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PubMed2002

Panzee, a copia-like retrotransposon from the grain legume, pigeonpea ( Cajanus cajan L.).

We report the isolation and characterization of a copia-like retrotransposon, Panzee, from pigeonpea ( Cajanus cajan). The 4947-bp Panzee element is AT rich (60%) and the integrated element is flanked by a target-site duplication of 5 bp. The structure of Panzee is that of a typical LTR-retrotransposon containing long terminal repeats (LTRs) which flank its internal region. The 5' LTR is 372 bp in length and the 3' LTR is 383 bp long. Both LTRs start with 5'-TG and end with CA-3' and have 4-bp terminal inverted repeats. The internal region between the LTRs contains two priming sites for DNA synthesis: the first, a 12-bp primer binding site complementary to initiator methionyl tRNA, is located adjacent to the 3' end of the 5' LTR and the other, a 12-bp polypurine tract lies just upstream to the 5' end of the 3' LTR. The putative polyprotein shows homology to all the proteins encoded by LTR retrotransposons, i.e. group-associated antigen ( gag), proteinase, endonuclease, reverse transcriptase (RT) and ribonuclease H (RNase H). However, the cloned copy of the element contains four frameshifts and a premature stop codon in its protein-coding domain. Genomic Southern hybridization experiments using probes derived from three different regions of the element show that Panzee or Panzee-related elements are present in high copy numbers in the pigeonpea genome. Analysis of transgenic tobacco plants containing the LTR:GUS construct shows that the 5' LTR of Panzee drives gene expression in this heterologous system in a tissue-specific manner. A phylogenetic tree constructed using reverse transcriptase sequences places Panzee in the copia group of retrotransposons.

Amino Acid Sequence↗

PubMed1996

Optimal sequencing strategies for surveying molecular genetic diversity.

Two commonly used measures of genetic diversity for intraspecies DNA sequence data are based, respectively, on the number of segregating sites, and on the average number of pairwise nucleotide differences. Expressions are derived for their variance in the presence of intragenic recombination for a panmictic population of fixed size that is at neutral equilibrium at the region sequenced. We show that, in contrast to the slow decrease in variance with increasing sample size, if the recombination rate is nonzero, the asymptotic rate of decrease of variance with increasing sequence length, for fixed sample size, is quite rapid. In particular, it is close to that which would be obtained by sequencing independent chromosome regions. The correlation between measures of diversity from linked regions is also examined. For a given total number of bases sequenced in a particular region, optimal sequencing strategies are derived. These typically involve sequencing relatively few (three to 10) long copies of the region. Under optimal strategies, the variances of the two measures are very similar for most parameter values considered. Results concerning optimal sequencing strategies will be sensitive to gross departures from the underlying assumptions, such as population bottlenecks, selective sweeps, and substantial population substructure.

DNA↗

PubMed1993

Mitotic stability and meiotic variability of the (CAG)n repeat in the Huntington disease gene.

The gene causing Huntington's disease, an autosomal dominantly inherited, neurodegenerative disorder, has been identified recently. The corresponding mutation is involving an expansion in the number of (CAG)n repeats in the coding region of the Huntington's disease gene on chromosome 4. In this report, we demonstrate the length variation of the repeat in 513 non-HD chromosomes from normal individuals and HD patients showing 23 alleles with 11 to 33 repeats. Analyzing the inheritance of the (CAG)n stretch we found meiotic instability for HD alleles ([CAG]40 to [CAG]75) with a mutation frequency of approximately 0.7, while in 431 meioses of normal alleles only two expansions were identified. The risk of expansion during spermatogenesis is enhanced compared to oogenesis explaining juvenile onset by transmission from affected fathers. Further, the number of (CAG)n copies in an affected individual in relation to the sex of the transmitting parent was evaluated and no significant differences were found. No mosaicism or differences in the repeat lengths were observed in the DNA from different tissues including brain and lymphocytes of two HD patients indicating mitotic stability of the mutation. Therefore, the determination of the repeat number in the DNA of blood lymphocytes is probably representative of all tissues in a patient.

Alleles↗

PubMed1981

Pulmonary artery pressure monitoring in the surgical intensive care unit. Benefits vs difficulties.

The process of pulmonary artery pressure monitoring in 50 consecutive patients in the surgical intensive care unit was analyzed to determine the number and types of problems that occurred in relation to the benefit obtained. Twenty-six percent of the patients had a change in their cardiorespiratory therapy and their conditions were improved after the pressure data were obtained. Many technical and interpretative problems that tended to decrease the desirability of using pulmonary artery pressure monitoring were identified. Most problems could be avoided by carefully calibrating the monitor system, clearing the catheter system of air bubbles and blood clots, learning to property interpret pulmonary artery pressure tracings despite large respiratory variations, and obtaining a hard-copy printout of the pressure tracing with the simultaneous ECG signal. A protocol for avoiding many difficulties was developed.

Blood Pressure↗

PubMed1980

Complicated organization of a single repeated DNA sequence in the chicken genome is revealed by cloning.

The structural organization of a family of repeated DNA sequences in the chicken genome has been determined by hybridization of a cloned repeated DNA sequence to Southern blots of total DNA. The length of the cloned DNA fragment is 3600 nucleotide pairs. This fragment consists principally, if not entirely, of a single repeated DNA sequence occurring only once within the cloned fragment. In the chicken genome, the family of repeated DNA sequences homologous to the cloned sequence has a limited number of alternative forms. Some of the restriction fragments of total DNA to which the cloned sequence hybridizes correspond to those expected from the location of restriction endonuclease cleavage sites within the cloned sequence. There are also a limited number of other genomic restriction fragments, each present in multiple copies, to which the cloned sequence hybridizes but which do not relate in any obvious way to the length of the cloned sequence. These various restriction fragments differ from one another in that they appear to be present in unequal amounts in total DNA, and many of them do not contain the entire cloned sequence. This study provides some new information about the structure of repeated DNA sequences in the chicken genome. The copies of a repeated DNA sequence may differ from one another both by minor variations in nucleotide sequence (divergence) and in more substantial ways as would be expected to arise from processes such as insertion, deletion, and translocation. In addition to this description of a single cloned repeated DNA sequence from the chicken genome, this paper reports the cloning of more than 100 different restriction fragments of chicken DNA, each of which contains one or more repeated DNA sequences.

Animals↗

PubMed1990

Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1.

The complete nucleotide (nt) sequence of a 5.1-kb EcoRI DNA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAM beta 1 has been determined. Of the seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (Mr 57,380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-kb AccI-HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr 30,471) was therefore composed of large tracts of reiterated amino acid sequence (11 x VDP and 35 x TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 kDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of ORF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035), none was evident to any other characterised Gram+ plasmid.

Amino Acid Sequence↗

PubMed1983

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.

Animals↗

PubMed1994

Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine.

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH-H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.

Amino Acid Sequence↗

PubMed2000

Copepod development rates in relation to genome size and 18S rDNA copy number.

It is known that body sizes and temperature-independent developmental durations within two genera of calanoid Copepoda (Crustacea) are positively related to nuclear DNA contents of their somatic nuclei. Evidently because of the constraint of similar cell numbers among the species, (nucleotypic) effects of nucleus size on cell size and on cell-level processes are expressed at the whole-organism level. Here, we show that developmental durations of eight species of five genera are also negatively related to their greatly differing numbers of 18S rRNA genes per unit DNA. We propose that levels of rDNA iteration among copepods have been controlled by natural selection to regulate ribosome concentrations, therefore protein production and development rates, independently of the large variations in genome sizes, which are in turn adapted to regulating cell and therefore body sizes.

Animals↗

PubMed1997

A family of differentially amplified repetitive DNA sequences in the genus Beta reveals genetic variation in Beta vulgaris subspecies and cultivars.

Members of a highly abundant restriction satellite family have been isolated from the wild beet species Beta nana. The satellite DNA sequence is characterized by a conserved RsaI restriction site and is present in three of four sections of the genus Beta, namely Nanae, Corollinae, and Beta. It was not detected in species of the evolutionary old section Procumbentes, suggesting its amplification after separation of this section. Sequences of eight monomers were aligned revealing a size variation from 209 to 233 bp and an AT content ranging from 56.5% to 60.5%. The similarity between monomers in B. nana varied from 77.7% to 92.2%. Diverged subfamilies were identified by sequence analysis and Southern hybridization. A comparative study of this repetitive DNA element by fluorescent in situ hybridization and Southern analyses in three representative species was performed showing a variable genomic organization and heterogeneous localizations along metaphase chromosomes both within and between species. In B. nana the copy number of this satellite, with some 30,000 per haploid genome, is more than tenfold higher than in Beta lomatogona and up to 200 times higher than in Beta vulgaris, indicating different levels of sequence amplification during evolution in the genus Beta. In sugar beet (B. vulgaris), the large-scale organization of this tandem repeat was examined by pulsed-field gel electrophoresis. Southern hybridization to genomic DNA digested with DraI demonstrated that satellite arrays are located in AT-rich regions and the tandem repeat is a useful probe for the detection of genetic variation in closely related B. vulgaris cultivars, accessions, and subspecies.

DNA, Plant↗

PubMed1989

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.

Cell Differentiation↗

PubMedDate not supplied

Antigenic variation in Trypanosoma equiperdum.

Trypanosoma equiperdum is an African trypanosome that causes dourine in horses. Like the other African trypanosomes, T. equiperdum escapes elimination by the immune system of its host by using an elaborate system of antigenic variant. The trypanosomes are covered by a coat consisting of a single protein called the variable surface glycoprotein (VSG) that acts as the major trypanosome immunogen. As the host responds to one VSG, trypanosomes covered with another VSG become dominant. There is a loose order of appearance of these VSG during the infection. The factors that affect the timing of VSG expression and the effective size of the VSG repertoire in T. equiperdum are reviewed. The VSG genes are generally activated by a process of duplicative transposition involving the duplication of a silent VSG gene and inserting a copy of the gene into an expression site. The order of VSG expression is related to the amount of homology between the silent gene and the expression site. The genes expressed late in infection lack extensive homology with the expression site and depend on homology with the gene in the expression site. The genes coding for VSG expressed late in infection are hybrid genes because of this mode of transfer. This transfer mechanism allows the trypanosome to create complex VSG genes from parts of several different silent genes that are each pseudogenes. Additionally, data are presented showing that only a limited portion of the VSG is actually seen by the host immune system. These factors indicate that the effective VSG repertoire is greater than the number of VSG genes in the trypanosome genome.

Animals↗

PubMed2002

Yfm1, a multicopy marker specific for the Y chromosome and beneficial for forensic, population, genetic, and spermatogenesis-related studies.

A recently developed microsatellite marker on the Y chromosome, Yfm1, which was originally cloned from a cosmid clone mapped near the DAZ (Deleted in AZoospermia) genes, was used to classify Y chromosomes using an automatic sequencer. Yfm1 could detect multicopies on Y chromosomes in a single polymerase chain reaction, showing four main classes, A, A*, B, and C, according to the number of copies and peak patterns. Compound haplotype analysis of the Y chromosome using the Yfm1 marker with three other biallelic markers on the Y chromosome, SRY, DXYS5Y, and YAP, resulted in nine different haplotypes among different populations, including Japanese. Haplotype II (defined by YAP insertion) observed in the Japanese population was consistently associated with Yfm1 class A or A*, which showed the lowest number of copies of Yfm1. Haplotypes III and IV were consistently associated with Yfm1 class B. On the other hand, haplotype I showed a variety of Yfm1 patterns that were dubbed class C when not appropriately classified as A, A*, or B. These relationships among Yfm1 microsatellite and Y-specific biallelic markers could supply useful population genetic information. Moreover, because we have already shown that men with haplotype II have significantly lower spermatogenic ability than those with other haplotypes, Yfm1 class A or A* with the least number of copies may be related to the haplotype II-specific structure of the Y chromosome, such as deletion of DAZ or DAZ repeats, reflecting the lower spermatogenic abilities of Japanese haplotype II men. Thus, Yfm1 represents a very useful marker for analysis of genetic structure in different populations and studies on Y chromosome lineage-specific genotype-phenotype correlations.

Alleles↗

PubMed2006

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies.

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

Bacteria↗

PubMed1994

Molecular genetic analyses of the Tyrolean Ice Man.

An approximately 5000-year-old mummified human body was recently found in the Tyrolean Alps. The DNA from tissue samples of this Late Neolithic individual, the so-called "Ice Man," has been extracted and analyzed. The number of DNA molecules surviving in the tissue was on the order of 10 genome equivalents per gram of tissue, which meant the only multi-copy sequences could be analyzed. The degradation of the DNA made the enzymatic amplification of mitochondrial DNA fragments of more than 100 to 200 base pairs difficult. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples of the body. This sequence showed that the mitochondrial type of the Ice Man fits into the genetic variation of contemporary Europeans and that it was most closely related to mitochondrial types determined from central and northern European populations.

Animals↗

PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗

PubMed2026

Novel epigenetic loci identified from an epigenome-wide association study underlying brain structural changes in bipolar disorder.

BACKGROUND: DNA methylation influences gene-environment interactions and brain development in bipolar disorder (BD). We aimed to identify BD-associated epigenetic loci and examine their associations with brain structural variation. METHODS: We conducted an epigenome-wide association study (BD group, n = 90; healthy controls group, n = 161) to identify BD-associated DNA methylation loci, and we additionally performed copy number alteration and functional enrichment analyses. The correlations between epigenetic loci and cortical thickness (CT) were assessed using Pearson's partial correlation analysis, and the co-methylation effect of the epigenetic loci identified in the neuroimaging-epigenetic analysis was investigated. FINDINGS: A total of 156 differentially methylated positions (DMPs) and 7 differentially methylated regions were identified, and the genes associated with them were observed to be enriched in biological processes related to muscle hypertrophy and neuronal activity. Significant correlations between the methylation levels of 13 DMPs associated with three genes (miR886, PLEC1, and ICAM5) and the CT of the right postcentral gyrus and inferior frontal gyrus were identified. Specifically, 10 DMPs associated with the CpG island in the upstream region of the miR886 gene showed negative correlations with the right postcentral gyrus CT, implicating miR886-associated CpG-island methylation in regional cortical thinning. CONCLUSION: Epigenetic changes might play an important role in brain structural changes in BD. These multimodal findings nominate miR886-related methylation as a candidate molecular correlate of cortical thinning and warrant replication and mechanistic follow-up in larger, state-diverse cohorts.

Humans↗

PubMed2004

Spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract.

The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant Lactobacillus murinus ASF361 present at 10(5) to 10(7) cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10(8) to 10(10) cells/g of tissue. The variation between the three mice was small for most ASF strains, except for Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.

Animals↗