Estimating Meiotic Exchange Patterns from Recombination Data: An Application to Humans

We present analytical methods to estimate the recombinational history of chromosomes in a human population. Our analysis, similar to those utilized in Drosophila, can be used to construct meiotic maps based upon crossover frequencies observed in family data. We apply this method of exchange estimation to a population of paternally and maternally inherited chromosomes 21. The patterns of chromosomal exchange estimated by this type of analysis are comparable to those obtained by the more technically difficult method of cytologically counting chiasmata among human male meiotic events (sperm). This type of analysis can be applied to both male and female meiosis, circumventing many technical problems inherent to cytological counting. Moreover, the distribution of exchange locations along a chromosome for each exchange type (i.e., single, double, or triple exchanges) can be examined individually, an advantage compared to examination of genetic maps that only provide a summary of these distributions. We discuss how this analysis can be used to examine various assumptions concerning meiotic exchange in humans and investigate properties of the analysis that contribute to the accuracy of the results.     

Synaptonemal complexes in mammals. I. The nature and mechanism of the formation of centric chromosome fusions (Robertsonian translocations)

The nature and mechanism of formation of chromosomes' centric fusion were studied in mammals using the spreading method and silver staining of pachytene chromosomes. The male mice carriers of Robertsonian translocations Rb (8, 17) 1IEM, animals with standard karyotypes and F1 hybrids were used. It was concluded that the centric fusions resulted from translocation events and do not differ from other simmetric structural rearrangements of the exchange types.     

Chiasmata and chromosome breakages are related to crossing over in Drosophila ananassae males.

A cytogenetic analysis of male crossing over in Drosophila ananassae revealed that cytological exchanges resulted in genetic crossing over, and that chiasma frequency and the genetic recombination correlated positively in chromosomes 2 and 3. Furthermore, the frequency of chromosome breakages correlated positively with chiasma frequency. Paracentric inversion heterozygosity had no detectable influence on the chromosome pairing or exchange events within the inversion loop at meiosis. Scoring of the chiasma demonstrated that males homozygous for the previously mapped enhancers of male crossing over had low frequencies of chiasmata, whereas higher frequencies of chiasmata were observed in males heterozygous for enhancers. The results presented here indicate that the genetic factors controlling male crossing over are involved in the origin of chromosome breakages and in exchange events.     

Sex preferences in Jeju pony foals (Equus caballus) for mutual grooming and play-fighting behaviors

We investigated mutual grooming by Jeju pony (Equus caballus) foals to determine whether male foals preferentially interact with potential future sexual partners or competitors. We predicted that relative to female foals, male foals would exchange grooming more frequently with young mares and that in general, foals would mutually groom more frequently with the opposite sex rather than the same sex. Observing 53 foals between April and October 1998, we recorded 113 mutual grooming events. Male foals exchanged grooming with yearling mares more frequently than with their mother, while female foals exchanged grooming with their mother more frequently than with yearling mares. Contrary to the prediction, foals were not more likely to mutually groom with a foal of the opposite sex than with a foal of the same sex. In our study, 21 instances of play-fighting behavior followed mutual grooming between peers. Relative to intersexual grooming events, play-fighting was more likely to follow intrasexual mutual grooming, and male foals were much more likely to play fight than female foals. These results provide evidence that Jeju pony foals develop and maintain social relationships at the earliest stage of their lives. We suggest that early social experiences might influence social bonding later when the male foal begins to form a harem after separation from its mother.     

Meiosis: how male flies do meiosis

In meiosis in male fruitflies, chromosome pairing events do not facilitate genetic exchange, but rather create bivalents that can be sequestered to discrete pockets of the prophase nucleus. This assignment of homologs to a common pocket likely facilitates the orientation of homologous centromeres to opposite poles.     

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.     

Genetic control of mammalian meiotic recombination. I. Variation in exchange frequencies among males from inbred mouse strains

Genetic background effects on the frequency of meiotic recombination have long been suspected in mice but never demonstrated in a systematic manner, especially in inbred strains. We used a recently described immunostaining technique to assess meiotic exchange patterns in male mice. We found that among four different inbred strains--CAST/Ei, A/J, C57BL/6, and SPRET/Ei--the mean number of meiotic exchanges per cell and, thus, the recombination rates in these genetic backgrounds were significantly different. These frequencies ranged from a low of 21.5 exchanges in CAST/Ei to a high of 24.9 in SPRET/Ei. We also found that, as expected, these crossover events were nonrandomly distributed and displayed positive interference. However, we found no evidence for significant differences in the patterns of crossover positioning between strains with different exchange frequencies. From our observations of >10,000 autosomal synaptonemal complexes, we conclude that achiasmate bivalents arise in the male mouse at a frequency of 0.1%. Thus, special mechanisms that segregate achiasmate chromosomes are unlikely to be an important component of mammalian male meiosis.     

RAD52-Independent Mitotic Gene Conversion in SACCHAROMYCES CEREVISIAE Frequently Results in Chromosomal Loss

We have examined spontaneous, interchromosomal mitotic recombination events between his4 alleles in both Rad+ and rad52 strains of Saccharomyces cerevisiae. In Rad+ strains, 74% of the His+ prototrophs resulted from gene conversion events without exchange of flanking markers. In diploids homozygous for the rad52-1 mutation, the frequency of His+ prototroph formation was less than 5% of the wild-type value, and more than 80% of the gene conversion events were accompanied by an exchange of flanking markers. Most of the rad52 intragenic recombination events arose by gene conversion accompanied by an exchange of flanking markers and not by a simple reciprocal exchange between the his4A and his4C alleles. There were also profound effects on the kinds of recombinant products that were recovered. The most striking effect was that RAD52-independent mitotic recombination frequently results in the loss of one of the two chromosomes participating in the gene conversion event.     

Isolation of renal brush-border membrane vesicles by a low-speed centrifugation; effect of sex hormones on Na+-H+ exchange in rat and mouse kidney

Na+-H+ exchange in rat and mouse renal brush-border membrane vesicles was studied by fluorescence quenching of the delta pH indicator, acridine orange. Brush-border membrane vesicles were isolated by a modified Mg/EGTA-precipitation method at low speed centrifugation (8000 X g). The enzymatic characteristics of these membrane vesicles were similar to those obtained by the original high-speed centrifugation method (Biber et al. (1981) Biochim. Biophys. Acta, 647, 169-176). The rates of Na+-H+ exchange in renal brush-border membrane vesicles from male and female rats were similar. Neither ovariectomy nor treatment of ovariectomized rats with estradiol or testosterone changed the activity of Na+-H+ exchanger. The rates of Na+-H+ exchange in the mouse were smaller than in the rat indicating the existence of species differences. Na+-H+ exchange in mouse renal brush-border membranes exhibit strong sex differences, the rates in the male being higher than in the female. Castration of male mice led to a decrease in Na+-H+ exchange to values found in females. Treatment of castrated mice with estradiol had no effect. In contrast, treatment with testosterone increased the rate of the exchanger by more than 100%. The effect of testosterone was restricted to the Vmax of the Na+-H+ exchanger, whereas the apparent Km for Na+ remained unchanged. Na+-dependent D-glucose transport in mouse renal luminal membranes exhibited also sex differences due to the potent stimulatory effect of testosterone. Therefore, Na+-H+ exchange and Na+-dependent D-glucose transport in the mouse kidney are under control of androgen hormones. This effect could be in close connection with the wellknown renotropic action of androgens in the mouse.     

Hybrid dysgenesis in Drosophila: The mechanism of T-007-induced male recombination

Summary The term hybrid dysgenesis describes a syndrome of genetic effects which sometimes results when Drosophila melanogaster from wild populations are outcrossed; this syndrome often includes male recombination as well as enhanced rates of genic and chromosomal mutation, sterility, and transmission ratio distortion. In this study, we have examined the mechanism of T-007-induced male recombination by genetically characterizing third chromosomes generated by an exchange in a well-marked euchromatic region. Most recombinant chromosomes were sequentially normal, and no recessive lethal events at the point of exchange were recovered. The results demonstrate that although some recombinants may be generated by nonhomologous chromosome (or chromatid) breakage and reunion, the predominant effect of T-007 is through an enhanced rate of normal mitotic exchange. The rate of mitotic exchange is also increased by ionizing radiation and chemical mutagens; we suggest that the common factor in all three cases is the induction of single strand breaks.     

The Relationship between P Elements and Male Recombination in Drosophila Melanogaster

P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.     

PHYLOGENETIC ESTIMATION OF PLASMID EXCHANGE IN BACTERIA

The existence of differential horizontal gene transfer may be assessed by comparing the phylogenetic trees derived from two different genes. We use this concept to estimate quantitatively the amount of plasmid exchange that has occurred in a bacterial population. By means of computer simulations we studied the effect of gene transfer on the topological distortion between two phylogenetic trees: one obtained from an euchromosomal gene and another from a plasmid-borne sequence, which may be subjected to horizontal transfer. The basic assumptions of our simulations were (a) that plasmid exchange had occurred recently (after the last population split); and (b) that either the amount of chromosomal horizontal exchange was negligible or that it was only a fraction of the amount of plasmid exchange in which case we will be estimating relative amounts of plasmid transfer. We found that the topological difference between two such trees is a function of the number of plasmid exchange events that have occurred. It can be explained by a logistic model that relates the average distortion index between two trees (d_T) to the number of transfer events (x). The behavior remains the same under different conditions that were tested (symmetry of the topology, number of taxa in the tree, effect of reconstruction errors, mutation after plasmid transfer). We have also tried our method on empirical data from the literature and estimated the amount of gene transfer that may have occurred among Sym plasmids in agricultural field populations of Rhizobium leguminosarum biovar phaseoli. We found that between 15.77 to 29.98% of all genetic types in these populations have been either the source or the target of a plasmid transfer event. When the comparisons were made among trees derived exclusively from plasmid probes this value dropped to 2.00%. Phylogenetic trees derived from symbiotic and nonsymbiotic sequences were also used to infer the number of gene transfer events among 11 isolates from R. galegae. The estimated number of transfer events of symbiotic sequences was 10.515 (although we do not know out of how many genetic types). We concluded that intraspecific transfer of symbiotic sequences is widespread in these two species of the genus Rhizobium.     

Differential histone modifications mark mouse imprinting control regions during spermatogenesis

Only some imprinting control regions (ICRs) acquire their DNA methylation in the male germ line. These imprints are protected against the global demethylation of the sperm genome following fertilisation, and are maintained throughout development. We find that in somatic cells and tissues, DNA methylation at these ICRs is associated with histone H4-lysine-20 and H3-lysine-9 trimethylation. The unmethylated allele, in contrast, has H3-lysine-4 dimethylation and H3 acetylation. These differential modifications are also detected at maternally methylated ICRs, and could be involved in the somatic maintenance of imprints. To explore whether the post-fertilisation protection of imprints relates to events during spermatogenesis, we assayed chromatin at stages preceding the global histone-to-protamine exchange. At these stages, H3-lysine-4 methylation and H3 acetylation are enriched at maternally methylated ICRs, but are absent at paternally methylated ICRs. H4 acetylation is enriched at all regions analysed. Thus, paternally and maternally methylated ICRs carry different histone modifications during the stages preceding the global histone-to-protamine exchange. These differences could influence the way ICRs are assembled into specific structures in late spermatogenesis, and may thus influence events after fertilisation.     

Kinetic measurements of gas exchange in the intact pulmonary microcirculation.

The kinetics of gas exchange are monitored in an isolated perfused lung preparation contained within a plethysmograph. The lungs are perfused with buffer, and there is no gas exchange until a 2.0-ml bolus of reactant is injected into the perfusion system. Subsequent gas exchange produces a pressure transient that is related to the corresponding volume of exchanged gas. The observed rate of volume change is the result of two separate processes: 1) the rate of gas exchange during transit through the capillary bed and 2) the distribution of vascular transit times between the point of injection and the capillary bed. The latter is assessed by a control injection containing a dissolved inert gas that is liberated in the alveoli as the bolus enters the capillary bed. Analysis of the experimental curves permits the separation of these two processes. A model of exchange kinetics indicates that this method has the capability of measuring kinetic events occurring during gas exchange in the microcirculation under physiological conditions.     

Biological Ion Exchanger Resins: I. Quantitative Electrostatic Correspondence of Fixed Charge and Mobile Counter Ion

Utilizing Escherichia coli as the prototype of an ion-accumulating cell, the ion exchange isotherm is introduced as a concise method of characterizing biological ion exchange events. The ion exchange isotherm for the alkali cation exchange, K ↔ Na, is described. The total charge profile of this bacterium is compiled and compared for bacteria in the Na form and in the K form. Macromolecule fixed charge was found to provide 80% of the counter ions that pair with potassium. Therefore, in its physiological state, 80% of the cell potassium in E. coli is associated with an ion exchange site on a macromolecule. The primary cation exchange sites are found to be about equally divided between carboxylate and phosphate sites indicating that E. coli is a bifunctional resin with respect to cation exchange. During substrate-dependent cation accumulation (“active transport”), phosphate esters and organic acids were shown to accumulate. One may conclude that the role of intermediate metabolism in “active transport” is to increase the ion exchange capacity of the biological resin by the production of charged metabolites that sorb to the framework of the resin.     

A selective method for studying primary sex chromosome non-disjunction in females and males and X-Y exchange in males of Drosophila melanogaster including a demonstration of euchromatic X-Y exchange

A selective method was developed, based on negative complementation of the Abruptex alleles of the Notch focus, for studying primary sex chromosome non-disjunction in females and males of Drosophila melanogaster and X-Y exchange in males. The results show that the frequency of primary non-disjunction of structurally normal X chromosomes was lower than the frequency of X-derY non-disjunction in males. Double exchange between the X and the derY chromosome in the male occurs with a frequency of at least 0.091%. Single exchanges are naturally expected to occur with even higher frequency. Exchanges were interestingly at least partly of cuchromatic nature. The origin of these exchanges is at least partly of gonial origin.     

Evidence of P-element-induced sister-chromatid exchange in a ring-X chromosome in Drosophila, with implication for a high rate of formation of hybrid elements

Activation of a single incomplete P element induces recombination at a rate of approximately 0.5-1% in the male germline of Drosophila. Male recombination rises by an order of magnitude to approximately 20% if homologous P elements are involved. The high rate of recombination suggests the possibility that sister-chromatid exchange (SCE) might be elevated to a similar extent, since homologous P elements must always be present in sister chromatids. This possibility was tested by recombining a single P element onto a ring-X chromosome and using sex-ratio distortion to measure the loss of the ring-X due to SCE in the male germline. The results confirmed a rate of loss comparable to that expected with homologous elements, although the rate of loss was variable. Both SCE and recombination results are consistent with the "hybrid element insertion" model, in which the left and right ends from different elements associate, providing that insertion occurs preferentially in the vicinity of a P-element end. For autosomes, hybrid element formation may thus occur at a much higher rate than the 0.5-1% implied by single element recombination, with only a small minority of hybrid element excision events being resolved by recombination.     

A plasmid model to study genetic recombination in yeast

Chimeric plasmids have been constructed containing two heteroallelic mutant copies of the yeast HIS3 gene as an inverted repetition. Intramolecular exchange events between these two allelic mutant copies are capable of generating a wild-type allele. Plasmids containing two mutant heteroalleles have been transformed into appropriate his3^? yeast strains, and the frequency of exchange events generating His⁺ prototrophs has been measured during mitotic division. After 20 generations of growth under nonselective conditions, between 0.1 and 1 % of the transformed yeast cells become His⁺ prototrophs. This percentage decreases at least ten-fold in a strain with a rad52 mutation. Plasmid molecules having undergone exchange events have been isolated from yeast cells and have been examined after transfer to Escherichia coli. Physical examination shows that less than 10 % of the plasmids having undergone genetic exchange have also undergone an internal reciprocal recombination event as evidenced by reorientation of linked restriction sites. The remainder of the plasmids having undergone genetic exchange do not exhibit reciprocal recombination. Characterization of the individual allelic copies within a plasmid having undergone exchange reveals that in 24 of 25 examples only one of the two HIS3 copies has become wild type, and that either copy is equally likely to become wild type. We conclude that the model plasmid we have constructed undergoes intramolecular genetic exchange events and will be useful for studying genetic recombination.     

A cytogenetic study of development in mechanically disrupted pairs of Tetrahymena thermophila

We examined the nuclear behavior of mating Tetrahymena cells that had been mechanically disrupted at various times throughout conjugation. Disruption was achieved by agitating conjugating Tetrahymena in the presence of 0.1-3 mm glass beads. Two minutes of agitation with 1 mm beads yielded optimal pair disruption (70%) with high viability (92%). Disrupting pairs between 0-4.7 h after the initiation of mating produced mostly disrupted conjugants in which development was aborted. However, as many as 20% of these early disrupted conjugants completed development even without their mating partners. After 5 h the percentage of disrupted conjugants completing development increased dramatically, reaching 80% by 6.7 h. These results support a model suggesting that events associated with nuclear exchange and fusion 5 h into conjugation trigger a commitment to completion of the postzygotic developmental program. The early conjugants that completed development following disruption suggest that development can be sustained even in the absence of a mating partner. This represents a novel method of bringing the micronuclear genome into macronuclear expression with minimal cytoplasmic exchange between partners. We discuss these results in light of a model relating cortical and nuclear signaling events that reciprocally drive conjugal development.