Mixture Of Wild-type Research Articles

Characterization of a trp RNA-binding Attenuation Protein (TRAP) Mutant with Tryptophan Independent RNA Binding Activity

TRAP ( trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism ( trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.

Wild-type and 'a' epitope variants in chronic hepatitis B virus carriers positive for hepatitis B surface antigen and antibody.

This study aims to examine the genomic variants of the 'a' epitope in chronic hepatitis B virus (HBV) carriers positive for both hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs). Eighteen HBV carriers were studied. Hepatitis B virus (HBV) DNA was extracted and the 'a' epitope region was amplified and sequenced. Eighteen Chinese asymptomatic HBV carriers were studied. There were 13 patients who were positive for both HBsAg and anti-HBs. Of these, one patient had only wild-type HBV, three had a viral mixture, and five had only 'a' epitope variant HBV. Of the three patients with a viral mixture, all had variants in the less conserved region (123-137). Of the five patients with pure HBsAg mutants, three had variants in the less conserved region while two had variants in the highly conserved region. In this study with a limited number of patients, the serum alanine aminotransferase (ALT) levels were higher in patients with wild-type HBV, compared with those with either 'a' epitope variants or a viral mixture consisting of wild type and variants. Eight of the nine (89%) patients positive for both HBsAg and anti-HBs harbored an 'a' epitope variant. The lower ALT levels seen in patients who had either pure 'a' epitope variant or a mixture of wild type and mutants suggest that a closer monitoring of these 'a' epitope variants should be required, as patients carrying these infectious viral strains may remain asymptomatic.

74th ENMC international workshop: mitochondrial diseases 19-20 november 1999, Naarden, the netherlands.

Disease due to mtDNA defects is increasingly recognised and approximately 1 in 8000 persons carry these mutations. Despite significant scientific advances, there has been limited progress in the clinical management and genetic counselling of these patients [1]. Mitochondrial DNA (mtDNA) is maternally inherited. Thousands of copies of mtDNA are present in each nucleated somatic cell and in normal individuals these have a virtually identical sequence (homoplasmy). In many patients with mtDNA disease, there is a mixture of wild type and mutant genomes in the same cell – a situation termed heteroplasmy. The current workshop focussed specifically on the problems and solutions for genetic counselling of heteroplasmic mtDNA disorders.

Expression of an estrogen receptor alpha variant protein in cell lines and tumors

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of ≈60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERΔ7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERΔ7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERΔ7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERΔ7P in vivo suggests that it may have some biological function. ERΔ7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE).

We extended our development of the means to measure point mutations at the DNA level to the problem of detecting TP53 gene variants in the area around tumors where mutant fractions are expected to be as low as one mutant per 1000 wild-type (WT) sequences. We met this criterion by using the following methods. The TP53 exon 8 sequence was amplified from genomic DNA samples under conditions of high polymerase fidelity using a fluorescein-labeled primer. This mixture of WT and mutant sequences was converted to heteroduplexes of mutant and WT sequences by melting and re-annealing. The mixture was resolved by capillary gel electrophoresis under appropriate constant denaturing conditions. Using laser-induced fluorescence, we found that fractions as low as 1/1000 could be detected without any prior enrichment for mutant sequences, and that the melting properties of the amplified DNA will leave "fingerprints" in the electropherogram that can be used to identify the specific mutation. This method is fast and robust and should be applicable to clinical analyses in which rapid scanning for any mutation in an exonic sequence is important.

Determination of human immunodeficiency virus RNA in plasma and cellular viral DNA genotypic zidovudine resistance and viral load during zidovudine-didanosine combination therapy

Eleven human immunodeficiency virus (HIV)-infected subjects on long-term zidovudine (ZDV) therapy had didanosine (ddI) added to their antiretroviral regimen. HIV RNA in plasma was quantitated by branched-DNA signal amplification assay. Peripheral blood mononuclear cell (PBMC) HIV viral DNA was quantitated by PCR. The relative amounts of wild-type (WT) sequence, ddI resistance-associated codon changes (reverse transcriptase [RT] gene codon 65 K-->R [RT K65R], RT 174V, RT I135K/T/V, and RT M184I/V), and ZDV resistance-associated codon change (RT T215Y/F) from HIV RNA in plasma and RT T215Y/F from PBMC viral DNA were determined by differential hybridization of PCR products from 10 of 11 subjects. All subjects had evidence of RT T215Y/F mutation in both RNA in plasma and PBMC DNA at baseline. Subjects with a mixture of WT and RT T215Y/F HIV RNA in plasma at baseline demonstrated a decline in RNA levels in plasma after the addition of ddI. However, after 6 months of ZDV-ddI therapy, WT HIV RNA in plasma was undetectable in all subjects who had demonstrated a mixture at baseline. Subjects with only RT T215Y/F RNA present in plasma at baseline remained so and demonstrated no decline in RNA levels in plasma. In all subjects, no significant changes in PBMC DNA viral load and RT T215Y/F or WT levels were seen. HIV RNA in plasma demonstrated a significantly higher RT T215Y/F mutant/WT ratio than that of PBMC viral DNA, both at baseline and after ZDV-ddI combination therapy in all subjects. No subjects developed mutations associated with ddI resistance at codons 65, 74, 135, and 184 during this study. This study suggests that determination of relative amounts of RT T215Y/F and WT species from HIV RNA in plasma at baseline may be predictive of virologic response during ZDV-ddI combination therapy.

An analysis of subunit exchange in the dimeric DNA-binding and DNA-bending protein, TF1

TF1 is the Bacillus subtilis bacteriophage-encoded dimeric type II DNA-binding protein. This relative of the eubacterial HU proteins and of the Escherichia coli integration host factor binds preferentially to 5-(hydroxymethyluracil)-containing DNA. We have examined the dynamics of exchange of monomer subunits between molecules of dimeric TF1. The analysis takes advantage of the fact that replacement of phenylalanine with arginine at amino acid 61 in the β-loop ‘arm’ of TF1 alters DNA-bending and -binding properties, generating DNA complexes with distinctively different mobilities in gel electrophoresis. New species of DNA-protein complexes were formed by mixtures of wild type and mutant TF1, reflecting the formation of heterodimeric TF1, and making the dynamics of monomer exchange between TF1 dimers accessible to a simple gel retardation analysis. Exchange was rapid at high protein concentrations, even at 0°C, and is proposed to be capable of proceeding through an interaction of molecules of TF1 dimer rather than exclusively through dissociation into monomer subunits. Evidence suggesting that DNA-bound TF1 dimers do not exchange subunits readily is also presented.