Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

Higher-order unfolding of satellite heterochromatin is a consistent and early event in cell senescence.

Epigenetic changes to chromatin are thought to be essential to cell senescence, which is key to tumorigenesis and aging. Although many studies focus on heterochromatin gain, this work demonstrates large-scale unraveling of peri/centromeric satellites, which occurs in all models of human and mouse senescence examined. This was not seen in cancer cells, except in a benign senescent tumor in vivo. Senescence-associated distension of satellites (SADS) occurs earlier and more consistently than heterochromatin foci formation, and SADS is not exclusive to either the p16 or p21 pathways. Because Hutchinson Guilford progeria syndrome patient cells do not form excess heterochromatin, the question remained whether or not proliferative arrest in this aging syndrome involved distinct epigenetic mechanisms. Here, we show that SADS provides a unifying event in both progeria and normal senescence. Additionally, SADS represents a novel, cytological-scale unfolding of chromatin, which is not concomitant with change to several canonical histone marks nor a result of DNA hypomethylation. Rather, SADS is likely mediated by changes to higher-order nuclear structural proteins, such as LaminB1.

Whitefly-mediated transmission of cotton leaf curl Multan betasatellite: evidence for betasatellite encapsidation in coat protein of helper begomoviruses.

Cotton leaf curl Multan betasatellite (CLCuMB) is responsible for symptom expression of a devastating disease of cotton in the Indian subcontinent. CLCuMB depends on helper virus replication-associated protein for its replication and on viral coat protein (CP) for its encapsidation. However, no direct evidence of encapsidation of CLCuMB in viral CP has been available. In the present study, non-viruliferous whiteflies were placed on tomato plants that had been agroinoculated with infectious clones of an Iranian isolate of tomato yellow leaf curl virus (TYLCV-[Ab]) and CLCuMB for an acquisition access period of 72 h and then transferred to healthy tomato seedlings at the 3- to 4-leaf stage. Typical symptoms of TYLCV-[Ab] appeared on inoculated seedlings 30-45 days post-inoculation. The presence of TYLCV-[Ab] and CLCuMB DNAs in symptomatic test plants and viruliferous whiteflies was confirmed by PCR analysis using specific primers and DIG Southern blotting. Furthermore, the possibility of CLCuMB DNA encapsidation in TYLCV-[Ab] CP within infected plants was examined by immunocapture PCR. The results showed that CLCuMB DNA was encapsidated in TYLCV-[Ab] CP. Whitefly-mediated transmission of CLCuMB in the presence of helper virus is additional evidence for encapsidation of CLCuMB by TYLCV-[Ab] CP.

Megasatellites: a new class of large tandem repeats discovered in the pathogenic yeast Candida glabrata.

Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell-cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.

Effect of location, organization, and repeat-copy number in satellite-DNA evolution.

Here, we analyze the evolutionary dynamics of a satellite-DNA family in an attempt to understand the effect of factors such as location, organization, and repeat-copy number in the molecular drive process leading to the concerted-evolution pattern found in this type of repetitive sequences. The presence of RAE180 satellite-DNA in the dioecious species of the plant genus Rumex is a noteworthy feature at this respect, as RAE180 satellite repeats have accumulated differentially, showing a distinct distribution pattern in different species. The evolution of dioecious Rumex gave rise to two phylogenetic clades: one clade composed of species with an ancestral XX/XY sex chromosome system and a second, derived clade of species with a multiple sex-chromosome system XX/XY(1)Y(2). While in the XX/XY dioecious species, the RAE180 satellite-DNA is located only in a small autosomal locus, the RAE180 repeats are present also in a small autosomal locus and additionally have been massively amplified in the Y chromosomes of XX/XY(1)Y(2) species. Here, we have found that the RAE180 repeats of the autosomal locus of XX/XY species are characterized by intra-specific sequence homogeneity and inter-specific divergence and that the comparison of individual nucleotide positions between related species shows a general pattern of concerted evolution. On the contrary, both in the autosomal and the Y-linked loci of XX/XY(1)Y(2) species, ancestral variability has remained with reduced rates of sequence homogenization and of evolution. Thus, this study demonstrates that molecular mechanisms of non-reciprocal exchange are key factors in the molecular drive process; the satellite DNAs in the non-recombining Y chromosomes show low rates of concerted evolution and intra-specific variability increase with no inter-specific divergence. By contrast, freely recombining loci undergo concerted evolution with genetic differentiation between species as occurred in the autosomal locus of XX/XY species. However, evolutionary periods of rapid sequence change might alternate with evolutionary periods of stasis with variability remaining by the reduced action of molecular mechanisms of non-reciprocal exchange as occurred in XX/XY(1)Y(2) species, which could depend on repeat-copy number and the processes involved in their amplification.

Extrachromosomal circular DNA in eukaryotes: possible involvement in the plasticity of tandem repeats.

Extrachromosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms, and has been noted for more than 3 decades. eccDNA occurs in normal tissues and in cultured cells, is heterogeneous in size, consists of chromosomal sequences and reflects plasticity of the genome. Two-dimensional (2D) gel electrophoresis has been adapted for the detection and characterization of eccDNA. It shows that most eccDNA consists of chromosomal tandem repeats, both coding genes and satellite DNA and is organized as circular multimers of the repeating sequence. 2D gels were unable to detect dispersed repeats within the population of eccDNA. eccDNA, organized as circular multimers, can be formed de novo in Xenopus egg extracts, in the absence of DNA replication. These findings support a mechanism for the formation of eccDNA that involves intra-chromosomal homologous recombination between tandem repeats and looping-out. Furthermore, eccDNA appears to undergo extrachromosomal replication via a rolling circle mechanism. Hence, the formation of eccDNA from arrays of tandem repeats may cause deletions, and the possible re-integration of rolling-circle replication products could expand these arrays. This review summarizes recent experimental data which characterizes eccDNA in several organisms using 2D gel electrophoresis, and discusses its possible implications on the dynamics of chromosomal tandem repeats.

Interaction with a host ubiquitin-conjugating enzyme is required for the pathogenicity of a geminiviral DNA beta satellite.

DNA beta is a single-stranded satellite DNA which encodes a single gene, betaC1. To better understand the role of betaC1 in the pathogenicity of DNA beta, a yeast two-hybrid screen of a tomato cDNA library was carried out using betaC1 from Cotton leaf curl Multan virus (CLCuMV) DNA beta as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between betaC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the betaC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-beta-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing betaC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of betaC1 with SlUBC3 is required for DNA-beta-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

Roles and interactions of begomoviruses and satellite DNAs associated with okra leaf curl disease in Mali, West Africa.

Okra leaf curl disease (OLCD) is a major constraint on okra (Abelmoschus esculentus) production in West Africa. Two monopartite begomoviruses (okra virus-1 and okra virus-2), a betasatellite and a DNA1 satellite are associated with OLCD in Mali. Okra virus-1 is an isolate of okra yellow crinkle virus (OYCrV), okra virus-2 is a recombinant isolate of cotton leaf curl Gezira virus (CLCuGV) and the betasatellite is a variant of cotton leaf curl Gezira betasatellite (CLCuGB). Cloned DNA of OYCrV and CLCuGV were infectious and induced leaf curl symptoms in Nicotiana benthamiana plants, but did not induce OLCD in okra. However, when these clones were individually co-inoculated with the cloned CLCuGB DNA, symptom severity and viral DNA levels were increased in N. benthamiana plants and typical OLCD symptoms were induced in okra. The CLCuGB was also replicated by, and increased symptom severity of, three monopartite tomato-infecting begomoviruses, including two from West Africa. The sequence of the DNA1 satellite was highly divergent, indicating that it represents a distinct West African lineage. DNA1 replicated autonomously, and replication required the DNA1-encoded Rep protein. Although DNA1 reduced helper begomovirus DNA levels, symptoms were not attenuated. In the presence of CLCuGB, DNA levels of the helper begomoviruses and DNA1 were substantially increased. Together, these findings establish that OLCD in Mali is caused by a complex of monopartite begomoviruses and a promiscuous betasatellite with an associated parasitic DNA1 satellite. These findings are discussed in terms of the aetiology of OLCD and the evolution of new begomovirus/satellite DNA complexes.

DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts.

Macrosatellite DNA is composed of large repeat units, arranged in tandem over hundreds of kilobases. The macrosatellite repeat DXZ4, localized at Xq23-24, consists of 50-100 copies of a CpG-rich 3-kb monomer. Here I report that on the active X chromosome (Xa), DXZ4 is organized into constitutive heterochromatin characterized by a highly organized pattern of H3K9me3. DXZ4 is expressed from both strands and generates an antisense transcript that is processed into small RNAs that directly correlate with H3K9me3 nucleosomes. In contrast, on the inactive X chromosome (Xi) a proportion of DXZ4 is packaged into euchromatin characterized by H3K4me2 and H3K9Ac. The Xi copy of DXZ4 is bound by the chromatin insulator, CTCF, within a sequence that unidirectionally blocks enhancer-promoter communication. Immediately adjacent to the CTCF-binding site is a bidirectional promoter that, like the sequence flanking the CTCF-binding region, is completely devoid of CpG methylation on the Xi. As on the Xa, both strands are expressed, but longer antisense transcripts can be detected in addition to the processed small RNAs. The euchromatic organization of DXZ4 on the otherwise heterochromatic Xi, its binding of CTCF, and its function as a unidirectional insulator suggest that this macrosatellite has acquired a novel function unique to the process of X chromosome inactivation.

Molecular variation of satellite DNA beta molecules associated with Malvastrum yellow vein virus and their role in pathogenicity.

Previous studies have found that the diversity of begomovirus-associated DNA beta satellites is related to host and geographical origin. In this study, we have cloned and sequenced 20 different isolates of DNA beta molecules associated with Malvastrum yellow vein virus (MYVV) isolated from Malvastrum coromandelianum plants in different geographical locations of Yunnan Province, China. Analyses of their molecular variation indicate that the satellites are clustered together according to their geographical location but that they have only limited sequence diversity. Infectivity tests using infectious clones of MYVV and its associated DNA beta molecule indicate that MYVV DNA beta is indispensable for symptom induction in Nicotiana benthamiana, N. glutinosa, Petunia hybrida, and M. coromandelianum plants. Furthermore, we showed that MYVV interacts functionally with heterologous DNA beta molecules in N. benthamiana plants.

DNA hypomethylation and human diseases.

Changes in human DNA methylation patterns are an important feature of cancer development and progression and a potential role in other conditions such as atherosclerosis and autoimmune diseases (e.g., multiple sclerosis and lupus) is being recognised. The cancer genome is frequently characterised by hypermethylation of specific genes concurrently with an overall decrease in the level of 5 methyl cytosine. This hypomethylation of the genome largely affects the intergenic and intronic regions of the DNA, particularly repeat sequences and transposable elements, and is believed to result in chromosomal instability and increased mutation events. This review examines our understanding of the patterns of cancer-associated hypomethylation, and how recent advances in understanding of chromatin biology may help elucidate the mechanisms underlying repeat sequence demethylation. It also considers how global demethylation of repeat sequences including transposable elements and the site-specific hypomethylation of certain genes might contribute to the deleterious effects that ultimately result in the initiation and progression of cancer and other diseases. The use of hypomethylation of interspersed repeat sequences and genes as potential biomarkers in the early detection of tumors and their prognostic use in monitoring disease progression are also examined.

Geminivirus disease complexes: the threat is spreading.

Symptom-modulating DNA satellites associated with geminiviruses have come to our attention only recently but have proven to be widespread, associated with many diseases throughout the Old World, and economically significant, particularly in developing countries. Recent developments are elucidating the role played by these novel molecules in pathogenicity and in overcoming host plant defense. Further investigation into the promiscuous nature of these satellites and their ability to recruit further begomoviruses indicates that regions not yet affected by such begomovirus-satellite complexes are at great risk.

Phloem specific promoter from a satellite associated with a DNA virus.

DNAbeta is a satellite molecule associated with some monopartite begomoviruses and encodes a single gene (betaC1), which is highly conserved in position and size among DNAbeta molecules. A 955 nt fragment of Tomato yellow leaf curl China virus (TYLCCNV) DNAbeta, upstream of the translation start site of betaC1 gene was tested for its promoter activity with gus as a reporter gene. Analysis of beta-glucuronidase (GUS) activity following transient expression assays indicated that the 955 nt fragment had promoter activity and that 3'-deletions of 399 or 173 nt of the fragment resulted in complete loss of its promoter activity. The 5'-deletions of 782 or 556 nt of the fragment, however, did not affect its activity. Histochemical staining revealed that this fragment can be used to express gus gene specifically in phloem tissue of stably transformed tobacco plants. Further studies have indicated that a 173 nt segment from 3'-end of the 955 nt fragment was responsible for basic promoter activity and phloem-specific expression.

Subviral agents associated with plant single-stranded DNA viruses.

Begomoviruses (family Geminiviridae) are responsible for many economically important crop diseases worldwide. The majority of these diseases are caused by bipartite begomovirus infections, although a rapidly growing number of diseases of the Old World are associated with monopartite begomoviruses. With the exception of several diseases of tomato, most of these are caused by a monopartite begomovirus in association with a recently discovered essential satellite component (DNA-beta). These begomovirus/satellite disease complexes are widespread and diverse and collectively infect a wide variety of crops, weeds and ornamental plants. Non-essential subviral components (DNA-1) originating from nanoviruses are frequently associated with these disease complexes, and there are tantalizing hints that further novel satellites may also be associated with some begomovirus diseases. DNA-beta components can be maintained in permissive plants by more than one distinct begomovirus, reflecting less stringent requirements for trans-replication that will undoubtedly encourage diversification and adaptation as a consequence of component exchange and recombination. In view of their impact on agriculture, there is a pressing need to develop a more comprehensive picture of the diversity and distribution of the disease complexes. A greater understanding of how they elicit the host response may provide useful information for their control as well as an insight into plant developmental processes.

Preservation and high sequence conservation of satellite DNAs suggest functional constraints.

Due to a high evolutionary turnover many satellite DNAs are restricted to a group of closely related species. Here we demonstrate that the satellite DNA family PSUB, abundant in the beetle Palorus subdepressus, is distributed in a low number of copies among diverse taxa of Coleoptera (Insecta), some of them separated for an evolutionary period of up to 60 Myr. Comparison of PSUB cloned from the species Tribolium brevicornis with the PSUB family previously characterized in Palorus subdepressus revealed high sequence conservation and absence of fixed species-specific mutations. The most polymorphic sites are those with ancestral mutations shared among clones of both species. Since the ancestral mutations contribute significantly to overall diversity, it could be proposed that a similar mutational profile already existed in an ancestral species. The pattern of variability along the satellite monomer is characterized by the presence of conserved and variable regions. The nonrandom pattern of variability as well as the absence of sequence divergence is also discerned for PRAT satellite DNA, cloned previously from two Palorus species and a distantly related Pimelia elevata. Since PRAT and PSUB are present in parallel in diverse taxa of Coleoptera, we propose that their long evolutionary preservation suggests a possible functional significance. This indication is additionally supported not only by the high evolutionary conservation of the sequences, but also by the presence of significantly conserved and variable regions along the monomers.

Pathogenicity and stability of a truncated DNAbeta associated with Tomato yellow leaf curl China virus.

DNAbeta molecules are single-stranded satellite DNA associated with monopartite begomoviruses (family Geminiviridae). DNAbeta possesses a C1 gene on the complementary strand, which has a conserved position and size. To better understand the function of C1 gene in virus infection, a C1 deletion DNAbeta associated with a Tomato yellow leaf curl China virus (TYLCCNV) isolate was constructed. Co-agroinoculation with TYLCCNV showed the truncated DNAbeta was infectious in Nicotiana benthamiana and N. glutinosa plants but not in N. tabacum Samsun, N. tabacum and Lycopersicon esculentum plants. The wild-type TYLCCNV DNAbeta co-agroinoculated with TYLCCNV caused systemic infection in all the above hosts. Results of Southern blot analysis indicate that C1 gene is not required for TYLCCNV and DNAbeta replication. However, the presence of C1 gene in DNAbeta can increase both TYLCCNV and DNAbeta accumulation in infected plants. The truncated TYLCCNV DNAbeta was stable in N. benthamiana and N. glutinosa plants.

Variable and hierarchical size distribution of L1-retroelement-enriched CENP-A clusters within a functional human neocentromere.

Human neocentromeres are fully functional centromeres that arise epigenetically from non-centromeric precursor sequences that are devoid of alpha-satellite DNA. Using chromatin immunoprecipitation (ChIP) and BAC-array analysis, we have previously described a 330 kb binding domain for CENP-A (a histone H3 variant that confers centromere-specific nucleosomal property) at the 10q25 neocentromere found on a chromosome 10-derived marker chromosome mardel(10). For the further detailed analysis of the CENP-A-associated chromatin, we have generated a high-resolution genomic array consisting of PCR fragments with an average size of 8 kb, providing an approximately 20-fold increment in analytical resolution. ChIP and PCR-array analysis reveals seven distinct CENP-A-binding clusters within the 330 kb domain, demonstrating the interspersion of CENP-A-associated nucleosomal blocks within the neocentromeric chromatin. Independent ChIP-PCR analysis verified this distribution profile and indicated that histone H3-containing nucleosomes directly intervene the CENP-A-binding clusters. The CENP-A-binding clusters are uneven in size, with the central cluster (>50 kb) being significantly larger than the flanking ones (10-30 kb), and the flanking clusters arranged in an interesting hierarchical and symmetrical configuration of alternating larger and smaller sizes around the central cluster. In silico sequence analysis indicates an approximately 2.5-fold increase in the prevalence of L1 retroelements within the CENP-A-binding clusters when compared with the non-CENP-A-binding regions. These results provide insight into the possible role of retroelements in determining the positioning of CENP-A binding at human neocentromeres, and that a hierarchical and symmetrical arrangement of CENP-A-binding clusters of varying sizes may be an important structural requirement for mammalian kinetochore assembly and/or to provide stability to withstand polar microtubule forces.

A DNAbeta associated with Tomato yellow leaf curl China virus is required for symptom induction.

We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNAbeta molecules. To investigate the biological role of DNAbeta, full-length infectious clones of viral DNA and DNAbeta of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAbeta was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAbeta of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the betaC1 gene of TYLCCNV-Y10 DNAbeta was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAbeta molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the betaC1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of betaC1 transcripts, while transgenic plants transformed with the betaC1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a betaC1 gene is adequate for induction of symptoms of viral infection in the absence of virus.

Localisation of centromeric proteins to a fraction of mouse minor satellite DNA on a mini-chromosome in human, mouse and chicken cells.

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear how centromere sites are specified on chromosomes in vertebrates. We have previously introduced a mini-chromosome, named ST1, into a variety of cell lines including human HT1080, mouse LA9 and chicken DT40. This mini-chromosome, segregating faithfully in these cells, contains mouse minor and major, and human Y alpha-satellite DNA repeats. In this study, after determining the organisation of the satellite repeats, we investigated the location of the centromere on the mini-chromosome by combined immunocytochemistry and fluorescence in situ hybridisation analysis. Centromeric proteins were consistently co-localised with the minor satellite repeats in all three cell lines. When chromatin fibres were highly stretched, centromeric proteins were only seen on a small portion of the minor satellite repeats. These results indicate that a fraction of the minor satellite repeats is competent in centromere function not only in mouse but also in human and chicken cells.

The role of CENP-B and alpha-satellite DNA: de novo assembly and epigenetic maintenance of human centromeres.

The centromere is an essential functional domain responsible for the correct inheritance of eukaryotic chromosomes during cell division. Eukaryotic centromeres include the highly conserved centromere-specific histone H3 variant, CENP-A, which has provided a powerful tool for investigating the recruitment of centromere components. However, the trigger that targets CENP-A to a specific genomic locus during centromere assembly remains unknown. Although, on rare occasions, CENP-A chromatin may assemble at non-centromeric DNA, all normal human centromeres are assembled and maintained on alpha-satellite (alphoid) DNA. The importance of alphoid DNA and CENP-B binding sites (CENP-B boxes), typical of normal human centromere DNA configurations, has been demonstrated through their requirement in de novo centromere assembly and Human Artificial Chromosome (HAC) assays. Mechanisms to link the centromere tightly to specific genomic sequences exist in humans and the two yeast species.