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Differential lineage-specific amplification of transposable elements is responsible for genome size variation in Gossypium.

The DNA content of eukaryotic nuclei (C-value) varies approximately 200,000-fold, but there is only a approximately 20-fold variation in the number of protein-coding genes. Hence, most C-value variation is ascribed to the repetitive fraction, although little is known about the evolutionary dynamics of the specific components that lead to genome size variation. To understand the modes and mechanisms that underlie variation in genome composition, we generated sequence data from whole genome shotgun (WGS) libraries for three representative diploid (n = 13) members of Gossypium that vary in genome size from 880 to 2460 Mb (1C) and from a phylogenetic outgroup, Gossypioides kirkii, with an estimated genome size of 588 Mb. Copy number estimates including all dispersed repetitive sequences indicate that 40%-65% of each genome is composed of transposable elements. Inspection of individual sequence types revealed differential, lineage-specific expansion of various families of transposable elements among the different plant lineages. Copia-like retrotransposable element sequences have differentially accumulated in the Gossypium species with the smallest genome, G. raimondii, while gypsy-like sequences have proliferated in the lineages with larger genomes. Phylogenetic analyses demonstrated a pattern of lineage-specific amplification of particular subfamilies of retrotransposons within each species studied. One particular group of gypsy-like retrotransposon sequences, Gorge3 (Gossypium retrotransposable gypsy-like element), appears to have undergone a massive proliferation in two plant lineages, accounting for a major fraction of genome-size change. Like maize, Gossypium has undergone a threefold increase in genome size due to the accumulation of LTR retrotransposons over the 5-10 Myr since its origin.

Base Sequence↗

High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

Adult↗

Analysis of genomic diversity and evolution of the low-level antiseptic resistance gene smr in Staphylococcus aureus.

A multidrug efflux pump specified by the smr gene mediates low-level antiseptic resistance in staphylococci. We analyzed the genomic diversity of smr and its gene cassette, a structural unit containing smr and terminal direct repeats (DRs), in 22 clinical strains of Staphylococcus aureus isolated over 9 years in a Japanese hospital. Although open reading frames (ORFs) of all the smr genes examined were identical to those reported previously (e.g., qacD in pSK41), smr gene cassettes were classified into three groups (types 1, 2, and 3). The type 1 cassette had an identical genetic organization to that found in the plasmid pSK41, a putative prototype of the smr gene cassette, which contains DRs flanking smr. In the type 2 cassette, the rep gene and a putative replication nick site were found upstream of smr, between a SSOA (single-strand origin) sequence and DR1c, which are components of the type 1 cassette DR. In the type 3 cassette detected in a single strain, IS431 was located between the 3' end of smr and DR. It was suggested by genomic comparison that type 2 and type 3 cassettes might have been derived from the type 1 cassette via insertion of foreign DNA sequence, and that the type 2 cassette might be a precursor form of some previously reported smr cassettes, such as those in pSK108 and pNVH99. Although MICs of antiseptics and ethidium bromide were generally the same among strains having type 1, 2, or 3 smr gene cassette, the type 1 cassette was detected most frequently. Moreover, the copy number of the smr gene in the type 2 cassette was found to be much higher than that in the type 1 or type 3 cassette.

Anti-Bacterial Agents↗

Human inherited marker chromosome 22 short-arm enlargement: investigation of rDNA gene multiplicity, Ag-band size, and acrocentric association.

The banding characteristics of an extreme variant familial chromosome 22 short-arm enlargement are described. Ag-AS staining for nucleolar-organizer regions, identified two areas of rDNA actively coding for 18S and 28S rRNA, the one being a broad distal Ag-band and the other a narrower centromeric Ag-band. The DNA in the major portion of the enlarged short arm was highly methylated, as shown by the binding of antibodies to 5-methylcytidine after UV-denaturation of chromosomal DNA. Mean Ag-band size on the aberrant 22p+ correlated with the mean number of 22p+ associations. Association of 22p+ was no greater than that of other acrocentrics, in spite of a presumed excess number of rDNA gene copies. This case represents only the second such normal variant defined by these techniques.

Cell Division↗

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.

Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.

Bacterial Proteins↗

Quantitation of HTLV-I proviral load by a TaqMan real-time PCR assay.

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood mononuclear cells (PBMCs). The HTLV-I copy number was referred to the actual amount of cellular DNA by means of the quantitation of the albumin gene. Ten copies of HTLV-I DNA could be detected with 100% sensitivity, and the assay had a wide range of at least 5 log(10). Intra- and inter-assay reproducibility was evaluated using independent extractions of PBMCs from an HTLV-I-infected patient (coefficients of variation, 24 and 7% respectively). The performance of this TaqMan PCR assay, coupled with its high throughput, thus allows reliable routine follow-up of HTLV-I proviral load in infected patients. Preliminary results using clinical samples indicate a higher proviral load in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers, and also suggest the usefulness of this quantitative measurement to assess the etiological link between HTLV-I and adult T-cell leukaemia/lymphoma-like syndromes.

Base Sequence↗

Mechanisms for chromosome and plasmid segregation.

The fundamental problems in duplicating and transmitting genetic information posed by the geometric and topological features of DNA, combined with its large size, are qualitatively similar for prokaryotic and eukaryotic chromosomes. The evolutionary solutions to these problems reveal common themes. However, depending on differences in their organization, ploidy, and copy number, chromosomes and plasmids display distinct segregation strategies as well. In bacteria, chromosome duplication, likely mediated by a stationary replication factory, is accompanied by rapid, directed migration of the daughter duplexes with assistance from DNA-compacting and perhaps translocating proteins. The segregation of unit-copy or low-copy bacterial plasmids is also regulated spatially and temporally by their respective partitioning systems. Eukaryotic chromosomes utilize variations of a basic pairing and unpairing mechanism for faithful segregation during mitosis and meiosis. Rather surprisingly, the yeast plasmid 2-micron circle also resorts to a similar scheme for equal partitioning during mitosis.

Adenosine Triphosphatases↗

Quantification of human ooplasmic mitochondria.

It is likely that there is an association between the fitness of mitochondria and their ability to support normal cellular function. Oocytes are greatly enriched in the number of mitochondria as they support essential developmental processes such as oocyte maturation and embryonic development, while their replication is deferred until gastrulation. The mitochondrial DNA (mtDNA) copy number in 87 human oocytes from 29 patients was evaluated after DNA extraction and real-time quantitative polymerase chain reaction (PCR). The average mtDNA copy number was 795,000 (+/- 243,000) in metaphase II oocytes. mtDNA content varied considerably between oocytes, even within the same patient. No relationship was found between mtDNA copy number and maternal age. The findings suggest that mtDNA replication is fully accomplished by the germinal vesicle stage in the fully developed oocyte.

DNA, Mitochondrial↗

Rodent BC1 RNA gene as a master gene for ID element amplification.

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.

Animals↗

The relationship between DNA structural variation and activities of P elements in P and Q strains of Drosophila melanogaster.

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of p pi 25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

Animals↗

Expression of retinoid X receptor transcripts and their significance for developmental competence in in vitro-produced pre-implantation-stage bovine embryos.

Retinoid X receptors are frequently implicated in their role in embryonic development. However, there is no report regarding their expression in embryos with different qualities. In this study, expression pattern and levels of retinoid X receptor transcripts in different qualities and stages of in vitro-produced pre-implantation bovine embryos were examined using real-time polymerase chain reaction (PCR) techniques. Four independent cDNA pools per sample were prepared from mRNAs isolated from different developmental-stage embryos, after classifying them to be of good and bad quality. Primers were also designed to amplify products with PCR. Based on analysis, except retinoid X receptor gamma (RXRG) transcripts, the remaining two types (alpha and beta forms) were expressed in all samples with significantly higher (p < or = 0.05) levels until the eight-cell stage compared with the stages thereafter. However, the transcript levels for bad-quality embryos were consistently lower in all cases in comparison with good-quality embryos. RXRG was identified in 39% of the samples, but all in the pre-embryonic genome activation development stage. Therefore, strong expression patterns of these transcripts in earlier stages indicates their possible role during the maternal phase of embryo development, and the variations of copy number in embryos with different qualities substantiates their possible candidacy as potential quality markers. Moreover, identifying the transcript variations in different qualities and expression of RXRG at these stages is a novel input to the pre-implantation-stage knowledge.

Animals↗

Evidence for limited molecular genetic heterogeneity as defined by allelotyping and clonal analysis in nine metastatic breast carcinomas.

To investigate genetic intratumor heterogeneity, 42 samples of nine primary breast carcinomas and 29 related lymph node metastases were examined for DNA ploidy status, allelotype, and X chromosome inactivation pattern. Two primary breast carcinomas showed DNA index heterogeneity and five contained a single DNA aneuploid tumor stemline, whereas the two remaining primary tumors were solely DNA diploid. Most primary DNA tumor stemlines recurred in lymph node metastases (9 of 11). The allelotype, constructed with 31 different probes mapping to 23 different chromosome arms showed allelic imbalances on nearly all chromosome arms investigated. All tumors contained multiple allelic imbalances (range, 3-12). An allelic imbalance present in a primary tumor was consistently present in all DNA samples of that primary tumor and also in all DNA samples of related lymph node metastases, irrespective of DNA index heterogeneity. X chromosome inactivation pattern analysis with probe M27 beta (DXS255) confirmed the presence of clonal tumor cell populations in these tumors at the time of diagnosis. Densitometry of autoradiograms, which by eye showed retention of heterozygosity, revealed a narrow clustering of allelic imbalance factors between 1.0 and 1.4. In contrast, autoradiograms visually showing an allelic imbalance exhibited a marked interprobe, intertumor and intratumor variation in allelic imbalance factors. No relation between densitometry results and DNA ploidy status was found. Thus, at the time of diagnosis, an advanced primary breast carcinoma consists of a clonal tumor cell population with an established complement of allelic imbalances in all parts of the primary tumor and in the related lymph node metastases. Secondary to the establishment of allelic imbalances, intratumor heterogeneity for the copy number of involved alleles may develop, which in turn probably precedes metastasis.

Adult↗

Transposable element-induced response to artificial selection in Drosophila melanogaster: molecular analysis of selection lines.

Artificial selection lines for abdominal bristle score of Drosophila melanogaster established from P-M hybrid dysgenic crosses showed increases in selection response, heritability and phenotypic variance compared to similar lines started from nondysgenic crosses. To determine whether this increased genetic variance could be due to enhanced transposition of P elements following the dysgenic cross, the cytological locations (sites) of P elements were determined by in situ hybridization for the whole genome of samples of 20 individuals from the parental P strain, 20 individuals from each of the eight dysgenic selection lines, and ten individuals from each of the eight nondysgenic selection lines. Variation among and within the selection lines and the parental P strain in P element insertion sites was exceptionally high. A total of 601 sites were identified, but there was no difference in total number of sites per line, mean number of sites per individual, mean copy number per individual, or site frequency between dysgenic and nondysgenic selection lines, or between lines selected for high and low bristle score. Transposition following nondysgenic crosses may explain additional observations of accelerated selection responses in nondysgenic selection lines. It was not possible to deduce which, if any, of the several hundred insertions in the dysgenic selection lines were responsible for their extreme bristle phenotypes.

Animals↗

Proliferation-dependent expression and phosphorylation of pRB in B cell non-Hodgkin's lymphomas: dependence on RB1 copy number.

Some studies have suggested that a significant fraction of non-Hodgkin's lymphomas (NHL) do not express pRB protein, possibly due to deletions of RB1. We examined RB1/centromere 17 copy number by fluorescent in situ hybridisation, and pRB expression/phosphorylation by immunohistochemistry (IHC) and immunoblotting (IB) in 66 cases of B cell NHL. Thirteen cases had lost one RB1 copy relative to centromere 17 copy number and total DNA content. Case 458/88 had no RB1 copies. pRB levels were heterogeneous as assessed by IB (0.04-1.12 relative units), but all tumours, except for case 458/88, expressed pRB localised to the nucleus in >75% of the tumour cells by IHC. The fraction of phosphorylated pRB was correlated with pRB expression (r(2)= 0.56, P < 0.001). The 14 cases with loss of RB1 had lower pRB expression (median 0.25) than those without (median 0.48, P < 0.001), but a correlation with S phase fraction (r(2) = 0.43, P < 0.001; previously published data for tumour-specific S phase and apoptotic fractions) indicated that the variation in pRB expression was due to differences in proliferative activity. Furthermore, the regression lines for pRB expression vs S phase fraction were not different for the cases with or without loss of one RB1 copy (P = 0.5). Cases 154/88 (one RB1 copy) and 258/88 (two RB1 copies), in addition to case 458/88, had low expression of (hypophosphorylated) pRB (0.04, 0.08 and 0.04), despite their high S phase fractions (21%, 17% and 21%). There was no association between pRB expression/RB1 copy number and apoptotic fraction. Neither pRB expression nor loss of RB1 had prognostic value, but cases 154/88, 258/88, and 458/88 had short survival times (5, 3 and 46 months, respectively) compared to the others (median survival: 44 months, P = 0.03). It is suggested that pRB expression and function are normal in 63 of 66 NHL cases, including 12 of 13 lymphomas with loss of one RB1 allele.

Alleles↗

Atypical mitochondrial DNA from the deep-sea scallop Placopecten magellanicus.

The mitochondrial DNA of most metazoan animals is highly conserved in size, averaging about 17 kilobase paris (kbp). The mitochondrial DNA from the deep-sea scallop Placopecten magellanicus, in contrast, has been found to be approximately 34 kbp long. It is also highly variable in size from individual to individual and is unusual in the extent of its size variation. Mitochondrial DNAs from individuals collected at the same site differ by as much as 7 kbp. The size variation is due largely to differences in the number of copies of a tandemly repeated 1.2-kbp element.

Journal Article↗

The control of natural variation in cytosine methylation in Arabidopsis.

We explore the extent and sources of epigenetic variation in cytosine methylation in natural accessions of the flowering plant, Arabidopsis thaliana, by focusing on the methylation of the major rRNA gene repeats at the two nucleolus organizer regions (NOR). Our findings indicate that natural variation in NOR methylation results from a combination of genetic and epigenetic mechanisms. Genetic variation in rRNA gene copy number and trans-acting modifier loci account for some of the natural variation in NOR methylation. Our results also suggest that divergence and inheritance of epigenetic information, independent of changes in underlying nucleotide sequence, may play an important role in maintaining natural variation in cytosine methylation.

Arabidopsis↗

Comparing tandem repeats with duplications and excisions of variable degree.

Traditional sequence comparison by alignment employs a mutation model comprised of two events, substitutions and indels (insertions or deletions) of single positions. However, modern genetic analysis knows a variety of more complex mutation events (e.g., duplications, excisions, and rearrangements), especially regarding DNA. With ever more DNA sequence data becoming available, the need to accurately compare sequences which have clearly undergone more complicated types of mutational processes is becoming critical. Herein we introduce a new method for pairwise alignment and comparison of sequences with respect to the special evolution of tandem repeats: substitutions and indels of single positions and, additionally, duplications and excisions of variable degree (i.e., of one or more repeat copies simultaneously) are taken into account. To evaluate our method, we apply it to the spa VNTR (variable number of tandem repeats) cluster of Staphylococcus aureus, a bacterium of high medical importance.

Algorithms↗

Intraspecific length heterogeneity of the rDNA-IGR in Arabidopsis thaliana due to homologous recombination.

A new heterogeneity of the rDNA spacer of Arabidopsis thaliana, resulting from variation in copy number of the so-called 'C' repeat located downstream of the presumptive polymerase I promoter, is reported. Variation is shown to occur within and between ecotypes. PCR analysis and sequence comparison suggests that the observed length heterogeneity is due to homologous recombination.

Arabidopsis↗