The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Spermatogenesis in an infertile XYY man

Summary Testicular histology and meiosis has been studied in an XYY male patient identified at an infertility clinic. This man was found to have an XYY sex chromosome complement in 15% of spermatogonial metaphases. There was no clear evidence of 2 Y chromosomes at diakinesis but there appeared to be a slight excess of sperm with a fluorescent Y body.     

Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft     

Snow depth manipulation and its influence on soil frost and water dynamics in a northern hardwood forest

Climate change will likelyresult in warmer winter temperatures leading toless snowfall in temperate forests. Thesechanges may lead to increases in soil freezingbecause of lack of an insulating snow cover andchanges in soil water dynamics during theimportant snowmelt period. In this study, wemanipulated snow depth by removing snow for twowinters, simulating the late development of thesnowpack as may occur with global warming, toexplore the relationships between snow depth,soil freezing, soil moisture, and infiltration.We established four sites, each with two pairedplots, at the Hubbard Brook Experimental Forest(HBEF) in New Hampshire, U.S.A. and instrumentedall eight plots with soil and snow thermistors,frost tubes, soil moisture probes, and soillysimeters. For two winters, we removed snowfrom the designated treatment plots untilFebruary. Snow in the reference plots wasundisturbed. The treatment winters (1997/1998 and1998/1999) were relatively mild, withtemperatures above the seasonal norm and snowdepths below average. Results show the treatedplots accumulated significantly less snow andhad more extensive soil frost than referenceplots. Snow depth was a strong regulator ofsoil temperature and frost depth at all sites.Soil moisture measured by time domainreflectometry probes and leaching volumescollected in lysimeters were lower in thetreatment plots in March and April compared tothe rest of the year. The ratio of leachatevolumes collected in the treatment plots tothat in the reference plots decreased as thesnow ablation seasons progressed. Our data showthat even mild winters with low snowfall,simulated by snow removal, will result inincreased soil freezing in the forests at theHBEF. Our results suggest that a climate shifttoward less snowfall or a shorter duration ofsnow on the ground will produce increases insoil freezing in northern hardwood forests.Increases in soil freezing will haveimplications for changes in soil biogeochemicalprocesses.     

Characterizing the complexity of enzymes on the basis of their mechanisms and structures with a bio-computational analysis

Enzymes are basically composed of 20 naturally occurring amino acids, yet they catalyse a dizzying array of chemical reactions, with regiospecificity and stereospecificity and under physiological conditions. In this review, we attempt to gain some understanding of these complex proteins, from the chemical versatility of the catalytic toolkit, including the use of cofactors (both metal ions and organic molecules), to the complex mapping of reactions to proteins (which is rarely one-to-one), and finally the structural complexity of enzymes and their active sites, often involving multidomain or multisubunit assemblies. This work highlights how the enzymes that we see today reflect millions of years of evolution, involving de novo design followed by exquisite regulation and modulation to create optimal fitness for life.     

Human histone gene organization : Identification of a histone gene polymorphism prevalent in a black population

Analysis of the restriction enzyme digests of total genomic DNAs from a broad spectrum of human cell lines and from individuals with different genetic backgrounds, by hybridization with a series of cloned human histone sequences, indicated restriction site polymorphisms (RSPs) for two adjacent human histone genes which reside on chromosome 1. In most cell lines and individuals examined we observed a single 2.05 kb H4 histone HindIII fragment and a 7.0 kb H3 histone HindIII fragment. In contrast, the polymorphisms were manifested as a 2.15 kb H4 HindIII fragment and a 9.1 kb H3 HindIII fragment. From population studies, we were able to show that there is no linkage disequilibrium between these two polymorphic restriction sites. Nor was there any apparent correlation between the presence of the H3/H4 histone polymorphisms and maintenance of the transformed karyotype, passage in culture, transformation or tumor progression. These chromosome 1 H3 and H4 histone gene polymorphisms are common in the American Black population and, in our survey of individuals, were not found in the American Caucasian population. Among the American Blacks studied, the frequency of the H3 HindIII(-) allele is 43% and of the H4 HindIII(-) allele 30%. In limited family studies, we were unable to detect recombination between these two physically linked alleles.     

TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro,preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc.     

Quantification of endothelin receptor subtypes in peripheral tissues reveals downregulation of ET(A) receptors in ET(B)-deficient mice

We have previously shown that in homozygous endothelin (ET)(B)-/- deficient mice, ET(A) receptor density is significantly downregulated in the brain by 45%. In these mice, plasma ET-1 levels are elevated. Our aim was to use quantitative autoradiography to establish the distribution of ET receptor subtypes in peripheral tissues from wild-type mice and to measure the density of the ET(A) subtype in ET(B)-/- knockout animals. Our second aim was to test whether deletion of ET(B) receptors, which is associated with elevated plasma levels of ET-1, would also reduce ET(A) expression in the periphery. In longitudinal sections from wild-type mice, the highest densities of ET(A) receptors localized to major organs including the ventricle of the heart, lung, and liver parenchyma. High densities of ET(A) receptors were detected in the smooth muscle layer of the vasculature such as intrarenal vessels as well as the smooth muscle layer and epithelial cells of the gastrointestinal tract. In these tissues, the ET(A) subtype was more abundant, representing between 60% and 100% of the ET receptors. ET(B) receptors predominated in the medulla of kidney, with high densities also localizing to glomeruli within the cortex and to the sinusoids from the liver. Lower densities of ET(B) receptors were also present in the lung, heart, liver, and the smooth muscle layer of the gastrointestinal tract. In ET(B)-/- knockout mice, ET(B) receptors were not detected as expected by either ligand binding or immunocytochemistry. The pattern of ET(A) receptor distribution in the ET(B)-/- knockout mice was similar to the controls, but the density of ET(A) receptors was significantly reduced in the lung by 39%. Diminished responses to the endogenous agonist after repeated stimulation are an important feature of G-protein signaling, preventing potential damage to the overstimulated cell, and it is likely that downregulation occurs in response to higher circulating levels of ET-1.