Search results for "DNA polymerase"

Virus replication and virion export in X-deficient hepatitis B virus transgenic mice

2002

The function of the X protein (pX) in the replication cycle of mammalian hepadnaviruses is enigmatic. Using tissue culture experiments it has been shown that the X gene product is not central to hepatitis B virus (HBV) replication and virion export. However, at present it is still unclear whether this also applies to the in vivo situation. Using a terminally redundant X-deficient HBV DNA construct, transgenic mice were established that exhibited high-level expression of the viral core protein in liver and kidneys. Importantly, replicative DNA intermediates and mature viral genomes could be detected in the liver and serum of these mice, respectively. These findings indicate that, in the in v…

The significance of the pre-S region of the hepatitis B virus

1986

Molecular hybridization techniques in current diagnosis of chronic hepatitis B in childhood.

1992

Following the cloning and sequencing of the hepatitis B virus genome, molecular hybridization techniques have been established to detect hepatitis B virus (HBV) DNA in serum and liver tissue. Analyses can be performed by dot blot, Southern blot and in situ hybridization. HBV DNA is regarded to be the most sensitive marker of viral replication and infectivity which was previously related to the presence of hepatitis B e antigen in serum and hepatitis B core antigen in liver cells. In liver tissue different molecular patterns can be recognized as free viral DNA and integrated sequences. Furthermore, introduction of the polymerase chain reaction allows the detection of very small amounts of vi…

Multiple approaches to identify bacteria in archaeological waterlogged wood

2013

This study was carried out in collaboration with Soprintendenza del Mare (SM) that started, since 2004, to plan and realize underwater archaeological parks, such as in the Sicilian islands of Pantelleria (Gadir), Levanzo (Cala Minnola), Ustica (Falconiera), Panarea (Basiluzzo) and Filicudi (Capo Graziano). In situ conservation, as well as virtual exhibitions of the same topics, can contribute to ensure the protection and best fruition of underwater cultural heritage. The focus of this study was the identification of bacterial colonies in waterlogged wood samples from the rostrum of a excellent workmanship, that is very likely one of the wrecks attributed to Sextus Pompey fleet (36 BC) and d…

IN SITU HYBRIDIZATION FOR DETECTION OF HEPATITIS B VIRUS GENOMES IN LIVER TISSUE OF CHRONIC INFECTED CHILDREN

1990

Detection of hepatitis B virus (HBV)-DNA in the liver of chronic infected patients is presently the most sensitive marker of viral replication and infectivity. In situ hybridization (ISH) allows the direct visualization of HBV infected liver cells and distribution of the viral sequences. This study was done to establish ISH and correlate the findings with conventional markers for HBV infection. Methods. Liver biopsies of 50 patients (28 ♂, 22 ♀) aged 0.5-20 years (mean 10.3) with various histological diagnoses were tested by 1SH. The HBV-DNA probe was labeled by nick translation with 35S-CTP to a specific activity of 3-5×108 cpm/μg DNA. Results. HBV-DNA/mRNA could be demonstrated in 38 pati…

The elemental role of iron in DNA synthesis and repair

2017

Iron is an essential redox element that functions as a cofactor in many metabolic pathways. Critical enzymes in DNA metabolism, including multiple DNA repair enzymes (helicases, nucleases, glycosylases, demethylases) and ribonucleotide reductase, use iron as an indispensable cofactor to function. Recent striking results have revealed that the catalytic subunit of DNA polymerases also contains conserved cysteine-rich motifs that bind iron–sulfur (Fe/S) clusters that are essential for the formation of stable and active complexes. In line with this, mitochondrial and cytoplasmic defects in Fe/S cluster biogenesis and insertion into the nuclear iron-requiring enzymes involved in DNA synthesis a…

A method for rapid generation of competitive standard molecules for RT-PCR avoiding the problem of competitor/probe cross-reactions.

1995

The analysis of gene expression is a widespread issue in a growing number of fields such as molecular genetics, immunology, and medical diagnostics. The ideal method for mRNA detection should be fast, inexpensive, sensitive, and reliable. Well-elaborated standard methods such as Northern hybridization, Sl-mapping, and RNAse protection are useful and recommended, but only reverse transcription PCR (RT-PCR) gives the highest possible sensitivity required. For many issues it is necessary not only to detect a distinct mRNA but to compare changes in mRNA levels. The use of RT-PCR for such semiquantitative and quantitative approaches resolves problems attributable to the intrinsic property of PCR…

Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mi…

1980

Abstract The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase α. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10 8 nucleated cells in the respective fraction) were determined. TdT activity was found to decrease markedly during ageing. Mature…

POLE, POLD1, and NTHL1: the last but not the least hereditary cancer-predisposing genes

2021

POLE, POLD1, and NTHL1 are involved in DNA replication and have recently been recognized as hereditary cancer-predisposing genes, because their alterations are associated with colorectal cancer and other tumors. POLE/POLD1-associated syndrome shows an autosomal dominant inheritance, whereas NTHL1-associated syndrome follows an autosomal recessive pattern. Although the prevalence of germline monoallelic POLE/POLD1 and biallelic NTHL1 pathogenic variants is low, they determine different phenotypes with a broad tumor spectrum overlapping that of other hereditary conditions like Lynch Syndrome or Familial Adenomatous Polyposis. Endometrial and breast cancers, and probably ovarian and brain tumo…

Influence of template inactivators on the binding of DNA polymerase to DNA.

1974

The agents daunomycin, ethidium bromide, distamycin A and cytochrome c inhibit DNA dependent DNA polymerase I (E. coli) reaction competitively to DNA. The influence of these template inactivators on the binding of DNA polymerase to native as well as denatured DNA has been determined by affinity chromatography. Cytochrome c blocks the binding of the enzyme to double-stranded and to single-stranded DNA Sepharose. In contrast to these results daunomycin, ethidium bromide or distamycin A reduce the binding affinity only with denatured DNA Sepharose as matrix. These data are discussed with respect to the modification by template inactivators of the affinity of DNA to the different binding sites …