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PubMed1994

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

Base Sequence↗

PubMed2005

Copy-number polymorphisms: mining the tip of an iceberg.

Copy-number polymorphisms (CNPs) represent a greatly underestimated aspect of human genetic variation. Recently, two landmark studies reported genome-wide analyses of CNPs in normal individuals and represent the beginning of an understanding of this type of large-scale variation. Future array-CGH-based CNP analyses should include standard criteria on a common microarray platform. It is only when parallel analyses of CNPs and SNPs are performed in an integrated format that we will obtain a global picture of our genetic diversity.

Chromosomes, Human, Pair 22↗

PubMed2003

Divergence and transcriptional analysis of the division cell wall (dcw) gene cluster in Neisseria spp.

Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3' of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox-like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5' of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw-associated CREE, the first demonstration of active promoter generation at the 5' end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components.

Bacterial Proteins↗

PubMed2005

A graphical simulation model of the entire DNA process associated with the analysis of short tandem repeat loci.

The use of expert systems to interpret short tandem repeat DNA profiles in forensic, medical and ancient DNA applications is becoming increasingly prevalent as high-throughput analytical systems generate large amounts of data that are time-consuming to process. With special reference to low copy number (LCN) applications, we use a graphical model to simulate stochastic variation associated with the entire DNA process starting with extraction of sample, followed by the processing associated with the preparation of a PCR reaction mixture and PCR itself. Each part of the process is modelled with input efficiency parameters. Then, the key output parameters that define the characteristics of a DNA profile are derived, namely heterozygote balance (Hb) and the probability of allelic drop-out p(D). The model can be used to estimate the unknown efficiency parameters, such as pi(extraction). 'What-if' scenarios can be used to improve and optimize the entire process, e.g. by increasing the aliquot forwarded to PCR, the improvement expected to a given DNA profile can be reliably predicted. We demonstrate that Hb and drop-out are mainly a function of stochastic effect of pre-PCR molecular selection. Whole genome amplification is unlikely to give any benefit over conventional PCR for LCN.

Computer Graphics↗

PubMed2003

Cryptons: a group of tyrosine-recombinase-encoding DNA transposons from pathogenic fungi.

A new group of transposable elements, which the authors have named cryptons, was detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. These elements are unlike any previously described transposons. An archetypal member of the group, crypton Cn1, is 4 kb in length and is present at a low but variable copy number in a variety of C. neoformans strains. It displays interstrain variations in its insertion sites, suggesting recent mobility. The internal region contains a long gene, interrupted by several introns. The product of this gene contains a putative tyrosine recombinase near its middle, and a region similar in sequence to the DNA-binding domains of several fungal transcription factors near its C-terminus. The element contains no long repeat sequences, but is bordered by short direct repeats which may have been produced by its insertion into the host genome by recombination. Many of the structural features of crypton Cn1 are conserved in the other known cryptons, suggesting that these elements represent the functional forms. The presence of cryptons in ascomycetes and basidiomycetes suggests that this is an ancient group of elements (>400 million years old). Sequence comparisons suggest that cryptons may be related to the DIRS1 and Ngaro1 groups of tyrosine-recombinase-encoding retrotransposons.

Amino Acid Sequence↗

PubMed1996

Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance.

We present a fluorescent-PCR-based technique to assay genomic sequence copy number and transcriptional abundance. This technique relies on the ability to follow fluorescent PCR progressively in real time during the exponential phase of the reaction so that quantitative PCR is accomplished. We demonstrated the ability of this technique to quantitate both known deletions and amplifications of loci that have been measured previously by other methods, and to measure transcriptional abundance. Using an efficient variant of the fluorescent-PCR technology, we can monitor transcription semiquantitatively. The ability to detect all amplifications and deletions at any single copy locus by PCR makes this the technique of choice to assay genomic sequence copy number anomalies in birth defects and cancers. The ability to detect variations in transcript abundance enables this technique to fashion a time and tissue analysis of transcription.

Chromosome Aberrations↗

PubMed2001

Genotyping Encephalitozoon cuniculi by multilocus analyses of genes with repetitive sequences.

Encephalitozoon cuniculi infects a wide range of mammalian hosts. Three genotypes based on the number of GTTT repeats in the internal transcribed spacer (ITS) of the rRNA have been described, of which genotypes I and III have been identified in humans. In this study, the genetic diversity of E. cuniculi was examined at the polar tube protein (PTP) and spore wall protein I (SWP-1) loci. Nucleotide sequence analysis of the PTP gene divided 11 E. cuniculi isolates into three genotypes in congruence with the result of analysis of the ITS of the rRNA gene. The three PTP genotypes differed from one another by the copy number of the 78-bp central repeat as well as point mutations. These E. cuniculi isolates also differed from one another in the number of 15- and 36-bp repeats in the SWP-1 gene. In addition, some E. cuniculi isolates had heterogeneous copies of the SWP-1 gene with various numbers of repeats. Intragenotypic variation was also observed at the SWP-1 locus. Based on the length polymorphism and sequence diversities of the PTP and SWP-1 genes, two simple PCR tests were developed to differentiate E. cuniculi in clinical samples.

Animals↗

PubMed2006

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.

AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 multiply 10(4)/mL, 6.3 multiply 10(2)/mL and 1.6 multiply 10(3)/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 multiply 10(9)/mL, 2.08 multiply 10(6)/mL and 4.40 multiply 10(7)/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10(5)/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

Base Sequence↗

PubMed1992

Fragile-X syndrome: unique genetics of the heritable unstable element.

The fragile site at Xq27.3 is an unstable microsatellite repeat, p(CCG)n. In fragile-X syndrome pedigrees, this sequence exhibits variable amplification, the length of which correlates with fragile-site expression. There is a direct relationship between increased p(CCG)n copy number and propensity for instability: individuals having large amplifications exhibit somatic variation due to increased instability. The instability of the p(CCG)n repeat, when transmitted through affected pedigrees, explains the unusual segregation patterns of fragile-X phenotype, referred to as the Sherman paradox. All individuals of fragile-X genotype were found (where testing was possible) to have a parent with amplified p(CCG)n repeat, indicating that few, if any, cases of fragile-X syndrome are not familial.

Blotting, Southern↗

PubMed2006

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by real-time RT-PCR assay using self-quenched fluorogenic primers.

HIV-1 viral load is a basic marker to evaluate the severity of HIV-1 related diseases and to monitor the effectiveness of treatment. A method based on real-time RT-PCR technology has been developed to quantify HIV-1 RNA using self-quenched fluorogenic primers known as LUX primers. They were used in this study to recognize a low variable gag region of subtype E and B consensus sequences. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with 10 fold serial dilutions of synthetic HIV-gag RNA. A broad range linear relationship (10 to 10(6) copies/ml) was observed between the number of PCR cycles needed to detect a fluorescent signal and the number of RNA copies. The intra- and inter-assay coefficients of variation were 0.72 to 2.54% and 3.14 to 8.83%, respectively, thus indicating good reproducibility. Thirty out of fifty HIV-infected individual plasma samples were quantified by this method and compared with the AMPLICOR HIV-1 Monitor assay, which is widely considered the reference technique for HIV-RNA viral load measurement. The results indicate that the AMPLICOR HIV-1 Monitor assay and real-time RT-PCR using LUX primers are in good agreement (mean difference in log10 copies/ml+/-2 standard deviations = 0.21+/-1.34).

HIV Infections↗

PubMed2006

Human SULT1A1 gene: copy number differences and functional implications.

SULT1A1, which catalyzes the sulfate conjugation of a wide variety of natural and synthetic compounds, is genetically polymorphic. Biochemical and pharmacogenetic studies have demonstrated that individual variation in the level of enzyme activity is inherited. Common single-nucleotide polymorphisms (SNPs) located in the open reading frame and in the 5'-flanking region (5'-FR) may account for a portion of this individual variation. In this study, we demonstrate the presence of SULT1A1 gene deletions and duplications, representing an additional source of variability in the metabolic activity of this enzyme. A quantitative multiplex PCR assay was used to measure the extent of copy number differences and the frequency of these events in different populations. An analysis of DNA from 362 Caucasian-American and 99 African-American showed the presence of 1 to approximately 5 copies of SULT1A1 in individual samples: 5% of Caucasian subjects contained a single copy of the gene and 26% had three or more copies, while 63% of African-American subjects had three or more copies. Analysis of the genomic region surrounding the SULT1A1 gene in three separate cases with a deletion demonstrated that the entire SULT1A1 gene was affected. Reporter assays, constructed for each of the various 5'-FR SNP haplotypes, suggest that these may also play a role in SULT1A1 activity. However, the variability in the level of enzyme activity among 23 human platelet and 267 human liver samples was best explained by gene copy number differences when all sources of genetic variability were considered (P < 0.0001). Overall, these observations have obvious implications for the effectiveness of SULT1A1 as a drug and hormone metabolizing enzyme and its potential role as a risk factor for disease.

Adult↗

PubMed2003

Real-time PCR assay of individual human immunodeficiency virus type 1 variants in coinfected human lymphoid tissues.

In the course of infection, human immunodeficiency virus type 1 (HIV-1) mutates, diverging into a "swarm" of viral quasispecies, and the predominance of CCR5- or CXCR4-utilizing quasispecies is strongly associated with the pattern of disease progression. Quantification of CCR5- and CXCR4-utilizing viruses in viral swarms is important in the investigation of the mechanisms of this phenomenon. Here, we report on a new real-time PCR-based methodology for the evaluation of replication of individual CCR5- and CXCR4-utilizing variants. The assay is highly reproducible, with a coefficient of variation of <3%, and it accurately estimates the numbers of virus-specific RNA copies even when their difference in the mixture is 2 orders of magnitude. We demonstrate that replications of CCR5- and CXCR4-utilizing variants can be evaluated and distinguished in experimentally coinfected human lymphoid tissue. The assay we developed may facilitate study of the mechanisms of the R5-to-X4 switch in viral swarms in human tissues infected with HIV-1.

Base Sequence↗

PubMed2003

Genetic and cytogenetic analyses of breast cancer yield different perspectives of a complex disease.

Genomic instability in breast cancer results in low-level changes in DNA copy number, a significant but poorly understood mechanism underlying the genetic heterogeneity of this disorder. Two different approaches, loss of heterozygosity (LOH) and comparative genomic hybridization (CGH), have been used to probe the genetics of breast cancer evolution. LOH is a locus specific method that detects the variation in the parental origin of DNA, but is not quantitative. CGH provides a genome-wide accounting of the magnitude of DNA copy number changes, but not parental origin. Both methods have identified complex and heterogeneous patterns of DNA losses, duplications, and amplifications during breast cancer evolution. LOH and CGH technologies interrogate very distinct mechanisms driving breast tumor evolution, yet are seldom used in parallel to profile specimens. Thus, the relative significance of genetic versus numerical variations of DNA in breast cancer evolution remains undefined. This review will attempt to summarize some of the successes of these investigations, highlight some complex and confounding observations emerging from these studies, and discuss the potential of these studies to improve our understanding of breast cancer biology and treatment.

Breast Neoplasms↗

PubMed1996

Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309).

Many tandemly repeated minisatellite loci display extreme levels of length variation as a consequence of high rates of spontaneous germline mutation altering repeat copy number. Direct screening for new allele lengths by small-pool PCR has shown that instability at the human minisatellite locus MS205 (D16S309) is largely germline specific and usually results in the gain or loss of just a few repeat units. Structural analysis of the order of variant repeats has shown that these events occur preferentially at one end of the tandem array and can result in complex rearrangements including the inter-allelic transfer of repeat units. In contrast, putative mutants recovered from somatic DNA occur at a substantially lower rate and are simple and non-polar in nature. Germline mutation rates vary considerably between alleles, consistent with regulation occurring in cis. Although examination of DNA sequence polymorphisms immediately flanking the minisatellite reveals no definitive associations with germline mutation rate variation, differences in rate may be paralleled by changes in mutation spectrum. These findings help to explain the diversity of MS205 allele structures in modern humans and suggest a common mutation pathway with some other minisatellites.

Alleles↗

PubMed1996

Is point mutagenesis a mechanism for antigenic variation in Trypanosoma brucei?

Antigenic variation in African trypanosomes proceeds by switching between different variant surface glycoprotein (VSG) molecules, whose extensive epitope differences enable evasion of antibody responses. Each trypanosome has approximately 1000 basic copy VSG genes inside chromosomes and a subset located at telomeres. Switching usually involves different individual basic copy genes being duplicated, as an expression linked copy, into a transcriptionally active site. In a few cases expression linked copies with a number of point mutations have been observed, leading to the suggestion that point mutagenesis provides another mechanism of antigenic variation. The most extensive example is a VSG gene that is normally activated in the metacyclic population in the tsetse fly, but the point mutations were detected in expression linked copies generated during bloodstream infection, after prolonged growth and selection. It was suggested that particularly telomeric or metacyclic VSG genes might undergo point mutagenesis during expression linked copy formation. To test this we have cloned 3 trypanosomes very soon after they had generated, during mouse infection, expression linked copies of the metacyclic VSG gene ILTat 1.22 and have detected only a single point mutation which is present in one expression linked copy, but not the corresponding basic copy, gene. This mutation does not prevent binding of a neutralizing antibody. Extensive VSG gene point mutagenesis may be a consequence merely of prolonged growth and extensive selection. There is not a single reported case of a point mutated VSG presenting a completely new set of exposed epitopes, suggesting point mutagenesis is unlikely to be an authentic mechanism for antigenic variation.

Animals↗

PubMedDate not supplied

Quantifying Aotus monkey cytokines by real-time quantitative RT-PCR.

Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-beta and IFN-gamma). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/microl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.

Animals↗

PubMed1998

Linkage of two distinct AT-rich minisatellites at multiple loci in the genome of Theileria parva.

Minisatellite tandem repeat elements are well known components of vertebrate genomes, but have not yet been extensively characterized in lower eukaryotes. We describe two unusual, AT-rich minisatellites of the protozoan parasite Theileria parva whose sequences are unrelated to the G/C-rich i minisatellite superfamily' of vertebrate and plant genomes. The T. parva tandem repeats, one with a conserved sequence T2-5ACACA (6-17 copies), and the other with a 6-bp core sequence of either ACTATA or TATACT associated with additional variable sequences in repeats of 10-17bp (3-7 copies), were closely linked at more than 20 sites in the T. parva genome, separated by 390, 510 and 660bp at three loci analysed in detail. Such linkage is without precedent in minisatellites so far analysed in other organisms. The minisatellite loci were widely dispersed on 13 out of 33 genomic SfiI fragments, on all four T. parva chromosomes and did not exhibit a telomeric bias in their distribution. Analysis of flanking sequences revealed no obvious conserved sequences between the five loci, or other multicopy repeat sequences outside the minisatellite regions. The T2-5 ACACA minisatellite was highly effective as a multilocus fingerprinting probe for discrimination of T. parva isolates. Analysis of two individual minisatellite loci revealed variation between the genomic DNAs of two T. parva isolates in the copy number of the constituent repeats within the array, similar to that typical of vertebrate minisatellites. 1998 Elsevier Science B.V.

Animals↗

PubMed1999

Tsessebe, Topi and Tiang: three distinct Tc1-like transposable elements in the malaria vector, Anopheles gambiae.

Three distinct types of Tc1-family transposable elements have been identified in the malaria vector, Anopheles gambiae. These three elements, named Tsessebe, Topi and Tiang, have the potential to encode transposases that retain most of the conserved amino acids that are characteristic of this transposon family. However, all three are diverged from each other by more than 50% at the nucleotide level. Full-length genomic clones of two types, Topi and Tsessebe, have been isolated and fully sequenced. The third, Tiang, is represented only by a 270 bp, PCR-amplified fragment of the transposase coding region. The Topi and Tsessebe elements are 1.4 kb and 2.0 kb in length, respectively, and differ in the length of their inverted terminal repeats (ITRs). The Topi elements have 26 bp ITRs, whereas the Tsessebe clones have long ITRs ranging in length from 105 to 209 bp, with the consensus being about 180 bp. This difference is due primarily to variation in the length of an internal stretch of GT repeats. The copy number and location of these elements in ovarian nurse cell polytene chromosomes varies greatly between element subtypes: Topi elements are found at between 17-31 sites, Tsessebe at 9-13 and Tiang at 20 euchromatic sites, in addition to several copies of these elements in heterochromatic DNA. The copy number and genomic insertion sites of these transposons varies between A. gambiae strains and between member species of the A. gambiae complex. This may be indicative of transpositionally active Tc1-like elements within the genome.

Amino Acid Sequence↗