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Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli-H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam- E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.
BACKGROUND - . Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects.
RESULTS - . We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyze plasmid fusion between the univector - a plasmid containing the gene of interest - and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3' end gene fusions using homologous recombination in Escherichia coli.
CONCLUSIONS - . Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.
During a study designed to identify changes in the genomes that are observed in mutant populations of pseudorabies virions, a thymidine kinase-defective population of virions which contains genomes that possess inverted repeated sequences of unequal sizes has been identified. This population of virions has been used to ascertain the mechanism and rates of equalization of the repeats. Results showed that when the virions were passed in cell cultures at high multiplicities of infection (either once or several times), one-half of the molecules had repeats of equal size and one-half had repeats of unequal size. This result is to be expected if conditions allowing a high degree of intermolecular recombination involving exchange of repeats exist. The process of intermolecular exchange of inverted repeats is rapid and the DNA molecules were at equilibrium by the time a virion had produced a small plaque. If the virions were passed in cell culture at low multiplicities of infection, they acquired genomes with equal-size repeats. This is probably due to segregation of the virions which had acquired genomes with equal repeats by recombination; in the absence of coinfection of the cells with virions containing genomes with different repeat sizes, heterogeneity of the repeats could not be generated. The fact that virions containing genomes with equal repeats are usually isolated from nature can thus be attributed to equalization of the repeats which results from intermolecular recombination, followed by segregation. A special mechanism ensuring equalization of repeats, such as one repeat acting as a template for the synthesis (or repair) of the other, need therefore not be invoked.
Mammalian cells possess mechanisms that allow unrelated sequences to recombine (illegitimate recombination), This is evidenced by the high rate of recombination between largely non-homologous sequences after DNA transfection. We have analysed the integrated viral sequences present in the polyoma transformed cell line 82-Rat. Within the single insert of integrated viral sequences there are two regions where multiple recombination events have occurred. The recombination events are particularly interesting as there was no obvious prior selection for their occurrence, and thus they may accurately reflect a normal mechanism of cellular recombination. A total of five recombinant joins have been sequenced. Our results, reported here, indicate that multiple recombinant events occur within small regions (about 50 bp) and that very short homologous stretches (3-4 bp) participate in joining two non-homologous sequences. This suggests that factors other than sequence homologies drive certain recombination events. These results have implications for site-directed recombination following the addition of exogenous DNA.
Only 8% of the sequences of the genomes of pseudorabies (PRV) and herpes simplex (type 1) (HSV) viruses are homologous. These homologous sequences have been shown previously to be distributed throughout most of the genomes of the two viruses. By means of blot hybridization of restriction fragments of HSV-1 DNA to cloned, nick-translated restriction fragments of PRV DNA, it was possible to compare the location on the genomes of these viruses of the homologous regions. The results showed that the genome of PRV is, for the most part, colinear with the IL arrangement of the genome of HSV-1. An inversion or translocation of sequences mapping on the PRV genome between 0.07 and 0.39 map units was observed on the genome of one of these viruses. A comparison of the map positions of five genes with known functions confirmed these findings. The genes coding for the major immediate-early protein, the major capsid protein, and the thymidine kinase occupy similar positions on the genome of PRV and on the genome of HSV-1 in the IL arrangement. However, the genes for DNA polymerase and for the major DNA binding protein appear to be inverted relative to one another on the genomes of the two viruses.
EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.
The DNA sequences at and around the junctions between viral and cellular DNA in the polyoma virus transformed mouse cell line, TS-A-3T3, have been determined. No common sequence specificity or structural features at the joins have been observed. The sequence indicates that the 94K truncated large T antigen found in TS-A-3T3 cells is a hybrid protein in which the carboxy-terminal 19 amino acids are encoded by adjacent host sequences. Moreover, the three early region transcripts initiated in viral sequences are also hybrid in nature and appear to utilize a host polyadenylation signal associated with the hexanucleotide, AATAAA, found 100 bp beyond a viral-host join.
The structure of gene sequences coding for the mRNA of human chorionic gonadotropin (hCG) alpha-subunit was investigated by Southern blot analysis of genomic DNAs using a cloned, full length cDNA probe. While four hormones, lutropin, follitropin, thyrotropin, and choriogonadotropin, have homologous alpha-subunits, only one gene that bears hCG-alpha sequences could be detected per haploid complement. The structure of this single gonadotropin alpha-subunit gene, which contains intervening sequences, is the same in DNA from first trimester and term placentae. However polymorphism was observed for the presence of a HindIII site and of an Eco RI site in the gene's 3' flanking sequences. The organization of hCG-alpha sequences in several trophoblastic and nontrophoblastic tumor cell lines, which produce hCG subunits, was also examined. In each, the same gene copy number and structure were seen as in normal tissue. Thus, the characteristics of ectopic alpha-subunit expression in these cells seem not to be determined by DNA rearrangements.
During the first round of the replication of pseudorabies virus DNA, replicating DNA is mainly in the form of circles. The main origin of replication is located inthe region of the molecule bearing the inverted repeat. Replication proceeds unidirectionally from the origin.