Biomedical subjects

J Paszkowski

Publications and source records attributed to J Paszkowski.

46 records · Page 3Linked to original sources

PubMed1990

Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration, its inheritance and factors affecting these processes in Nicotiana plumbaginifolia.

Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).

Benzamides↗

PubMed1990

Recognition efficiency of Dicotyledoneae-specific promoter and RNA processing signals in rice.

Heterologous gene expression experiments have shown that genes of Monocotyledoneae are often not transcribed in Dicotyledoneae, or produce pre-mRNA that is inefficiently or aberrantly processed. It is however not known how correctly and efficiently dicotyledon-specific gene expression signals are recognized in cells of Monocotyledoneae. Here we address this question using tobacco (Nicotiana tabacum) and rice (Oryza sativa) protoplasts transformed with the same hybrid gene constructs. Constructs including the nptII protein coding sequence fused to Cauliflower Mosaic Virus (CaMV) promoter and polyadenylation signals were used to obtain stably transformed cell lines of tobacco and rice. In one of the constructs the nptII coding region is interrupted by a modified intron-3 sequence from the soybean phaseolin gene. Although the mean number of hybrid gene copies integrated into the rice genome was on average 5- to 10-fold higher than in tobacco, the steady-state transcript level was 3 times lower. A lower level of transcript was also observed in transient expression experiments. The amount of the mature mRNA was not influenced by the presence of the intron. The phaseolin intron was processed in rice with high efficiency and an accuracy indistinguishable from that seen in tobacco.

Base Sequence↗

PubMed1990

Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.

Blotting, Southern↗

PubMed1988

Gene targeting in plants.

Although the generation of transgenic plants is now routine, the integration of foreign genetic information has so far been at random sites in the genome. We now present evidence for directed integration into a predicted location in the host plant genome. Protoplasts of transgenic tobacco (Nicotiana tabaccum) plants carrying copies of a partial, non-functional drug-resistance gene in the nuclear DNA were used as recipients for DNA molecules containing the missing part of the gene. Molecular and genetic data confirm the integration of the foreign DNA through homologous recombination within overlapping parts of the protein coding region, resulting in the formation of an active gene in the host chromosome. This approach is referred to as gene targeting. The gene targeting frequency (the number of drug-resistant clones resulting from gene correction compared to the number of resistant clones from parallel experiments with a similar non-interrupted hybrid gene) was 0.5-4.2x10. These experiments demonstrate the possibility of producing transgenic plants with desired modifications to a specific nuclear gene.

Journal Article↗

PubMed1985

Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer.

Two clones of N. tabacum, transformed to kanamycin resistance by direct transfer to protoplasts of a hybrid gene, consisting of the protein coding region from the bacterial gene for aminoglycoside phosphotransferase under the control of 5'/3' expression signals from cauliflower mosaic virus gene VI, in the bacterial plasmid pUC8, have been subjected to a detailed genetic crossing analysis accompanied by Southern blot analysis and enzyme activity assays of representative offspring. The genetic data obtained from large populations of R1/F1 and R2/F2 offspring as well as from more than 20 subclones of each of the original of the hybrid gene was stably integrated into chromosomal DNA of the original transformants, (b) that the gene normally was stably maintained during clonal proliferation, (c) that normally it is transmitted in a regular fashion (with exceptions) to sexual offspring, and (d) that it is inherited as a single dominant trait. Data from DNA hybridisation and enzyme assays confirm this interpretation. The functional gene is integrated together with several non-functional copies and bacterial plasmid sequences, which are inherited as one block together with the functional gene.

Brassica↗

PubMed1985

Chimeric vector construction for higher-plant transformation.

A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene.

Chimera↗

PubMed1984

Direct gene transfer to plants.

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F(1) generation.

Journal Article↗

PubMed1980

Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.

Adenine↗

PubMed1979

RNA synthesis in newly isolated and cultivated mesophyll protoplasts.

Tobacco leaf mesophyll protoplasts exhibit low incorporation of [3H]uridine and 32P into RNA, up to 12 h of cultivation, irrespective of the presence of phytohormones. After 24 h of cultivation a dramatic increase in RNA synthesis is observed; it is the highest in the heterogeneous nucleoplasmic RNA fraction. The protoplasts cultivated in the absence of phytohormones show lower incorporation of precursors.

Culture Techniques↗

PubMedDate not supplied

Prospects for the precise engineering of plant genomes by homologous recombination.

The targeting of chromosomal genes via homologous recombination (HR) is an essential tool of reverse genetics as applied for the functional assay of genes within complex genomes. However, in higher plants, foreign DNA integrates almost exclusively at random, non-homologous sites. A variety of environmental parameters known to influence levels of HR do not increase targeting frequencies when combined in gene-targeting experiments. The identification of cellular factors that may control the level of chromosomal HR in plant somatic cells is required. Plant genes encoding proteins similar to those involved in HR in other organisms can be found in the expanding sequence databases. Evidence for evolutionary conservation should help to decipher mechanisms of plant HR and possibly detect limiting factors. At present, however, only one genetic locus influencing levels of chromosomal recombination in plants has been well defined. Here we summarise current knowledge of HR and the status of gene targeting (GT) in plants, focusing on genetic approaches to molecular factors regulating HR levels.

DNA Damage↗