Trans-centromere effects on meiotic recombination in the zebrafish

We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.     

Physical distribution of recombination in B-genome chromosomes of tetraploid wheat

Summary Several studies have indicated a noncorrespondence between genetic and physical distances in wheat chromosomes. To study the physical distribution of recombination, polymorphism for C-banding patterns was used to monitor recombination in 67 segments in 11 B-genome chromosome arms of Triticum turgidum. Recombination was absent in proximal regions of all chromosome arms; its frequency increased exponentially with distance from the centromere. A significant difference was observed between the distribution of recombination in physically short and physically long arms. In physically short arms, recombination was almost exclusively concentrated in distal segments and only those regions were represented in their genetic maps. In physically long arms, while a majority of the genetic distance was again based upon recombination in distal chromosome segments, some interstitial recombination was observed. Consequently, these regions also contributed to the genetic maps. Such a pattern of recombination, skewed toward terminal segments of chromosomes, is probably a result of telomeric pairing initiation and strong positive chiasma interference. Interference averaged 0.81 in 35 pairs of adjacent segments and 0.57 across the entire recombining portions of chromosome arms. The total genetic map lengths of the arms corresponded closely to those expected on the basis of their metaphase-I chiasma frequencies. As a consequence of this uneven distribution of recombination there can be a 153-fold difference (or more) in the number of DNA base pairs per unit (centiMorgan) of genetic length.     

CENP-B controls centromere formation depending on the chromatin context

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.     

A telomere-mediated chromosome fragmentation approach to assess mitotic stability and ploidy alterations of Leishmania chromosomes

We have used a telomere-associated chromosome fragmentation strategy to induce internal chromosome-specific breakage of Leishmania chromosomes. The integration of telomeric repeats from the kinetoplastid Trypanosoma brucei into defined positions of the Leishmania genome by homologous recombination can induce chromosome breakage accompanied by the deletion of the chromosomal part that is distal to the site of the break. The cloned telomeric DNA at the end of the truncated chromosomes is functional and it can seed the formation of new telomeric repeats. We found that genome ploidy is often altered upon telomere-mediated chromosome fragmentation events resulting in large chromosomal deletions. In most cases diploidy is either preserved, or partial trisomic cells are observed, but interestingly we report here the generation of partial haploid mutants in this diploid organism. Partial haploid Leishmania mutants should facilitate studies on the function of chromosome-assigned genes. We also present several lines of evidence for the presence of sequences involved in chromosome mitotic stability and segregation during cell cycle in this parasitic protozoan. Telomere-directed chromosome fragmentation studies in Leishmania may constitute a useful tool to assay for centromere function.     

ERCC1/XPF Protects Short Telomeres from Homologous Recombination in Arabidopsis thaliana

Many repair and recombination proteins play essential roles in telomere function and chromosome stability, notwithstanding the role of telomeres in “hiding” chromosome ends from DNA repair and recombination. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and is the subject of considerable interest in studies of recombination. In contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants mutated for either XPF (AtRAD1) or ERCC1 (AtERCC1) orthologs, which develop normally and show wild-type telomere length. However, in the absence of telomerase, mutation of either of these two genes induces a significantly earlier onset of chromosomal instability. This early appearance of telomere instability is not due to a general acceleration of telomeric repeat loss, but is associated with the presence of dicentric chromosome bridges and cytologically visible extrachromosomal DNA fragments in mitotic anaphase. Such extrachromosomal fragments are not observed in later-generation single-telomerase mutant plants presenting similar frequencies of anaphase bridges. Extensive FISH analyses show that these DNAs are broken chromosomes and correspond to two specific chromosome arms. Analysis of the Arabidopsis genome sequence identified two extensive blocks of degenerate telomeric repeats, which lie at the bases of these two arms. Our data thus indicate a protective role of ERCC1/XPF against 3′ G-strand overhang invasion of interstitial telomeric repeats. The fact that the Atercc1 (and Atrad1) mutants dramatically potentiate levels of chromosome instability in Attert mutants, and the absence of such events in the presence of telomerase, have important implications for models of the roles of recombination at telomeres and is a striking illustration of the impact of genome structure on the outcomes of equivalent recombination processes in different organisms.     

Crossing-over across the centromere in the mating-type chromosome ofNeurospora crassa

The frequency of crossing-over in the two short regions on opposite arms, adjacent to the centromere of the mating-type chromosome ofNeurospora crassa is controlled, independently in each arm, by at least two genes with equal and additive effect. These genes segregate on inbreeding and cause great variability in both the frequency of recombination and the frequency of second-division segregation of loci situated within these regions. Recombination values between loci situated beyond these sensitive regions is not affected; the relative increase or decrease in their centromere distances may be attributed to change in the recombination frequency about the centromere only.     

Searching for a Common Centromeric Structural Motif: Drosophila Centromeric Satellite DNAs Show Propensity to form Telomeric-Like Unusual DNA Structures

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physical distribution of translocation breakpoints in homoeologous recombinants induced by the absence of the Ph1 gene in wheat and triticale

The physical distribution of translocation breakpoints was analyzed in homoeologous recombinants involving chromosomes 1A, 1B, 1D of wheat and 1R of rye, and the long arms of chromosome 7S of Aegilops speltoides and 7A of wheat. Recombination between homoeologues was induced by removal of the Ph1 gene. In all instances, translocation breakpoints were concentrated in the distal ends of the chromosome arms and were absent in the proximal halves of the arms. The relationship between the relative distance from the centromere and the relative homoeologous recombination frequency was best explained by the function f(x)=0.0091e^0.0592x. The pattern of recombination in homoeologous chromosomes was essentially the same as in homologues except that there were practically no double exchanges. Among 313 recombinant chromosomes, only one resulted from a double crossing-over. The distribution of translocation breakpoints in translocated arms indicated that positive chiasma interference operated in homoeologous recombination. This implies that the reduction of the length of alien chromosome segments present in translocations with wheat chromosomes may be more difficult than the production of the original recombinants.     

Recombination map of the common shrew, Sorex araneus (Eulipotyphla, Mammalia)

The Eurasian common shrew (Sorex araneus L.) is characterized by spectacular chromosomal variation, both autosomal variation of the Robertsonian type and an XX/XY(1)Y(2) system of sex determination. It is an important mammalian model of chromosomal and genome evolution as it is one of the few species with a complete genome sequence. Here we generate a high-precision cytological recombination map for the species, the third such map produced in mammals, following those for humans and house mice. We prepared synaptonemal complex (SC) spreads of meiotic chromosomes from 638 spermatocytes of 22 males of nine different Robertsonian karyotypes, identifying each autosome arm by differential DAPI staining. Altogether we mapped 13,983 recombination sites along 7095 individual autosomes, using immunolocalization of MLH1, a mismatch repair protein marking recombination sites. We estimated the total recombination length of the shrew genome as 1145 cM. The majority of bivalents showed a high recombination frequency near the telomeres and a low frequency near the centromeres. The distances between MLH1 foci were consistent with crossover interference both within chromosome arms and across the centromere in metacentric bivalents. The pattern of recombination along a chromosome arm was a function of its length, interference, and centromere and telomere effects. The specific DNA sequence must also be important because chromosome arms of the same length differed substantially in their recombination pattern. These features of recombination show great similarity with humans and mice and suggest generality among mammals. However, contrary to a widespread perception, the metacentric bivalent tu usually lacked an MLH1 focus on one of its chromosome arms, arguing against a minimum requirement of one chiasma per chromosome arm for correct segregation. With regard to autosomal chromosomal variation, the chromosomes showing Robertsonian polymorphism display MLH1 foci that become increasingly distal when comparing acrocentric homozygotes, heterozygotes, and metacentric homozygotes. Within the sex trivalent XY(1)Y(2), the autosomal part of the complex behaves similarly to other autosomes.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.     

The origin of tertiary monosomics in the tomato

Six aneuploid tomato plants with 2n–1=23 chromosomes were observed in populations grown from the seedlings treated with thermal neutrons and from seeds treated with X-rays. Four of the aneuploids were tertiary monosomics in which, as a result of centromeric interchanges between two different chromosomes, two whole arms were missing from the complement and two arms connected at the centromere. In one aneuploid, as a result of centromeric breakage, the two short arms of a homologous pair were missing from the complement and the two long arms connected to the long arm and the short arm respectively of another chromosome in which breakage had occurred also at the centromere. In one aneuploid, the interchange has occurred in the arms, and not in the centromere. Here the aneuploid condition is due to the loss of an arm with a centromere and a short piece of the other arm.In most of the tertiary monosomics the missing arms were either the short arms of sub-metacentric chromosomes or any of the arms of metacentric chromosomes. However, in one case the long arms of two submetacentric chromosomes were lost from the complement. That in spite of such large chromosomal deletions the sporophyte can survive, may be due to the fact that the aberrant plants are mostly chimeras.This study was part of a project resulting from a contract between the Association Euratom-I.T.A.L., and the Agricultural University of Wageningen.     

Telomeric DNA allocation in chromosomes of common shrew (Sorex araneus, eulipotyphla)

Recently, we displayed an Iberian shrew species (Sorex granarius) with telomere structures unusual for mammals. Long telomeres on the short acrocentric arms contain an average of 213 kb of telomere repeats, whereas the other chromosomal ends have only 3.8 kb (Zhdanova et al., 2005; 2007). However, it is not clear whether these telomeres are typical for all shrew species or only for S. granarius. S. granarius and common shrew Sorex araneus are sibling species. In this study, using modified Q-FISH we demonstrated that telomeres in S. araneus from various chromosomal races distinguished by their number of metacentrics contain 6.8–15.2 kb of telomeric tracts. The S. araneus telomere lengths appear to correspond to telomere lengths in the majority of both shrew species and wild mammals, whereas S. granarius has telomeres with unique or rare structures. Using DNA and RNA high-specific modified probes to telomeric repeats (PNA and LNA), we showed that interstitial telomeric sites in S. araneus chromosomes contain mainly telomeric DNA and that their localization coincide with some evolutionary breakpoints. Interstitial telomeric DNA in S. granarius chromosomes was not revealed. Thus, the distribution of telomeric DNA may be significantly different, even in closely related species whose chromosomes are composed of almost identical chromosomal arms.     

Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid^tet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.     

Genetic linkage between C-bands and storage protein genes in chromosome 1B of tetraploid wheat

Summary Genetic mapping of polymorphic C-bands allows direct comparisons between genetic and physical maps. Eleven C-bands and two seed storage protein genes on chromosome 1B, polymorphic between Langdon durum and four accessions of T. dicoccoides, were used to study the distribution of recombination along the entire length of the chromosome. Recombination in the short arm was almost completely restricted to the satellite, two-thirds of the arm's length from the centromere; the Gli-B1 gene was found to be tightly linked to the telomeric C-band. In the long arm, the distal 51.4% of the arm accounted for 88% of recombination; the proximal half of the arm accounted for the remaining 12%. While the amount of crossing-over differed significantly between the four T. dicoccoides 1B chromosomes, there were no significant differences in the relative distributions of crossing-over along the chromosome. Consequently, the genetic maps obtained from the four individual T. dicoccoides chromosomes were combined to yield a consensus map of 14 markers (including the centromere) for the chromosome.     

Relevance of histone acetylation and replication timing for deposition of centromeric histone CENP-A

A centromere-specific variant of histone H3, centromere protein A (CENP-A), is a critical determinant of centromeric chromatin, and its location on the chromosome may determine centromere identity. To search for factors that direct CENP-A deposition at a specific chromosomal locus, we took advantage of the observation that CENP-A, when expressed at elevated levels, can get incorporated at ectopic sites on the chromosome, in addition to the centromere. As core histone hypoacetylation and DNA replication timing have been implicated as epigenetic factors that may be important for centromere identity, we hypothesized that the sites of preferential CENP-A deposition will be distinguished by these parameters. We found that, on human dicentric chromosomes, ectopically expressed CENP-A preferentially incorporates at the active centromere only, despite the fact that the levels of histone acetylation and replication timing were indistinguishable at the two centromeres. In CHO cells, ectopically expressed CENP-A is preferentially targeted to some, but not all telomeric regions. Again, these regions could not be distinguished from other telomeres by their acetylation levels or replication timing. Thus histone acetylation and replication timing are not sufficient for specifying the sites of CENP-A deposition and likely for centromere identity.     

Genetic and physical analyses of the centromeric and pericentromeric regions of human chromosome 5: recombination across 5cen

Human centromeres are poorly understood at both the genetic and the physical level. In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19. This result was obtained by pulsed-field gel electrophoresis after cutting genomic DNA with restriction endonucleases NcoI (chromosome 5) and BamHI (chromosome 19). We could thus define a highly polymorphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere. The centromeric region of chromosome 5 was then analyzed in full detail. We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides. The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far. Two genes and several putative cDNAs could be precisely located close to the centromere. Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen. They were used to define STSs that could serve as physical anchor points at the junction of 5cen with the p and q arms. Some STSs were placed on a radiation hybrid map. One was polymorphic and could therefore be used as a second centromeric genetic marker at the p arm boundary of 5cen. We could thus estimate recombination rates within and around the centromeric region of chromosome 5. Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected between the two markers located at the two extremities of the centromere. In its immediate vicinity, 5cen indeed exerts a direct negative effect on meiotic recombination within the proximal chromosomal DNA. This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the entire chromosome 5. Rates then become close to the average when one goes further within the arms. Finally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombination around 5cen is essentially occurring in the female lineage.     

Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae.

To elucidate yeast chromosome structure and behavior, we examined the breakage of entangled chromosomes in DNA topoisomerase II mutants by hybridization to chromosomal DNA resolved by pulsed-field gel electrophoresis. Our study reveals that large and small chromosomes differ in the nature and distribution of their intertwinings. Probes to large chromosomes (450 kb or larger) detect chromosome breakage, but probes to small chromosomes (380 kb or smaller) reveal no breakage products. Examination of chromosomes with one small arm and one large arm suggests that the two arms behave independently. The acrocentric chromosome XIV breaks only on the long arm, and its preferred region of breakage is approximately 200 kb from the centromere. When the centromere of chromosome XIV is relocated, the preferred region of breakage shifts accordingly. These results suggest that large chromosomes break because they have long arms and small chromosomes do not break because they have small arms. Indeed, a small metacentric chromosome can be made to break if it is rearranged to form a telocentric chromosome with one long arm or a ring with an "infinitely" long arm. These results suggest a model of chromosomal intertwining in which the length of the chromosome arm prevents intertwinings from passively resolving off the end of the arm during chromosome segregation.