Recombination and chiasmata: few but intriguing discrepancies.

The paradigm that meiotic recombination and chiasmata have the same basis has been challenged, primarily for plants. High resolution genetic mapping frequently results in maps with lengths far exceeding those based on chiasma counts. In addition, recombination between specific homoeologous chromosomes derived from interspecific hybrids is sometimes much higher than can be explained by meiotic chiasma frequencies. However, almost the entire discrepancy disappears when proper care is taken of map inflation resulting from the shortcomings of the mapping algorithm and classification errors, the use of dissimilar material, and the difficulty of accurately counting chiasmata. Still, some exchanges, especially of short interstitial segments, cannot readily be explained by normal meiotic behaviour. Aberrant meiotic processes involving segment replacement or insertion can probably be excluded. Some cases of unusual recombination are somatic, possibly premeiotic exchange. For other cases, local relaxation of chiasma interference caused by small interruptions of homology disturbing synaptonemal complex formation is proposed as the cause. It would be accompanied by a preference for compensating exchanges (negative chromatid interference) resulting from asymmetry of the pairing chromatid pairs, so that one side of each pair preferentially participates in pairing. Over longer distances, the pairing face may switch, causing the normal random chromatid participation in double exchanges and the relatively low frequency of short interstitial exchanges. Key words : recombination frequency, map length, chiasmata, discrepancy, chromatid interference.     

Analysis of exchanges in differentially stained meiotic chromosomes of Locusta migratoria after BrdU-substitution and FPG staining

Differential staining of the sister-chromatids of meiotic chromosomes of Locusta migratoria was achieved following abdominal implantation of BrdU tablets and fluorescent plus Giemsa (FPG) staining of fixed and squashed testicular follicles. This paper presents a detailed analysis of crossover exchanges between light and dark chromatids in monochiasmate bivalents. Approximately half the bivalents studied had visible exchanges of dark and light chromatids associated with the chiasmata, as expected if chiasmata originate by breakage and rejoining exchange events between randomly selected non-sister chromatids. In all the bivalents studied the visible crossover exchanges coincided exactly with chiasmata thus showing that chiasma movement (terminalisation) does not occur subsequent to crossing-over in Locusta migratoria, and that chiasmata are therefore accurate indicators of crossing over. It was noted that a proportion (9.5%) of chiasmata were associated with apparently anomalous exchanges of dark and light chromatids which could not be explained by conventional crossing-over. Various hypotheses for the origin of these anomalous exchanges are considered.     

Distribution of chiasmas in the 2d and 6th chromosomes involved in Robertsonian translocations in male mice

The distribution of chiasmata in 2 and 6 chromosomes in males homozygous for Rb(2.6)4Iem and Rb(8.17)1Iem was studied. Chiasmata were shown to distribute along chromosomes non-randomly, exchanges occurring in telomeric regions. Chiasmata distribution is substantially different for the cases of one and two chiasmata per bivalent. The main cause for these differences is supposed to be strong positive chiasmata interference (the position of the first chiasma may determine the position of the second one). The centromere blocks this effect, so chiasma in one arm does not interfere with that in the second arm. It has been shown that the frequency of double exchanges depended on not only the distance between markers under study, but also on marker position in the chromosome.     

Further correlations between chiasmata and U-type exchanges in rye meiosis

Earlier studies have demonstrated convincing correlations between the distribution patterns of chiasmata and of U-type exchanges within bivalents. On the basis of this evidence and other considerations it has been proposed that these contrasting meiotic exchange events are related in origin, and that U-type exchanges, giving rise to bridge and fragment configurations, arise as errors in crossing-over and chiasma formation. This hypothesis is given further consideration in the present report and further correlated distributions of chiasmata and U-type exchanges are presented. These correlations involve the distribution patterns of exchanges between bivalents and between the two arns of one particular bivalent which is consistently marked by the presence of localised neocentric activity. The relationships of these exchange distributions to chromosome length are also investigated and as a result it becomes clear that a mutual dependence of the two types of exchange on chromosome length cannot account for the observed correlations. The total evidence relating to the hypothesis of a causal connection between chiasmata and U-type exchanges is reviewed and critically assessed.     

Meiosis VI: A reinterpretation of tritium thymidine labelling of meiotic chromosomes,supporting the chiasmatype hypothesis

Re-examination of data relating chiasmata per bivalent to tritium thymidine switch points per chromatid in male grasshoppers suggests that they are consistent with the chiasmatype hypothesis: that the number of chiasmata is equal to the number of crossover exchanges.     

The analysis of exchanges in tritium-labelled meiotic chromosomes

The autoradiographic analysis of exchanges in tritium-labelled meiotic chromosomes is potentially a useful approach to the study of meiotic exchange events since this method differentially labels meiotic chromatids along their entire length. The main problem encountered in earlier autoradiographic studies is that of distinguishing label exchanges generated at chiasmata from label exchanges generated by sister chromatid exchange. This problem was overcome in the present study by the choice of a meiotic system (male meiosis of Stethophyma grossum) where chiasmata are limited to just one proximally localised chiasma in each bivalent. This system allows the positive identification of chiasma-generated label exchanges and demonstrates convincingly the origin of chiasmata through breakage and rejoining of homologous non-sister chromatids. Sister chromatid exchanges are also readily detected in labelled meiotic chromosomes of this species, where they occur with a mean frequency of 0.35 per chromosome. This frequency is similar to that found in mitotic spermatogonial cells and the exchanges are randomly distributed both within and between chromosomes. These features of meiotic sister chromatid exchanges suggest that they are unrelated to non-sister chiasmatic exchanges and they probably have no special meiotic significance.     

Mitotic crossing-over and segregation in man

Summary Although clear genetic evidence of mitotic crossing-over is lacking in man, observations of mitotic chiasmata in normal cells (0.1–1 per 1000) and in Bloom's syndrome (BS) cells (5–150 per 1000) demonstrate its occurrence. That mitotic chiasmata are true exchanges is concluded from the occurrence of heteromorphic bivalents and the pattern of sister chromatid exchanges in mitotic bivalents. Several observations demonstrate that chiasmata are different in principle from chromatid translocations which simply happen to take place at homologous loci. For example, the ratio of adjacent exchanges to mitotic chiasmata is 1/20–1/60, whereas this ratio is approximately 1:1 for chromatid translocations. Furthermore, mitotic chiasmata make up a very high proportion of total quadriradials (QRs): 48% in normal untreated cells and 90% in BS cells.Close proximity of homologous chromosomes promotes mitotic crossing-over. Thus in normal diplochromosomes, the incidence is increased a hundred-fold as compared to diploid cells. However, closeness of homologues is not the only factor promoting crossing-over; the BS gene specifically promotes exchanges between homologous segments as shown by the roughly 15-fold increase of chiasmata in BS diplochromosomes as compared to normal diplochromosomes.Mitotic chiasmata are distributed extremely nonrandomly in different chromosomes and chromosome segments. The preferred sites are short Q-dark regions, 3p21, 6p21, 11q13, 12q13, 17q12, and 19p13 or q13 being veritable hot spots. Our preferred hypothesis is that the hot spots have higher gene densities than other regions. Consequently they are active and extended in interphase which would promote their pairing and chiasma formation.Segregation after mitotic corssing-over in satellite stalks can be demonstrated by means of distinct satellites. In a BS patient there were 31 different patterns for Q-bright satellites in 58 cells. Segregation after presumed crossing-over has also been seen in three dicentric chromosomes with one centromere inactivated. Recombination in satellite stalks in BS resulted in 12/58 cells homozygous for Q-bright satellites. In two of these cells, two chromosomes were homozygous for Q-bright satellites, and in one cell, three chromosomes were homozygous. This high degree of homozygosity which obviously applies to other chromosome regions too, may explain the high incidence of malignant disease in BS on the assumption that cancer is caused by recessive genes.     

Double insertion of homogeneously staining regions in chromosome 1 of wild Mus musculus musculus: effects on chromosome pairing and recombination

An examination of the meiotic pattern of chromosome 1 isolated from a feral mouse population and containing a double insertion (Is) of homogeneously staining regions (HSRs) was carried out. The region delineated by the proximal breakpoint of Is(HSR;1C5) 1Icg and the distal breakpoint of Is(HSR;1E3)2Icg is desynapsed during the early pachytene stage and heterosynapsed at the midpachytene, as shown by electron microscopic analysis of synaptonemal complexes. The HSRs have no effect on the segregation of chromosome 1 in heterozygous mice. The lack of homosynapsis in the region under study causes chiasmata redistribution in heteromorphic bivalents. In normal males, single chiasmata are located in the medial part of the chromosome. In heterozygotes, this segment is heterosynapsed and unavailable for recombination. This leads to a significant decrease in the frequency of bivalents bearing single chiasmata. The total number of chiasmata per bivalent is much higher in heterozygous males than in normal ones. The recombination frequency between proximal markers fz and In also is higher in heterozygous animals. The increase in the total chiasma number in the heteromorphic bivalent is due to the addition of double chiasmata located mostly at precentromeric and pretelomeric regions of the chromosome.     

Segregation of tritium-labeled DNA at meiosis in chorthippus

A technique is described which gives well-defined label on meiotic chromosomes which incorporated tritiated thymidine two or more mitotic cycles prior to meiosis. It is shown that the distribution of label on meiotic chromosomes can be used to determine in which mitotic cycles label was incorporated. It is concluded that sister chromatid exchanges are common prior to meiosis, and that chiasmata do not show crossing-over of labeled material as is expected if the chiasmata are the result of breakage and rejoining or if chiasmata and crossovers are not related.     

Chiasma-based models of multilocus recombination: increased power for exclusion mapping and gene ordering

Cytological evidence indicates that the number of chiasmata which can occur on any given chromosome arm is limited. In this paper a Monte-Carlo simulation study is used to compare the power of a model of multilocus recombination parameterized in terms of chromosome-specific chiasma distributions with the traditional model parameterized by recombination fractions in adjacent intervals. Two specific gene mapping problems are considered: excluding a test locus from a given chromosome map and ordering a test locus with respect to a fixed map of syntenic marker loci. We show that the chiasma-based models require significantly fewer observations to exclude or order a test locus and that they are quite robust to errors in specifying the underlying true chiasma distribution.     

DISTRIBUTION OF TRITIUM-LABELED DNA AMONG CHROMOSOMES DURING MEIOSIS : I. Spermatogenesis in the Grasshopper

Thymidine-H³ of high specific activity was used to study the distribution of labeled chromatids during meiotic divisions in spermatocytes of a species of grasshopper (Orthoptera). The distribution is regularly semiconservative as has been shown previously for mitosis, i.e., all chromatids are labeled after incorporation of thymidine-H³ into DNA at premeiotic interphase. If incorporation occurs at the interphase preceding this one, the chromosomes arrive at meiotic divisions with the equivalent of one chromatid of each homologue labeled. Chromatid exchanges occur at a frequency which is very nearly that predicted on the assumption that each chiasma represents an exchange between homologous chromatids. However, the exchanges are randomly distributed among chromosomes in a size group, whereas chiasmata are not. A quantitative analysis of the frequency and pattern of exchanges indicates that most of these result from breakage and reciprocal exchange between homologous chromatids. Sister chromatid exchanges are much less frequent and may be limited to premeiotic stages.     

Features of crossing-over in virus-infected tomato

The evidence of increased crossing over rate in tomato hybrids infected with TAV (Tomato aspermy virus), PVX (Potato virus X), TMV (Tobacco mosaic virus), TMV+PVX indicates the recombinogenic effect of viral infection. Cytological studies of the early diakinesis in healthy and virus-infected tomato revealed significant changes in chiasma number and position. The most significant changes were established for bivalents with two interstitial chiasmata and with one terminal and one interstitial. The data obtained indicate redistribution of the chiasmata position and induction of additional exchanges. The virus-induced recombination is segment-specific and depends on the host plant genotype, virus infection and the interaction between them.     

Functional interactions between BLM and XRCC3 in the cell

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.     

Quantitative analysis of sister chromatid exchanges in the cell]

Assuming a random nature of distribution of sister chromatid exchanges (SCE) in a karyotype, the formulae have been obtained allowing the calculation of the number of SCE that are overlooked because of a limited resolving power of the SCE detection method. The results obtained mean that the actual number of SCE is more than the observed one, the part of overlooked exchanges being increased with the heightening of the SCE level. Taking into account overlook exchanges, the formula has been obtained that makes possible the calculation of the expected number of SCE observed in any group of chromosomes. These results were applied in the analysis of the SCE distribution among chromosomes. A better conformity has been obtained between the expected results and the observed ones, than under the assumption that the observed SCE are distributed in proportion to the lengths of chromosomes. The obtained formulae are of use in interpreting the lack of the observed SCE in small chromosomes and the excess of them in large ones.     

Models of multilocus recombination: nonrandomness in chiasma number and crossover positions.

Cytological evidence indicates that genetic interference can be partitioned into two empirical components: nonrandomness in the number of chiasmata that occur and nonrandomness in the locations of multiple chiasmata. Previous studies have incorporated the first effect into genetic models for analyzing multipoint data. An extension to this approach is presented which allows for the second component of interference by modeling the probability density function of the locations of multiple crossovers. Results of reanalyses of multilocus data for the Drosophila X chromosome show that models that incorporate only the first effect give a better fit to these data than do standard mapping functions and that the extended model significantly improved the fit by decreasing the predicted frequency of multiple crossovers in nearby regions. Our results demonstrate that chiasma-based models of multilocus recombination, which are unique in incorporating direct estimates of the frequency of multiple crossovers for a chromosome region, can provide a powerful and realistic means of accounting for genetic interference when applied to the problems of gene localization, locus ordering, and exclusion mapping.     

Characterization of the sequence element directing translation reinitiation in RNA of the calicivirus rabbit hemorrhagic disease virus

The calicivirus minor capsid protein VP2 is expressed via reinitiation of protein synthesis after termination of translation of the preceding VP1 gene. A sequence element of about 80 nucleotides denoted "termination upstream ribosomal binding site" (TURBS) (25) is crucial for reinitiation. Deletion mapping in the TURBS of a rabbit calicivirus identified two short sequence motifs that were crucial for VP2 expression. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18S rRNA. Single-residue exchanges in this motif severely impaired reinitiation when they affected the putative rRNA binding, whereas an exchange preserving complementarity had only a minor effect. Single exchanges in motif 2 were rather well tolerated, but the introduction of double exchanges almost blocked VP2 expression. In contrast, the deletion analyses showed that the RNA between the two motifs is of minor importance. The distance between motif 2 and the start site was found to be important, since deletions of increasing length in this sequence or upstream positioning of the start codon reduced VP2 expression stepwise to low levels, whereas multiple-nucleotide exchanges in this region were tolerated. The low flexibility of the arrangement of TURBS motif 2 and the start codon stand in marked contrast to the requirements with regard to the location of the stop codon of the preceding VP1 gene, which could be moved far downstream with continuous reduction, but without loss, of VP2 translation. The sequence mapping resulted in a refined model of the reinitiation mechanism leading to VP2 expression.     

Induction of sister-chromatid exchanges in cultured human cells by an organophosphorous insecticide: malathion.

Because malathion is a widely used organophosphorous insecticide, the effects of non-toxic concentrations (2.5--40 micrograms/ml) on sister-chromatid exchange (SCE) frequencies were determined. Human fetal fibroblasts were exposed once or twice to malathion, with 20 h between exposures. A single exposure to a concentration of 40 micrograms/ml resulted in a highly significant increase in the number of SCEs. After a double exposure, a concentration of 20 micrograms/ml induced an even greater increase in SCE frequencies. Comparison of Sce frequencies after single and double exposures indicated a cumulative effect; the number of exchanges at concentrations of 5 micrograms/ml or higher was significantly greater after the double exposure. An analysis of SCEs by chromosome group showed that exchanges were distributed approximately according to chromosome length.     

Mitotic chiasmata in human diplochromosomes

Summary Three placental tissue cultures of spontaneous human abortions showed an unusually high frequency of metaphases with diplochromosomes. In 62 such cells, nine configurations were interpreted as mitotic chiasmata between the two sister chromosomes of a diplochromosome. One U-type exchange between two sister chromosomes was also found. This differs significantly from the 1:1 ratio of adjacent and alternate exchanges in translocations, thus supporting the idea that mitotic chiasmata are in principle different from chromatid translocations. The hypothesis is put forward that the frequency of homologous exchanges is determined by the intimacy of pairing which ranges from meiotic pairing through sister chromatid association, through sister chromosome association in diplochromosomes to accidental pairing of homologous regions in diploid cells.     

Chiasma frequency and distribution in a maize family segregating for k10 and trisomy 10

Chiasma frequency was determined from total chiasma counts, and the distribution of these exchanges was determined by the ratio of proximal to distal chiasmata. No effect of trisomy 10 could be demonstrated. Confirmation was obtained of earlier work showing more proximal and fewer distal chiasmata in K10 plants than in controls. However, diakinesis data failed to confirm the ability of K10 to increase total chiasmata as suggested from metaphase I results.     

Localization of genes on mouse chromosomes by comparing the genetic map and chiasma distribution

It is possible to determine chromosomal position of the genes having definite genetic localization, using chiasmata distribution along the chromosome. This approach was used for subchromosomal mapping of the house mouse genes. It was shown that the chiasmata distributions are different for different chromosomes. The positions of some gene on chromosomes 1, 2, 17 and 19 were determined. The results coincide with those on subchromosomal gene mapping using chromosome translocations and in situ hybridization.