ABSTRACT
A study was conducted in two primary health facilities in Kigali, Rwanda, to determine whether dried blood spots (DBS) used for quality control of HIV testing would give comparable results with serum after being stored for a period of 14 days and 30 days at ambient temperature. DBS and serum specimens were collected from patients undergoing HIV testing. ELISA performed on serum at baseline (gold standard) was compared with DBS results. The study included a total of 491 patients, comprising 92 (19%) males and 399 (81%) females with a median age of 27 years. A total of 148 individuals (30%) were HIV-positive. The average ambient temperature under which DBS specimens were stored at the health facilities was 23 degrees C (range 18-25 degrees C). The kappa statistic at 14 days and 30 days was 0.99 (99.4% agreement) and 0.98 (99.2% agreement), respectively, signifying almost 'perfect agreement (P<0.001)' with the gold standard. In a resource-limited sub-Saharan African country embarking on scaling-up of HIV testing, DBS stored at ambient conditions for up to 1 month were found to be a useful and robust tool to perform quality control of rapid HIV testing at the health centre level.
Subject(s)
HIV Seropositivity/blood , HIV-1 , RNA, Viral/blood , Adult , Blood Specimen Collection , Enzyme-Linked Immunosorbent Assay , Female , HIV Seropositivity/virology , Humans , Male , Quality Control , Reagent Kits, Diagnostic , Rwanda , Sensitivity and Specificity , Temperature , Time Factors , Young AdultABSTRACT
Adenovirus type 12 induces four fragile sites upon infection of human cells. The U2 locus, consisting of up to 20 tandem repeats of a 5.8-kbp monomer, maps at the most sensitive of these sites at 17q21-22. We have previously shown that an artificial U2 locus integrated into the human genome generates a new virus-induced fragile site. To determine which elements within the U2 monomer are responsible for fragility, we constructed loci consisting of tandem repeats of subfragments of the U2 monomer. With this approach, we demonstrate that a transcriptionally competent U2 gene is necessary and sufficient for virus-induced fragility and that no other element within the 5.8-kbp monomer contributes to this effect.
Subject(s)
Adenovirus Infections, Human/genetics , Adenoviruses, Human/genetics , Chromosome Fragility , Chromosomes, Human, Pair 17 , RNA, Small Nuclear/genetics , Chromosome Fragile Sites , Chromosome Mapping , DNA Damage , Genes , Humans , In Situ Hybridization, Fluorescence , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Heat-shock treatment of Rhizobium meliloti cells causes major enhancement in the synthesis of several proteins with apparent molecular weights in the range of 58-60 kDa. Using the polymerase chain reaction and degenerate oligodeoxyribonucleotide primers for conserved regions of the 60-kDa heat-shock protein (HSP60) or GroEL protein family, a 0.6-kb probe for the R. meliloti hsp60 gene was prepared. Southern blot analysis of R. meliloti DNA digested with different restriction enzymes and hybridized to R. meliloti hsp60 probes indicated the presence of between four and five hsp60 or groEL in this species. From the cloning and sequencing of several of these fragments, we have been able to deduce the complete nucleotide sequences of three groEL in R. meliloti. The deduced amino acid (aa) sequences of these proteins show extensive similarity to each other (78-85% aa identity) and to other GroEL homologues. In the upstream regions of two of the groEL, but not the third, open reading frames corresponding to GroES proteins were also identified. Analysis of various prokaryotic GroEL sequences suggests that the multiple groEL of R. meliloti have evolved by means of gene duplication events within this or a related group of organisms. Results presented in this paper also show that some of the groEL in R. meliloti are located on the two megaplasmids present in these cells. The presence of multiple GroEL homologues in R. meliloti suggests a possible role of the GroEL or HSP60 chaperonins in the nodulation (symbiosis) and nitrogen fixation processes.
Subject(s)
Bacterial Proteins/genetics , Heat-Shock Proteins/genetics , Sinorhizobium meliloti/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Chaperonin 60 , Cloning, Molecular , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Heat-Shock Proteins/metabolism , Hot Temperature , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Restriction Mapping , Sequence Homology, Amino AcidABSTRACT
Using degenerate oligonucleotide primers for conserved regions of HSP60, a 0.6 kilobase fragment of Clostridium perfringens DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C. perfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) family of bacterial and eukaryotic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone HSP60 gene/cDNA from any species.
