the base pairs formed by nearest-neighbor nucleotides affects the frequency of incorporation of noncomplementary nucleotides. Misincorporation frequency is increased when the nearest-neighbor nucleotides form more stable base pairs with the corresponding nucleotides in the template and is decreased when they form less stable base pairs. The stability of the base pair formed by a nucleotide either preceding (5' to) or following (3' to) a misincorporated nucleotide influences misincorporation frequency, but by different mechanisms. The stability of base pairs formed by preceding nucleotides affects the rate of insertion of mismatched nucleotide but does not protect the mismatched nucleotide from removal by the 3' to 5' exonuclease activity. In contrast, the stability of a base pair formed by a following nucleotide determines whether a misincorporated nucleotide is extended or excised by affecting the ability of the enzyme to edit errors of incorporation.     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.     

Peptide maps and N-terminal sequences of polypeptides from early region 1A of human adenovirus 5

Experiments exploring the reasons for a multiplicity of products from early region 1A of adenovirus 5 are described. Labeled early region 1A products from wild-type virus were synthesized in infected cells and in a cell-free system programmed with mRNA from infected cells, immunoprecipitated specifically with an antipeptide serum, E1A-C1, directed against the C-terminal sequence of E1A products, and separated by gel electrophoresis. Two-dimensional maps of [35S]methionine-labeled peptides were consistent with antigens of 52,000 daltons (52K) and 48.5K being from the 13S mRNA and antigens of 50K, 45K, and 35K from the 12S mRNA. Partial N-terminal sequences of 52K, 50K, 48.5K, and 45K synthesized in vitro showed that each of these antigens was initiated at the predicted ATG at nucleotide 560 in the DNA sequence. These results eliminate multiple initiation sites and proteolytic cleavage at the N-terminal end as sources of antigen diversity. Peptide maps and N-terminal sequences were obtained in a similar way for E1A products from the Ad5 deletion mutant dl1504, which lacks the normal initiator codon. As predicted, these polypeptides are initiated at the next ATG, 15 codons downstream in the wild-type sequence. These results are discussed in relation to Kozak's ribosomal scanning model.     

HLA Haplotypes with C4B5; evidence for further allelic heterogeneity

Twenty-three individuals from various disease groups and normal controls were identified by immunofixation with anti-C4, C4-dependent lysis, determination of Rg (Rodgers) and Ch (Chido) phenotypes, and immunoblotting with C4-specific mouse monoclonal antibody. We found that one haplotype predominates with the C4B ^* 5 allele, HLA-A11, B22(55), Cw3, Bf ^* S, C4A ^* 4B ^* 5, which also carries the Ch ^{1,–2, 3} haplotype. The B5 allotype was also found with HLA-1360, HLA-1335 in Caucasoids, and HLA-B18 in non-Caucasoids; these carried the Ch ^{–1, –2, –3} haplotype. Our results are in accord with an earlier report of two B5 subtypes, B5Rg⁺ and B5Rg^– (Roos et al. 1984). The specificity of the mouse monoclonal antibodies IC4 and 21312 had been previously related to C4A and C4B, respectively, but our results suggest that they relate more closely to Rg and Ch determinants.     

Production of ochratoxin a,citrinin, and ergosterol byPenicillium viridicatum in autoclaved and non-autoclaved wheat at low temperature

Wheat (moisture content: 26%) was autoclaved or left untreated, inoculated with conidia ofPenicillium viridicatum and stored at 10°C. The fungus grew on both substrates and was the dominant mould on the non-autoclaved grain. Autoclaving resulted in an earlier onset of ergosterol, ochratoxin A, and citrinin production due to accelerated mould growth. Yield of ochratoxin A increased while citrinin slightly decreased in autoclaved wheat.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in -glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.     

‘Milk’ secretion for embryogenesis in a viviparous cockroach

The brood sac of viviparous Diploptera punctata is a typical insect integumentary gland which secretes a ‘milk’ containing protein and carbohydrate to nourish the developing embryos. During gestation the secretory cells proliferate organelles of protein synthesis and secretion and brood sac wet weight, protein content, synthetic activity and secretory output increase five- to six-fold ; a maximum of 0.4 mg protein was collected in 24 hr from one brood sac in a later stage of gestation. Following parturition, when secretory activity ceases, these parameters fall markedly, and the secretory cells decrease their mass by autophagic regression. Acid phosphatase has been located histochemically in autolysomes and assayed in brood sac homogenates; activity reaches a maximum five days after parturition.     

Influence of Oxygen on Development of Nitrate Respiration in Bacillus stearothermophilus

A denitrifying mutant of Bacillus stearothermophilus NCA 2184, strain 2184-D, was used to explore the development of nitrate respiration in relation to oxygen respiration. Aerobically grown wild-type cultures could acquire the ability to use nitrate as a result of selection of nitrate-respiring mutants by the presence of nitrate and a reduced oxygen tension. Fluctuation analysis has revealed that the frequency of occurrence of the nitrate-respiring mutant is about 7.5 x 10(-8) per bacterium per generation. Nitrate reductase and nitrite reductase appeared to be induced sequentially in strain 2184-D by the addition of nitrate. The formation of both of these enzymes was repressed by oxygen so that cells grown aerobically with nitrate possessed a low basal level of nitrate reducatase and exhibited no denitrification. The rate of synthesis of nitrate reductase increased quickly after addition of nitrate and removal of oxygen. It then declined to a lower steady-state level. Cells grown anaerobically with nitrate retained approximately 30 to 40% of the respiratory activity of aerobically grown cells. Aeration of anaerobically grown cells in the presence of amino acids increased the respiratory activity to normal aerobic levels. This aeration promoted rapid degradation of the existing nitrate reductase with or without the added amino acids.