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PubMed1997

Biogeographic analysis of the Uhu and LOA elements in the Hawaiian Drosophila.

Two transposable elements have been isolated from the Hawaiian Drosophila, the Uhu and LOA elements. The Uhu element has been shown to be present in a group of closely related species, the planitibia subgroup of the picture-winged Drosophila. This study examines the distribution of the Uhu element in several other subgroups of the picture-winged Drosophila, as well as the modified-mouthparts and antopocerus groups of the Hawaiian Drosophila. The LOA element has a much more limited distribution, having only been found in representatives of the planitibia and grimshawi subgroups of the picture-winged Drosophila. For both the Uhu and LOA elements there is an inverse correlation between the copy number of the element and the age of the island on which the species is endemic, i.e., species endemic to the Island of Hawaii, the youngest island, have the highest copy number, while species endemic to Kauai, the oldest island, have the lowest copy number of the element. The correlation suggests there is a relationship between speciation and the activity of transposable elements.

Animals↗

PubMed1993

The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates.

Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product. We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E. Using Northern blotting, primer extension analysis, [32P]GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI. Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria.

Adenosine Monophosphate↗

PubMed1984

The cohesive population genetics of molecular drive.

The long-term population genetics of multigene families is influenced by several biased and unbiased mechanisms of nonreciprocal exchanges (gene conversion, unequal exchanges, transposition) between member genes, often distributed on several chromosomes. These mechanisms cause fluctuations in the copy number of variant genes in an individual and lead to a gradual replacement of an original family of n genes (A) in N number of individuals by a variant gene (a). The process for spreading a variant gene through a family and through a population is called molecular drive. Consideration of the known slow rates of nonreciprocal exchanges predicts that the population variance in the copy number of gene a per individual is small at any given generation during molecular drive. Genotypes at a given generation are expected only to range over a small section of all possible genotypes from one extreme (n number of A) to the other (n number of a). A theory is developed for estimating the size of the population variance by using the concept of identity coefficients. In particular, the variance in the course of spreading of a single mutant gene of a multigene family was investigated in detail, and the theory of identity coefficients at the state of steady decay of genetic variability proved to be useful. Monte Carlo simulations and numerical analysis based on realistic rates of exchange in families of known size reveal the correctness of the theoretical prediction and also assess the effect of bias in turnover. The population dynamics of molecular drive in gradually increasing the mean copy number of a variant gene without the generation of a large variance (population cohesion) is of significance regarding potential interactions between natural selection and molecular drive.

Biological Evolution↗

PubMed2001

Species and recombination effects on DNA variability in the tomato genus.

Population genetics theory predicts that strong selection for rare, beneficial mutations or against frequent, deleterious mutations reduces polymorphism at linked neutral (or weakly selected) sites. The reduction of genetic variation is expected to be more severe when recombination rates are lower. In outbreeding species, low recombination rates are usually confined to certain chromosomal regions, such as centromeres and telomeres. In contrast, in predominantly selfing species, the rarity of double heterozygotes leads to a reduced effective recombination rate in the whole genome. We investigated the effects of restricted recombination on DNA polymorphism in these two cases, analyzing five Lycopersicon species with contrasting mating systems: L. chilense, L. hirsutum, L. peruvianum, L. chmielewskii, and L. pimpinellifolium, of which only the first three species have self-incompatibility alleles. In each species, we determined DNA sequence variation of five single-copy genes located in chromosomal regions with either high or low recombination rate. We found that the mating system has a highly significant effect on the level of polymorphism, whereas recombination has only a weak influence. The effect of recombination on levels of polymorphism in Lycopersicon is much weaker than in other well-studied species, including Drosophila. To explain these observations, we discuss a number of hypotheses, invoking selection, recombination, and demographic factors associated with the mating system. We also provide evidence that L. peruvianum, showing a level of polymorphism (almost 3%) that is comparable to the level of divergence in the whole genus, is the ancestral species from which the other species of the genus Lycopersicon have originated relatively recently.

DNA↗

PubMed1986

Random assignment of treatments in a 96-well (8 X 12) microtiter plate. A practical method.

A computer program, written in BASIC, has been developed to assign treatments strictly at random to the wells of a microtiter plate. A hard copy is printed of the treatment numbers with their assigned wells, followed by an 8 X 12 matrix of the treatment numbers. When a microtiter plate is placed over the matrix these numbers may be viewed directly through the wells. A program listing is reproduced. The effects of randomization on within-plate variation have been investigated and compared with two systematic methods of assignment.

Analysis of Variance↗

PubMed1991

Evolutionary lines among Salmonella enteritidis phage types are identified by insertion sequence IS200 distribution.

A survey was made of the presence, copy number and location of the Salmonella-specific DNA insertion element IS200, within the genomes of the 27 phage type strains of Salmonella enteritidis. All the phage type strains contained copies of IS200 revealed by genomic Southern blot hybridizations with a 300-bp DNA probe internal to the element. Restriction site variation around IS200 insertion sites was examined. Three fundamental patterns of hybridization corresponding to chromosomal IS200 loci were found. In terms of population genetics, these 'IS200 profiles' correspond to clonal lineages of recent evolutionary origin, and underline the phage-typing scheme for epidemiological subdivision of S. enteritidis. The molecular analysis is consistent with genetic selection pressures which are apparent in the observed epidemiological distribution of S. enteritidis, since each clonal lineage contained one of the phage types of major clinical importance in the U.K.

Biological Evolution↗

PubMed1999

Soluble tumor necrosis factor receptor type II (sTNFRII) in HIV-infected patients: relationship with the plasma level of HIV-1 RNA.

The value of soluble receptor for tumor necrosis factor type II (sTNFRII) as a strong and early predictor of HIV disease progression was suggested. Recently it has been reported that sTNFRII may provide an indication of the HIV load. In this work we focused on the relationship between sTNFRII and HIV burden in 95 HIV-1+ patients without AIDS grouped according to the 1993 classification of the CDC as group A, n = 55, and group B, n = 40. Compared with healthy controls, higher values of sTNFRII were obtained in all groups of HIV-1 infected patients (P < 0.001), but we found no inverse correlation between sTNFRII and CD4+ lymphocyte counts in CDC group A and B of the disease, and no correlation with log RNA copy number in patients with CD4 T-cell counts > 499/microl. A correlation was obtained between sTNFRII and the viral load in patients with CD4 T-cell counts ranging from 200 to 499/microl, but only in CDC group B patients (P < 0.01, n = 26). There was no correlation between the variations of sTNFRII and HIV-1 RNA levels in 19 CDC group A and 15 CDC group B clinically stable patients in the course of a short follow up. The plasma level of sTNFRII do not appear as a valuable surrogate marker of the plasma level of HIV-1 RNA in patients. Further investigations are needed to define the mechanism of the raised level of sTNFRII in HIV-1 infected patients.

Adult↗

PubMed2003

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND: A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES: A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN: Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS: Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION: All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

Cell Line↗

PubMed2006

Recurrent gene amplification and soft selective sweeps during evolution of multidrug resistance in malaria parasites.

When selection is strong and beneficial alleles have a single origin, local reductions in genetic diversity are expected. However, when beneficial alleles have multiple origins or were segregating in the population prior to a change in selection regime, the impact on genetic diversity may be less clear. We describe an example of such a "soft" selective sweep in the malaria parasite Plasmodium falciparum that involves adaptive genome rearrangements. Amplification in copy number of genome regions containing the pfmdr1 gene on chromosome 5 confer resistance to mefloquine and spread rapidly in the 1990s. Using flanking microsatellite data and real-time polymerase chain reaction determination of copy number, we show that 5-15 independent amplification events have occurred in parasites on the Thailand/Burma border. The amplified genome regions (amplicons) range in size from 14.7 to 49 kb and contain 2-11 genes, with 2-4 copies arranged in tandem. To examine the impact of drug selection on flanking variation, we genotyped 48 microsatellites on chromosome 5 in 326 parasites from a single Thai location. Diversity was reduced in a 170- to 250-kb (10-15 cM) region of chromosomes containing multiple copies of pfmdr1, consistent with hitchhiking resulting from the rapid recent spread of selected chromosomes. However, diversity immediately flanking pfmdr1 is reduced by only 42% on chromosomes bearing multiple amplicons relative to chromosomes carrying a single copy. We highlight 2 features of these results: 1) All amplicon break points occur in monomeric A/T tracts (9-45 bp). Given the abundance of these tracts in P. falciparum, we expect that duplications will occur frequently at multiple genomic locations and have been underestimated as drivers of phenotypic evolution in this pathogen. 2) The signature left by the spread of amplified genome segments is broad, but results in only limited reduction in diversity. If such "soft" sweeps are common in nature, statistical methods based on diversity reduction may be inefficient at detecting evidence for selection in genome-wide marker screens. This may be particularly likely when mutation rate is high, as appears to be the case for gene duplications, and in pathogen populations where effective population sizes are typically very large.

ATP-Binding Cassette Transporters↗

PubMed1995

Variations of two repetitive DNA sequences in several Triticeae genomes revealed by polymerase chain reaction and sequencing.

Genomes of Triticeae were analyzed using PCR with synthesized primers that were based on two published repetitive DNA sequences, pLeUCD2 (pLe2) and 1-E6hcII-1 (L02368),which were originally isolated from Thinopyrum elongatum. The various genomes produced a 2240 bp PCR product having high homology with the repetitive DNA pLe2. The PCR fragments produced from different genomes differed mainly in amplification quantity and in base composition at 89 variable sites. On the other hand, amplification products from the primer set for L02368 were of different sizes and nucleotide sequences. These results show that the two repetitive DNA sequences have different evolutionary significance. ple2 is present in all genomes tested, although differences in copy number and nucleotide sequence are notable. L02368 is more genome specific, i.e., fewer genomes possess this family of repetitive sequences. It was concluded that the repetitive sequence pLe2 family is an ancient one that existed in progenitor genome prior to divergence of annual and perennial genomes. In contrast, sequences similar to L02368 have only evolved following genome divergence.

Base Sequence↗

PubMed2006

Serotonin transporter gene promoter polymorphism predicts SSRI response in generalized social anxiety disorder.

OBJECTIVES: To determine whether variation in the serotonin transporter gene promoter (5HTTLPR) influences the efficacy of selective serotonin reuptake inhibitors (SSRIs) in generalized social anxiety disorder (GSAD). METHODS: Consecutive series of N=32 patients with DSM-IV GSAD for whom DNA and standardized outcome data from a 12-week SSRI trial were available. After ensuring that neither clinical response [clinical global impression of change scale (CGI-C)] nor 5HTTLPR genotype was confounded by ethnicity or sex, we determined whether the number of copies (0, 1, or 2) of hi-risk alleles using either the diallelic L-S system or the triallelic La-Lg-S system, predicted response and change in Liebowitz social anxiety scale (LSAS) and brief social phobia scale (BSPS) scores during SSRI treatment. RESULTS: Twenty-one patients (66%) were responders to SSRI (i.e., CGI-C much or very much improved). A trend was seen for a linear association between 5HTTLPR genotype and likelihood of response to SSRI: diallelic classification L/L 7/8 (88%), L/S 12/18 (67%), S/S 2/6 (33%), p=0.051; triallelic classification L'/L' 4/5 (80%), L'/S' 14/19 (74%), S'/S' 3/8 (38%), p=0.093. Reduction in LSAS (and BSPS) scores during SSRI treatment was significantly (p<0.02) associated with 5HTTLPR genotype using either the diallelic or triallelic classification. CONCLUSIONS: Variation in a functional polymorphism known to influence serotonin reuptake is associated with SSRI response in patients with GSAD. Independent replication in larger samples is required before the predictive utility of this information can be confirmed and generalized to clinical settings.

Adult↗

PubMed2003

Genetic variation in organisms with sexual and asexual reproduction.

The genetic variation in a partially asexual organism is investigated by two models suited for different time scales. Only selectively neutral variation is considered. Model 1 shows, by the use of a coalescence argument, that three sexually derived individuals per generation are sufficient to give a population the same pattern of allelic variation as found in fully sexually reproducing organisms. With less than one sexual event every third generation, the characteristic pattern expected for asexual organisms appear, with strong allelic divergence between the gene copies in individuals. At intermediary levels of sexuality, a complex situation reigns. The pair-wise allelic divergence under partial sexuality exceeds, however, always the corresponding value under full sexuality. These results apply to large populations with stable reproductive systems. In a more general framework, Model 2 shows that a small number of sexual individuals per generation is sufficient to make an apparently asexual population highly genotypically variable. The time scale in terms of generations needed to produce this effect is given by the population size and the inverse of the rate of sexuality.

Gene Frequency↗

PubMed1996

Hypothesis: hypersensitive plasmid copy number control for ColE1.

Initiation of replication of the plasmid ColE1 is primed by the cis-acting RNA II. Copy numbers are regulated by inhibition of RNA II by the antisense RNA I, whose concentration is proportional to the plasmid concentration. This inhibition is enhanced by a protein. Rom, and takes place during a time set by the transcription of 250 bases of the gene for RNA II. When this transcription is dominated by several steps of about equal duration, the probability for RNA II to prime DNA replication is approximately determined by e-constant[RNA I]. For large values of the "constant" small changes in [RNA I] give large variations in the priming probability. It is shown, first, that this type of mechanism can reduce the rate of plasmid loss and enable single copies of ColE1 to duplicate at a well-defined time in the cell cycle; second, that when the rate of initiation of transcription of RNA II increases, plasmid losses decrease and the distribution of single copy duplication times becomes narrower; third, that the action of Rom may further reduce plasmid losses and further narrow the distribution of duplication times in the single-copy case.

Bacterial Proteins↗

PubMed2004

Quantification of virulence-associated gene transcripts in epidemic methicillin resistant Staphylococcus aureus by real-time PCR.

The control of Staphylococcus aureus virulence gene expression is complex and few data are available for the epidemic methicillin resistant S. aureus clones circulating in the UK. Quantitative real-time PCRs were developed for key gene transcripts involved in S. aureus infection (RNAIII, hla and spa) and for the 16S rRNA. These assays were applied to log and stationary phase cultures of the important EMRSA strains. To correct for inconsistencies in extract yield, results were calculated as ratios using the 16S rRNA values as denominator. The quantitative assays were sensitive and reproducible. The number of copies of each transcript present differed greatly between the EMRSA strains tested. Strains within an EMRSA clone or type gave similar results. High levels of RNAIII transcripts were not consistently linked to elevated levels of hla transcripts or to low levels of the spa transcript. In addition, strains showed significant variations in their patterns of induction (or repression) of all three transcripts. A complex interplay exists between the regulatory factors that control the expression of proteins required for colonisation and survival in the host. The transcript level data suggest that this pattern shows great diversity among the currently important EMRSA strains.

Bacterial Proteins↗

PubMed1998

An alternative PCR assay for quantifying mitochondrial DNA in crude preparations.

A method is described for the quantification of mitochondrial DNA present in crude biological preparations. A known copy number of a standard is amplified in the presence of inactivated target DNA so as to determine the overall efficiency of the PCR process in a particular sample. In this way any inhibitory and/or stimulatory substances present in sample preparations can be taken into account. To reduce tube-to-tube variations product DNA quantification is limited to small cycle numbers. Using this method quantitations of DNA amounts in different crude preparations can be compared.

Animals↗

PubMed2006

[Using real-time polymerase chain reaction to quantitate bcr-abl mRNA].

BACKGROUND & OBJECTIVE: When leukemia patients achieve complete remission after chemotherapy, a few tumor cells still exist in other tissues outside bone marrow, which is called minimal residual disease (MRD), and it is the base of relapse. To cure leukemia, we should not only find MRD in time, but also quantitate MRD for instructing treatment and predicting prognosis. This study was to establish a real-time reverse transcription-polymerase chain reaction (RT-PCR) system to quantitate bcr-abl mRNA. METHODS: bcr-abl mRNA in leukemia cell line K562 was amplified by RT-PCR. T-A clone was used to construct the combined plasmid to be standard template; the standard curve of bcr-abl oncogene was drawn. bcr-abl mRNA in bone marrow samples of 16 chronic myelocytic leukemia (CML) patients was quantitated by this method. The sensitivity, stability, and repetition of this method were evaluated. RESULTS: The sensitivity is 10 copies of the recombined plasmid. The coefficient variations (CV) of repetition and stability were 2.19% and 3.21%. The correlation (R) of the standard curve was 0.984. The median bcr-abl level of the 16 CML patients was 4.58x10(4) kb/microg RNA. CONCLUSIONS: Real-time PCR has high sensitivity, repetition, and specificity. It can quantitate the copy number of bcr-abl oncogene.

Bone Marrow↗

PubMed2003

Regulation of toxA by PtxR in Pseudomonas aeruginosa PA103.

Exotoxin A (ETA) production in Pseudomonas aeruginosa requires the regulatory locus regAB. Pseudomonas aeruginosa PA103 produces significantly higher levels of ETA than the prototypic strain PAO1 does, partly because of differences in the regAB locus. Other factors that contribute to this variation are not known. We previously described the P. aeruginosa gene ptxR that positively regulates production of ETA through regAB. ETA production was enhanced but still iron regulated in the PAO1 strain PAO1-XR that carries two copies of ptxR on its chromosome. Here we determine whether ptxR regulation of ETA is different in PA103. In contrast to PAO1-XR, ETA activity produced by PA103-2R, a PA103 strain carrying two copies of ptxR, is enhanced tenfold and partially deregulated in the presence of iron. Real-time PCR transcriptional analysis showed that the copy number of toxA mRNA in PA103-2R is significantly higher than in PA103 in both the presence and absence of iron, yet no similar increase in either regAB or ptxR mRNA copy number was detected. The integrated plasmid together with adjoining DNA was retrieved from the PA103-2R chromosome to determine whether integration-induced DNA changes played a role in this phenotype. Introduction of the retrieved plasmid in PA103 produced a phenotype similar to that of PA103-2R. Sequence analysis of the plasmid revealed the loss of 322 bp within the region 3' of ptxR. A plasmid construct carrying a 4-bp insertion in this same region produced in PA103 a phenotype similar to that of PA103-2R. Our results suggest that the effect of ptxR on toxA expression is different in PA103 than in PAO1 and that this variation in PA103-2R does not occur solely through regAB. Changes within the region 3' of ptxR are critical for the production of the unique PA103-2R phenotype, which occurs in trans and requires intact ptxR, but is not caused by ptxR overexpression.

3' Untranslated Regions↗

PubMedDate not supplied

Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization.

Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

Algorithms↗