Single Crossovers Research Articles

Mobile Element Integration Reveals a Chromosome Dimer Resolution System in Legionellales.

In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The dimers must be resolved before cell separation to ensure genomic stability and cell viability. Dimer resolution is achieved by the broadly conserved dif/Xer system, which catalyzes one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here, we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (here named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Finally, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids. IMPORTANCE The maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers after they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif within the same molecule, leading to rapid site-specific recombination and conversion back into chromosome monomers. The apparent absence of chromosome dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens.

Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster.

Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. Here, we test for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. We show that winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. We ascribe the observed differences to indirect selection on recombination caused by directional selection on desiccation tolerance. Our findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8-10 generations).

Global Assessments of the HY-2B Measurements and Cross-Calibrations with Jason-3

The HY-2B satellite was successfully launched on 25 October 2018. One of the main payloads of the HY-2B was a radar altimeter. In the present study, the quality of the HY-2B along-track sea surface heights (SSH), significant wave heights (SWH), and sea surface wind speeds (SSWS) were assessed, including their precision and accuracy. In order to achieve this goal, the mono-mission metrics of the HY-2B were analyzed and compared with those of the Jason-2 and Jason-3 over the same periods of time. The results of both direct comparison and cross comparison methods were presented in this study. The comparison results indicated that the quality of the HY-2B satellite’s geophysical data records (GDRs) data was excellent, with 95% of the sea surfaces effectively observed between 82 degrees north and south latitudes. In addition, the standard deviation of the sea level anomalies (SLA) at the single mission crossovers was 4.6 cm to 5.8 cm, and at the dual-crossovers with Jason-3, the standard deviation was determined to be 5.1 cm to 5.8 cm. The accuracy levels of the significant wave heights and products of the HY-2B satellite radar altimeter were observed to be greater than 0.3 m and 1.4 m/s (STD), respectively. Therefore, it was concluded in this study that the data quality and system performance of the HY-2B satellite were excellent and stable, and could be widely utilized in such fields as global sea-level change monitoring, wave numerical assimilation predictions etc.

Pervasive gene conversion in chromosomal inversion heterozygotes.

Chromosomal inversions shape recombination landscapes, and species differing by inversions may exhibit reduced gene flow in these regions of the genome. Though single crossovers within inversions are not usually recovered from inversion heterozygotes, the recombination barrier imposed by inversions is nuanced by noncrossover gene conversion. Here, we provide a genomewide empirical analysis of gene conversion rates both within species and in species hybrids. We estimate that gene conversion occurs at a rate of 1×10-5 to 2.5×10-5 converted sites per bp per generation in experimental crosses within Drosophila pseudoobscura and between D.pseudoobscura and its naturally hybridizing sister species D.persimilis. This analysis is the first direct empirical assessment of gene conversion rates within inversions of a species hybrid. Our data show that gene conversion rates in interspecies hybrids are at least as high as within-species estimates of gene conversion rates, and gene conversion occurs regularly within and around inverted regions of species hybrids, even near inversion breakpoints. We also found that several gene conversion events appeared to be mitotic rather than meiotic in origin. Finally, we observed that gene conversion rates are higher in regions of lower local sequence divergence, yet our observed gene conversion rates in more divergent inverted regions were at least as high as in less divergent collinear regions. Given our observed high rates of gene conversion despite the sequence differentiation between species, especially in inverted regions, gene conversion has the potential to reduce the efficacy of inversions as barriers to recombination over evolutionary time.

Third Chromosome Balancer Inversions Disrupt Protein-Coding Genes and Influence Distal Recombination Events in Drosophila melanogaster.

Balancer chromosomes are multiply inverted chromosomes that suppress meiotic crossing over and prevent the recovery of crossover products. Balancers are commonly used in Drosophila melanogaster to maintain deleterious alleles and in stock construction. They exist for all three major chromosomes, yet the molecular location of the breakpoints and the exact nature of many of the mutations carried by the second and third chromosome balancers has not been available. Here, we precisely locate eight of 10 of the breakpoints on the third chromosome balancer TM3, six of eight on TM6, and nine of 11 breakpoints on TM6B. We find that one of the inversion breakpoints on TM3 bisects the highly conserved tumor suppressor gene p53—a finding that may have important consequences for a wide range of studies in Drosophila. We also identify evidence of single and double crossovers between several TM3 and TM6B balancers and their normal-sequence homologs that have created genetic diversity among these chromosomes. Overall, this work demonstrates the practical importance of precisely identifying the position of inversion breakpoints of balancer chromosomes and characterizing the mutant alleles carried by them.

Sex chromosome synapsis and recombination in male guppies.

Guppy X and Y chromosomes represent an early stage in sex chromosome divergence. Synapsis and recombination between X and Y chromosomes attract special attention because recombination suppression promotes their differentiation, but previous studies have given contradictory results. Linkage analysis indicated that recombination between X and Y was extremely rare (<10%) and occurred in the medial part of the Y chromosome, while cytological analysis demonstrated regular association between the distal ends of the X and Y at diakinesis. In this study, we examine pairing and recombination between X and Y chromosomes using immunolocalization of MLH1 to mark recombination nodules, and genomic in situ hybridization with a male DNA probe to identify the Y-specific heterochromatic region. Pairing between X and Y is initiated distally. Single crossovers were detected in 87% of XY synaptonemal complexes, most often in the distal region and less frequently in a median position indicating that end-to-end associations between X and Y are chiasmatic. Thus, we suggest that the very low frequency of recombination detected by linkage analysis in a previous study resulted from a lack of informative markers in distal regions.

GISH INVESTIGATION OF CROSSOVER EVENTS DURING MEIOSIS OF INTERSPECIFIC HYBRIDS OF LILY

Crossover not only establishes proper segregation of omologous/homoeologous chromosomes during meiosis I, but also promotes genetic diversity though creating new recombination of maternal and paternal alleles, which is essential for introgression breeding. In this paper, crossovers between non-sister chromatids were observed during meiosis of interspecific hybrids of Lilium longiflorum × Asiatic (LA) using genomic in situ hybridization (GISH). Results showed that different types of crossing over events, viz. single, two strand double, three strand double, four strand double crossovers, occurred between non-sister chromatids during meiosis of selected interspecific lily hybrids. From an analysis of a total of 637 pairs of half-bivalents it was evident that a large majority (65%) was two strand single crossovers, 5.5% were two strands double, 3.0% three strand double, 9.3% four strand double and 17.3% were multiple crossing over events. In one specific genotype which showed the existence of a reciprocal translocation, the frequency of multiple crossovers was relatively high, which might alter the map distances of markers on the translocated chromosomes.

Further evidence for the theory that crossover interference in &amp;lt;i&amp;gt;Drosophila melanogaster&amp;lt;/i&amp;gt; is dependent on genetic rather than physical distance between adjacent crossover points

The effect of heat shock on certain meiotic parameters in Drosophila melanogaster was studied in the cv – v – f region of the X chromosome of females homozygous for the mus309 mutation, deficient in DNA double-strand break repair, or those of wild type. The heat shock in the wild females caused the frequencies of the single crossovers and double crossovers and all the map lengths to decrease while crossover interference remained unchanged. In the mus309 mutants all parameters, crossover interference included, remained unchanged despite the heat shock treatment. However, the mus309 mutation had a significant effect on all meiotic parameters both in the females not given the heat shock and in the heat shocked females with the exception that the recombination frequency of the v and f markers was the same in both genotypes in the females not given the heat shock. It seems that the heat shock treatment has an effect on crossing over which is independent on the mus309 gene and affecting the occurrence of crossing over itself. On the other hand, the mus309 gene has an effect on crossing over which is independent of the heat shock treatment and affects some precondition of crossing over. This precondition is probably the choice between two routes of the repair of double-strand DNA breaks known to be controlled by the mus309 gene. As explained in the discussion, the results are in accordance with the genetic models of interference in which interference depends on genetic distance between the crossover points, but in contradiction with physical models where interference is dependent on physical distance between the crossover points.

Further evidence for the theory that crossover interference in &amp;lt;i&amp;gt;Drosophila melanogaster&amp;lt;/i&amp;gt; is dependent on genetic rather than physical distance between adjacent crossover points

The effect of heat shock on certain meiotic parameters in Drosophila melanogaster was studied in the cv – v – f region of the X chromosome of females homozygous for the mus309 mutation, deficient in DNA double-strand break repair, or those of wild type. The heat shock in the wild females caused the frequencies of the single crossovers and double crossovers and all the map lengths to decrease while crossover interference remained unchanged. In the mus309 mutants all parameters, crossover interference included, remained unchanged despite the heat shock treatment. However, the mus309 mutation had a significant effect on all meiotic parameters both in the females not given the heat shock and in the heat shocked females with the exception that the recombination frequency of the v and f markers was the same in both genotypes in the females not given the heat shock. It seems that the heat shock treatment has an effect on crossing over which is independent on the mus309 gene and affecting the occurrence of crossing over itself. On the other hand, the mus309 gene has an effect on crossing over which is independent of the heat shock treatment and affects some precondition of crossing over. This precondition is probably the choice between two routes of the repair of double-strand DNA breaks known to be controlled by the mus309 gene. As explained in the discussion, the results are in accordance with the genetic models of interference in which interference depends on genetic distance between the crossover points, but in contradiction with physical models where interference is dependent on physical distance between the crossover points.

Effect of temperature on crossing over in the &amp;lt;i&amp;gt;mus&amp;lt;/i&amp;gt;309 mutant, deficient in DNA double-strand break repair, of &amp;lt;i&amp;gt;Drosophila melanogaster&amp;lt;/i&amp;gt; suggests a mechanism for crossover interference

Crossing-over frequencies, crossover interference, recombination frequencies and map distances were compared in the cv-v-f region of the X chromosome of Drosophila melanogaster in females bearing either wild type 3rd chromosomes (control) or having the DNA double-strand break repair deficient mus309D2/mus309D3 mutant constitution in the 3rd chromosomes (experiment), and raised in three different temperatures viz. 18。C, 25。C and 29。C. In addition, the fecundity of the females was also measured. In the control crosses none of the mean values of the parameters measured was dependent on the temperature, whereas in the experimental crosses all the parameters, except for the frequency of true single crossovers in the cv-v interval, the recombination frequency of the v and f markers, and the coefficient of coincidence, changed due to the effect of temperature. When comparing the genotypes studied, a significant difference between them was observed in all the parameters measured, apart from the frequency of the true single cross-overs in the cv-v interval. These results support the counting number model of the mechanism of interference based on the genetic distance, but are in contradiction with the models based on physical distance.

Derivation and Characterization of Recombinants of the Lr54/Yr37 Translocation in Common Wheat

The wild relatives are an important source of new genes for the genetic improvement of wheat. Leaf and stripe rust resistance genes Lr54 and Yr37 occur on an Aegilops kotschyi-derived chromosomal translocation that had apparently replaced wheat chromosome arm 2DL. The alien chromatin also includes the locus of a gene for reduced plant height (H), which appears to be different from Rht8 on chromosome arm 2DS. The introgressed genes were mapped relative to homoeologous wheat marker loci following the induction of chromosome pairing in translocation heterozygotes that lacked the Ph1 locus. Ten recombined Lr54/Yr37 translocation chromosomes were derived and characterized with micro- satellite, AFLP and SCAR markers. The data suggested that there was significant homoeology between the full-length translocated segment and the wheat 2DL chromosome arm. The recombined translocations apparently resulted from single crossovers during which the distal end of the long arm of the translocation chromosome was replaced with wheat chroma- tin. Recombinant (Lr54/Yr37-74) retained the least alien chromatin and both resistance genes, yet had lost the reduced plant height gene. A polymorphic AFLP fragment was converted into a dominant SCAR marker to detect rec. #74. In addition three wheat microsatellite loci that map to the introgressed region provide a useful recessive marker system to detect Lr54/Yr37. The shortened translocation could be useful in breeding and may be used for continued, closer mapping of the resistance genes.

The effect of the mus309 mutation, defective in DNA double-strand break repair, on crossing over in Drosophila melanogaster suggests a mechanism for the centromere effect of crossing over

The mus309 gene in Drosophila melanogaster encodes a RecQ helicase which is involved in DNA double-strand break (DSB) repair. In a brood pattern analysis, it was observed that in mus309 mutant females, the frequency of single crossovers in the central cv-v interval of the X chromosome was reduced in young females but returned to the level of the wild type control as the females aged. In the proximal v-f interval, the frequency of single crossovers was increased during the entire experimental period. In particular, it was observed that the frequency of double crossovers, as well as the coefficient of coincidence first increased but then gradually decreased, finally reaching the level of the control flies, as the females aged. Map distances increased due to the mus309 mutation in both gene interval studies, but they did not change as the females aged, a result suggesting that the mus309 gene controls the distribution of DSBs to be repaired as crossovers instead of non-crossovers. The results suggest a mechanism for the centromere effect of crossing over in Drosophila, viz the fact the frequency of meiotic crossing over reduces with the age of the female, and that the reduction is more pronounced the closer the interval is to the proximal heterochromatin of the chromosome arm. According to the model suggested, the centromere effect is simply a matter of the balance between different pathways of the repair of the DSBs of DNA.

The effect of themus309mutation, defective in DNA double-strand break repair, on crossing over inDrosophila melanogastersuggests a mechanism for interference

The mus309 gene in Drosophila melanogaster encodes a RecQ helicase which is involved in DNA double-strand break (DSB) repair. In a brood pattern analysis, it was observed that in mus309 mutant females, the frequency of single crossovers in the central cv - v interval of the X chromosome was decreased in young females but returned to the level of the wild type control as the females aged. In the proximal v - f interval, the frequency of single crossovers was increased during the whole experimental period. In particular, it was observed that the frequency of double crossovers, as well as the coefficient of coincidence first increased but then gradually decreased, finally reaching the level of the control flies, as the females aged. Map distances increased due to the mus309 mutation in both gene interval studies, but they did not change as the females aged, a result suggesting that the mus309 gene controls the distribution of DSBs to be repaired as crossovers instead of non-crossovers. The results are consistent with the hypothesis that in general the DSBs are initially independently distributed on the chromosome but non-randomly repaired so that the distribution of crossovers in the mutant flies becomes uniform, but uneven in wild-type flies. The results are consistent with the counting number model of crossover interference, based on genetic distance. On the other hand, the data are not consistent with the reaction-diffusion model based on physical distance. Consequently, the view that crossover interference in Drosophila is tightly tied to genetic distance is supported.

Single-crossover recombination in discrete time

Modelling the process of recombination leads to a large coupled nonlinear dynamical system. Here, we consider a particular case of recombination in discrete time, allowing only for single crossovers. While the analogous dynamics in continuous time admits a closed solution (Baake and Baake in Can J Math 55:3-41, 2003), this no longer works for discrete time. A more general model (i.e. without the restriction to single crossovers) has been studied before (Bennett in Ann Hum Genet 18:311-317, 1954; Dawson in Theor Popul Biol 58:1-20, 2000; Linear Algebra Appl 348:115-137, 2002) and was solved algorithmically by means of Haldane linearisation. Using the special formalism introduced by Baake and Baake (Can J Math 55:3-41, 2003), we obtain further insight into the single-crossover dynamics and the particular difficulties that arise in discrete time. We then transform the equations to a solvable system in a two-step procedure: linearisation followed by diagonalisation. Still, the coefficients of the second step must be determined in a recursive manner, but once this is done for a given system, they allow for an explicit solution valid for all times.

Analysis of the meiosis in the F1 hybrids of Longiflorum × Asiatic (LA) of lilies (Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modern lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the F(1) hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC(1) progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC(1) progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F(1) hybrids of Longiflorum x Asiatic (LA) hybrids in Lilium. Therefore, the meiotic process of ten F(1) LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that: at metaphase I, the homoeologous chromosome pairing among different F(1) hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell (PMC), and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all bivalents were disjoined and most univalents were divided. Both the disjoined bivalents (half-bivalents) and the divided univalents (sister chromatids) moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the F(1) LA hybrids and the variation of their BC(1) progenies, but also provided a new method to analyze crossover types in other F(1) interspecific hybrids as well.

Single-Crossover Dynamics: Finite versus Infinite Populations

Populations evolving under the joint influence of recombination and resampling (traditionally known as genetic drift) are investigated. First, we summarize and adapt a deterministic approach, as valid for infinite populations, which assumes continuous time and single crossover events. The corresponding nonlinear system of differential equations permits a closed solution, both in terms of the type frequencies and via linkage disequilibria of all orders. To include stochastic effects, we then consider the corresponding finite-population model, the Moran model with single crossovers, and examine it both analytically and by means of simulations. Particular emphasis is on the connection with the deterministic solution. If there is only recombination and every pair of recombined offspring replaces their pair of parents (i.e., there is no resampling), then the expected type frequencies in the finite population, of arbitrary size, equal the type frequencies in the infinite population. If resampling is included, the stochastic process converges, in the infinite-population limit, to the deterministic dynamics, which turns out to be a good approximation already for populations of moderate size.

Phenotypic Variation and Natural Selection at Catsup, a Pleiotropic Quantitative Trait Gene in Drosophila

Quantitative traits are shaped by networks of pleiotropic genes . To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the level of quantitative trait nucleotides (QTNs). Catecholamines up (Catsup), which encodes a negative regulator of tyrosine hydroxylase , the rate-limiting step in the synthesis of the neurotransmitter dopamine, is a pleiotropic quantitative trait gene in Drosophila melanogaster. We used association mapping to determine whether the same or different QTNs at Catsup are associated with naturally occurring variation in multiple quantitative traits. We sequenced 169 Catsup alleles from a single population and detected 33 polymorphisms with little linkage disequilibrium (LD). Different molecular polymorphisms in Catsup are independently associated with variation in longevity, locomotor behavior, and sensory bristle number. Most of these polymorphisms are potentially functional variants in protein coding regions, have large effects, and are not common. Thus, Catsup is a pleiotropic quantitative trait gene, but individual QTNs do not have pleiotropic effects. Molecular population genetic analyses of Catsup sequences are consistent with balancing selection maintaining multiple functional polymorphisms.

Discontinuities and unsynapsed regions in meiotic chromosomes have a cis effect on meiotic recombination patterns in normal human males

During meiosis, homologous chromosome pairing is essential for subsequent meiotic recombination (crossover). Discontinuous chromosome regions (gaps) or unsynapsed chromosome regions (splits) in the synaptonemal complex (SC) indicate anomalies in chromosome synapsis. Recently developed immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with fluorescence in situ hybridization (using centromere-specific DNA probes) to identify bivalents with gaps/splits and to examine the effect of gaps/splits on meiotic recombination patterns during the pachytene stage of meiotic prophase from three normal human males. Gaps were observed only in the heterochromatic regions of chromosomes 9 and 1, with 9q gaps accounting for 90% of these events. Most splits were also found in chromosomes 9 and 1, with 58% of splits occurring on 9q. Gaps and splits significantly altered the distribution of MLH1 foci on the SC. On gapped SC 9q, the frequency of MLH1 foci was decreased compared with controls, and single 9q crossovers tended toward a more distal distribution. Furthermore, the larger the gap the more distal the location of the MLH1 focus closest to the q arm's telomere. MLH1 foci on split SC 9 had distributions similar to those of gapped SC 9; however, splits did not change the frequencies of MLH1 foci on SC 9. This is the first demonstration that gaps and splits have an effect on meiotic recombination in humans.

Generating molecular diversity by homologous recombination in Escherichia coli

We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of beta-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (10(10)) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range.

Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display.

Homologous recombination in yeast can be exploited to reliably generate libraries of >10(7) transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5' homology to the cut vector and the second scFv PCR product lacked 3' homology to the cut vector, or both PCR products had both 5' and 3' homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 10(4)-10(5) transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast-displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site-specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast.