Biomedical subjects
Publications and source records attributed to A Depicker.
A new hypervariable tandem repeat was isolated from the pig genome and characterized by DNA sequence. The use of this DNA fragment as a probe in order to follow allelic segregation and DNA fingerprinting in pigs, horses and rabbits is documented.
We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500- to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units.
We have constructed an Agrobacterium-mediated plant vector,pHTT88,which upon integration into the plant chromosomes can produce transcriptional fusions between upstream control elements in the plant genome and the gene for neomycin phosphotransferase II (npt-II). The vector is based on a juxtaposition of a T-DNA right border to the coding sequence of npt-II. Using this system it is possible to isolate kanamycin-resistant plant calli where the site of integration is suitably positioned downstream of plant transcription signals. We have demonstrated that the NPT-II activity of such tobacco transformants shows different tissue-specific patterns of regulation in the regenerated plants. Among eight transformants analyzed, we have isolated one gene fusion expressed only in roots and others expressed only in stem. The results demonstrate that a gene fusion vector can be used effectively in plants to identify and characterize regulatory elements.
Explore the source record for details and available documents.
Molecular cloning has been used to isolate the ends of that portion of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which has been designated T-DNA and which has been transferred to the genome of tobacco crown gall tumor cells. Analysis of the DNA sequences of the plant border clones compared with the corresponding sequences of the Ti plasmid suggests that the mechanism of transferred DNA integration and subsequent stabilization is precise at the right border and imprecise on the left. The T-DNA junction occurs within a variation of a single base pair (bp) on the right but varies over at least 70 bp on the left. In addition, there are several sequences which are repeated near the ends of the T-DNA region in the Ti plasmid. Seemingly, there is no specificity with regard to the site of integration in the plant genome.
The DNA sequence of the nopaline synthase gene (nos) from Agrobacterium tumefaciens Ti plasmid pTiT37 and adjacent regions up to the right border of the T-DNA was determined. The 5' and 3' termini of the polyadenylated nos mRNA, isolated from a T37 tobacco teratoma tumor line, were localized by S1 mapping. The final mRNA is unspliced, encoded by a region of about 1450 bp, and specifies an open reading frame of 413 amino acids. Potential transcriptional signals in the 5' flanking DNA, such as CATAAA ("TATA box") and GGTCACTAT ("CAT box"), bear close resemblance to other eukaryotic promoters. Two putative polyadenylation signals, AATAAA and AATAAT, are found about 135 and 50 bp from the 3' end, respectively. This study may provide information for the development of expression vectors for genes in plant cells; moreover, the structural gene can be used as an easy screenable marker.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted 'opines'). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Ti-plasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.
Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA) have been cloned from transformed plant cells of tobacco. Detailed mapping with restriction enzymes and nucleotide sequence analysis of two independent clones were used to study the molecular structure of the ends of the T-DNA. One clone contains the two ends of the T-DNA joined together; the other contains one end of the T-DNA joined to repetitive plant DNA sequences. These studies provide direct evidence that the T-DNA can be integrated into the plant genome. In addition, the data suggest that in the plant, T-DNA can be tandemly repeated. Sequence analysis of the junction of crown gall clone 1 reveals several direct repeats as well as an inverted repeat; these structures may be involved in the transfer of the DNA from Agrobacterium to plant cells.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi::RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8 sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.
Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.
The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10(-7) transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum frequency of 3.5 10(-7) transformants per total recipient population was obtained. Agrobacterium Ti-plasmids were introduced in the strain GV3100 with a maximal efficiency of 4.5 10(-8). These experiments provide further evidence that the Ti-plasmid is responsible for the oncogenic properties of A tumefaciens and for its capacity to induce "opine" synthesis in Crown-gall plant cells.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.