High-level recombination specific to polyomavirus genomes targeted to the integration-transformation pathway.

An unusually high incidence of interviral recombination was found in the process of integration of the polyomavirus genome concomitant with neoplastic transformation of nonpermissive cells. Transformants were isolated after mixed infections of Fischer rat cells with two mutants lacking restriction endonuclease sites and were analyzed for the presence of unselected integrated recombinant restriction fragments. A large fraction of the transformants isolated (38% of the 64 transformed cell lines studied) contained recombinant viral genomes that had undergone recombination in a 1.3-, 1.7-, or 3.6-kilobase-pair interval. More than 90% of these recombinant transformants showed evidence of crossovers in multiple intervals. To our knowledge, the recombination frequencies observed in these experiments represent the highest frequencies of homologous recombination reported for a mitotic mammalian system that does not involve transfection. In contrast to the elevated level of recombination in the integrated viral genomes, no evidence of recombination was obtained among the replicated unintegrated pool of viral genomes isolated from the same population of infected cells from which the recombinant transformants were derived. Either of two hypotheses can provide an explanation for the segregated recombination: either recombination occurs at elevated levels in a small, recombination-prone fraction of the population destined to become transformed, or recombination occurs only among those viral genomes which are engaged in the process of integration and thus interact with a recombinogenic host machinery (for example, the host scaffold). We favor the latter hypothesis.     

Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene

Summary Cochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.     

Simian virus 40 integration sites in the genome of virus-transformed mouse cells.

To gain information on the specificity of simian virus 40 (SV40) integration in the genome of transformed cells, mouse 3T3 cells were transformed by a temperature-sensitive (ts) SV40 mutant, using high multiplicity of infection (MOI). Transformed cells were superinfected with wild-type (wt) virus at high MOI. Clones were isolated and fused with permissive BSC-1 cells to promote virus rescue. All rescued viruses were of the ts type only. When the high-MOI transformants were infected with 3H-labeled wt SV40, the amount of radioactivity associated with their nuclear fraction was found to be similar to that of 3T3 cells. 3T3 cells were then transformed by ts SV40 at low MOI and superinfected by wt virus at high MOI. Upon fusion with BSC-1 cells, most clones produced both ts and wt virus. These results suggest that the number of stable SV40 integration sites in the 3T3 genome is limited, since they can be saturated by transformation at high MOI. When the MOI is low, the sites are not saturated and a subsequent infection can lead to integration.     

Replicative cis-advantage of polyomavirus regulatory region mutants in different murine cell lines.

To determine the relative growth advantages of polyomavirus regulatory region mutants selected from Friend leukemic and neuroblastoma cells persistently infected with polyomavirus A2 wild type, different murine cell lines, some of which are capable of further differentiation in vitro, were used as hosts in mixed infection and transfection. The tests allowed the determination, through the measurement of cis-advantage in replication, of the most effective polyomavirus regulatory region constitutions for a given cell line and, in some cases, for specific stages of cell differentiation. Furthermore, different domains of the viral regulatory region were shown to have different effects--positive, neutral, or negative--depending on the host analyzed.     

Stable allele replacement and unstable non-homologous integration events during transformation of Ascobolus immersus

An homologous transformation system for the filamentous fungus Ascobolus immersus has been developed, based on the complementation of a met2 mutation by the wild-type (wt) allele gene encoding homoserine O-transacetylase. Transformation of A. immersus met2 mutants occurs with moderate frequencies (about 50 transformants per microgram input DNA). Analysis of the DNA of the met2+ transformants showed that transformation resulted either in a single integration of the donor DNA into the genome by many different nonhomologous recombination events or in the substitution of the endogenous met2 mutation by the wt transforming allele. The relative frequencies of both events depended on the vector sequences carrying the cloned met2 gene. Whereas the substitution event led, as expected, to genetically stable transformants, the non-homologous integration was always associated with a strong instability when transformants were crossed and underwent meiosis.     

Episomal amplification or chromosomal integration of the viral genome: alternative pathways in hamster polyomavirus-induced lymphomas.

The state and expression of the hamster polyomavirus genome in a large panel of virus-induced lymphomas have been investigated. The viral genome is present within tumor cells either as abundant nonrandomly deleted extrachromosomal copies or as a single copy integrated into cellular DNA. We show that these two physical states are likely to be functionally equivalent: first, deletion and integration of the viral genome both inactivate the late coding region; second, the amount of viral early RNAs yielded by a single integrated copy appears to be very similar to that associated with several thousands of extrachromosomal copies of the viral genome. These data underline two essential requisites for hamster polyomavirus to become lymphomagenous: suppression of the late coding functions of the viral genome and expression of the viral oncogenes above a threshold level.     

The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Transgenic loci obtained after Agrobacterium tumefaciens -mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6–30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20–36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4–6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24–31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells.     

Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.     

Relationship of simian virus 40 tumor antigens to virus-induced mutagenesis.

We analyzed the mutation frequency to 8-azaguanine (8AZ) resistance in rat FR3T3 cells acutely infected with simian virus 40 wild type and tsA and early deletion mutants and in a series of temperature-sensitive (N) and temperature-insensitive (A) transformants derived from Chinese hamster lung (CHL) cells. Upon acute infection, the frequency of mutation to 8AZ resistance was raised at most by two- to eightfold over the spontaneous frequency, and it was independent of the presence of a functional 90,000-molecular-weight T antigen or 20,000-molecular-weight t antigen or both. Similarly, in the stable transformants of CHL cells, no correlation was found between functional T antigens and mutation to 8AZ resistance. It therefore seems unlikely that simian virus 40-induced transformation results from any mutagenic activity of this virus.     

盐藻肌动蛋白基因启动子驱动的bar基因表达作为核转化筛选标记

运用基因组步行方法克隆盐藻肌动蛋白基因5′上游调控序列,发现相对于ATG上游-573和-424bp的位置上分别有75bp长的两个重复序列。没有典型的TATA盒,但有两个TATA样结构、一个CCAAT结构和一个与GCTC(G／C)AAGGC一致的序列。以700bp的盐藻肌动蛋白基因启动子区序列驱动bar基因的表达作为转化盐藻的筛选标记。转化的藻细胞暗光恢复24h后,在含0．5μg／mL除草剂的培养基中常规培养生长1周,然后将细胞平铺于含0．5μg／mL除草剂的固体培养基上继续筛选培养。约20d后从固体培养板上挑选出5个藻落并作了进一步培养和分析。结果显示,5个转化藻中携带bar嵌合基因的整合位点均位于核基因组内。Southern blotting分析表明,仅有一个转化藻整合单拷贝的bar基因,而另外4个转化藻株则包含多个拷贝bar基因片段,提示盐藻核基因转化主要是外源基因的随机整合,外源基因在转化盐藻中的整合拷贝数并不影响其除草剂抗性。RT-PCR方法证明了bar基因在转化藻中的转录。5个转化藻在含除草剂的液体培养基中维持生长了至少7个月,表明核基因转化的稳定性。     

Transformation and mutagenesis of the nematode-trapping fungus Monacrosporium sphaeroides by restriction enzyme-mediated integration (REMI)

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants microg(-1) per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.     

Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions.

DNA transformations of mouse tissue culture cells and mouse embryos were carried out by iontophoretic microinjection of DNA. Iontophoresis involves the use of an externally applied electric current to expel DNA molecules from the injection micropipette into the impaled cell (microelectrophoresis). Restriction analysis of transformants obtained by using this procedure demonstrated that this method gave strikingly different results from those previously obtained by others with transformants generated by pressure injection. First, tandem insertions were never observed in any of our transformants. Second, despite the lack of tandem insertions, we have obtained transformants which have integrated many copies of the injected DNA sequences, probably via a comparably large number of independent integration events. And third, in one transformant, an injected BamHI restriction fragment was found to have been integrated in its entirety; this included the preservation of the terminal BamHI recognition sequence. Based on these observations, we discuss the potential usefulness of iontophoretic DNA microinjections in DNA transformation studies that are focused either on the analysis of the regulation of gene expression or on the targeting of DNA sequences into the eucaryotic cell genome.     

Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts.

Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.     

Induction of polyomavirus-specific CD8(+) T lymphocytes by distinct dendritic cell subpopulations

Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8(+) T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8(+) T-cell responses in vivo.     

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.     

The RecJ DNase strongly suppresses genomic integration of short but not long foreign DNA fragments by homology-facilitated illegitimate recombination during transformation of Acinetobacter baylyi

Homology-facilitated illegitimate recombination (HFIR) promotes genomic integration of foreign DNA with a single segment homologous to the recipient genome by homologous recombination in the segment accompanied by illegitimate fusion of the heterologous sequence. During natural transformation of Acinetobacter baylyi HFIR occurs at about 0.01% of the frequency of fully homologous recombination. The role of the 5' single-strand-specific exonuclease RecJ in HFIR was investigated. Deletion of recJ increased HFIR frequency about 20-fold compared with wild type while homologous recombination was not affected. Illegitimate fusion sites were predominantly located within 360 nucleotides away from the homology whereas in wild type most fusion sites were distal (500-2500 nucleotides away). RecJ overproduction reduced the HFIR frequency to half compared with wild type, and transformants with short foreign DNA segments were diminished, leading to on average 866 foreign nucleotides integrated per event (682 in wild type, 115 in recJ). In recJ always the 3' ends of donor DNA were integrated at the homology whereas in wild type these were 3' or 5'. RecJ apparently suppresses HFIR by degrading 5' non-homologous DNA tails at the post-synaptic stage. We propose that the RecJ activity level controls the HFIR frequency during transformation and the amount of foreign DNA integrated per event.