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PubMed2002

Feast and famine in plant genomes.

Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a 'one-way ticket to genomic obesity', as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.

Chromosomes↗

PubMed2003

Tandemly repeated DNA sequences and centromeric chromosomal regions of Arabidopsis species.

Despite their common function, centromeric DNA sequences are not conserved between organisms. Most centromeres of animals and plants so far investigated have now been shown to consist of large blocks of tandemly repeated satellite sequences that are embedded in recombination-deficient heterochromatic regions. This central domain of satellite sequences that is postulated to mediate spindle attachment is surrounded by pericentromeric sequences incorporating various classes of repetitive sequences often including retroelements. The centromeric satellite DNA sequences are amongst the most rapidly evolving sequences and pose some fundamental problems of maintaining function. In this overview, we will discuss work on centromeric repetitive sequences in Arabidopsis thaliana and its relatives, and highlight some of the common features that are emerging when analysing closely related species.

Arabidopsis↗

PubMed2003

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.

Amino Acid Oxidoreductases↗

PubMedDate not supplied

[The tissue-specific methylation of human-specific endogenous retroviral long terminal repeats].

A possible involvement of retroelements in the epigenetic regulation of human gene expression was considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results correlated with published data on the tissue-specific changes in the expression level of human genes adjacent to the LTRs under study. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Arabidopsis↗

PubMed2001

Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation.

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.

Alleles↗

PubMed2004

Whole-genome fractionation rapidly purifies DNA from centromeric regions.

The condensed centromeric regions of higher eukaryotic chromosomes contain satellite sequences, transposons and retroelements, as well as transcribed genes that perform a variety of functions. These chromosomal domains nucleate kinetochores, mediate sister chromatid cohesion and inhibit recombination, yet their characterization has often lagged behind that of chromosome arms. Here, we describe a whole-genome fractionation technique that rapidly identifies bacterial artificial chromosome (BAC) clones derived from plant centromeric regions. This approach, which relies on hybridization of methylated genomic DNA, revealed BACs that correspond to the genetically mapped and sequenced Arabidopsis thaliana centromeric regions. Extending this method to other species in the Brassicaceae family identified centromere-linked clones and provided genome-wide estimates of methylated DNA abundance. Sequencing these clones will elucidate the changes that occur during plant centromere evolution. This genomic fractionation technique could identify centromeric DNA in genomes with similar methylation and repetitive DNA content, including those from crops and mammals.

Arabidopsis↗

PubMed2006

Dancin' deaminase.

New biochemical experiments with the retroelement restriction protein APOBEC3G indicate that it processively deaminates single-stranded DNA cytosines by a unique jumping and sliding mechanism.

APOBEC-3G Deaminase↗

PubMed2005

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture.

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post-transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

DNA, Complementary↗

PubMed2000

Single nucleotide polymorphisms (SNPs) within Bov-A2 SINE in the second intron of bovine and buffalo k-casein (CSN3) gene.

The genetic polymorphism of an entire Bov-A2 element located in the second intron of the buffalo and bovine k-casein (CSN3) gene was investigated by amplification and sequencing of PCR products. Single nucleotide polymorphisms were detected. A PCR-RFLP method was developed to detect an A or G mutation at position 605 of bovine Bov-A2 element which creates a BfaI polymorphic site. The frequencies of the B allele, with the BfaI site, were for 0.275, 0.775, 0.750, 0.975, respectively, for Italian Holstein Friesian, Grey Alpine, Friuli Red Pied and Reggio bovine breeds. The mutation rate (substitutions and deletions/insertions per nucleotide site per year) was 2.5 x 10(-9) for Bov-A2 sequences in the second intron of CSN3. The comparison with other Bov-A2 elements suggests that this retroelement might be an important source of single nucleotide polymorphism for analysis of Bovidae genomes.

Animals↗

PubMed1999

Masc2, a gene from Ascobolus encoding a protein with a DNA-methyltransferase activity in vitro, is dispensable for in vivo methylation.

We have shown previously that masc1, a gene encoding a putative C5-DNA-methyltransferase (MTase), was necessary for the de novo 'Methylation Induced Premeiotically' (MIP) process and sexual reproduction in Ascobolus, whereas it was dispensable for maintenance methylation. A second MTase gene from Ascobolus, masc2, encodes a protein, Masc2, which possesses the large amino-terminal part characteristic of eukaryotic maintenance MTases. In vitro assays have shown that Masc2 displays a methylation activity, suggesting that it might be the MTase responsible for maintenance methylation. To check its function in vivo, we engineered a disruption of the masc2 gene. The resulting mutant strains did not exhibit any particular phenotype during either vegetative growth or sexual reproduction. Neither the masc2 mutation nor the double masc1 masc2 mutation had any detectable effect upon the maintenance of the pre-existing methylation of single gene copies previously subjected to MIP, natural retroelement-like repeats and tandemly repeated rDNA. The masc2 mutation did not alter either MIP or the other de novo methylation process that operates in vegetatives cells. Nor did it impair the meiotic process of methylation transfer. These results suggest that at least a third MTase gene responsible for maintenance and vegetative de novo methylation is present in Ascobolus.

Ascomycota↗

PubMed2003

Methylation of histone H3 in euchromatin of plant chromosomes depends on basic nuclear DNA content.

Strong methylation of lysine 4 (K4) and low methylation of lysine 9 (K9) have been proposed as modifications of histone H3, typical for transcriptionally active euchromatin and the opposite for inactive heterochromatin. We have analysed the correlation between the global distribution of histone H3, methylated at either lysine 4 or lysine 9, and of microscopically detectable euchromatic or heterochromatic regions in relation to genome size for 24 plant species. Two different distribution patterns of methylated (K9)H3 (Met(K9)H3) were found that depend on genome size. For most species with small genomes (1C <500 Mbp), including Arabidopsis thaliana, strong methylation of (K9)H3 was restricted to constitutive heterochromatin. Species with larger genomes showed a uniform distribution of Met(K9)H3. Contrary to this and regardless of the genome size, methylated (K4)H3 (Met(K4)H3) was found to be enriched within the euchromatin of all species. Transcriptionally less active B chromosomes showed the same patterns as basic A chromosomes. We thus propose that large genomes with high amounts of dispersed repetitive sequences (mainly retroelements) have to silence these sequences and therefore display epigenetic modifications such as methylation of DNA and (K9)H3 also within euchromatic regions.

Cell Nucleus↗

PubMed2005

Diverse DNA transposons in rotifers of the class Bdelloidea.

We surveyed the diversity, structural organization, and patterns of evolution of DNA transposons in rotifers of the class Bdelloidea, a group of basal triploblast animals that appears to have evolved for millions of years without sexual reproduction. Representatives of five superfamilies were identified: ITm (IS630/Tc/mariner), hAT, piggyBac, helitron, and foldback. Except for mariners, no fully intact copies were found. Mariners, both intact and decayed, are present in high copy number, and those described here may be grouped in several closely related lineages. Comparisons across lineages show strong evidence of purifying selection, whereas there is little or no evidence of such selection within lineages. This pattern could have resulted from repeated horizontal transfers from an exogenous source, followed by limited intragenomic proliferation, or, less plausibly, from within-host formation of new lineages under host- or element-based selection for function, in either case followed by eventual inactivation and decay. Unexpectedly, the flanking sequences surrounding the majority of mariners are very similar, indicating either insertion specificity or proliferation as part of larger DNA segments. Members of all superfamilies are present near chromosome ends, associated with the apparently domesticated retroelement Athena, in large clusters composed of diverse DNA transposons, often inserted into each other, whereas the examined gene-rich regions are nearly transposon-free.

Acari↗

PubMed1991

Single-step selection for Ty1 element retrotransposition.

The yeast retrotransposon Ty1 has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms. The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation. The HIS3AI sequences were inserted into a Ty1 element such that the intron is on the sense strand of the Ty1 element; therefore, splicing and retrotransposition of marked Ty1 transcripts can give rise to His+ cells. Fusion of the Ty1-H3mHIS3AI element to the inducible GAL1 promoter resulted in a high frequency of histidine prototrophs upon galactose induction. Moreover, spontaneous His+ revertants derived from strains containing genomic TymHIS3AI elements are a result of retrotransposition. By using this assay, we estimated the Ty1 transposition rate to be between 3 x 10(-7) and 1 x 10(-5) transpositions per Ty1 element per generation. Variations in the transposition rate of individual Ty1 elements are correlated with the relative abundance of their transcripts.

Blotting, Northern↗

PubMed1992

In vivo production of a stable single-stranded cDNA in Saccharomyces cerevisiae by means of a bacterial retron.

Gram-negative bacteria such as Myxococcus xanthus, Stigmatella aurantiaca, and Escherichia coli contain retroelements called retrons. Retrons consist of the msr-msd region and the gene for reverse transcriptase (RT), which are essential for the production of the branched RNA-linked ms-DNA (multicopy single-stranded DNA). In this study, we attempted to produce msDNA in the yeast Saccharomyces cerevisiae. Retron Ec67 from E. coli, which is responsible for the production of msDNA-Ec67, was cloned under the GAL10 promoter in a 2-microns-based plasmid. msDNA thus produced was detected by extending the 3' end of the msDNA by avian myeloblastosis virus RT. This yielded a main product of 117 nucleotides. Treatment of this product with RNase A resulted in a DNA of 105 nucleotides. These results are in good agreement with the structure of msDNA-Ec67. The production of msDNA-Ec67 was further confirmed by Southern blot hybridization. The msDNA production was dependent upon the bacterial RT gene in the clone and was increased severalfold when the RT gene of retron Ec67 was placed in front of the msr-msd region. The potential of msDNA as a eukaryotic vector producing a stable single-stranded DNA as well as RNA is discussed.

Base Sequence↗

PubMed1994

Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences.

Certain retrovirus and retrotransposons display strong biases in the selection of host DNA sites for integration. To probe the possibility that simple tethering of the retroelement integrase protein to a target DNA site is sufficient to direct integration, the activities of a hybrid composed of human immunodeficiency virus 1 integrase and lambda repressor were analyzed. In in vitro reactions containing several target DNAs, the lambda repressor-integrase hybrid was found to direct integration selectively to targets containing lambda operators. Addition of lambda repressor blocked selective integration, indicating that binding to the operators was required. The lambda repressor-integrase hybrid protein directed integration primarily to sites near the operators on the same face of the B-DNA helix, indicating that target DNA was probably captured by looping out the intervening sequences. Such hybrid integrase proteins may be useful for directing retroviral integration to specific sequences in vivo.

Bacteriophage lambda↗

PubMed1998

Grass genomes.

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Journal Article↗

PubMed1999

The distribution and copy number of copia-like retrotransposons in rice (Oryza sativa L.) and their implications in the organization and evolution of the rice genome.

We used 22 fragments corresponding to the reverse transcriptase domain of copia-like retrotransposons as representatives to study the organization and distribution of these elements in the rice genome. The loci detected by these 22 fragments were assigned to 47 locations in the molecular-linkage map involving all 12 chromosomes. The distributional features of copia-like retrotransposons found in the rice genome indicated that (i) the loci detected were located mainly in one arm of each chromosome; (ii) one fragment usually detected several loci that were mapped to similar locations of different chromosomes; (iii) retrotransposons sharing high identity in nucleotide sequences were usually assigned to similar locations of the chromosomes; and (iv) concurrences of multiple loci, detected by different fragments, in similar locations or stretches of different chromosomes were common in the rice genome. We also determined that the copy number of copia-like retrotransposons in rice genome may be as low as approximately 100 per haploid genome. The restricted distribution, along with low copy number, suggested that copia-like retrotransposons in rice were relatively inactive during evolution compared with those in other plants. The distributional features of the copia-like retrotransposons suggested the existence of possible lineages among the rice chromosomes, which in turn suggested that chromosome duplication and diversification may be a mechanism for the origin and evolution of the rice chromosomes. The information provided by fine mapping of the retroelements in the genetic linkage map may also be useful for gene tagging and molecular cloning.

Journal Article↗