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1.
Schneeberger RG Zhang K Tatarinova T Troukhan M Kwok SF Drais J Klinger K Orejudos F Macy K Bhakta A Burns J Subramanian G Donson J Flavell R Feldmann KA 《Functional & integrative genomics》2005,5(4):240-253
Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5 and 3 flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5 upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in March 2005 with corrections to Dr. Tatarinovas name. 相似文献
2.
Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique 总被引:3,自引:0,他引:3
Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique.
The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type
adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments.
Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the
integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred
in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual
plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic
Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored
PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant,
indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied. 相似文献
3.
A plasmid rescue technique for the recovery of plant DNA disrupted by T-DNA insertion 总被引:6,自引:1,他引:5
Arabidopsis mutants generated by insertion of the T-DNA from Ti plasmid 3850∶1003 serve as a starting point for the isolation of novel
genes. The disrupted plant DNA can be recovered using a plasmid rescue technique utilizing high efficiency electroporation.
Rescued plasmids are resistant to ampicillin and contain an origin of replication from pBR322. Plasmids generated from either
the left or right border of the T-DNA that carry flanking DNA sequences can be identified by analyzing the products of restriction
enzyme digests on agarose gels. The plasmids with flanking sequences can then serve as a starting point for cloning plant
sequences that share homology to the DNA at the point of T-DNA insertion. 相似文献
4.
Kan Wang Chris Genetello Marc Van Montagu Patricia C. Zambryski 《Molecular & general genetics : MGG》1987,210(2):338-346
Summary We present a detailed analysis of the function of the right and left T-DNA border regions of the nopaline Ti plasmid of Agrobacterium tumefaciens. An avirulent deletion of the right border of the nopaline Ti plasmid (pGV3852) was used as an acceptor for 14 different T-DNA border constructs. The functional activities of these constructs were assayed by their ability to restore virulence, i.e. transformation on inoculated plants. Tumorigenicities were measured in several independent experiments over a 2 year period and the statistical significance of their relative levels was evaluated. The data indicate: (i) the entire sequence of the 25 bp direct repeat of the T-DNA is required to provide an efficient substrate for mediating T-DNA transfer events; deletion derivatives of either the conserved or the vaiable domain of the repeat are defective in T-DNA transfer; (ii) while the 25 bp direct repeat alone can promote the T-DNA transfer, the flanking sequences of the repeats enhance (on the right) or attenuate (on the left) their activity; and (iii) tumorigenicity measurements vary depending on the plant assay system: potato discs are more sensitive than wounded tobacco leaves in detecting differences in T-DNA border activity. 相似文献
5.
A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome 总被引:14,自引:0,他引:14
T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants. 相似文献
6.
Studies in several plants have shown that Agrobacterium tumefaciens T-DNA can integrate into plant chromosomal DNA by different mechanisms involving single-stranded (ss) or double-stranded
(ds) forms. One mechanism requires sequence homology between plant target and ssT-DNA border sequences and another double-strand-break
repair in which preexisting chromosomal DSBs “capture” dsT-DNAs. To learn more about T-DNA integration in Solanum lycopersicum we characterised 98 T-DNA/plant DNA junction sequences and show that T-DNA left border (LB) and right border transfer is
much more variable than previously reported in Arabidopsis thaliana and Populus tremula. The analysis of seven plant target sequences showed that regions of homology between the T-DNA LB and plant chromosomal
DNA plays an important role in T-DNA integration. One T-DNA insertion generated a target sequence duplication that resulted
from nucleolytic processing of a LB/plant DNA heteroduplex that generated a DSB in plant chromosomal DNA. One broken end contained
a captured T-DNA that served as a template for DNA repair synthesis. We propose that most T-DNA integrations in tomato require
sequence homology between the ssT-DNA LB and plant target DNA which results in the generation of DSBs in plant chromosomal
DNA. 相似文献
7.
Ortega D Raynal M Laudié M Llauro C Cooke R Devic M Genestier S Picard G Abad P Contard P Sarrobert C Nussaume L Bechtold N Horlow C Pelletier G Delseny M 《Comptes rendus biologies》2002,325(7):773-780
Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions. 相似文献
8.
9.
Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis 总被引:6,自引:0,他引:6
Linda A. Castle Deena Errampalli Tammy L. Atherton Linda H. Franzmann Elizabeth S. Yoon David W. Meinke 《Molecular & general genetics : MGG》1993,241(5-6):504-514
Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis. 相似文献
10.
Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice 总被引:22,自引:0,他引:22
Jeong DH An S Park S Kang HG Park GG Kim SR Sim J Kim YO Kim MK Kim SR Kim J Shin M Jung M An G 《The Plant journal : for cell and molecular biology》2006,45(1):123-132
We have generated 47,932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T(1) or T(2) plants will be provided to the scientific community. 相似文献
11.
Robert A. Rubin 《Molecular & general genetics : MGG》1986,202(2):312-320
Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (TL-) and right (TR-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the TL-DNA or the TR-DNA are fully tumorigenic. Analogous tmr constructs containing only the TL-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the TR-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the TL-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine TL-DNA left border is defective in this DNA-transfer process. 相似文献
12.
Zhang J Guo D Chang Y You C Li X Dai X Weng Q Zhang J Chen G Li X Liu H Han B Zhang Q Wu C 《The Plant journal : for cell and molecular biology》2007,49(5):947-959
We isolated 13 804 T-DNA flanking sequence tags (FSTs) from a T-DNA insertion library of rice. A comprehensive analysis of the 13 804 FSTs revealed a number of features demonstrating a highly non-random distribution of the T-DNA insertions in the rice genome: T-DNA insertions were biased towards large chromosomes, not only in the absolute number of insertions but also in the relative density; within chromosomes the insertions occurred more densely in the distal ends, and less densely in the centromeric regions; the distribution of the T-DNA insertions was highly correlated with that of full-length cDNAs, but the correlations were highly heterogeneous among the chromosomes; T-DNA insertions strongly disfavored transposable element (TE)-related sequences, but favored genic sequences with a strong bias toward the 5' upstream and 3' downstream regions of the genes; T-DNA insertions preferentially occurred among the various classes of functional genes, such that the numbers of insertions were in excess in certain functional categories but were deficient in other categories. The analysis of DNA sequence compositions around the T-DNA insertion sites also revealed several prominent features, including an elevated bendability from -200 to 200 bp relative to the insertion sites, an inverse relationship between the GC and TA skews, and reversed GC and TA skews in sequences upstream and downstream of the insertion sites, with both GC and TA skews equal to zero at the insertion sites. It was estimated that 365 380 insertions are needed to saturate the genome with P = 0.95, and that the 45 441 FSTs that have been isolated so far by various groups tagged 14 287 of the 42 653 non-TE related genes. 相似文献
13.
M. C. Failla F. Maimone A. De Paolis P. Costantino M. Cardarelli 《Plant molecular biology》1990,15(5):747-753
The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part [1,2]. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots. 相似文献
14.
Stephen L. Sain Kwabena K. Oduro Douglas B. Furtek 《Plant Cell, Tissue and Organ Culture》1994,37(3):243-251
Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer. 相似文献
15.
To investigate the various integration patterns of T-DNA generated by infection withAgrobacterium, we developed a vector (pRCV2) for the effective T-DNA tagging and applied it to tobacco (Nicotiana tabacum cv. Havana SR1). pRCV2 was constructed for isolating not only intact T-DNA inserts containing both side borders of T-DNA,
but also for partial T-DNA inserts that comprise only the right or left side. We also designed PCR confirmation primer sets
that can amplify in several important regions within pRCV2 to detect various unpredictable integration patterns. These can
also be used for the direct inverse PCR. Leaf disks of tobacco were transformed withAgrobacterium tumefaciens LBA4404 harboring pRCV2. PCR and Southern analysis revealed the expected 584 bp product for thehpt gene as well as one of 600 bp for thegus gene in all transformants; one or two copies were identified for these integrated genes. Flanking plant genomic DNA sequences
from the transgenic tobacco were obtained via plasmid rescue and then sequenced. Abnormal integration patterns in the tobacco
genome were found in many transgenic lines. Of the 17 lines examined, 11 contained intact vector backbone; a somewhat larger
deletion of the left T-DNA portion was encountered in 4 lines. Because nicking sites at the right border showed irregular
patterns when the T-DNA was integrated, it was difficult to predict the junction regions between the vector and the flanking
plant DNA. 相似文献
16.
T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation 总被引:5,自引:0,他引:5
De Buck Sylvie De Wilde Chris Van Montagu Marc Depicker Ann 《Molecular breeding : new strategies in plant improvement》2000,6(5):459-468
Transgenic Arabidopsis and tobacco plants (125) derived from seven Agrobacterium-mediated transformation experiments were screened by polymerase chain reaction and DNA gel blot analysis for the presence of vector `backbone' sequences. The percentage of plants with vector DNA not belonging to the T-DNA varied between 20% and 50%. Neither the plant species, the explant type used for transformation, the replicon type nor the selection seem to have a major influence on the frequency of vector transfer. Only the border repeat sequence context could have an effect because T-DNA vector junctions were found in more than 50% of the plants of three different transformation series in which T-DNAs with octopine borders without inner border regions were used. Strikingly, many transgenic plants contain vector backbone sequences linked to the left T-DNA border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. We assume that integration into the plant genome of complete vector backbone sequences could be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model would imply that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer. 相似文献
17.
18.
M. Fladung 《Molecular & general genetics : MGG》1999,260(6):574-581
The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into
the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is
available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events.
In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. × tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one
of these transgenic lines (Esch5:35S-rolC-##1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed
that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders,
and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C
to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located
within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed
plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive
from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional
truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this
truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special
feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently
observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of
single-stranded DNA during mitotic cell divisions.
Received: 9 June 1998 / Accepted: 6 October 1998 相似文献
19.
Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction 总被引:18,自引:0,他引:18
Colwyn M. Thomas David A. Jones James J. English Bernard J. Carroll Jeffrey L. Bennetzen Kate Harrison Alan Burbidge Gerard J. Bishop Jonathan D. G. Jones 《Molecular & general genetics : MGG》1994,242(5):573-585
We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis. 相似文献