The Arabidopsis HOP2 Gene Has a Role in Preventing illegitimate Exchanges between Nonhomologous Chromosomes

Meiotic homologous chromosomes pair up and undergo crossing over. In many eukaryotes both intimate pairing and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via Homologous Recombination (HR). In these organisms, two key proteins are the recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 have been identified as proteins that assist RAD51 and DMC1 and are needed to promote stabilized pairing. We have probed the nature of the genetic lesions seen in hop2 mutants and looked at the role of HOP2 in the fidelity of genetic exchanges. Using γH2AX as a marker for unrepaired DSBs we found that hop2-1 and mnd1 mutants have different appearance/disappearance for DSBs than wild type, but all DSBs are repaired by mid-late pachytene. Therefore, the bridges and fragments seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Studying Arabidopsis haploid meiocytes we found that wild type haploids produced the expected five univalents, but hop2-1 haploids suffered many illegitimate exchanges that were stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing nonhomologous associations. We also found evidence that HOP2 plays a role in preventing illegitimate exchanges during repair of radiation-induced DSBs in rapidly dividing petal cells. Thus, HOP2 plays both a positive role in promoting homologous chromosome synapsis and a separable role in preventing nonhomologous chromosome exchanges. Possible mechanisms for this second important role are discussed.

RNAi and Ino80 complex control rate limiting translocation step that moves rDNA to eroding telomeres

The discovery of HAATIrDNA, a mode of telomerase-negative survival in which canonical telomeres are replaced with ribosomal DNA (rDNA) repeats that acquire chromosome end-protection capability, raised crucial questions as to how rDNA tracts ‘jump’ to eroding, nonhomologous chromosome ends. Here we show that HAATIrDNA formation is initiated and limited by a single translocation that juxtaposes rDNA from Chromosome (Chr) III onto subtelomeric elements (STE) on Chr I or II; this rare reaction requires the RNAi pathway and the Ino80 nucleosome remodeling complex (Ino80C), thus defining an unforeseen relationship between these two machineries. The unique STE-rDNA junction created by this initial translocation is efficiently copied to the remaining STE chromosome ends, without the need for RNAi or Ino80C, forming HAATIrDNA. Intriguingly, both the RNAi and Ino80C machineries contain a component that plays dual roles in HAATI subtype choice. Dcr1 of the RNAi pathway and Iec1 of the Ino80C both promote HAATIrDNA formation as part of their respective canonical machineries, but both also inhibit formation of the exceedingly rare HAATISTE (in which STE sequences mobilize throughout the genome and assume chromosome end protection capacity) in non-canonical, pathway-independent manners. This work provides a glimpse into a previously unrecognized crosstalk between RNAi and Ino80C in controlling unusual translocation reactions that establish telomere-free linear chromosome ends.

Nonhomologous Chromosome Pairing in Aegilops-Secale Hybrids

Intergeneric hybrids and amphidiploid hybrids from crosses of Aegilopstauschii and Secale cereale were produced using young embryo rescue. The hybrids showed complete sets of both parental chromosomes. The dihaploid plants showed an average meiotic pairing configuration of 10.84 I + 1.57 II + 0.01 III. Genomic in situ staining revealed 3 types of bivalent associations, i.e. D-D, R-R and D-R at frequencies of 8.6, 8.2 and 83.3%, respectively. Trivalents consisted of D-R-D or R-D-R associations. These results suggested that both intra- and intergenomic chromosome homology were contributed to chromosome pairing. Derived amphidiploids with 2n = 28 paired at metaphase I of meiosis as 4.51 I + 11.70 II + 0.03 III. Chromosome pairing of amphidiploids appeared more or less regular, i.e. bivalent-like with some trivalent configurations.

Directed synthesis of a segmental chromosomal transposition: an approach to the study of chromosomes lethal to the gametophyte generation of maize.

Because of the haploid nature of the gametophyte generation of plants, most mutations that are lethal or detrimental to the gametophytes cannot be recovered. Our laboratory is currently developing several techniques to overcome this situation. In this paper, a procedure is described to generate directed segmental chromosomal transpositions. The method involves recovery of recombinants between reciprocal translocation overlaps such that one region of the genome is inserted into a nonhomologous chromosome in a predetermined and directed manner. This duplicated segment then could serve to cover deficiencies or mutations, lethal to the gametophytes, in the region from whence it originated. The manipulation of segmental chromosomal transpositions for analyzing mutants lethal or detrimental to the gametophyte generation is discussed. The procedure to generate transpositions, the translocations between normal A and supernumerary B chromosomes that generate deficiencies in the male gametes, the r-X1 chromosome that generates deficiencies in the female gametes and other techniques available in maize form a system to analyze gametophyte lethal mutations.

A test for equilocality of supernumerary heterochromatin distribution in grasshoppers

The presence of extra segments in four or more nonhomologous chromosome pairs led us to test the applicability of the principle of the principle of equilocality of heterochromatin distribution to supernumerary heterochromatin. In Chorthippus brunneus, which carries extra segments on all chromosomes except the L3, the 13 heterochromatic variants occupy all possible locations with 3 proximal, 5 interstitial, and 5 distal. This indicates that equilocality does not seem to apply to the supernumerary heterochromatin in this species. On the other hand, the four extra segments found in Chorthippus parallelus and five of the seven extra heterochromatic segments found in Chorthippus binotatus are all located distally on the M5–S8 chromosomes. Thus for these groups of chromosomes the extra heterochromatic segments do appear to show a tendency for an equilocal distribution. Key words: Chorthippus, heterochromatin.

HOMOLOGOUS CHROMOSOME PAIRING REMAINS AN UNSOLVED PROBLEM: A TEST OF A POPULAR HYPOTHESIS UTILIZING MAIZE MEIOSIS

ABSTRACT A recently proposed and popular model for the mechanism of meiotic homologue pairing relies on prior association of nonhomologous chromosome arms of most similar length. According to this model, the diploid complement is organized into two genomic linear chains, each containing the various heterologues in the same sequence. At meiosis, then, appression of the two genomic chains could presumably readily accomplish homologue pairing. This model fails in its simplest form when observations of meiotic pairing of homologues in heterozygotes for arm length alterations are compared with computer-simulated predictions of the model. Contrary to predictions of the model, heterozygotes for arm length changes were found to exhibit only small frequencies of homologue-pairing failure, and this only for a single homologue pair in each case. It is difficult to conceive of a reasonable modification of this model that would be consistent with the observations.