Replication-associated activities of purified human papillomavirus type 11 E1 helicase

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.     

Self-organization of an oscillatory neural system

Hebbian dynamics is used to derive the differential equations for the synaptic strengths in the neural circuitry of the locomotive oscillator. Initially, neural connection are random. Under a specified arborization hypothesis relating to the density of neural connections, the differential equations are shown to model the self-organization and the stability of the oscillator.     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

ABNORMAL GUARD CELL DEVELOPMENT IN AN OLIVE NECROTIC MUTANT OF MAIZE

A study of a mutant variety of Zea mays (ON8147) revealed that the mutant plants, in contrast with normal maize plants, do not exhibit a light-induced increase in the rate of transpiration, and that the ontogeny of the stomatal complex is abnormal. In later stages of differentiation, the guard cells of mutant plants deteriorate, leaving the mature stomata with only the two subsidiary cells. The subsidiary cells in stomata of mutant leaves are similar to those of normal leaves with respect to their capacity to accumulate K⁺ in the dark, but they do not lose K⁺ in the light, as do subsidiary cells of stomata of nonmutant plants. It is suggested that impairment of guard cell function causes death of the mutant plant seedlings primarily by restricting CO₂ entry into the leaf.     

Tissue-specific heterogeneity in DNA replication patterns of human X chromosomes

Regional DNA replication kinetics in human X chromosomes have been analysed using BrdU-33258 Hoechst-Giemsa techniques in five cell types from human females: amniotic fluid cells, fetal and adult skin fibroblasts, and fetal and adult peripheral lymphocytes. In all cell types, the late-replicating X chromosome can be distinguished from its active, earlyreplicating homologue, and both the early and late X exhibit temporally and regionally characteristic internal sequences of DNA replication. The replication pattern of the early X in amniotic fluid cells and skin fibroblasts is similar to that of the early X in lymphocytes, although certain discrete regions are later-replicating in these monolayer tissue culture cells than are the corresponding regions in lymphocytes. However, DNA replication kinetics in late X chromosomes from amniotic fluid cells and skin fibroblasts are strikingly different from those observed in lymphocytes with respect both to the initiation and termination of DNA synthesis. The predominant late X pattern observed in 80–95% of lymphocytes, in which replication terminates in the long arm in bands Xq21 and Xq23, was never seen in amniotic fluid cells or skin fibroblasts. Instead, in these cell types, bands Xq25 and Xq27 are the last to complete DNA synthesis, while bands Xq21 and Xq23 are earlier-replicating; this pattern is similar to the alternative replication sequence observed in 5–20% of lymphocyte late X chromosomes. This replication sequence heterogeneity is consistent with the existence of tissue-specific influences on the control of DNA replication in human X chromosomes.     

Quantitative and qualitative analysis of cellular prion protein (PrPC) expression in bovine somatic tissues

The host encoded cellular prion protein (PrP^C) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrP^C can undergo conversion into a conformationally-altered isoform (PrP^Sc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Understanding the tissue-specific expression of PrP^C is crucial considering that cells expressing high levels of PrP^C bear a risk for conversion and accumulation of PrP^Sc. In the present study, fifteen bovine somatic tissues were analyzed for PrP^C expression by quantitative western blot and immunohistochemistry. Quantitative western blot analysis revealed highest expression of PrP^C in cerebellum, obex and spinal cord. Intermediate levels were detected in thymus, intestine, nerve, heart and spleen, and lower levels in lung, muscle, kidney, lymph node, skin, pancreas and liver. Immunohistochemical analysis detected intense cellular-specific PrP^C staining in neurons, thymocytes and lymphocytes. PrP^C was also detected in the enteric wall, pancreatic islets of langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli and dermal epithelial cells. This study demonstrated the quantitatively varied, wide-spread, tissue- and cell-specific expression pattern of PrP^C in bovine somatic tissues. The importance of this study is to lay the foundation for understanding the tissue-specific expression of PrP^C and to consider the potential participation of more bovine tissues in the transmission of BSE infection.Key words: cellular prion protein (PrP^C), protein expression, bovine somatic tissues, BSE, western blot, immunohistochemistry