Site-specific insertion of a SINE-like element, Cp1, into centromeric tandem repeats from Chironomus pallidivittatus

A SINE-like dispersed element, Cp1, from the dipteran Chironomus pallidivittatus was found to show site-specific insertion into two different centromeric tandem repeats. The insertions result in identical target site duplications of nine base-pairs. In contrast, extracentromeric Cp1 elements, which are polymorphic and degenerate, are previously known to be surrounded by different target site duplications. The intracentromeric Cp1 is uniform in structure and contains a single pol III unit, upstream of which 87 bp arms of a palindrome surround a 103 bp unique sequence. The numbers of Cp1 elements per centromere were determined in microdissected material and were found to be in the range of five to ten units per centromere. The well-defined insertion properties, correlated to chromosomal localization, suggest that Cp1 is likely to be a component of importance for the centromere. Similarities of Cp1 and its parts to functionally identified centromeres in Saccharomyces cerevisiae and Schizosaccharomyces pombe are discussed.     

Tiggers and DNA transposon fossils in the human genome

We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

Molecular structure of a functional Drosophila centromere

Centromeres play a critical role in chromosome inheritance but are among the most difficult genomic components to analyze in multicellular eukaryotes. Here, we present a highly detailed molecular structure of a functional centromere in a multicellular organism. The centromere of the Drosophila minichromosome Dp1187 is contained within a 420 kb region of centric heterochromatin. We have used a new approach to characterize the detailed structure of this centromere and found that it is primarily composed of satellites and single, complete transposable elements. In the rest of the Drosophila genome, these satellites and transposable elements are neither unique to the centromeres nor present at all centromeres. We discuss the impact of these results on our understanding of heterochromatin structure and on the determinants of centromere identity and function.     

A stable marker chromosome with a cryptic centromere: evidence for centromeric sequences associated with an inverted duplication

Centromere activation, an important mechanism in karyotype evolution, is occasionally observed in some human chromosome rearrangements. We report a possible occurrence of centromere activation in a marker chromosome containing an atypical centromere associated with an inverted duplication of the region 14q32 --> qter. The marker chromosome's reduced centromere lacks both the alpha and beta satellite sequences usually found at normal centromeres. In an attempt to identify the centromeric sequences, the marker chromosome was flow-sorted and amplified by a degenerate oligonucleotide primer polymerase chain reaction. Reverse chromosome painting experiments showed that the marker chromosome contains sequences that are unique to the distal region of chromosome 14, as well as a low copy number of (centromeric) sequences that are also highly represented in the centromeres of chromosomes 18 and 19. These data suggest the activation of a novel centromere in the 14q32 --> qter region, very likely consequent to the duplication of the region itself.     

Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

Structural requirements and dynamics of mitosin-kinetochore interaction in M phase

Mitosin is a 350-kDa human nuclear protein which transiently associates with centromeres and spindle poles in M phase. Ultrastructure studies reveal that it is located at the outer kinetochore plate. In this work, we explored the detailed structural basis and dynamics of the mitosin-kinetochore interaction. Two major regions important for targeting to centromeres were identified by analyzing different deletion mutants expressed in CHO cells: (i) the "core region" between amino acids 2792 and 2887, which was essential for the centromere localization of mitosin; and (ii) the internal repeats between residues 2094 and 2487, which cooperated with the core region to achieve strong mitosin-kinetochore interaction. The core region is characteristic of two leucine zipper motifs. Deletion of either motif abolished the centromere localization activity. In addition, Cys2864, adjacent to the second motif, was also essential for the activity of the core region. In contrast, the internal repeats alone were insufficient for centromere localization. We propose that this region may serve as a regulatory domain to facilitate interaction of the core region with the kinetochore. We showed that mitosin molecules entering nuclei after nuclear envelope breakdown (NEBD) were not assembled onto kinetochores efficiently, suggesting that the mitosin-kinetochore interaction is stabilized prior to NEBD. This result supports the idea of an ordered process for kinetochore assembly. Our data also suggest that mitosin might interact with chromatin in interphase. Evidence for coordinated regulation between the centromere-targeting and the putative chromatin-binding activities is also provided.     

Sequence analysis of an 80 kb human neocentromere

We previously described the cloning of an 80 kb DNA corresponding to the core protein-binding domain of a human chromosome 10-derived neocentromere. Here we report the complete sequence of this DNA (designated NC DNA) and its detailed structural analysis. The sequence is devoid of human centromeric alpha-satellite DNA and the pericentric beta- and gamma-satellites, the ATRS and 48 bp repeat DNA. One copy of a sequence that is related to the CENPB box motif is present, and a number of copies of other pericentric sequences including pJalpha and classical satellites I and III are present but both their relative sparsity and non-tandem organization suggest that each sequence, on its own, is unlikely to mimic any role the sequence may have in the normal centromere. The DNA-binding motifs of the architectural and regulatory proteins HMGI and topoII have a normal abundance and random distribution, implying that these sequences are not key functional elements. The total A + T content of the sequence is not notably different from that of the human genome, but an abundance of AT-rich islands and a biased distribution of these islands within the NC sequence are clearlydiscernible and may be functionally significant. Substantial amounts of transposable elements and low copy number tandem repeats, including several that are highly AT- and purine-rich, are also present and may act as functional elements. One of the AT-rich tandemrepeats (AT28) may form interesting structures and is described in detail. The defined features show only a loose resemblance to the structures of known centromeres, highlighting the possibility that, rather than a conserved primary sequence, it is the overallcomposition and distribution patterns of various unknown functional elements, or any 'ordinary' DNA under appropriate epigenetic influences, that determine centromere formation and function. This is the firstdetailed analysis of a neocentromere DNA and provides a basis for comparison against future sequences.     

Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts

Short mobile elements are present in different recombined forms as interspersed GC-rich islands between AT rich centromeric 155 bp tandem repeats in the dipteran Chironomus pallidivittatus . The basic element is 80 bp long, has a pronounced invert repeat structure and contains a 17 bp segment similar to the CENP-B box in mammals. The element inserts into a specific site of the 155 bp repeat in a defined orientation surrounded by 2 bp direct repeats. The total number per genome of the main variant is <20. Elements can be present in all centromeres from C.pallidivittatus and the sibling species Chironomus tentans with pronounced differences in distribution within and between species.     

Identification of trans-acting genes necessary for centromere function in Drosophila melanogaster using centromere-defective minichromosomes

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centro mere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.     

Engineering mammalian chromosomes

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.     

Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria

A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.     

The centromere enhancer mediates centromere activation in Schizosaccharomyces pombe

The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat. We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function. A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy. The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S. pombe. This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region. Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function. An S. pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores. These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated.     

Formation of de novo centromeres and construction of first-generation human artificial microchromosomes

We have combined long synthetic arrays of alpha satellite DNA with telomeric DNA and genomic DNA to generate artificial chromosomes in human HT1080 cells. The resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in the absence of selection for up to six months in culture, bind centromere proteins specific for active centromeres, and are estimated to be 6-10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes. We conclude that this strategy results in the formation of de novo centromere activity and that the microchromosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This first-generation system for the construction of human artificial chromosomes should be suitable for dissecting the sequence requirements of human centromeres, as well as developing constructs useful for therapeutic applications.     

Telomere and LINE-like elements at the termini of the Chlorella chromosome I

The telomeres of Chlorella chromosomes consisted of 5'-TTTAGGG repeats, which are exactly the same as those of higher plants. This sequence was reiterated approximately 70 times at both termini of chromosome I. Subtelomeric sequences next to the telomeres were totally different between the right and left arms. On the left-side subtelomeric region, polyA associated LINE (long interspersed element)-like elements were found tandemly repeated just next to the telomeric repeats. It is very interesting to compare this unique structure with telomeres of Drosophila, where a transposable element play a major role in forming telomerase-generated repeats. We propose a mechanism of transposon-mediated healing of a broken chromosomal end that would operate in the unicellular green alga Chlorella.