Double Recombination Event Research Articles

Summary report: Missing data and pedigree and genotyping errors.

Genetic epidemiology is faced with mapping complex traits to genes with relatively small effects whose phenotypes may be modulated by temporal factors. To do this, detailed and accurate data must be available on families, perhaps collected over time. The Framingham Heart Study data supplied to Genetic Analysis Workshop 13 (GAW13), along with its simulated counterpart, contain longitudinal measurements and genomic scan data on 2,885 individuals in 330 families, and offer an opportunity to examine data quality and completeness issues as they affect analytical conclusions. Six GAW13 contributions applied methods to deal with missing data, both phenotypic and genotypic, at a single time point and longitudinally, and with possible errors in pedigree structure and genotypes. The methods included missing phenotypic data imputation by Markov chain Monte Carlo sampling, propensity scoring, regression, and adjusted mean values, as well as the assessment of transmission-disequilibrium tests when missing marker data may be allele-specific. Pedigree structural errors were found by genome-wide allele-sharing probabilities, while Mendelian consistent genotype errors were evaluated through likelihoods of double-recombination events. Each of the methods reviewed here offered insights into how to better take advantage of large, time-dependent, familial data sets. However, no one of them dealt with the longitudinal and familial aspects simultaneously. Overall, more consideration needs to be given to the effects that missing data and data errors have on our ability to map complex traits efficiently and accurately.

Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY.

Phosphorylation of circadian clock proteins represents a major regulatory step that controls circadian clocks. In Neurospora, the circadian clock protein FREQUENCY (FRQ) is progressively phosphorylated over time, and its level decreases when it is hyperphosphorylated. In this study, we showed that most of the kinase activity phosphorylating FRQ in vitro was calcium/calmodulin-dependent, and the endogenous FRQ in the Neurospora extracts was phosphorylated by a Ca/CaM-dependent kinase-like activity. From Neurospora cell extracts, an approximately 50-kDa Ca/CaM-dependent kinase (CAMK-1) that can specifically phosphorylate FRQ was purified. In vitro, this kinase accounts for near half of the FRQ kinase activity, and it can phosphorylate the FRQ region that contains the three known functionally important phosphorylation sites. To understand the function of camk-1 in vivo, it was disrupted in Neurospora by gene replacement. After germination from ascospores, the camk-1 null strains grew slowly, indicating that CAMK-1 plays an important role in growth and development of Neurospora. This phenotype was transient however, revealing redundancy in the system. Analysis of the camk-1 null strain revealed that the deletion of camk-1 affected phase, period, and light-induced phase shifting of the circadian conidiation rhythm. Taken together, our results suggest that multiple kinases may phosphorylate FRQ in vivo.

Extent and Distribution of Linkage Disequilibrium in Three Genomic Regions

The positional cloning of genes underlying common complex diseases relies on the identification of linkage disequilibrium (LD) between genetic markers and disease. We have examined 127 polymorphisms in three genomic regions in a sample of 575 chromosomes from unrelated individuals of British ancestry. To establish phase, 800 individuals were genotyped in 160 families. The fine structure of LD was found to be highly irregular. Forty-five percent of the variation in disequilibrium measures could be explained by physical distance. Additional factors, such as allele frequency, type of polymorphism, and genomic location, explained <5% of the variation. Nevertheless, disequilibrium was occasionally detectable at 500 kb and was present for over one-half of marker pairs separated by <50 kb. Although these findings are encouraging for the prospects of a genomewide LD map, they suggest caution in interpreting localization due to allelic association.

Transgenic or plant expression vector-mediated recombination of Plum Pox Virus.

Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region of Zucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections in Nicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3'-nontranslated region (3'-NTR) (plant line 17.27. 4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3'-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants.

Three-partner conversion induced by the P-element transposase in Drosophila melanogaster.

Conversion of one P-derived transposon into another has already been shown to occur with a measurable frequency. However, the mechanism responsible for such replacements has remained controversial. We previously proposed a mechanism involving three partners. We assumed that after excision of the P-element inserted at the target site, the double-strand break was repaired using, first, the homologous P sequences on the sister chromatid, and second, a remote template, the donor P-derived transposon. However, two other mechanisms have been proposed. The first involves two partners only, the broken end and the remote template, while the second involves transposition of the donor into the target P-element, followed by a double recombination event. Here we describe the conversion of a defective P-element using as a remote template an enhancer-trap element that is itself unable to transpose because it lacks 21 bp at its 5' end. This result makes it possible to exclude the possibility that this conversion event occurred after transposition. The new allele was molecularly and genetically characterized. The occurrence of a polymorphism at position 33 of the P-element sequence and of an imperfect copy of the template on the 3' side of the converted transposon confirmed that the sister chromatid was absolutely necessary as a partner for repair. Our results show that targeting of a marked P-element is possible, even when this element is unable to transpose. This provides a means of improving recovery of conversion events by eliminating unwanted transpositions catalyzed by the P transposase.

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene.

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.

Recombination with Host Transgenes and Effects on Virus Evolution: An Overview and Opinion

This commentary relates to the work by M. Borja et al. (M. Borja, T. Rubio, H. B. Scholthof, and A. O. Jackson, MPMI 12:153-162, 1999) that shows that wild-type virus can be restored frequently by double recombination events between a tomato bushy stunt virus mutant with deletions inactivating the coat protein gene and a coat protein transgene. Here, we focus on evidence suggesting that new viruses might evolve via recombination with transgenes used for disease resistance, and discuss the potential effects of widespread use of these sources of resistance on virus evolution. We argue that the benefits arising from using transgenic sources of resistance for virus disease control outweigh potential negative consequences of evolution of novel hybrid viruses with destructive disease potential.

Haplotype analysis in the Collaborative Study on the Genetics of Alcoholism data: double recombinants.

The presence of close double recombinants in genotyping data may help identify genotyping errors. Alternatively, putative double recombinants may be associated with genetic mechanisms that may be related to disease. Phase-known apparent double recombination events were identified in the Collaborative Study on the Genetics of Alcoholism data, and compared to the sex-specific genetic map at each region. A number of double recombinants occurred within a short genetic distance. Also, in some families multiple double recombinants were observed flanking the same genetic marker, both suggesting possible genotyping error. An excess of paternal double recombinants was identified, which is consistent with reports of sex-specific differential meiotic interference.

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se.

Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus.

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hohdatsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425-433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40-44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5' half of ORF1b of strain 79-1146.

Generation of targeted nonpolar gene insertions and operon fusions in Pasteurella haemolytica and creation of a strain that produces and secretes inactive leukotoxin.

An efficient method for targeted gene inactivation and generation of chromosomal gene fusions in Pasteurella haemolytica has been devised and used to create an lktC::cat operon fusion by allelic exchange at the leukotoxin gene cluster (lktCABD). A copy of the lktC gene was insertionally inactivated by using a nonpolar, promoterless cat cassette and then delivered into P. haemolytica on a shuttle vector. Plasmid incompatibility was used to detect clones where double recombination events had occurred at the chromosomal locus. The insertion in lktC did not affect expression of the downstream genes, and the mutant strain secreted an antigenic proleukotoxin that was neither leukotoxic nor hemolytic. Expression of the lktC gene in trans restored the wild-type phenotype, confirming that LktC is required for activation of the proleukotoxin to the mature leukotoxin. Construction of the lktC::cat operon fusion allowed us to quantitate leukotoxin promoter activity in P. haemolytica and to demonstrate that transcription was maximal during early logarithmic growth phase but was reduced following entry into late logarithmic phase. This allelic exchange system should be useful for future genetic studies in P. haemolytica and could potentially be applied to other members of Haemophilus-Actinobacillus-Pasteurella family, where genetic manipulation is limited.

A novelDRB1*13allele (DRB1*1327) on a DR17, DQ2 haplotype with aDRB1*0301sequence motif in the 2nd hyperpolymorphic region

In a sample from a Netherlands Caucasian, we found a new DRB1*13 allele (DRB1*1327). The nucleotide sequence of the second exon of the novel allele was identical to DRB1*1301 except for a single productive base substitution changing codon 26 from TTC to TAC, encoding phenylalanine and tyrosine, respectively. The new allele shares sequence with DRB1*03011 from codons 5 to 66. The haplotype carrying the new allele was, from known linkage disequilibria, deduced to be DRB1*1327, DRB3*0101, DQA1*05011, DQB1*0201, i.e. similar to the DR17, DQ2 haplotype, which suggests that the DRB1*1327 allele has arisen by a double recombination event between a DR13 donor haplotype and a DR17, DQ2 recipient haplotype.

Insertion of a short Alu sequence into the hMSH2 gene following a double cross over next to sequences with chi homology

Alu repeat sequences and other multiple copy repetitive elements are present throughout the human genome and are active in promoting recombination. It is believed that reverse transcription of transcribed Alu repeats followed by chromosomal integration has been responsible for the wide dispersion and high copy number of these sequences. During studies on the hMSH2 gene we have used RT-PCR to amplify from peripheral blood lymphocytes a cDNA species in which 553 base pairs of hMSH2 cDNA have been deleted to be replaced by a short 36 base pair Alu sequence as a result of a genomic insertion/deletion event. The 36 base pair Alu insert is homologous to a 26 base pair Alu sequence previously implicated in the promotion of recombination and contains the GCTGG motif which is part of the prokaryotic chi sequence. A second chi-like sequence is also located within the deleted hMSH2 region. Both chi-like sequences are located within 4 bp of the two 4-bp regions of cross over containing the insertion/deletion breakpoints. This suggests that a double recombination event has occurred, providing direct evidence for the recombinogenic activity of this Alu element. Furthermore, it suggests that chi-like sequences may define recombination hotspots as in prokaryotes.

Plasmids for ectopic integration in Bacillus subtilis

Plasmids have been constructed that allow integration by a double recombination event at the thrC locus of the Bacillus subtilis (Bs) chromosome. These plasmids can be used either for construction of merodiploid strains and complementation analysis, or for construction of transcriptional fusions to the Escherichia coli lacZ gene. The plasmids contain an antibiotic (An) marker selectable in Bs, as well as an additional An marker outside of the region that can recombine into the chromosome. When used in conjunction with recipient strains containing a third An marker at their thrC locus, these plasmids allow easy identification of transformants issued from a marker exchange event without additional Campbell-type integration. The existing plasmids used for ectopic integration at the amyE locus have been modified similarly.

Design of cassette baculovirus vectors for the production of therapeutic antibodies in insect cells

Background: Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. Objectives: To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. Study design: Two transfer vectors, pBHUCκ and pBHuγl, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain Ca gene or heavy chain Cγl sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. Results: Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. Conclusion: Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.

Development of a Heterodimer Plasmid System for the Introduction of Heterologous Genes into Streptococci

We have previously constructed a model secretion system for oral streptococci using the secretory domain of theStreptococcus mutansGS-5gtfB gene. As an initial step in developing systems for secreting protein fusions containing a glucan-binding polypeptide from streptococci, a DNA fragment corresponding to the glucan-binding domain (GBD) of the glucosyltransferase-S enzyme from strain GS-5 was fused to thegtfB secretory domain. However, it was not possible to clone the hybrid gene intoEscherichia coli–streptococcal shuttle plasmids usingE. colias host cells. Integration of the hybrid GBD gene into theStreptococcus gordoniichromosome was directly accomplished utilizing a strategy involving a novel heterodimeric plasmid system. The heterodimer was constructed by ligating together two plasmids each containing DNA fragments homologous with a corresponding region of theS. gordoniichromosome flanking the hybrid GBD gene following a double crossover recombination event. However, this single-copy integrant secreted limited quantities of the GBD protein. In order to achieve high-level expression of the protein, the hybrid GBD gene was integrated into resident plasmids inS. gordoniifollowing construction of intermediate heterodimeric plasmids. These plasmids contained the GBD gene flanked by sequences homologous to regions of the resident plasmids. The presence of the GBD protein in culture fluids from transformants harboring the multicopy plasmids demonstrated the secretion of the functional GBD protein. The strategy successfully developed for secreting the GBD in the streptococci should be adaptable for other organisms for which gene transfer systems are available. In addition, these systems will allow the direct introduction of genetic constructs when the constructs cannot be stably maintained in shuttle vectors within intermediate hosts such asE. coli.

A 37-Marker PCR-Based Genetic Linkage Map of Human Chromosome 9: Observations on Mutations and Positive Interference

Refinement of an "index" marker genetic linkage map of human chromosome 9 using the CEPH reference pedigrees has been achieved through the addition of 11 markers to the previous map of 26 markers. Five of the 11 markers added to the map are new markers of the GATA repeat type, 1 is a complex repeat, and the remaining 5 as well as the original 26 markers are all GT/CA repeats. Twelve definite and five probable mutations were detected in this analysis and were more common for the GATA repeats than the GT/CA repeats. Strong evidence for positive interference was seen over the length of the chromosome, but there were significantly more double recombination events in the pericentromeric region than elsewhere, suggesting that interference is less strong in that region.

Methane monooxygenase mutants ofMethylosinus trichosporiumconstructed by marker-exchange mutagenesis

Methylosinus trichosporium OB3b synthesizes a soluble cytoplasmic methane monooxygenase when grown in copper-depleted medium and a membrane-bound particulate methane monooxygenase under copper-replete conditions. The genes encoding the hydroxylase component of soluble methane monooxygenase, carried on a plasmid in Escherichia coli, were insertionally inactivated using a kanamycin cassette and transferred back into M. trichosporium by conjugation. Marker-exchange mutagenesis, via a double homologous recombination event, yielded a soluble methane monooxygenase-negative mutant which grew only on methane using the particulate methane monooxygenase during copper-replete growth conditions, thus proving that the two methane oxidation systems in this methanotroph are genetically distinct.

Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis

Methylosinus trichosporium OB3b synthesizes a soluble cytoplasmic methane monooxygenase when grown in copper-depleted medium and a membrane-bound particulate methane monooxygenase under copper-replete conditions. The genes encoding the hydroxylase component of soluble methane monooxygenase, carried on a plasmid in Escherichia coli, were insertionally inactivated using a kanamycin cassette and transferred back into M. trichosporium by conjugation. Marker-exchange mutagenesis, via a double homologous recombination event, yielded a soluble methane monooxygenase-negative mutant which grew only on methane using the particulate methane monooxygenase during copper-replete growth conditions, thus proving that the two methane oxidation systems in this methanotroph are genetically distinct.

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon

A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MGS267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF-deficient strain produced a Lac − phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF-deficient strain resulting in a Lac + phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.