Structure of the chromosome VII centromere region in Neurospora crassa: degenerate transposons and simple repeats

DNA from the centromere region of linkage group (LG) VII of Neurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical of Neurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (approximately 16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3' end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full-length but nonmobile copia-like element, designated Tcen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tgl2, shows similarity to the Ty3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the repeat-induced point mutation (RIP) process. Three new simple DNA repeats have also been identified in the LG VII centromere region. While Tcen elements map exclusively to centromere regions by restriction fragment length polymorphism analysis, the defective Tad elements appear to occur most frequently within centromeres but are also found at other loci including telomeres. The characteristics and arrangement of these elements are similar to those seen in the Drosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements, is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those of most single-cell yeasts.     

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.     

Distribution of T1, Q, Pegasus and mariner transposable elements on the polytene chromosomes of PEST, a standard strain of Anopheles gambiae

The chromosomal locations of four families of transposable elements, T1, Q, Pegasus and mariner, have been determined by in situ hybridization to polytene chromosomes of ovarian nurse cells of the mosquito Anopheles gambiae. As part of this effort, we have developed a vigorous pink-eyed laboratory strain of A. gambiae (PEST), rendered homozygous standard for chromosomal inversions on all autosomes. Ten different individuals of this strain were studied with each transposable element probe. The average number of hybridization sites per genome was 83.9 for T1, 63.4 for Q, 31.5 for Pegasus and 64.7 for mariner, excluding pericentric and centromeric regions. However, some degree of polymorphism was observed within each family such that, considering all ten individuals, 94 different sites were detected for T1, 82 sites for Q, 45 sites for Pegasus and 71 sites for mariner. The mean occupancy per site varied from 0.70 (Pegasus) to 0.91 (mariner), which, while significantly higher than that seen for transposable elements in natural populations of Drosophila melanogaster, is comparable to that seen in established laboratory stocks. In addition, these element families were not randomly distributed. All but Pegasus were concentrated in centromeric heterochromatin and centromere-proximal euchromatin, most showed a deficit of hybridization sites in the distal section of chromosomes, and a significant proportion of sites were coincident between families. These results provide the first detailed examination of the cytogenetic location of transposable elements in a nondrosophilid insect, and, through comparison with the behavior of transposable elements in Drosophila, may provide insight into the interaction between elements and host. The mapped elements are also expected to serve as landmarks useful in integrating the developing physical map of the PEST strain with the chromosomal banding pattern.     

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.     

The product of proliferation disrupter is concentrated at centromeres and required for mitotic chromosome condensation and cell proliferation in Drosophila

Homozygosity for a null mutation in the proliferation disrupter (prod) gene of Drosophila causes decreased mitotic index, defects of anaphase chromatid separation, and imperfect chromosome condensation in larval neuroblasts and other proliferating cell populations. The defective condensation is especially obvious near the centromeres. Mutant larvae show slow growth and massive cell death in proliferating cell populations, followed by late larval lethality. Loss of prod function in mitotic clones leads to the arrest of oogenesis in the ovary and defective cuticle formation in imaginal disc derivatives. The prod gene encodes a novel 301-amino-acid protein that is ubiquitously expressed and highly concentrated at the centric heterochromatin of the second and third mitotic chromosomes, as well as at > 400 euchromatic loci on polytene chromosomes. We propose that Prod is a nonhistone protein essential for chromosome condensation and that the chromosomal and developmental defects are caused by incomplete centromere condensation in prod mutants.     

Induction of centromeric activity in maize by suppressor of meiotic drive 1

The Abnormal chromosome 10 (Ab10) in maize causes normally-quiescent blocks of heterochromatin called knobs to function as meiotic centromeres. Under these circumstances genetic markers associated with knobs exhibit meiotic drive, i.e., they are preferentially transmitted to progeny. Here we describe a mutation called suppressor of meiotic drive (smd1) that partially suppresses meiotic drive, and demonstrate that smd1 causes a quantitative reduction in the mobility of knobs on the meiotic spindle. We conclude that Smd1 encodes a product that is necessary for the activation of ectopic centromeres, and that meiotic drive occurs as a consequence of the resulting change in chromosome movement. As a genetic system, Ab10 offers a new and powerful approach for analyzing centromere/kinetochore function.     

Evolutionary links between telomeres and transposable elements

Transposable elements are abundant in the genomes of higher organisms but are usually thought to affect cells only incidentally, by transposing in or near a gene and influencing its expression. Telomeres of Drosophila chromosomes are maintained by two non-LTR retrotransposons, HeT-A and TART. These are the first transposable elements with identified roles in chromosome structure. We suggest that these elements may be evolutionarily related to telomerase; in both cases an enzyme extends the end of a chromosome by adding DNA copied from an RNA template. The evolution of transposable elements from chromosomal replication mechanisms may have occurred multiple times, although in other organisms the new products have not replaced the endogenous telomerase, as they have in Drosophila. This is somewhat reminiscent of the oncogenes that have arisen from cellular genes. Perhaps the viruses that carry oncogenes have also arisen from cellular genetic systems.     

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.     

Centromeres, checkpoints and chromatid cohesion

An emerging view is that the formation of active centromeres is modulated in an epigenetic manner reflecting the association of centromeres with heterochromatin. Support for this comes from studies on fission yeast centromeres, the properties of human neocentromeres and dicentric chromosomes, and analyses of Drosophila minichromosome deletion derivatives. A link has been established between tension across kinetochores and the phosphorylation status of kinetochore components. Vertebrate homologues of yeast MAD2 have recently been isolated and localized to kinetochores, indicating that components of the spindle integrity checkpoint are conserved. The linkage between sister chromatids is only dissolved at anaphase during mitotic and meiotic divisions. Phenotypic and localization data combined with their pattern of rapid degradation at anaphase have implicated several yeast and Drosophila proteins in aspects of sister chromatid cohesion.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

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.     

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.     

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.     

Exaggerated precocious centromere separation in cells of a human breast cancer line treated with a green tea extract

In a breast cancer cell line, MDA-MB-468, established in our laboratory, an average of 3% of the mitotic cells exhibited a phenomenon known as centromere splaying, which is a characteristic feature of cells of patients with Roberts syndrome. However, centromere splaying in cells of Roberts syndrome patients is limited to i) the centromere region and ii) chromosomes with large amounts of heterochromatin. When the breast cancer cells were treated with an extract of green tea GTE-TP91, up to 45% of the metaphases were observed to exhibit this behavior; and the precocious centromere separation was highly exaggerated, affecting all chromosomes in such metaphases. Apparently, as the sister centromeres continued to pull apart, they carried the chromatids with them, except for the telomere regions, giving a ring-like configuration. Eventually, the sister chromatids became completely separated. Whether this bizarre phenomenon was induced by the polyphenols contained in this green tea extract GTE-TP91 is not known, but this phenomenon, upon further investigation, may throw some light on chromosomal proteins, centromere behavior, telomere behavior and related questions.     

INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13-amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11-amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha). Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.     

Enigma of Y chromosome degeneration: neo-Y and neo-X chromosomes of Drosophila miranda a model for sex chromosome evolution

Y chromosome degeneration is characterized by structural changes in the chromosome architecture and expansion of genetic inertness along the Y chromosome. It is generally assumed that the heteromorphic sex chromosome pair has developed from a pair of homologues. Several models have been suggested. We use the unique situation of the secondary sex chromosome pair, neo-Y and neo-X (X2), in Drosophila miranda to analyze molecular mechanisms involved in the evolutionary processes of Y chromosome degeneration. Due to the fusion of one of the autosomes to the Y chromosome (about 2 Mya), a neo-Y chromosome and a neo-X chromosome, designated X2, were formed. Thus, formerly autosomal genes are inherited now on a pair of sex chromosomes in D. miranda. Analyzing DNA sequences from the X2 and neo-Y region, we observed a massive accumulation of DNA insertions on the neo-Y chromosome. From the analysis of several insertion elements, we present compelling evidence that the first step in Y chromosome degeneration is driven by the accumulation of transposable elements, especially retrotransposons. An enrichment of these elements along an evolving Y chromosome could account for the switch from a euchromatic into a heterochromatic chromatin structure.