Spiroplasma citri virus SpV1-derived cloning vector: deletion formation by illegitimate and homologous recombination in a spiroplasmal host strain which probably lacks a functional recA gene.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for expressing an epitope of the P1 adhesin protein from Mycoplasma pneumoniae in S. citri (A. Marais, J. M. Bové, S.F. Dallo, J. B. Baseman, and J. Renaudin, J. Bacteriol. 175:2783-2787, 1993). We have now studied the structural instability of the recombinant RF leading to loss of the DNA insert. Analyses of viral clones with deletions have shown that both illegitimate and homologous recombination were involved in deletion formation. For one such clone, deletion has occurred via a double crossing-over exchange between the circular free viral RF and SpV1 viral sequences present in the S. citri host chromosome. The homologous recombination process usually requires the RecA protein. However, characterization of the recA gene of the S. citri R8A2 host strain revealed that over two-thirds of the open reading frame of the recA gene was deleted from the C-terminal part, indicating that this particular strain is probably RecA deficient.     

A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.     

Spiroplasma citri Virus SpV1: Characterization of Viral Sequences Present in the Spiroplasmal Host Chromosome

Spiroplasma citri virus SpV1-R8A2B is a naked, rod-shaped virus with a circular, single-stranded DNA genome of 8273 bp. SpV1-related sequences were detected in the chromosomal DNA of all S. citri strains tested. Southern blot hybridization analyses revealed that several copies of most, if not all, SpV1 ORFs are present in the chromosome of S. citri strain R8A2. For further study of the integrated viral sequences, a genomic DNA library of S. citri R8A2 was constructed, and two cloned chromosomal DNA fragments containing SpV1 viral sequences were studied by comparison with the free viral genome of SpV1-R8A2B. One fragment seems to contain a full-length viral genome, while the other contains only parts of the viral genome. In both fragments, the left and right ends of viral sequences consist of 31-bp inverted repeat sequences, those which are facing each other at nucleotide 4737 in the circular viral genome. In addition, both fragments contain the SpV1-ORF3, encoding a putative transposase, immediately upstream of the right repeat. These data suggest that the SpV1-ORF3 and the repeat sequences could be parts of an IS-like element of chromosomal origin.     

Integration and excision of SV40 DNA from the chromosome of a transformed cell

The single insertion of SV40 DNA present in the genome of the 14B line of transformed rat cells has been cloned in procaryotic vectors. Analysis of the clones reveals a complex arrangement of viral sequences in which a small tract of DNA is inverted with respect to the major insertion. The nucleotide sequences at the two junctions show sharp transitions between cellular and viral sequences. The sequences which flank the viral insertion have been used as probes to clone the corresponding genomic sequences from the DNA of untransformed rat cells. Analysis of the structure of these clones shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration. When 14B cells are fused with uninfected simian cells a heterogeneous set of low molecular weight superhelical DNAs containing viral sequences is generated. These have been cloned in procaryotic vectors and their structures have been analyzed. All of them span the origin of SV40 DNA replication and are colinear with various segments of the integrated viral DNA and its flanking sequences. The shorter molecules contain part of the integrated viral genome and cellular sequences from one side of the insertion. They were therefore generated by recombination between the viral DNA and its flanking cellular sequences. The longer molecules contain cellular sequences from both sides of the insertion as well as an entire copy of the integrated viral DNA. They were therefore generated by recombination between the flanking cellular sequences. These results argue strongly against the involvement of specific excision enzymes, and rather are discussed in terms of a model involving replication of the integrated viral DNA followed by recombination for release of integrated viral sequences.     

Relationship between integrated and nonintegrated viral DNA in rat cells transformed by polyoma virus

Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose presence is under the control of the viral A gene. To understand the mechanism of production of the "free" viral DNA, we have characterized the DNA species produced by several rat lines transformed by wild-type virus or by ts-a polyoma virus and compared them with the integrated viral sequences. Every cell line tested yielded a characteristic number of discrete species of viral DNA. The presence of defectives was a very common occurrence, and these molecules generally carried deletions mapping in the viral "late" region. The production of multiple species of free viral DNA was not due to heterogeneity of the transformed rat cell population, and its pattern did not change upon fusion with permissive mouse cells. Analysis of the integrated viral DNA sequences in the same cell lines showed, in most cases, a full head-to-tail tandem arrangement of normal-size and defective molecules. The free DNA produced by these lines faithfully reflected the integrated species. This was true also in the case of a cell line which contained a viral insertion corresponding to approximately 1.3 polyoma genomes, with each of the repeated portions of the viral DNA molecule carrying a different-size deletion. These results support the hypothesis that the free DNA derives from the integrated form through a mechanism of homologous recombination leading to excision and limited replication.     

Resolution of a polyomavirus-mouse hybrid replicon: release of genomic viral DNA.

RmI is a circular chimera containing 1.03 copies of polyomavirus DNA and 1,628 base pairs of mouse DNA, joined through direct and inverted repeat sequences. It is excised from the chromosome of a transformed cell via a site-specific recombination event that is dependent on the activation of the viral gene coding for large T antigen. RmI is shown here to be highly infectious for normal mouse cells. This infectivity reflects the ability of RmI to effectively yield unit-length viral DNA via intramolecular recombination. The effectiveness with which infectious viral DNA is produced from RmI is consistent with the idea that the underlying recombination event is site specific, rather than homologous or illegitimate.     

Characterization of in vivo recombination activities in the mouse embryo

Homologous recombination makes use of sequence homology to repair DNA and to rearrange genetic material. In mammals, these processes have mainly been characterized using cultured cell systems. We have developed an assay that allows us to quantitatively analyze homologous recombination in vivo in the mouse embryo. Transgenic mouse lines were generated by microinjection into a fertilized mouse ovum of a vector containing two homologous LINE-1 (L1) sequences arranged as a direct repeat: these sequences can recombine with each other and with endogenous L1 sequences before, during or after integration of the vector into the genome. Using a plasmid rescue procedure, we determined the composition of the integrated vector array in several transgenic mice and their descendants. Homologous recombination frequencies were found to be strikingly high, involving 70% of integrated vectors in some arrays, with homologous deletions being five times more frequent than gene conversion without crossing-over. Interestingly, non-homologous recombination was found to be much less frequent. We also found that endogenous L1 sequences could be involved in homologous recombination events in the mouse embryo, and that the integrated arrays could be modified from generation to generation by homologous recombination between the integrated L1 sequences.     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.     

Sequence requirements for DNA rearrangements induced by the terminal repeat of herpes simplex virus type 1 KOS DNA.

We investigated the sequence requirements for the site-specific DNA cleavages and recombinational genome isomerization events driven by the terminal repeat or a sequence of herpes simplex virus type 1 KOS DNA by inserting a series of mutated a sequences into the thymidine kinase locus in the intact viral genome. Our results indicate that sequences located at both extremities of the a sequence contribute to these events. Deletions entering from the Ub side of the a sequence progressively reduced the frequency of DNA rearrangements, and further deletion of the internal DR2 repeat array had an additional inhibitory effect. This deletion series allowed us to map the pac1 site-specific DNA cleavage signal specifying the S-terminal cleavage to a sequence that is conserved among herpesvirus genomes. Constructs lacking this signal were unable to directly specify the S-terminal cleavage event but retained a reduced ability to give rise to S termini following recombination with intact a sequences. Deletions entering from the Uc side demonstrated that the copy of direct repeat 1 located adjacent to the Uc region plays an important role in the DNA rearrangements induced by the a sequence: mutants lacking this sequence displayed a reduced frequency of novel terminal and recombinational inversion fragments, and further deletions of the Uc region had a relatively minor additional effect. By using a construct in which site-specific cleavage was directed to heterologous DNA sequences, we found that the recombination events leading to genome segment inversion did not occur at the sites of DNA cleavage used by the cleavage-packaging machinery. This observation, coupled with the finding that completely nonoverlapping portions of the a sequence retained detectable recombinational activity, suggests that inter-a recombination does not occur by cleavage-ligation at a single specific site in herpes simplex virus type 1 strain KOS. The mutational sensitivity of the extremities of the a sequence leads us to hypothesize that the site-specific DNA breaks induced by the cleavage-packaging system stimulate the initiation of recombination.     

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic “units,” suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33°C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of ~1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.     

Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.     

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene. The high frequencies of marker excision obtained with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event. Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida. This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.     

A third DNA polymerase from Spiroplasma citri and two other spiroplasmas.

Recently, two DNA polymerases (ScA and ScB) were isolated and characterized from Spiroplasma citri. We now have found a third DNA polymerase (ScC) not only in S. citri but also in the serologically related honeybee spiroplasma BC3 and the unrelated flower spiroplasma BNR1. Enzyme ScC is N-ethylmaleimide (NEM) sensitive. The three DNA polymerases from the honeybee spiroplasma seem to be similar to the respective enzymes of S. citri. However, whereas the NEM-resistant enzyme ScA from S. citri and that from the BC3 honeybee spiroplasma are retained on DEAE-cellulose and require 0.09 M KCl for elution, the NEM-resistant enzyme A from the flower spiroplasma BNR1 is not retained.     

DNA sequences at immunoglobulin switch region recombination sites.

The immunoglobulin heavy chain switch from synthesis of IgM to IgG, IgA or IgE is mediated by a DNA recombination event. Recombination occurs within switch regions, 2-10 kb segments of DNA that lie upstream of heavy chain constant region genes. A compilation of DNA sequences at more than 150 recombination sites within heavy chain switch regions is presented. Switch recombination does not appear to occur by homologous recombination. An extensive search for a recognition motif failed to find such a sequence, implying that switch recombination is not a site-specific event. A model for switch recombination that involves illegitimate priming of one switch region on another, followed by error-prone DNA synthesis, is proposed.     

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues

Horizontal gene transfer (HGT) is part of prokaryotic life style and a major factor in evolution. In principle, any combinations of genetic information can be explored via HGT for effects on prokaryotic fitness. HGT mechanisms including transformation, conjugation, transduction, and variations of these plus the role of mobile genetic elements are summarized with emphasis on their potential to translocate foreign DNA. Complementarily, we discuss how foreign DNA can be integrated in recipient cells through homologous recombination (HR), illegitimate recombination (IR), and combinations of both, site-specific recombination, and the reconstitution of plasmids. Integration of foreign DNA by IR is very low, and combinations of IR with HR provide intermediate levels compared to the high frequency of homologous integration. A survey of studies on potential HGT from various transgenic plants indicates very rare transfer of foreign DNA. At the same time, in prokaryotic habitats, genes introduced into transgenic plants are abundant, and natural HGT frequencies are relatively high providing a greater chance for direct transfer instead of via transgenic plants. It is concluded that potential HGT from transgenic plants to prokaryotes is not expected to influence prokaryotic evolution and to have negative effects on human or animal health and the environment.     

Inversion events in the HSV-1 genome are directly mediated by the viral DNA replication machinery and lack sequence specificity

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.     

'Illegitimate' recombination events in polyoma-transformed rat cells

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.