Single Crossovers Research Articles

Sequence homology-independent protein recombination (SHIPREC).

Genomic recombination is widely recognized as the principal mechanism by which proteins evolve new functions (). Having realized the importance of recombination in evolution, scientists have developed a variety of methods to mimic this phenomenon in the lab to create libraries of gene chimeras. While the ideal technique would allow for the recombination of any parental genes, most available methods are limited to recombining closely related sequences, i.e., those with ≥70% sequence identity, resulting in crossovers biased towards regions of high identity. This occurs because most recombination methods, e.g., Stemmer-shuffling (), StEP (), RACHITT (), and in vivo methods (), rely on template switching to generate chimeras. However, two methods have recently been described which are capable of generating chimeric libraries independent of sequence identity. Sequence homology-independent protein recombination (SHIPREC) () is described in this chapter; another method, incremental truncation for the creation of hybrid enzymes (ITCHY) (), is also described in this volume. Both methods are capable of generating chimeric libraries containing all possible single crossovers between the two parental genes.

Food-grade delivery system for controlled gene expression in Lactococcus lactis.

A food-grade system for the delivery of desired genes to Lactococcus lactis, their inducible expression, and their transfer to related strains was established. Based on the thermosensitive pG(+)host replicon, two types of plasmid vectors were constructed which contained sections of either the chromosomal leu operon of L. lactis or the tel operon from the lactococcal sex factor. Genes cloned into the leu or tel sequences of these vectors were delivered to the homologous regions of the chromosome or the sex factor through two single crossovers, leading to integration of the recombinant plasmids and subsequent excision of the vector portions. Inducible transcription of integrated genes was achieved by using the nisin-controlled expression (NICE) system. To establish the signal transduction genes nisRK in L. lactis, the vectors pLNG1363 (targeted to the chromosome) and pUK500 (targeted to the sex factor) were constructed. Fusions of six different peptidase genes (pep) from Lactobacillus delbrueckii with the nisin-inducible promoter P(nisA) were delivered to the sex factor with derivatives of the vector pUK300. Food-grade recombinants of L. lactis were constructed which had the nisRK genes and individual P(nisA)::pep fusions integrated either separately into the chromosome and the sex factor or simultaneously into the sex factor. With both types of recombinants, expression of P(nisA)::pep fusions after induction with nisin was demonstrated. Depending on the loci used for integration of nisRK, variable induction rates were observed. Furthermore, an engineered sex factor carrying a P(nisA)::pepI fusion was transfered by conjugation between two strains of L. lactis at a frequency of 4 x 10(-4).

Detection of interannual oceanographic signals and improvement of geopotential from inversion of single and dual satellite altimetry

Extensive analyses of long-term averaged altimetrically determined sea height differences at crossovers for the satellites Geosat, ERS-1 and Topex/Poseidon (single satellite crossovers) as well as between the pairs of satellite (dual-satellite crossovers) clearly show the effects of orbit errors arising from the geopotential model (JGM3) used in computing their pairs. Upon inversion for an adjusted geopotential, the residuals for the dual satellite crossovers with multi-year gaps reveal surprisingly strong and consistent signals which appear to be largely of interannual oceanographic origin. We show that the height differences are sensitive to a variety of other orbit, instrument, tide and even geodetic errors, generally at lower level of observability. The results of the inversion to harmonic geopotential coefficients to degree and order 50 as extension of JGM3 will be discussed.

A reinvestigation of non-disjunction resulting in 47, XXY males of paternal origin

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

The occurrence of double strand DNA breaks is not the sole condition for meiotic crossing over in Drosophila melanogaster.

Analysis of the interchromosomal effects of In(2L + 2R)Cy, In(3L + 3R)LVM and their joint effect on the frequencies of single and double crossovers in the cv-v-f region of the X chromosome as well as interference showed that both inversions, occurring separately, increased the frequency of single as well as double crossovers and the coefficient of coincidence. However, when the inversions occurred together the frequencies of single crossovers no longer increased, but the frequency of double crossovers, as well as the coefficient of coincidence did increase. These results indicate firstly that the interchromosomal effects influence some precondition of exchange, but that this precondition is not an occurrence of double strand DNA breaks. Thus, the occurrence of double strand DNA breaks is not the sole condition for crossing over in Drosophila melanogaster.

Occurrence of double strand DNA breaks and pairing of homologous chromosomes are preconditions of crossing over in Drosophila melanogaster, and its timing.

Brood pattern analysis of the effect of a heat shock (35 degrees C 24h) given to the parental females in presence and absence of In (2L + 2R) Cy in heterozygous condition in the second chromosome on recombination and interference in the cv-v-f region of the X chromosome showed the following: (i) In the absence of the inversion recombination frequencies in both gene intervals increased in the daily broods, which represent oocytes in which premeiotic DNA synthesis was occurring during the heat shock treatment. (ii) In the presence of the inversion recombination frequencies did not increase in any of the daily broods. (iii) The frequencies of double crossovers did not increase in any broods either in the presence or in the absence of inversion. (iv) The coefficient of coincidence changed in virtually all broods both in the presence and absence of the inversion, indicating that the heat shock affects the distribution of single crossovers. (v) The results showed that the effect of the heat shock on the coefficient of coincidence was similar during premeiotic DNA synthesis both in the absence and presence of the inversion, but different in the broods representing oocytes which were undergoing the first meiotic division, suggesting that crossing over occurs during this division, probably during pachytene.

FAUST: Multi-satellite orbital dynamics software

The orbital software FAUST with its multi-arc multi-satellite capability is illustrated for simultaneous computations of TOPEX/Poseidon and ERS using SLR, DORIS and PRARE tracking. The multi-arcs are bridged by single satellite crossovers with dual satellite crossovers making the connection between the different satellite orbits. The software has been used to generate a gravity field enhancement of JGM-3 utilising TOPEX/Poseidon and ERS-1 data. The field has been compared against JGM-3 for TOPEX/Poseidon, ERS and GFZ-1 orbits. Additional computations of GFZ-1 compare several thermospheric models for this low satellite.

Inferences from dual crossover analyses between concurrent satellite missions

Dual satellite crossovers or repeat pass data between different altimetric satellite missions provides a mechanism for resolution of constant and geographically correlated errors which are unobservable in single satellite crossovers. Through long-term analyses it is possible to characterise the form of the difference both temporally and spatially. In this study dual crossovers between TOPEX/Poseidon and the less accurate ERS datasets are analysed for errors in ERS with particular emphasis on the long-term stability of the altimetric datasets. Corresponding results are also presented from analysis of the ERS repeat pass data during the tandem period.

Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration.

Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified. A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to-tail tandem array. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus.

Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus.

We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.

ERS-1 radial position refinement by dual crossover analysis with TOPEX/Poseidon

Achieving orbital accuracies in the radial direction for ERS-1 commensurate with those for TOPEX/Poseidon is of utmost importance for the integration of the two altimeter data sets. This paper outlines a procedure whereby the radial orbit error for ERS-1 is recovered as a time series expansion in the form of a finite Fourier series with additional terms for atmospheric drag, solar radiation pressure, and initial state vector mismodelling. Using a least squares collocation method with constraints derived from the JGM2 gravity field co-variance matrix, the radial error is recovered using both dual crossovers and ERS-1 single satellite crossovers. Aggregate arcs are then used to derive the ERS-1 orbit error over the repeat period of 35 days. The results are presented in the improvement of fit in the dual crossover, ERS-1 crossover and altimetry data sets as well as the recovery of an altimeter bias for the two satellites.

RIP60 Dimers and Multiples of Dimers Assemble Link Structures at an Origin of Bidirectional Replication in the Dihydrofolate Reductase Amplicon of Chinese Hamster Ovary Cells

We show assembly of low and high multimers of HeLa cell nuclear protein, RIP60, at the origin of bidirectional replication (OBR) identified by Burhans, Vassilev, Caddle, Heintz and DePamphilis in Chinese hamster ovary cells. RIP60 binds a 5′-ATT-3′ reiterated sequence downstream of the OBR and a second, homologous ATT sequence of opposite orientation situated within the OBR zone. Specifically bound structures were studied by conventional electron microscopy (EM) and quantitative scanning transmission electron microscopy (STEM). Dimers and multiples of dimers link the downstream binding site that overlaps a bent DNA sequence and the homologous upstream OBR sequence, looping out 700 bp of intervening DNA. Superposed dimers are found at individual unlinked sites, stabilized presumably through protein-protein interaction, and such superposition appears to occur also in the basic link structure. Along the loop, single crossovers and extended twists are observed by conventional EM. By STEM, loop DNA is laterally compacted, with diameter and mass equivalent to double-duplex DNA strands. Supercoiled 736 bp and 5243 bp circular DNAs assume similar laterally compacted geometries that are mostly absent from relaxed forms. These observations parallel the compacted, interwound superhelices viewed by cryo-electron microscopy in vitrified solutions containing magnesium ions, and provide structural evidence in agreement with that from conventional EM for superhelical tension in RIP60 loop DNA. Loop superhelicity could arise as a topological response to linking and suggests a functional role for link formation.

Genetic mapping of the breast-ovarian cancer syndrome to a small interval on chromosome 17q12-21: exclusion of candidate genes EDH17B2 and RARA.

A susceptibility gene for hereditary breast-ovarian cancer, BRCA1, has been assigned by linkage analysis to chromosome 17q21. Candidate genes in this region include EDH17B2, which encodes estradiol 17 beta-hydroxysteroid dehydrogenase II (17 beta-HSD II), and RARA, the gene for retinoic acid receptor alpha. We have typed 22 breast and breast-ovarian cancer families with eight polymorphisms from the chromosome 17q12-21 region, including two in the EDH17B2 gene. Genetic recombination with the breast cancer trait excludes RARA from further consideration as a candidate gene for BRCA1. Both BRCA1 and EDH17B2 map to a 6 cM interval (between THRA1 and D17S579) and no recombination was observed between the two genes. However, direct sequencing of overlapping PCR products containing the entire EDH17B2 gene in four unrelated affected women did not uncover any sequence variation, other than previously described polymorphisms. Mutations in the EDH17B2 gene, therefore do not appear to be responsible for the hereditary breast-ovarian cancer syndrome. Single meiotic crossovers in affected women suggest that BRCA1 is flanked by the loci RARA and D17S78.

An examination of the effects of double-strand breaks on extrachromosomal recombination in mammalian cells.

We studied the effects of double-strand breaks on intramolecular extrachromosomal homologous recombination in mammalian cells. Pairs of defective herpes thymidine kinase (tk) sequences were introduced into mouse Ltk- cells on a DNA molecule that also contained a neo gene under control of the SV40 early promoter/enhancer. With the majority of the constructs used, gene conversions or double crossovers, but not single crossovers, were recoverable. DNA was linearized with various restriction enzymes prior to transfection. Recombination events producing a functional tk gene were monitored by selecting for tk-positive colonies. For double-strand breaks placed outside of the region of homology, maximal recombination frequencies were measured when a break placed the two tk sequences downstream from the SV40 early promoter/enhancer. We observed no relationship between recombination frequency and either the distance between a break and the tk sequences or the distance between the tk sequences. The quantitative effects of the breaks appeared to depend on the degree of homology between the tk sequences. We also observed that inverted repeats recombined as efficiently as direct repeats. The data indicated that the breaks influenced recombination indirectly, perhaps by affecting the binding of a factor(s) to the SV40 promoter region which in turn stimulated or inhibited recombination of the tk sequences. Taken together, we believe that our results provide strong evidence for the existence of a pathway for extrachromosomal homologous recombination in mammalian cells that is distinct from single-strand annealing. We discuss the possibility that intrachromosomal and extrachromosomal recombination have mechanisms in common.

The Huntington's disease candidate region exhibits many different haplotypes.

Analysis of 78 Huntington's disease (HD) chromosomes with multi-allele markers revealed 26 different haplotypes, suggesting a variety of independent HD mutations. The most frequent haplotype, accounting for about one third of disease chromosomes, suggests that the disease gene is between D4S182 and D4S180. However, the paucity of an expected class of chromosomes that can be related to this major haplotype by assuming single crossovers may reflect the operation of other mechanisms in creating haplotype diversity. Some of these mechanisms sustain alternative scenarios that do not require a multiple mutational origin for HD and/or its positioning between D4S182 and D4S180.

Generation and analysis of nonhomologous RNA-RNA recombinants in brome mosaic virus: sequence complementarities at crossover sites

All three single-stranded RNAs of the brome mosaic virus (BMV) genome contain a highly conserved, 193-base 3' noncoding region. To study the recombination between individual BMV RNA components, barley plants were infected with a mixture of in vitro-transcribed wild-type BMV RNAs 1 and 2 and an RNA3 mutant that carried a deletion near the 3' end. This generated a population of both homologous and nonhomologous 3' recombinant BMV RNA3 variants. Sequencing revealed that these recombinants were derived by either single or double crossovers with BMV RNA1 or RNA2. The primary sequences at recombinant junctions did not show any similarity. However, they could be aligned to form double-stranded heteroduplexes. This suggested that local hybridizations among BMV RNAs may support intermolecular exchanges.

High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions.

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.

High-Frequency Homologous Recombination between Duplicate Chromosomal Immunoglobulin μ Heavy-Chain Constant Regions

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.

Haplotypes that are mosaic for wild-type and t complex-specific alleles in wild mice.

Two outstanding problems pertaining to the population dynamics and evolution of the t complex in mice concern the frequency of t haplotypes in the wild and the degree to which these haplotypes recombine with their wild-type homologs. To address these problems, the frequency and distribution of several t complex-associated restriction fragment variants in wild mice were estimated. Sixty-four versions of chromosome 17 from wild-derived Mus musculus musculus and Mus musculus domesticus were examined with DNA probes for six loci within the t complex that exhibit restriction fragment variation. All six probes detect variants that have heretofore been found exclusively associated with the t complex. Haplotype analysis of wild-derived chromosomes revealed a high frequency (45.3%) of "mosaic" haplotypes with a mixture of t-specific and wild-type variants and only one haplotype with t-specific variants at all six loci. When 12 well-characterized t haplotypes isolated from diverse geographic regions were analyzed, only three had a complete set of t-specific restriction fragments for the six loci examined. The preponderance of mosaic haplotypes in both groups of mice can be explained by any one of the following hypotheses: genetic recombination between t haplotypes and their wild-type homologs, the persistence in wild populations of haplotypes that have descended from ancestral partial t haplotypes, or that the restriction fragment variants fixed in the ancestral t haplotype were also fixed in some wild-type haplotypes. There is evidence to support all three of these hypotheses in our data. The allelic composition of some mosaic haplotypes indicates that they may have been formed by segmental recombination, either double crossing over or gene conversion, rather than by simple single crossovers. The occurrence of indistinguishable mosaic haplotypes in both M. m. musculus and M. m. domesticus suggests that these haplotypes are ancestral rather than recently derived.

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster

The nucleotide sequences of nine genes corresponding to tRNA 4 Ser or tRNA 7 Ser of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA 4,7 Ser cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA 4 Ser and tRNA 7 Ser (which are 96% homologous), two appear to result from single crossovers between tRNA 4 Ser and tRNA 7 Ser genes, one is an apparent double crossover product, and the last differs from a tRNA 4 Ser gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA 7 Ser. Comparison of a pair of genes corresponding to tRNA 4 Ser from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA Ser genes, and suggest that at least some recombinational events may be intergenic conversions.