De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive. (c) 2005 Elsevier B.V. All rights reserved.

Effects of Molecular Length, Ortientation and Amino Acid Mutation on Kinetics of Selectin/Ligand interactions

Receptor/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes such as lymphocyte-mediated cytotoxicity, tumor metastasis and inflammatory reactionl. Selectin/carbohydrate ligand bindings have been found to mediate the fast rolling of leukocytes on activated endothelial monolayer. Kinetic rate and binding affinity constants are essential determinants of cell adhesion...

A human-specific mutation leads to the origin of a novel splice form of neuropsin (KLK8), a gene involved in learning and memory

Neuropsin (kallikrein 8, ELKS) is a secreted-type serine protease preferentially expressed in the central nervous system and involved in learning and memory. Its splicing pattern is different in human and mouse, with the longer form (type II) only express

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Moderate mutation rate in the SARS coronavirus genome and its implications

Background: The outbreak of severe acute respiratory syndrome (SARS) caused a severe global epidemic in 2003 which led to hundreds of deaths and many thousands of hospitalizations. The virus causing SARS was identified as a novel coronavirus (SARS-CoV) an

mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample f

Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in d

Evidence of gene conversion in the evolutionary process of the codon 41/42 (-CTTT) mutation causing beta-thalassemia in southern China

The 4-bp deletion (-CTTT) at codon 41/42 (CD41/42) of the human beta-globin gene represents one of the most common beta-thalassemia mutations in East Asia and Southeast Asia, which is historically afflicted with endemic malaria, thus hypothetically evolvi

Mutation analysis of a large Chinese pedigree with congenital preaxial polydactyly

Mutations in the long-range limb-specific cis-regulator (ZRS) could cause ectopic shh gene expression and are responsible for preaxial polydactyly (PPD). In this study, we analyzed a large Chinese isolated autosomal dominant PPD pedigree. By fine mapping

Mitochondrial DNA Haplogroups M7b1 ' 2 and M8a Affect Clinical Expression of Leber Hereditary Optic Neuropathy in Chinese Families with the m.11778G -> A Mutation

Leber hereditary optic neuropathy (LHON) is the most extensively studied mitochondrial disease, with the majority of the cases being caused by one of three primary mitochondrial DNA (mtDNA) mutations. Incomplete disease penetrance and gender bias are two

Phantom mutation hotspots in human mitochondrial DNA

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30000 published hypervariable segment 1 sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.