Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.     

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for -glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.     

A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Bam HI with ³²P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1-transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32–0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state.     

Replication and amplification of recombinant plasmid molecules as extra chromosomal elements in transformed mammalian cells

Mouse thymidine kinase negative (TK^?) L cells, F4-12B2TK^? Friend erythroleukemic cells and hamster BHKTK^? cells were transformed in the absence of carrier DNA with closed circular molecules of herpes simplex virus 1 TK DNA cloned in plasmid pAT153 (pTK-1). The physical status of donor DNA in the transformed cells was studied by Southern blot hybridization and spot hybridization techniques. Up to 65 copies of pTK-1 DNA molecules/cell were present in transformed cells grown under selective conditions. Whereas a steady increase in the number of pTK-1 copies/cell was found during the first few weeks of growth in selective medium, 2–8 months later copy numbers varied within the same cell line and their numbers rarely exceeded fifty copies/cell. Donor DNA sequences were found both in the Hirt precipitate and in the supernatant showing that some of the pTK-1 molecules existed in circular form. Many of the cell lines gave a Southern blot hybridization pattern indicative of pTK-1 sequences integrated into high molecular weight DNA as well as present in a circular configuration.     

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.     

Human T-Lymphocyte Transformation with Human T-Cell Lymphotropic Virus Type 2

Human T-cell lymphotrophic virus type 2 (HTLV-2), a common infection of intravenous drug users and subpopulations of Native Americans, is uncommon in the general population. In contrast with the closely related HTLV-1, which is associated with both leukemia and neurologic disorders, HTLV-2 lacks a strong etiologic association with disease. HTLV-2 does shares many properties with HTLV-1, including in vitro lymphocyte transformation capability. To better assess the ability of HTLV-2 to transform lymphocytes, a limiting dilution assay was used to generate clonal, transformed lymphocyte lines. As with HTLV-1, the transformation efficiency of HTLV-2 producer cells was proportionately related to the number of lethally irradiated input cells and was comparable to HTLV-1-mediated transformation efficiency. HTLV-2-infected cells were reproducibly isolated and had markedly increased growth potential compared to uninfected cells; HTLV-2 transformants required the continued presence of exogenous interleukin 2 for growth for several months and were maintained for over 2 years in culture. All HTLV-2-transformed populations were CD2 and/or CD3 positive and B1 negative and were either CD4⁺ or CD8⁺ populations or a mixture of CD4⁺ and CD8⁺ lymphocytes. Clonality of the HTLV-2 transformants was confirmed by Southern blot analysis of T-cell receptor β chain rearrangement. Southern blot analysis revealed a range of integrated full-length genomes from one to multiple. In situ hybridization analysis of HTLV-2 integration revealed no obvious chromosomal integration pattern.     

SV40 RNA: Filter hybridization for rapid isolation and characterization of rare RNAs

Modifications of the filter hybridization method for the measurement and isolation of simian virus 40 (SV40) RNA from transformed cells are described. The modified method used small (0.02 cm²) nitrocellulose filters with > 30 μg/cm² SV40 DNA applied following formaldehyde denaturation. The small volume and high DNA densities allowed hybridization to be completed in 2 h and washing after hybridization to be completed in 6 h. The washing reduced background to 1 × 10^?5 of input radioactivity without using nucleases. The efficiency of hybridization after washing was 40% or greater. These procedures have allowed the quantification of proportions of SV40 RNA in labeled RNA from transformed lines and the characterization of SV40 RNAs by electrophoresis and cell-free translation. A 3.7-kb SV40 RNA from SV80 cells was discovered in this work.     

IRAP,REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (H^chH^chAABB; 2n?=?6x?=?42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (H^chH^ch; 2n?=?2x?=?14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n?=?4x?=?28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

大赖草总DNA转化小麦的分子证据

用来自大麦组的４个高度重复序列克隆了ｐＨｖ７１６１,ｐＨｖ７１７８９,ｐＨｖ７１９１、ｐＨｖ７２９３,经地高辛和同位素２种方法标记后作为探针,对新疆大赖草（供体）、春麦７６１（受体）以及用大赖草总ＤＮＡ通过花粉管通道转化成功的大穗转化株基因组在高度严谨条件下进行了分子杂交。结果表明,这４个探针可以探查出基因内一种具有主串产重复单位的散在重复序列。比较受共体和转化体的杂交图谱,发现在转化株中出现了     

Inactivation of the nopaline synthase gene by double transformation: reactivation by segregation of the induced DNA

Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop⁺ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA transferred DNA - NPTII neomycin phosphotransferase II - uidA -glucuronidase - Km kanamycin - Gm gentamicin - nop⁺ nopaline positive - nop^– nopaline negative - MS medium, Murashige-Skoog medium     

Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment

It has been proposed that transgenic plants of cereals can be generated by inoculating florets with Agrobacterium at or near anthesis. This procedure is shown to lead to the production of embryos of wheat and barley with enhanced resistance to antibiotic selection. It has also been possible to recover plants of wheat, barley and maize that gave positive hybridization signals with probes produced from within the T-DNA of the Agrobacterium vector. However, no evidence was found for transmission of the bands detected by hybridization in the progeny of the putative transgenic plants nor could enzyme activity associated with the resistance genes be found in plant extracts. Furthermore, undigested genomic DNA from the plants that were positive when probed with the T-DNA, showed hybridization to bands smaller than the genomic DNA. It is suggested that the apparent transformation is an artifact of the procedure and does not reflect transformation of the plant nuclear genome.     

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD₆₀₀???0.6 and diluted to 10⁹ cells ml^?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml^?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T₀). Among T₁ seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ ²) test. Southern hybridization of genomic DNA from kanamycin-resistant T₁ transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T₁ transgenic plants, thereby confirming stable expression of the nptII gene.     

Towards Molecular Tagging of Karnal Bunt Resistance Gene(s) in Wheat

We describe preliminary results of cosegregation of a DNA probe with Karnal bunt resistance genets) in wheat. The tagging strategy utilized 4 uniformly resistant and 13 uniformly susceptible F³ families from a cross of Karnal bunt resistant (HD29) and susceptible (WL711) lines of Triticum aestivum. Bulked DNA of 8–10 plants in each family were tested by gel blot DNA hybridization. Thirty three per cent of the probes (11/33) detected restriction fragment length polymorphism (RFLP) between Karnal bunt resistant and susceptible lines. Probe Cxp1 from chromosome group 6 detected the resistant parent (HD29) polymorphic band in all the resistant family bulks but was missing in 6 uniformly susceptible family bulks. A similar pattern was observed with probe XKsu G34 from 1DL. The intensity of the resistant parent band in the susceptible family bulks was less as compared to the resistant family bulks. Probe Xksu H8 which mapped on chromosomes 2, 3, 5 and 7D on the T. tauschii map did not differentiate resistant family bulks from susceptible family bulks. Our results indicated that one or more resistance genets) are located on chromosome groups 1 and 6 of wheat.     

Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew

Several Triticum aestivum L.-Haynaldia villosa disomic 6VS/6AL translocation lines with powdery mildew resistance were developed from the hybridization between common wheat cultivar Yangmai 5 and alien substitution line 6V(6A). Mitotic and meiotic C-banding analysis, aneuploid analysis with double ditelosomic stocks, in situ hybridization, as well as the phenotypic assessment of powdery mildew resistance, were used to characterize these lines. The same translocated chromosome, with breakpoints near the centromere, appears to be present in all the lines, despite variation among the lines in their morphology and agronomic characteristics. The resistance gene, conferred by H. villosa and designated as Pm21, is a new and promising source of powdery mildew resistance in wheat breeding.This research was supported by grants from the National High-Tech R and D Program and the National Science and Technology Commission