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PubMed1993

Variability and evolution of highly repeated DNA sequences in the genus Beta.

Satellite DNA from wild beet species was separated from restriction endonuclease digested genomic DNA by polyacrylamide gel electrophoresis. Two nonhomologous HaeIII satellite DNA repeats were cloned from the wild beet Beta trigyna. The type I repeat is 140-149 bp long and AT rich, while the type II is 162 bp in size and GC rich. A third repetitive HaeIII element cloned from the related wild beet B. corolliflora was shown to be organized as a HinfI satellite DNA family in the cultivated beet B. vulgaris ssp. vulgaris and the wild beet B. vulgaris ssp. maritima. This type III satellite monomer is 149 bp long and contains a high number of short direct subrepeats. The monomer was found in different genomic organizations and copy numbers in all sections of the genus Beta indicating an amplification early in the phylogeny. The HaeIII repeats from B. trigyna are characterized by a lower variability and form long tandem arrays in the genomes of Corollinae species. The investigation of the distribution of all three sequence families provided data that may contribute to the solution of taxonomic problems of the genus Beta and be useful in the characterization of hybrids and derived lines with alien wild beet chromosomes.

Base Sequence↗

PubMed1984

Evolving sea urchin histone genes--nucleotide polymorphisms in the H4 gene and spacers of Strongylocentrotus purpuratus.

We present a comparison of spacer and coding sequences of histone gene repeats from four Strongylocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions : transversions : single-base-pair insertions/deletions was 3:2:2. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)n and (CT)n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of every S. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pairwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1-2 million years necessary for accumulation of the variation. The level of sequence variation found within the S. purpuratus population, for both tandemly repeated and single-copy genes, is 25%-50% of that found between S. purpuratus and S. drobachiensis.

Alleles↗

PubMed2006

Optimal gene trees from sequences and species trees using a soft interpretation of parsimony.

Gene duplication and gene loss as well as other biological events can result in multiple copies of genes in a given species. Because of these gene duplication and loss dynamics, in addition to variation in sequence evolution and other sources of uncertainty, different gene trees ultimately present different evolutionary histories. All of this together results in gene trees that give different topologies from each other, making consensus species trees ambiguous in places. Other sources of data to generate species trees are also unable to provide completely resolved binary species trees. However, in addition to gene duplication events, speciation events have provided some underlying phylogenetic signal, enabling development of algorithms to characterize these processes. Therefore, a soft parsimony algorithm has been developed that enables the mapping of gene trees onto species trees and modification of uncertain or weakly supported branches based on minimizing the number of gene duplication and loss events implied by the tree. The algorithm also allows for rooting of unrooted trees and for removal of in-paralogues (lineage-specific duplicates and redundant sequences masquerading as such). The algorithm has also been made available for download as a software package, Softparsmap.

Algorithms↗

PubMed2003

Nuclear DNA sequences detect species limits in ancient moa.

Ancient DNA studies have typically used multi-copy mitochondrial DNA sequences. This is largely because single-locus nuclear genes have been difficult to recover from sub-fossil material, restricting the scope of ancient DNA research. Here, we have isolated single-locus nuclear DNA markers to assign the sex of 115 extinct moa and, in combination with a mitochondrial DNA phylogeny, tested competing hypotheses about the specific status of moa taxa. Moa were large ratite birds that showed extreme size variation both within and among species. For some taxa, this large variation was hypothesized to represent sexual dimorphism, while for others it was argued to reflect the existence of different species. Our results show that moa were characterized by extreme reverse sexual dimorphism and as a result we have been able to clarify the number of moa species. For example, we show that the three recognized 'species' of Dinornis comprised only two monophyletic groups and that two of these 'species' comprised individuals of one sex only. This study also illustrates that single-locus nuclear DNA sequences can be consistently recovered from ancient material.

Animals↗

PubMed1993

Rapid assessment of single-copy nuclear DNA variation in diverse species.

We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin, MHC DQA, and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA), and in birds, reptiles and mammals (aldolase, H2AF, myoglobin). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A) or high (e.g. skink ALD-1) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.

Amino Acid Sequence↗

PubMed2005

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis.

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.

Animals↗

PubMed1994

Amplification of the ancient murine Lx family of long interspersed repeated DNA occurred during the murine radiation.

We identified and characterized the relics of an ancient rodent L1 family, referred to as Lx, which was extensively amplified at the time of the murine radiation about 12 million years ago, and which we showed was ancestral to the modern L1 families in rat and mouse. Here we have extended our analysis of the Lx amplification by examining more murine and nonmurine species for Lx sequences using both blot hybridization and the polymerase chain reaction for a total of 36 species. In addition we have determined the relative copy number and sequence divergence, or age, of Lx elements in representative murine genera. Our results show that while Lx sequences are confined to murine genera, the extent of the amplification was different in the different murine lineages, indicating that the amplification of Lx did not precede, but was coincident with, the murine radiation. The implications of our findings for the evolutionary dynamics of L1 families and the utility of ancestral amplification events for systematics are discussed.

Animals↗

PubMed2003

Downregulation of Trypanosoma brucei VSG expression site promoters on circular bacterial artificial chromosomes.

Trypanosoma brucei has about 20 telomeric variant surface glycoprotein (VSG) gene expression sites (ESs), which are downregulated in the insect form. We investigated the transcriptional behaviour of ES promoters on bacterial artificial chromosomes (BACs) containing two different ESs and their flanking regions on fragments of about 140kb. Four different BACs containing either the 221 or the VO2 ES were introduced into insect form T. brucei. The BACs replicated as circular episomes as shown using pulsed field gel (PFG) analysis of DNA exposed to increasing doses of gamma radiation, and digestion with Dam methylation-sensitive restriction enzymes. BAC copy number per cell varied from about 3 for the 221 ES BACs to about 15 for the VO2 ES BACs. Increasing drug selection pressure on the VO2 BAC T. brucei transformants resulted in amplification to about 80 BACs per cell. Although BACs were maintained in the absence of drug selection for at least 56 days, copy number fell and there was no evidence for centromere activity. ES promoters on small plasmid episomes introduced into insect form T. brucei in transient transfections are derepressed. In contrast, ES promoters on large BAC episomes are downregulated both on the original ES BACs, and on ES BACs selected for a drug marker driven by a rDNA promoter fused to the BAC vector. This indicates that downregulation of ES promoters in insect form T. brucei is influenced by genomic context, but does not necessitate proximity to a chromosome end.

Animals↗

PubMed1994

No association between alleles or genotypes at the dopamine transporter gene and schizophrenia.

The dopamine transporter gene (DAT1) is an important candidate gene for schizophrenia. A 40-bp VNTR (variable number of tandem repeats) polymorphism of DAT1 has been typed in 105 schizophrenic patients and 98 normal control subjects from Sichuan (China). Compared with allele frequencies for Caucasians reported in the literature, the Chinese population investigated showed a reduced frequency of the 9-copy allele and an increased frequency of the 10-copy allele. The observed frequency of genotypes was in agreement with the expected values according to Hardy-Weinberg equilibrium. No significant difference was found between patients and control subjects with regard to allele frequency, allele prevalence, and genotype counts. The results of the association study presented here are in agreement with the negative results of linkage analyses in schizophrenia pedigrees from Iceland (Kristbjarnarson et al., submitted) and from Utah (Byerley et al., 1993). Taken together, these studies suggest that variation in the dopamine transporter gene (DAT1) is unlikely to be a factor in the etiology of schizophrenia. The observed differences in allele frequencies between Chinese and Caucasian groups suggest that the human transporter gene might be useful for the construction of evolutionary trees in humans and primates as illustrated by Cavalli-Sforza's work (Mountain et al., 1992).

Adolescent↗

PubMed2006

Real-time GUS analysis using Q-PCR instrumentation.

The development of new technology within biological sciences has resulted in a number of real-time PCR instruments that have become essential tools within molecular biology. This equipment has facilitated high throughput analysis of samples and optimal information gathering of completed PCR reactions for example estimating the copy number of a gene of interest that is inserted into particular genomes. Real-time PCR instruments frequently come with optional filter sets, e.g. the ALEXA filter set which has parameters in common with excitation and emission wavelengths of sodium methyl umbelliferone (NaMU) widely used in beta-glucuronidase reporter gene assays. Using these filter sets it has been possible to quantify and measure gus A activity of Ulmus procera SR4 in real-time removing the necessity for aliquots of reactions to be stopped by pipetting into carbonate buffer for each time point. The introduction of real-time GUS analysis leads to faster, more accurate and reproducible assays with reduced potential for pipetting errors, requires fewer manipulations and encourages high throughput analysis of inter-individual gene expression variation.

Equipment Design↗

PubMed2005

A survey of homozygous deletions in human cancer genomes.

Homozygous deletions of recessive cancer genes and fragile sites are known to occur in human cancers. We identified 281 homozygous deletions in 636 cancer cell lines. Of these deletions, 86 were homozygous deletions of known recessive cancer genes, 17 were of sequenced common fragile sites, and 178 were in genomic regions that do not overlap known recessive oncogenes or fragile sites ("unexplained" homozygous deletions). Some cancer cell lines have multiple homozygous deletions whereas others have none, suggesting intrinsic variation in the tendency to develop this type of genetic abnormality (P < 0.001). The 178 unexplained homozygous deletions clustered into 131 genomic regions, 27 of which exhibit homozygous deletions in more than one cancer cell line. This degree of clustering indicates that the genomic positions of the unexplained homozygous deletions are not randomly determined (P < 0.001). Many homozygous deletions, including those that are in multiple clusters, do not overlap known genes and appear to be in intergenic DNA. Therefore, to elucidate further the pathogenesis of homozygous deletions in cancer, we investigated the genome landscape within unexplained homozygous deletions. The gene count within homozygous deletions is low compared with the rest of the genome. There are also fewer short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and low-copy-number repeats (LCRs). However, DNA within homozygous deletions has higher flexibility. These features may signal the presence of currently unrecognized zones of susceptibility to DNA rearrangement. They may also reflect a tendency to reduce the adverse effects of homozygous deletions by minimizing the number of genes removed.

Cell Line, Tumor↗

PubMed1996

Age-dependent silencing of globin transgenes in the mouse.

Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation.

Aging↗

PubMed2003

Candidate defense genes from rice, barley, and maize and their association with qualitative and quantitative resistance in rice.

Candidate genes involved in both recognition (resistance gene analogs [RGAs]) and general plant defense (putative defense response [DR]) were used as molecular markers to test for association with resistance in rice to blast, bacterial blight (BB), sheath blight, and brown plant-hopper (BPH). The 118 marker loci were either polymerase chain reaction-based RGA markers or restriction fragment length polymorphism (RFLP) markers that included RGAs or putative DR genes from rice, barley, and maize. The markers were placed on an existing RFLP map generated from a mapping population of 116 doubled haploid (DH) lines derived from a cross between an improved indica rice cultivar, IR64, and a traditional japonica cultivar, Azucena. Most of the RGAs and DR genes detected a single locus with variable copy number and mapped on different chromosomes. Clusters of RGAs were observed, most notably on chromosome 11 where many known blast and BB resistance genes and quantitative trait loci (QTL) for blast, BB, sheath blight, and BPH were located. Major resistance genes and QTL for blast and BB resistance located on different chromosomes were associated with several candidate genes. Six putative QTL for BB were located on chromosomes 2, 3, 5, 7, and 8 and nine QTL for BPH resistance were located to chromosomes 3, 4, 6, 11, and 12. The alleles of QTL for BPH resistance were mostly from IR64 and each explained between 11.3 and 20.6% of the phenotypic variance. The alleles for BB resistance were only from the Azucena parent and each explained at least 8.4% of the variation. Several candidate RGA and DR gene markers were associated with QTL from the pathogens and pest. Several RGAs were mapped to BB QTL. Dihydrofolate reductase thymidylate synthase co-localized with two BPH QTL associated with plant response to feeding and also to blast QTL. Blast QTL also were associated with aldose reductase, oxalate oxidase, JAMyb (a jasmonic acid-induced Myb transcription factor), and peroxidase markers. The frame map provides reference points to select candidate genes for cosegregation analysis using other mapping populations, isogenic lines, and mutants.

Aldehyde Reductase↗

PubMed2002

Size and complexity of the nuclear genome of the ectomycorrhizal fungus Paxillus involutus.

The basidiomycete Paxillus involutus is forming ectomycorrhizal symbiosis with a broad range of forest trees. Reassociation kinetics on P. involutus nuclear DNA indicated a haploid genome size of 23 Mb including 11% of repetitive DNA. A similar genome size (20 Mb) was estimated by genomic reconstruction analysis using three single copy genes. To assess the gene density in the P. involutus genome, a cosmid containing a 33-kb fragment of genomic DNA was sequenced and used to identify putative open reading frames (ORFs). Twelve potential ORFs were predicted, eight displayed significant sequence similarities to known proteins found in other organisms and notably, several homologues to the Podospora anserina vegetative incompatibility protein (HetE1) were found. By extrapolation, we estimate the total number of genes in the P. involutus haploid genome to approximately 7700.

Amino Acid Sequence↗

PubMed2002

Recognition of variant Rifin antigens by human antibodies induced during natural Plasmodium falciparum infections.

Antibodies from individuals living in areas where malaria is endemic are known to react with parasite-derived erythrocyte surface proteins. The major immunogenic and clonally variant surface antigen described to date is Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1), which is encoded by members of the multicopy var gene family. We report here that rifin proteins (RIF proteins), belonging to the largest known family of variable infected erythrocyte surface-expressed proteins, are also naturally immunogenic. Recombinant RIF proteins were used to analyze the antibody responses of individuals living in an area of intense malaria transmission. Elevated anti-rifin antibody levels were detected in the majority of the adult population tested, whereas the prevalence of such antibodies was much lower in malaria-exposed children. Despite the high degree of diversity between rif sequences and the high gene copy number, it appears that P. falciparum infections can induce antibodies that cross-react with several variant rifin molecules in many parasite isolates in a given community, and the immune response is most likely to be stable over time in a hyperendemic area. The protein was localized by fluorescence microscopy on the membrane of ring and young trophozoite-infected erythrocytes with antibodies from human immune sera with specificities for recombinant RIF protein.

Adolescent↗

PubMed1993

Molecular variation in trypanosomes.

Several species of the genus Trypanosoma cause parasitic diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. These parasites exhibit considerable intra-species genetic diversity and variation, which has complicated their taxonomic classification. This diversity and variation can be defined at the level of both the genome and of individual genes. The nuclear genome shows considerable inter- and intra-species plasticity in terms of chromosome number and size (molecular karyotype). The mitochondrial (kDNA) genome also varies considerably between species, especially in terms of minicircle size and organization. There is also considerable intra-specific sequence diversity in minicircles and within the Variable Region of the maxicircle. Restriction enzyme analysis of this diversity has lead to the concept of 'schizodemes'. At the gene level, isoenzyme analysis has proven very useful for strain and isolate identification, with the classification into numerous 'zymodemes'. Considerable antigenic diversity has also been identified in T. cruzi and T. brucei, with the development of 'serodemes' in the latter. In addition to this inter-strain diversity, African trypanosomes (T. brucei, T. congolense, and T. vivax) exhibit the phenomenon of antigenic variation, where individual parasites are able to express any one of hundreds of different copies of the Variant Surface Glycoprotein gene at any particular time. The molecular mechanisms underlying antigenic variation are now understood in considerable detail. The implication of this molecular diversity and variation are discussed in terms of trypanosome taxonomy and disease control.

Animals↗

PubMed1995

A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating.

A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan. Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added. These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation. Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies. In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism. Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus. Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed. Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells. Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8. The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures.

Cell Aggregation↗

PubMed2000

Association of estrogen receptor beta gene polymorphism with bone mineral density.

Chromosomal mapping of the human estrogen receptor beta (ERbeta) gene by fluorescence in situ hybridization (FISH) reveals that ERbeta is located at human chromosome 14, region q23-24.1, where the aberration of DNA copy number in the bone disorders is frequently involved. Then, we investigated the association between dinucleotide (cytosine-adenine; CA) repeat polymorphism located in the flanking region of ERbeta gene and bone mineral density (BMD) in 204 healthy postmenopausal Japanese women. The genotype was classified into "A" through "O" according to the number of the repeats, from 18 to 32. BMD was expressed in Z score (a deviation from the weight-adjusted average BMD of each age using the standard deviation as a unit). When we separate the subjects into two groups bearing at least one I allele (26 CA repeats) and those who did not, the former subjects had significantly higher Z score of L2-4 BMD (mean +/- standard deviation; 0.674 +/- 1.53 vs 0.128 +/- 1.38; P = 0.027). These data suggest that genetic variation at the ERbeta locus may be associated with some determinants for BMD and the possible involvements of this polymorphism in the cause of postmenopausal osteoporosis in Japanese women.

Base Sequence↗