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Centromeres in the genomic era: Unraveling paradoxes
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The centromeres of higher plants and animals share many common features, though current models fail to account for all aspects of centromere composition and function. This dilemma is likely to be resolved in the next few years in Arabidopsis where robust assays for centromere function are available and the sequence of the entire genome will be determined.
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... TEs and derived sequences comprise 22% of the D. melanogaster genome [121] and roughly half of the human genome [122]. They reside primarily in heterochromatic (repressive chromatin) regions in diverse species, from flies [123] to plants [124]. ...
Transposable elements (TEs) are ubiquitous genetic elements, able to jump from one location of the genome to another, in all organisms. For this reason, on the one hand, TEs can induce deleterious mutations, causing dysfunction, disease and even lethality in individuals. On the other hand, TEs can increase genetic variability, making populations better equipped to respond adaptively to environmental change. To counteract the deleterious effects of TEs, organisms have evolved strategies to avoid their activation. However, their mobilization does occur. Usually, TEs are maintained silent through several mechanisms, but they can be reactivated during certain developmental windows. Moreover, TEs can become de-repressed because of drastic changes in the external environment. Here, we describe the ‘double life’ of TEs, being both ‘parasites’ and ‘symbionts’ of the genome. We also argue that the transposition of TEs contributes to two important evolutionary processes: the temporal dynamic of evolution and the induction of genetic variability. Finally, we discuss how the interplay between two TE-dependent phenomena, insertional mutagenesis and epigenetic plasticity, plays a role in the process of evolution.
... Repeats derived from degenerated retroelements have often been found in centromeric locations in fungi and plants (Cambareri et al., 1998;Presting et al., 1998;Copenhaver et al., 1999). Whether these repeats are important components of centromeric function, either by nature of their sequences or by their epigenetic modifications, remains a matter for discussion (reviewed in Csink and Henikoff, 1998;Copenhaver and Preuss, 1999). One of the features proposed as a prerequisite for centromere function is late replication of these heterochromatic regions (reviewed in Csink and Henikoff, 1998). ...
Transcriptional gene silencing (TGS) frequently inactivates foreign genes integrated into plant genomes but very likely also suppresses an unknown subset of chromosomal information. Accordingly, RNA analysis of mutants impaired in silencing should uncover endogenous targets of this epigenetic regulation. We compared transcripts from wild-type Arabidopsis carrying a silent transgene with RNA from an isogenic transgene-expressing TGS mutant. Two cDNA clones were identified representing endogenous RNA expressed only in the mutant. The synthesis of these RNAs was found to be released in several mutants affected in TGS, implying that TGS in general and not a particular mutation controls the transcriptional activity of their templates. Detailed analysis revealed that the two clones are part of longer transcripts termed TSI (for transcriptionally silent information). Two major classes of related TSI transcripts were found in a mutant cDNA library. They are synthesized from repeats present in heterochromatic pericentromeric regions of Arabidopsis chromosomes. These repeats share sequence homology with the 3′ terminal part of the putative retrotransposon Athila. However, the transcriptional activation does not include the transposon itself and does not promote its movement. There is no evidence for a general release of silencing from retroelements. Thus, foreign genes in plants encounter the epigenetic control normally directed, at least in part, toward a subset of pericentromeric repeats.
... Moreover, these studies provide a platform for identification of the minimal sequence that provides centromere function. Such sequences might be spread across the entire genetically defined region, be concentrated at a discrete point, or exist as redundant copies within the centromere (26). ...
High-precision genetic mapping was used to define the regions that contain centromere functions on each natural chromosome
inArabidopsis thaliana. These regions exhibited dramatic recombinational repression and contained complex DNA surrounding large arrays of 180–base
pair repeats. Unexpectedly, the DNA within the centromeres was not merely structural but also encoded several expressed genes.
The regions flanking the centromeres were densely populated by repetitive elements yet experienced normal levels of recombination.
The genetically defined centromeres were well conserved among Arabidopsis ecotypes but displayed limited sequence homology between different chromosomes, excluding repetitive DNA. This investigation
provides a platform for dissecting the role of individual sequences in centromeres in higher eukaryotes.
... Thus, considering the chromosomal evolution model proposed by Wichman et al. (1991), based on the genomic distribution of heterochromatic sequences in equids, it is possible to hypothesize a direct relationship between the occurrence of non-Robertsonian rearrangements and the variation in the location and to quantify the As51 satellite DNA in interpopulation and interspecific diversification in the genus Astyanax. Heterochromatin is a state of chromatin rich in satellite DNA and transposable elements, as found in Drosophila melanogaster (Pimpinelli et al., 1995 ), Arabidopsis (Copenhaver & Preuss, 1999) and Zea mays (Ananiev et al., 1998). The origin of many satellite DNA families is unknown. ...
Constitutive heterochromatin makes up a substantial portion of the genome of eukaryotes and is composed mainly of satellite DNA repeating sequences in tandem. Some satellite DNAs may have been derived from transposable elements. These repetitive sequences represent a highly dynamic component of rapid evolution in genomes. Among the genus Astyanax, the As51 satellite DNA is found in species that have large distal heterochromatic blocks, which may be considered as derived from a transposable DNA element. In the present study, As51 satellite DNA was mapped through in situ fluorescent hybridization in the chromosomes of five species of the genus. The possible roles of this type of saltatory DNA type in the genome of the species are discussed, along with its use for the phylogenetic grouping of the genus Astyanax, together with other shared chromosomal characters. However, the number of As51 clusters is presented as a homoplastic characteristic, thereby indicating evident genomic diversification of species with this type of DNA.
The mechanics of centric misdivision Univalency and centric misdivision Susceptibility of chromosomes to misdivision Symmetry of breakage Fusion of broken chromosome ends Separation of centromeric functions and the minimum chromosome size Centric fi ssion-fusion versus Robertsonian translocations References
The invention is generally related to methods of generating plants transformed with novel autonomous mini-chromosomes. Mini-chromosomes with novel compositions and structures are used to transform plants cells which are in turn used to generate the plant. Methods for generating the plant include methods for delivering the mini-chromosome into plant cell to transform the cell, methods for selecting the transformed cell, and methods for isolating plants transformed with the mini-chromosome. Plants generated in the present invention contain novel genes introduced into their genome by integration into existing chromosomes.
Telomeres and centromeres are the most important functional elements in plant chromosomes, as in other eukaryotic chromosomes. Both elements are in general composed of repetitive DNA sequences, and binding or associated proteins. Recent findings showed that the telomere DNA sequences are conserved among all plant species except Allium and related species, and that they form high-order complexes together with special proteins for maintaining the functions. Although centromere-specific DNA sequences have been isolated in a wide range of plant species, almost no conservation was found in their DNA sequences. Exceptions are cereal centromeres, which contain common Ty3 / gypsy-type retrotransposon-like sequences. Recently, homologues to the genes encoding mammalian centromere proteins (CENPs) have been identified in maize and A. thaliana. This indicates that high-order structures of centromeres or kinetochore assemblies are conserved among eukaryotic organisms. The 180-bp centromeric repeat family of Arabidopsis thaliana is strongly suggested to play important roles for centromere functions. Since the organization of this family is similar to that of human alpha-satellite family, it is likely to be possible to build Arabidopsis thaliana artificial chromosomes (AtAC) with the same strategies used for constructing mammalian artificial chromosomes.
As a transcriptional coactivator and acetyltransferase, CREB-binding protein (CBP) is widely characterized due to its functions in cell proliferation and development. However, the activities of CBP in oocyte meiosis are not completely clear. Here we showed that the localization and expression of CBP changed regularly with the progression of porcine oocyte meiosis. The emergence of CBP in chromosomal domains is temporally coincident with the establishments of acetylated lysine 18 (AcH3/K18), lysine 23 (AcH3/K23) and dimethylated arginine 17 (dime-H3/R17) of histone H3 at meiotic stages from germinal vesicle breakdown (GVBD) to metaphase I (MI). Both CBP expression and these three histone modifications persisted to telophase I (TI). When trichostatin A (TSA) was used to enhance histone acetylations in porcine oocytes, we found that hyperacetylations of H3K18 and H3K23 occurred at meiotic stage from GVBD to TI, together with advanced and enhanced expression of CBP in the nucleus. In addition, disturbance of CBP activity by treatment with 2-Naphthol-AS-Ephosphate (KG-501, a drug targeting the KIX domain of CBP that disrupts the formation of CBP functional complex) led to synchronous decreases of CBP expression, AcH3/K18 and AcH3/K23 in chromosomal domains during oocyte meiosis. Therefore, these results indicate that the synchronous changes of CBP expression, AcH3/K18 and AcH3/K23 occur during porcine oocyte meiosis.
Nucleotide sequencing of the Arabidopsis genome is nearing completion, sequencing of the rice genome has begun, and large amounts of expressed sequence tag information are being obtained for many other plants. There are many opportunities to use this wealth of sequence information to accelerate progress toward a comprehensive understanding of the genetic mechanisms that control plant growth and development and responses to the environment.
A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.
Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is
characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML
CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular
minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself
sufficient for complete mitotic and meiotic centromere functions.
A linear mammalian artificial chromosome vector will require at least three functional elements: a centromere, two telomeres and replication origins. One route to generate such a vector is by the fragmentation of an existing chromosome. We have previously described the use of cloned telomeric DNA to generate and stably rescue truncated derivatives of a human X chromosome in a somatic cell hybrid. Further rounds of telomere-associated chromosome fragmentation have now been used to engineer a human X-derived minichromosome. This minichromosome is estimated to be < 10 Mb in size. In situ hybridization and molecular analysis reveal that the minichromosome has a linear structure, with two introduced telomere constructs flanking a 2.5 Mb alpha-satellite array. The highly truncated chromosome also retains some chromosome-specific DNA, originating from Xp11.21. There is no significant change in the mitotic stability of the minichromosome as compared with the X chromosome from which it was derived.
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The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.
Functional centromeric DNAs have now been isolated and characterized from both budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeasts. Artificial chromosomes containing these centromere DNA sequences segregate faithfully in both mitotic and meiotic cell divisions, but only in the parent organism. Structure-function analyses have revealed surprising fundamental differences between these two centromere classes. In the budding yeast centromeres, a 125-bp consensus DNA sequence contains all the information needed in cis to provide proper chromosome segregation. In contrast, the fission yeast centromeres each contain a long run (40 to 100 kb) of untranscribed repetitive DNA sequences arranged into a large inverted repeat, most of which is required for full centromere function. The fission yeast centromere-kinetochore appears to be a highly relevant experimental model for analysis of the mechanism of chromosome segregation in higher eukaryotes, in which the centromere regions often contain megabases of transcriptionally silent repetitive DNA sequences of unknown function.
A colony color assay that measures chromosome stability is described and is used to study several parameters affecting the mitotic maintenance of yeast chromosomes, including ARS function, CEN function, and chromosome size. A cloned ochre-suppressing form of a tRNA gene, SUP11, serves as a marker on natural and in vitro-constructed chromosomes. In diploid strains homozygous for an ochre mutation in ade2, cells carrying no copies of the SUP11 gene are red, those carrying one copy are pink, and those carrying two or more copies are white. Thus, the degree of red sectoring in colonies reflects the frequency of mitotic chromosome loss. The assay also distinguishes between chromosome loss (1:0 segregation) and nondisjunction (2:0 segregation). The most dramatic effect on improving mitotic stability is caused by increasing chromosome size. Circular chromosomes increase in stability through a size range up to approximately 100 kb, but do not continue to be stabilized above this value. However, linear chromosomes continue to increase in mitotic stability throughout the size range tested (up to 137 kb). It is possible that the mitotic stability of linear chromosomes is proportional to chromosome length, up to a plateau value that has not yet been reached in our synthetic constructions.
The centromeric DNA (CEN3) from yeast chromosome III has been isolated on a 1.6 kilobase-pair segment of DNA located near the centromere-linked CDC10 locus of Saccharomyces cerevisiae. When present on a plasmid carrying a yeast chromosomal replicator, CEN3 enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing CEN3 are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions.
The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system. Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat. The S. pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes. Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy. Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences. Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S. pombe.