Localization of translocation breakpoints in somatic metaphase chromosomes of barley

Karyotype analyses based on staining by acetocarmine followed by Giemsa N-banding of somatic metaphase chromosomes of Hordeum vulgare L. were carried out on 61 reciprocal translocations induced by X-irradiation. By means of computer-based karyotype analyses all of the 122 breakpoints could be localized to defined sites or segments distributed over the seven barley chromosomes. The pre-definition of translocations with respect to their rearranged chromosome arms from other studies rendered it possible to define the break positions even in translocations having exchanged segments equal in size and the breakpoints located distally to any Giemsa band or other cytological marker. The breakpoints were found to be non-randomly spaced along the chromosomes and their arms. All breaks but one occurred in interband regions of the chromosomes, and none of the breaks was located directly within a centromere. However, short and long chromosome arms recombined at random. An improved tester set of translocations depicting the known break positions of most distal location is presented.     

Chromosome segmental dosage analysis of maize morphogenesis using B-A-A translocations

The B–A–A translocations have enabled us to simultaneously assess the possible dosage-sensitive interactions of two nonhomologous chromosome segments in affecting maize plant development. Maize B–A–A translocations contain segments of two nonhomologous essential A chromosomes in tandem arrangement attached to a segment of the long arm of a supernumerary B chromosome. By utilizing the frequent nondisjunction of the B centromere at the second pollen mitosis we produced plants containing an extra copy of the two A chromosome segments. We compared these hyperploid plants with nonhyperploid plants by measuring leaf width, plant height, ear height, internode length, stalk circumference, leaf length, and tassel-branch number in 20 paired families that involved one of the chromosome arms 1S, 1L, 4L, 5S, and 10L. One or more of the seven measured traits displayed dosage sensitivity among 17 of the 20 B–A–A translocations, which included the involvement of chromosome arms 2L, 3L, 5L, 6L, and 7L. The most obvious effect of an increased dosage of the B–A–A translocation was a significant decrease in the traits in the hyperploid plants. These effects may be either the additive effects of hyperploidy for the two chromosome segments or a result of gene interaction between them.     

Common sequence motifs at the rearrangement sites of a constitutional X/autosome translocation and associated deletion.

Reciprocal chromosome translocations are common de novo rearrangements that occur randomly throughout the human genome. To learn about causative mechanisms, we have cloned and sequenced the breakpoints of a cytologically balanced constitutional reciprocal translocation, t(X;4)(p21.2;q31.22), present in a girl with Duchenne muscular dystrophy (DMD). Physical mapping of the derivative chromosomes, after their separation in somatic cell hybrids, reveals that the translocation disrupts the DMD gene in Xp21 within the 18-kb intron 16. Restriction mapping and sequencing of clones that span both translocation breakpoints as well as the corresponding normal regions indicate the loss of approximately 5 kb in the formation of the derivative X chromosome, with 4-6 bp deleted from chromosome 4. RFLP and Southern analyses indicate that the de novo translocation is a paternal origin and that the father's X chromosome contains the DNA that is deleted in the derivative X. Most likely, deletion and translation arose simultaneously from a complex rearrangement event that involves three chromosomal breakpoints. Short regions of sequence homology were present at the three sites. A 5-bp sequence, GGAAT, found exactly at the translocation breakpoints on both normal chromosomes X and 4, has been preserved only on the der(4) chromosome. It is likely that the X-derived sequence GGAATCA has been lost in the formation of the der(X) chromosome, as it matches an inverted GAATCA sequence present on the opposite strand exactly at the other end of the deleted 5-kb fragment. These findings suggest a possible mechanism which may have juxtaposed the three sites and mediated sequence-specific breakage and recombination between nonhomologous chromosomes in male meiosis.     

Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements.

The structural analysis of aberrant chromosomes is important for our understanding of the molecular mechanisms underlying chromosomal rearrangements. We have identified a number of diploid Saccharomyces cerevisiae clones that have undergone loss of heterozygosity (LOH) leading to functional inactivation of the hemizygous URA3 marker placed on the right arm of chromosome III. Aberrant-sized chromosomes derived from chromosome III were detected in approximately 8% of LOH clones. Here, we have analyzed the structure of the aberrant chromosomes in 45 LOH clones with a PCR-based method that determines the ploidy of a series of loci on chromosome III. The alterations included various deletions and amplifications. Sequencing of the junctions revealed that all the breakpoints had been made within repeat sequences in the yeast genome, namely, MAT-HMR, which resulted in intrachromosomal deletion, and retrotransposon Ty1 elements, which were involved in various translocations. Although the translocations involved different breakpoints on different chromosomes, all breakpoints were exclusively within Ty1 elements. Some of the resulting Ty1 elements left at the breakpoints had a complex construction that indicated the involvement of other Ty1 elements not present at the parental breakpoints. These indicate that Ty1 elements are crucially involved in the generation of chromosomal rearrangements in diploid yeast cells.     

Chromosomal Translocations Generated by High-Frequency Meiotic Recombination between Repeated Yeast Genes

We have examined meiotic and mitotic recombination between repeated genes on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae. The results of these experiments can be summarized in three statements. First, gene conversion events between repeats on nonhomologous chromosomes occur frequently in meiosis. The frequency of such conversion events is only 17-fold less than the analogous frequency of conversion between genes at allelic positions on homologous chromosomes. Second, meiotic and mitotic conversion events between repeated genes on nonhomologous chromosomes are associated with reciprocal recombination to the same extent as conversion between allelic sequences. The reciprocal exchanges between the repeated genes result in chromosomal translocations. Finally, recombination between repeated genes on nonhomologous chromosomes occurs much more frequently in meiosis than in mitosis.     

Karyotype Variability in Yeast Caused by Nonallelic Recombination in Haploid Meiosis

Chromosomes of altered size were found in the meiotic products of a haploid Saccharomyces cerevisiae strain by pulsed field gel electrophoretic separation of whole chromosomes. About 7% of haploid meioses produced chromosomes that differed by >/=10 kb from their wild-type counterparts. Chromosomes most often became enlarged or shortened due to recombination events between sister chromatids at nonallelic sequences. By this mechanism chromosome III acquired tandem arrays of up to eight extra copies of the ~100 kb MAT-HMR segment during repeated rounds of haploid meioses. Enlarged chromosomes III were unstable and changed their size during meiosis more often than remaining unchanged. Altered chromosomes appeared also as the products of intrachromatid recombination and of reciprocal translocations caused by ectopic recombination between nonhomologous chromosomes. In diploid meiosis, chromosomes of altered size occurred at least 10 times less frequently, whereas in mitotic cultures cells with altered karyotypes were virtually absent. The results show that various forms of ectopic recombination are promoted by the absence of allelic homologies.     

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.     

The t(4;8) is mediated by homologous recombination between olfactory receptor gene clusters, but other 4p16 translocations occur at random

The t(4;8)(p16;p23) is the second most common constitutional chromosomal translocation and is caused by an ectopic meiotic recombination between the olfactory receptor gene clusters (ORGC), located on chromosome 4p and 8p. Given that ORGCs are scattered across the genome and make-up about 0.1% of the human genome we reasoned that translocations between 4p16 and other chromosomes might be mediated by ectopic recombination between different ORGC. In 13 patients, we mapped the breakpoints of either a balanced or unbalanced translocation between chromosome 4p16 and different chromosomes. For all four t(4;8) cases, the breakpoints fall within the 4p and 8pter ORGC, confirming that non-allelic homologous recombination (NAHR) between the ORGC is the main mechanism of the t(4;8) formation. For the nine other translocations, the breakpoints on chromosome 4 mapped to different loci, one of them within the ORGC and in two flanking the ORGC. In these three cases, the translocation breakpoint at the reciprocal chromosome did not contain ORGC sequences. We conclude that only the t(4;8) is mediated by NAHR between ORGC.     

Two Types of Sites Required for Meiotic Chromosome Pairing in Caenorhabditis Elegans

Previous studies have shown that isolated portions of Caenorhabditis elegans chromosomes are not equally capable of meiotic exchange. These results led to the proposal that a homolog recognition region (HRR), defined as the region containing those sequences enabling homologous chromosomes to pair and recombine, is localized near one end of each chromosome. Using translocations and duplications we have localized the chromosome I HRR to the right end. Whereas the other half of chromosome I did not confer any ability for homologs to pair and recombine, deficiencies in this region dominantly suppressed recombination to the middle of the chromosome. These deletions may have disrupted pairing mechanisms that are secondary to and require an HRR. Thus, the processes of pairing and recombination appear to utilize at least two chromosomal elements, the HRR and other pairing sites. For example, terminal sequences from other chromosomes increase the ability of free duplications to recombine with their normal homologs, suggesting that telomere-associated sequences, homologous or nonhomologous, play a role in facilitating meiotic exchange. Recombination can also initiate at internal sites separated from the HRR by chromosome rearrangement, such as deletions of the unc-54 region of chromosome I. When crossing over was suppressed in a region of chromosome I, compensatory increases were observed in other regions. Thus, the presence of the HRR enabled recombination to occur but did not determine the distribution of the crossover events. It seems most likely that there are multiple initiation sites for recombination once homolog recognition has been achieved.     

Mapping RFLP Loci in Maize Using B-a Translocations

Plants hypoploid for specific segments of each of the maize (Zea mays L.) chromosomes were generated using 24 different B-A translocations. Plants carrying each of the B-A translocations were crossed as male parents to inbreds, and sibling progeny hypoploid or not hypoploid for specific chromosomal segments were recovered. Genomic DNAs from the parents, hypoploid progeny, and nonhypoploid (euploid or hyperploid) progeny for each of these B-A translocations were digested with restriction enzymes, electrophoresed in agarose gels, blotted onto reusable nylon membranes, and probed with nick-translated, cloned DNA fragments which had been mapped previously by restriction fragment length polymorphism (RFLP) analysis to the chromosome involved in the B-A translocation. The chromosomal segment on our RFLP map which was uncovered by each of the B-A translocations was determined. This work unequivocally identified the short and long arms of each chromosome on this map, and it also identified the region on each chromosome which contains the centromere. Because the breakpoints of the B-a translocations were previously known on the cytological and the conventional genetic maps, this study also allowed this RFLP map to be more highly correlated with these maps.     

Three autosomal chromosome translocations associated with repeated early embryonic loss (REEL) in the domestic horse (Equus caballus)

Repeated early embryonic loss (REEL) represents a considerable economic loss to the horse industry. Mares that experience REEL may be overlooked as potential carriers of a chromosome abnormality. Here we report three different autosomal translocations in Thoroughbred mares presented for chromosome analysis because of REEL. The karyotypes were 64,XX,t(1;21), 64,XX,t(16;22), and 64,XX,t(4;13), respectively. In order to confirm the chromosomes involved in the translocations, to map the breakpoints, and to determine if the translocations were reciprocal, genes surrounding the breakpoints were identified using existing maps and from the newly assembled horse genome sequence. Bacterial artificial chromosomes containing the genes of interest were identified and mapped to the translocation chromosomes by fluorescence in situ hybridization (FISH). FISH confirmed that the t(16;22) and t(4;13) translocations were reciprocal, while the t(1;21) was not. The breakpoints on horse chromosomes 1 and 16 appear to be the same or near breakpoints previously identified in translocations. These breakpoints are at the fusion boundary of human chromosomes 10 and 15 on horse chromosome 1 and at human chromosome 3p and 3q on horse chromosome 16. These sites may represent ancient breakpoints reused during equid evolution. Overall, chromosome abnormalities may have a greater influence on mare fertility than previously known. Thus, it is important to karyotype subfertile mares exhibiting REEL.     

Recombination in the B Chromosome of Maize to Produce a-B-a Chromosomes

The translocations between the supernumerary B chromosomes and the normal A chromosomes of maize provide a valuable tool for gene localizations, dosage studies and characterization of mutants as null, leaky or gain-of-function. A procedure is described, that relies on recombination in the B chromosome, for marking each of the various B-A translocations with a single dominant marker that will allow dosage classifications of individuals at the mature kernel stage. This marker is R-scm3, which conditions anthocyanin pigment in the aleurone of the endosperm and the scutellum of the embryo. A test for recombination in the B chromosome was conducted by crossing together two translocations, that were broken on opposite sides of the B centromere, and in different A chromosome arms, namely TB-1La and TB-10L18. An example was recovered that linked genetic markers on 1L and 10L to the B centromere. Cytological examination at pachytene of meiosis confirmed the new chromosomal linkage. The use of this procedure to produce a comprehensive set of uniformly marked B-A translocations is discussed.     

Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.     

The cytogenetic localization of the alcohol dehydrogenase-1 locus in maize

The alcohol dehydrogenase-1 (Adh) locus in maize has been positioned relative to thirteen reciprocal translocations that have breakpoints in the long arm of chromosome 1(1L). The methods of Gopinath and Burnham (1956) to produce interstitial segmental trisomy with overlapping translocations and of Rakha and Robertson (1970) to produce compound B-A translocations were coupled with the co-dominant nature of the ADH isozymes to allow the cytological placement. The results of several crosses are consistent with Adh being in the region of 0.80-0.90 of 1L.--The duplication that results from the overlap of translocations 1-3(5267) and 1-3(5242) and that includes Adh was studied with respect to meiotic segregation and pollen transmission. When heterozygous with normal chromosomes, a low level of recombination within the duplicated regions is detectable and the duplication and normals are recovered with equal frequencies through the female. In the pollen, the hyperploid grains cannot compete equally with the euploids in achieving fertilization.--The use of co-dominant heteromultimeric isozymes as genetic markers for the development of a series of interstitial segmental trisomics in maize is discussed.     

The heterochromatic B chromosome of maize: the segments affecting recombination

The B chromosome of maize is known to increase recombination in specific regions of the genome. In an attempt to determine the portion of the B responsible for crossover enhancement, translocations between the B and A chromosomes were used to dissect the B into four distinct segments. The effects of the segments on crossing over were studied in a sensitive region composed of chromatin from chromosomes 3 and 9. The relatively euchromatic chromomeres terminating the B lacked enhancement activity, but the remaining segments, all of which possess large amounts of heterochromatin, were capable of elevating recombination. There was no localization of activity to a specific heterochromatic region, however.     

The Effects of Translocations on Recombination Frequency in Caenorhabditis Elegans

In the nematode Caenorhabditis elegans, recombination suppression in translocation heterozygotes is severe and extensive. We have examined the meiotic properties of two translocations involving chromosome I, szT1(I;X) and hT1(I;V). No recombination was observed in either of these translocation heterozygotes along the left (let-362-unc-13) 17 map units of chromosome I. Using half-translocations as free duplications, we mapped the breakpoints of szT1 and hT1. The boundaries of crossover suppression coincided with the physical breakpoints. We propose that DNA sequences at the right end of chromosome I facilitate pairing and recombination. We use the data from translocations of other chromosomes to map the location of pairing sites on four other chromosomes. hT1 and szT1 differed markedly in their effect on recombination adjacent to the crossover suppressed region. hT1 had no effect on recombination in the adjacent interval. In contrast, the 0.8 map unit interval immediately adjacent to the szT1(I;X) breakpoint on chromosome I increased to 2.5 map units in translocation heterozygotes. This increase occurs in a chromosomal interval which can be expanded by treatment with radiation. These results are consistent with the suggestion that the szT1(I) breakpoint is in a region of DNA in which meiotic recombination is suppressed relative to the genomic average. We propose that DNA sequences disrupted by the szT1 translocation are responsible for determining the frequency of meiotic recombination in the vicinity of the breakpoint.     

Genomic rearrangements resulting in PLP1 deletion occur by nonhomologous end joining and cause different dysmyelinating phenotypes in males and females

In the majority of patients with Pelizaeus-Merzbacher disease, duplication of the proteolipid protein gene PLP1 is responsible, whereas deletion of PLP1 is infrequent. Genomic mechanisms for these submicroscopic chromosomal rearrangements remain unknown. We identified three families with PLP1 deletions (including one family described elsewhere) that arose by three distinct processes. In one family, PLP1 deletion resulted from a maternal balanced submicroscopic insertional translocation of the entire PLP1 gene to the telomere of chromosome 19. PLP1 on the 19qtel is probably inactive by virtue of a position effect, because a healthy male sibling carries the same der(19) chromosome along with a normal X chromosome. Genomic mapping of the deleted segments revealed that the deletions are smaller than most of the PLP1 duplications and involve only two other genes. We hypothesize that the deletion is infrequent, because only the smaller deletions can avoid causing either infertility or lethality. Analyses of the DNA sequence flanking the deletion breakpoints revealed Alu-Alu recombination in the family with translocation. In the other two families, no homologous sequence flanking the breakpoints was found, but the distal breakpoints were embedded in novel low-copy repeats, suggesting the potential involvement of genome architecture in stimulating these rearrangements. In one family, junction sequences revealed a complex recombination event. Our data suggest that PLP1 deletions are likely caused by nonhomologous end joining.     

A common breakpoint on 11q23 in carriers of the constitutional t(11;22) translocation

Structural chromosomal rearrangements occur commonly in the general population. Individuals that carry a balanced translocation are at risk of having unbalanced offspring; therefore, the frequency of translocations in couples with recurrent spontaneous abortions is higher than that in the general population. The constitutional t(11;22) translocation is the most common recurrent non-Robertsonian translocation in humans and may serve as a model to determine the mechanism that causes recurrent meiotic translocations. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat, termed "LCR22." To define the breakpoint on 11q23 and to ascertain whether this region shares homology with LCR22 sequences, we performed haplotype analysis on patients with der(22) syndrome. We found that the breakpoint on 11q23 occurred between two genetic markers, D11S1340 and APOC3-tetra, both being present within a single bacterial-artificial-chromosome clone. To determine whether the breakpoint occurred within the same region among a larger set of carriers, we performed FISH mapping studies. The breakpoints were all within the same clone, suggesting that this region may harbor sequences that are prone to breakage. We narrowed the breakpoint interval, in both derivative chromosomes from two unrelated carriers, to a 190-bp, AT-rich repeat, which indicates that this repeat may mediate recombination events on chromosome 11. Interestingly, the LCR22s harbor AT-rich repeats, suggesting that this sequence motif may mediate recombination events in nonhomologous chromosomes during meiosis.     

Allelic and Ectopic Interactions in Recombination-Defective Yeast Strains

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.     

POLYPLOIDY,DYSPLOIDY, AND CHROMOSOME PAIRING IN ECHEVERIA (CRASSULACEAE) AND ITS HYBRIDS

The 140+ species of Echeveria have more than 50 gametic chromosome numbers, including every number from 12 through 34 and polyploids to n = ca. 260. With related genera, they comprise an immense comparium of 200+ species that have been interconnected in cultivation by hybrids. Some species with as many as 34 gametic chromosomes include none that can pair with each other, indicating that they are effectively diploid, but other species with fewer chromosomes test as tetraploids. Most diploid hybrids form multivalents, indicating that many translocations have rearranged segments of the chromosomes. Small, nonessential chromosomal remnants can be lost, lowering the number and suggesting that higher diploid numbers (n = 30–34) in the long dysploid series are older. These same numbers are basic to most other genera in the comparium (Pachyphytum, Graptopetalum, Sedum section Pachysedum), and many diploid intergeneric hybrids show very substantial chromosome pairing. Most polyploid hybrids here are fertile, even where the parents belong to different genera and have very different chromosome numbers. This seems possible only if corresponding chromosomes from a polyploid parent pair with each other preferentially, strong evidence for autopolyploidy. High diploid numbers here may represent old polyploids that have become diploidized by loss, mutation, or suppression of duplicate genes, but other evidence for this is lacking. Most species occur as small populations in unstable habitats in an area with a history of many rapid climatic and geological changes, presenting a model for rapid evolution.