Absence of interparental recombination in multiplicity reconstitution from incomplete bacteriophage T4 genomes.

Interparental recombination between injected T4 DNA molecules is indetectable for incomplete petite phages (carrying a terminally deficient genome and therefore unable to circularize) as well as for genetically complete phages. The nonvialbe petite phages can individually replicate their DNA repeatedly, and they aso undergo multiplicity reconstitution, producing complete phages, provided that a host bacterium is infected by several petite particles that carry genetically complementary segments of DNA. The formation of complete phages in multiplicity reconstitution must be due to recombination among incomplete progeny fragments, i.e., partial replicas of the T4 genomes. It evidently does not result from interparental recombination. To test for interparental recombination, light bacteria (containing no bromouracil) were simultaneously infected in light medium with light radioactive phage in minority (usually less than one per cell) and heavy (bromouracil-labeled) phage in majority (usually about nine per cell). Any interparental recombination should, under these circumstances of infection, head to movement of the radioactive label of the minority light phage DNA to a position of higher density. That possibility was not observed.     

Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin

A fragment of DNA carrying the hitherto unisolated members of the cluster of genes (red) for biosynthesis of the red-pigmented antibiotic undecylprodigiosin of Streptomyces coelicolor A3(2) was isolated. This was done by cloning random fragments of S. coelicolor DNA into the closely related Streptomyces lividans 66 and recovering a clone that caused overproduction of undecylprodigiosin. The effect was probably due to the presence of the cloned redD gene, which functions as a positive regulator of the expression of the red cluster, activating the normally poorly expressed red genes of S. lividans. Two fragments from either end of the red cluster were cloned adjacent to each other on a low-copy-number Streptomyces vector. Double crossing-over occurring between these plasmid-borne sequences and the chromosomal copy of the same DNA in S. coelicolor led to isolation of the entire red cluster as a single cloned fragment. Isolation of antibiotic biosynthetic genes by the effects of an activator in a self-cloning experiment, and in vivo reconstitution of a large cluster of genes by homologous recombination, may turn out to be usefully generalizable procedures.     

Multiple origins of the human glycophorin Sta gene. Identification of hot spots for independent unequal homologous recombinations.

Human glycophorin Sta (HGpSta), one of the structural variants of erythrocyte membrane sialoglycoproteins, is encoded by a delta-alpha hybrid gene that arose from a single unequal crossover between the parent HGpB(delta) and HGpA(alpha) genes. We report here the identification of two new HGpSta genes (type A and type B) in four unrelated Sta heterozygotes from two ethnic groups. These Sta genes represent distinct genetic isoforms that differ from the previously reported Sta gene (type C) in the location of crossing-over sites. Comparison of nucleotide sequences among HGpB(delta), HGpA(alpha), and HGpSta type A genes revealed that the delta-alpha unequal crossover for the Sta type A gene occurred 110-246 base pairs downstream from pseudoexon III. In the crossing-over site of this Sta gene, an AT-rich sequence lying 3' to a nonameric palindrome was found to be highly similar to the lambda phage attachment site, att B, in inverted orientation. In the Sta type B gene, the delta-alpha crossing-over point was localized to an AG-rich sequence that is 302-490 base pairs downstream from pseudoexon III. Multiple lambda chi-like elements were identified at the crossover boundaries and within the breakpoint of this Sta gene. These results suggest strongly that recurrent and independent unequal recombination events have occurred in the formation of multiple Sta genes and that particular genomic sequences are important in defining the recombination sites for these homology-driven processes.     

The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events

In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu and Cdelta flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cmu and Cdelta regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu and Cdelta regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.     

Recombination between the X and Y chromosomes and the Sxr region of the mouse.

The Sxr (sex-reversed) region that carries a copy of the mouse Y chromosomal testis-determining gene can be attached to the distal end of either the Y or the X chromosome. During male meiosis, Sxr recombined freely between the X and Y chromosomes, with an estimated recombination frequency not significantly different from 50% in either direction. During female meiosis, Sxr recombined freely between the X chromosome to which it was attached and an X-autosome translocation. A male mouse carrying the original Sxra region on its Y chromosome, and the shorter Sxrb variant on the X, also showed 50% recombination between the sex chromosomes. Evidence of unequal crossing-over between the two Sxr regions was obtained: using five markers deleted from Sxrb, 3 variant Sxr regions were detected in 159 progeny (1.9%). Four other variants (one from the original cross and three from later generations) were presumed to have been derived from illegitimate pairing and crossing-over between Sxrb and the homologous region on the short arm of the Y chromosome. The generation of new variants throws light on the arrangement of gene loci and other markers within the short arm of the mouse Y chromosome.     

Cloning of V region fragments from mouse liver DNA and localization of repetitive DNA sequences in the vicinity of immunoglobulin gene segments.

Two different kappa light chain genes have previously been isolated from one mouse myeloma. The V (variable, abbreviations in ref. 2) gene segments of the two genes were now used to identify their germline counterparts in EcoRI digests of mouse liver DNA. In addition two sets of related V gene segments were found which hybridize with either of the two DNA probes. Five of the V region fragments of one set were cloned in a lambda phage vector and partially characterized by restriction mapping and Southern blot hybridization. Repetitive DNA sequences were found on each of the five fragments as well as on other cloned immunoglobulin gene containing fragments. Cross-hybridization between some but not all of the regions containing repetitive DNA sequences was observed.     

Mechanisms for gene conversion and homologous recombination: The double-strand break repair model and the successive half crossing-over model

Two mechanisms for gene conversion and homologous recombination were discussed. (1) The double-strand break repair model. A double-strand break is expanded to a gap, which is then repaired by copying a homologous sequence. The gene conversion is often accompanied by crossing-over of the flanking sequences. We obtained evidence for this model in Red pathway of bacteriophage lambda and RecE pathway of E. coli. (2) The successive half crossing-over model. Half crossing-over leaves one recombinant duplex and one or two end(s) out of two parental duplexes. The resulting ends are, in turn, recombinogenic. Successive rounds of the half crossing-over mechanism explains why apparent plasmid gene conversion in RecF pathway of E. coli is not accompanied by crossing-over. This model can explain chromosomal gene conversion if we assume that the donor is first replicated. Gene conversion during mating-type switching in yeast, antigenic variation in unicellular microorganisms, and chromosomal gene conversion in mammalian somatic cells are explained by this model. Distinguishing between these two mechanisms is important in understanding recombination in yeast and mammalian cells and also in its application to gene targeting.     

Cloning and expression of a chloramphenicol acetyltransferase gene in cytosine-substituted T4 bacteriophage

We have developed an efficient method for transferring foreign genes into the T4 phage genome. Any foreign genes inserted into the T4 uvsY gene cloned on plasmids can be transferred into a cytosine-substituted T4dC(delta NB5060) phage genome by a replacement type of recombination. To achieve this, we constructed chimeric plasmids which had a chloramphenicol acetyltransferase gene (cat) derived from transposon Tn9 inserted into the Bg/II site within the T4 uvsY gene on pBR322. The cat gene was then transferred by in vivo recombination into the T4dC(delta NB5060) phage genome. Moreover, it was demonstrated that the cat gene in the hybrid T4dC phage was expressed upon phage infection and development.     

Regeneration of herpesviruses from molecularly cloned subgenomic fragments.

The ability to manipulate the genomes of herpesviruses is of eminent importance for obtaining insight into gene function and regulation of gene expression of these complex viruses. Here we report the use of in vivo overlap recombination to generate pseudorabies virus mutants. Cotransfection of up to five overlapping cloned subgenomic fragments, which together constitute the entire genomic information of pseudorabies virus, results in the efficient reconstitution of virus. This allows the efficient introduction of multiple well-defined mutations in herpesvirus genomes in a single step, without any selection or screening for a particular phenotype.     

Sequence and deletion analysis of the recombination enhancement gene (ref) of bacteriophage P1: evidence for promoter-operator and attenuator-antiterminator control.

The ref gene of bacteriophage P1 stimulates recombination between two defective lacZ genes in the Escherichia coli chromosome (lac x lac recombination) and certain other RecA-dependent recombination processes. We determined the DNA sequence of the 5' portion of the ref gene and tested various regions for functionality by inserting DNA fragments lacking increasing amounts of 5' sequence into plasmid and lambda phage vectors and measuring the ability of the constructs to stimulate lac x lac recombination. The region found essential for Ref activity in the absence of external heterologous promoters encodes two presumptive promoters, pref-1 and pref-2, whose -10 regions fall in a nearly perfect 13-base-pair (bp) tandem repeat. The -10 region of the putative pref-1 is part of a phage P1 c1 repressor recognition sequence. The first two ATG codons in the ref reading frame are, respectively, 90 and 216 bp downstream from the putative promoter-operator region. Deletion analysis indicated that translation can initiate at either ATG (although neither is associated with a canonical ribosome-binding sequence) and that the 42 amino acids in between are not indispensable for Ref stimulation of lac x lac recombination. However, the shorter reading frame appears to encode a less active polypeptide. The 91-bp leader region between the putative promoter-operator and the first ATG contains 30 codons in frame with the ref structural sequence, but its frame can be shifted without affecting Ref activity. The leader region ends with an apparent rho-independent termination sequence (attenuator). Deletion of 18 bp of early leader sequence drastically reduced Ref activity, even when ref was driven by a heterologous promoter (plac). An 8-bp internal deletion in the putative attenuator sequence relieved this requirement for the early leader sequence. This latter observation, along with nucleotide complementarity between portions of the early leader and attenuator sequences, are consistent with preemption of attenuation by the early leader.     

Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.     

Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

Tandem-repetitive noncoding DNA: forms and forces

A model of sequence-dependent, unequal crossing-over and gene amplification (slippage replication) has been stimulated in order to account for various structural features of tandemly repeated DNA sequences. It is shown that DNA whose sequence is not maintained by natural selection will exhibit repetitive patterns over a wide range of recombination rates as a result of the interaction of unequal crossing-over and slippage replication, processes that depend on sequence similarity. At high crossing-over frequencies, the nucleotide patterns generated in the simulations are simple and highly regular, with short, nearly identical sequences repeated in tandem. Decreasing recombination rates increase the tendency to longer and more-complex repeat units. Periodicities have been observed down to very low recombination rates (one or more orders of magnitude lower than mutation rate). At such low rates, most of the sequences contain repeats which have an extensive substructure and a high degree of heterogeneity among each other; often higher-order structures are superimposed on a tandem array. These results are compared with various structural properties of tandemly repeated DNAs known from eukaryotes, the spectrum ranging from simple-sequence DNAs, particularly the hypervariable mini-satellites, to the classical satellite DNAs, located in chromosomal regions of low recombination, e.g., heterochromatin.     

Illegitimate recombination is a major evolutionary mechanism for initiating size variation in plant resistance genes

Current models for the evolution of plant disease resistance (R) genes are based on mechanisms such as unequal crossing-over, gene conversion and point mutations as sources for genetic variability and the generation of new specificities. Size variation in leucine-rich repeat (LRR) domains was previously mainly attributed to unequal crossing-over or template slippage between LRR units. Our analysis of 112 R genes and R gene analogs (RGAs) from 16 different gene lineages from monocots and dicots showed that individual LRR units are mostly too divergent to allow unequal crossing-over. We found that illegitimate recombination (IR) is the major mechanism that generates quasi-random duplications within the LRR domain. These initial duplications are required as seeds for subsequent unequal crossing-over events which cause the observed rapid increase or decrease in LRR repeat numbers. Ten of the 16 gene lineages studied contained such duplications, and in four of them the duplications served as a template for subsequent repeat amplification. Our analysis of Pm3-like genes from rice and three wheat species showed that such events can be traced back more than 50 million years. Thus, IR represents a major new evolutionary mechanism that is essential for the generation of molecular diversity in evolution of RGAs.     

A specialized transducing phage, lambda psrlA, for the sorbitol phosphotransferase of Escherichia coli K12

A specialized transducing phage for the srlA gene, specifying the sorbitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system, was constructed and its DNA was analysed by restriction endonuclease digestion. Phage construction involved four steps: (1) integration of lambda into the srlA gene; (2) selection of phage carrying (a) the left and (b) the right end of the srlA gene by means independent of the function of the new DNA acquired; (3) reconstitution of the srlA gene in a dilysogen of these two phage; and (4) the excision, using the heteroimmune lambdoid phage 21, of a plaque-forming srlA+ phage from the dilysogenic chromosome. Comparison of the DNA restriction digests of the transducing phage with those of its parents and of wild-type lambda revealed fragments consisting partly of lambda and partly of Escherichia coli DNA. The junction points in the intermediate phage define a site that must lie within the reconstituted gene of the final phage. This technique should be of general application in relating genes, cloned by our method, to DNA sequences.     

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.     

Gene conversion occurs within the mating-type locus of Cryptococcus neoformans during sexual reproduction

Meiotic recombination of sex chromosomes is thought to be repressed in organisms with heterogametic sex determination (e.g. mammalian X/Y chromosomes), due to extensive divergence and chromosomal rearrangements between the two chromosomes. However, proper segregation of sex chromosomes during meiosis requires crossing-over occurring within the pseudoautosomal regions (PAR). Recent studies reveal that recombination, in the form of gene conversion, is widely distributed within and may have played important roles in the evolution of some chromosomal regions within which recombination was thought to be repressed, such as the centromere cores of maize. Cryptococcus neoformans, a major human pathogenic fungus, has an unusually large mating-type locus (MAT, >100 kb), and the MAT alleles from the two opposite mating-types show extensive nucleotide sequence divergence and chromosomal rearrangements, mirroring characteristics of sex chromosomes. Meiotic recombination was assumed to be repressed within the C. neoformans MAT locus. A previous study identified recombination hot spots flanking the C. neoformans MAT, and these hot spots are associated with high GC content. Here, we investigated a GC-rich intergenic region located within the MAT locus of C. neoformans to establish if this region also exhibits unique recombination behavior during meiosis. Population genetics analysis of natural C. neoformans isolates revealed signals of homogenization spanning this GC-rich intergenic region within different C. neoformans lineages, consistent with a model in which gene conversion of this region during meiosis prevents it from diversifying within each lineage. By analyzing meiotic progeny from laboratory crosses, we found that meiotic recombination (gene conversion) occurs around the GC-rich intergenic region at a frequency equal to or greater than the meiotic recombination frequency observed in other genomic regions. We discuss the implications of these findings with regards to the possible functional and evolutionary importance of gene conversion within the C. neoformans MAT locus and, more generally, in fungi.     

A plasmid-based method to quantitate homologous recombination frequencies in gram-negative bacteria

A method is described which enables quantitative evaluation of the ability of gram-negative bacterial cells to perform homologous recombination between DNA molecules. This method is particularly useful in cases where the stringency of rec mutations is to be determined. The procedure is based on a wide-host-range vector (pRK404) in which two unequally truncated and overlapping fragments of the neo gene were cloned. When introduced into gram-negative bacteria either by transformation or by conjugation, molecules of this plasmid, pBX404-7, undergo unequal crossing-over leading to the restoration of a functional neo gene. The stringency of putative rec mutations can thus be determined by measuring the frequency at which kanamycin-resistant colonies appear in bacterial strains harboring pBX404-7.     

Analysis of a horse family with a crossing-over between the ELA complex and the A blood group system

A horse family in which a recombination occurred in the chromosome region coding for the serological specificities of the ELA complex and those of the A blood group system of a mare was further analysed by mixed lymphocyte reaction (MLR) and Southern blot hybridization. This family consisted of a stallion, a mare and five full sibs. The stallion and the mare were heterozygous for internationally recognized ELA specificities while only the mare was heterozygous for the A blood group system. MLR between all members of the family confirmed that the stallion possessed two different ELA haplotypes and suggested that recombination in the mare occurred outside the segment delimited by the ELA-A locus and the MLR region. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI), three human HLA probes (one of class I cDNA and two of class II probes), one cDNA (DR beta) and one genomic (DQ alpha). Class I and class II restriction fragments of the mare segregated in accordance to the ELA specificities and thus clearly confirming that the crossing-over did not occur between the ELA-A gene and the class I, class II region nor between DR beta and DQ alpha subsets. The A blood group genetic determinants would thus be situated outside the ELA region defined by class I and class II genes.