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J Paszkowski

Publications and source records attributed to J Paszkowski.

At least 37 records · Page 2Linked to original sources
PubMed1997

Arabidopsis thaliana mutants altered in homologous recombination.

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination. Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.

Arabidopsis↗
PubMed1997

Methylation of cytosines in nonconventional methylation acceptor sites can contribute to reduced gene expression.

Epigenetic silencing of gene expression is often correlated with extensive DNA methylation at cytosine residues in the promoter and the coding region of silenced genes. Increasing evidence indicates that, in such cases, DNA methylation can also occur in sequence contexts other than CG and CNG, resulting in genomic regions with almost complete modification of cytosines. Whether this nonconventional methylation at CNN sites also contributes to gene repression is not known. We constructed genes with a promoter and a coding region devoid of the conventional methylation acceptor sites CG and CNG in addition to constructs with the corresponding wild-type sequences containing these sites. We generated unmethylated and completely methylated DNA by the polymerase chain reaction and performed expression assays in plant protoplasts. Quantification of transcript levels by RNase protection assay demonstrated that DNA methylation at positions other than CG or CNG sites contributes to the reduction in gene expression.

Amino Acid Sequence↗
PubMed1997

High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter.

Two selectable marker genes harbouring the bar coding region but differing in their promoters were compared in an Arabidopsis thaliana transformation assay using in planta infiltration with Agrobacterium tumefaciens. Surprisingly, in four Arabidopsis ecotypes examined, the 1' promoter from the right T-DNA was superior to the most commonly used 35S promoter of cauliflower mosaic virus (CaMV). The ecotype Wassilewskija gave the highest transformation frequencies, with an average of between 5.3 and 6.3% of the seedlings subjected to the selection. This is approximately 30-fold higher than previously reported results. Analysis of T-DNA integration patterns in single transformed plants or pooled populations revealed independent T-DNA integration events in each case. Results show that the 1' promoter is an attractive alternative to the 35S promoter for the generation of T-DNA insertion lines. The 1' promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology-mediated gene silencing.

Agrobacterium tumefaciens↗
PubMed1997

Mutants of Arabidopsis thaliana hypersensitive to DNA-damaging treatments.

A simple screening method was developed for the isolation of Arabidopsis thaliana mutants hypersensitive to X-ray irradiation. The root meristem was used as the target for irradiation with sublethal doses of X rays, while protection of the shoot meristem by a lead cover allowed the rescue of hypersensitive individuals. We isolated nine independent X-ray-hypersensitive mutants from 7000 M2 seedlings. Analysis of three chosen mutants (xrs4, xrs9 and xrs11) showed that alterations in single recessive alleles are responsible for their phenotypes. The mutations are not allelic but linked and map to chromosome 4, suggesting mutations in novel genes as compared to previously mapped mutant alleles. Importantly, hypersensitivity to X rays was found to correlate with hypersensitivity to the DNA-alkylating agent mitomycin C, which provokes interstrand crosslinks, and/or to methyl methanesulfonate, which is known as a radiomimetic chemical. These novel phenotypes suggest that the mutants described here are altered in the repair of DNA damage, most probably by recombinational repair.

Arabidopsis↗
PubMed1997

[45 years of experience in surgical treatment for cancer of the large intestine].

The aim of the study was to evaluate the results of surgical treatment of colorectal cancer during 45 years of existence of the Department. In this time 1478 cases of rectal cancer and 1008 cases of colon cancer were operated on. Most commonly the diseases occurred between the sixth and seventh decade of live (32.8%). Histology revealed tubular adenocarcinoma in 83.3% of the cases, mucinous adenocarcinoma in 13.5% and other types in 3.2%. The choice of the surgical procedure was based on the individual characteristics of each case, including: localisation of the tumor, histological type, clinical staging, sex of the patient, obesity and overall operative risk. The tumor was localised less then 10 cm above the anal verge in 70% of the patients with rectal cancer. Well differentiated carcinomas (G1) were seen in 32% of the patients, moderately differentiated carcinomas (G2) in 57% and poorly differentiated (G3) in 11%. Curative resections were performed in 64.7% of the patients (1608 cases) and palliative procedures in 35.3% of the patients (878 cases). The mortality rate after curative surgery was 6% and after palliative procedures 5%. The use of combined therapy consisting of surgical treatment and chemo- or radiotherapy allowed for obtaining five years survival rate of 57.4%, local recurrences were seen in 21% of patients. Analysing our own material we evaluated the radicality of different types of operations and the possibility of preserving the sphincter apparatus.

Adenocarcinoma↗
PubMed1997

[Combined treatment of large bowel cancer].

During the years 1989 to 1994, in the Department of General Surgery we performed surgery on 800 cases of large bowel cancer. The cases comprised 412 men (51.5%) and 388 women (48.5%), aged 25 to 85, the most numerous age group ranging from 50 to 70 (60%). Radical surgery was carried out on 501 cases (62.7%) and palliative surgery on 299 cases (37.3%). In cases with histopathological evaluation pTNM-T1N0M0 or T2N0M0 we refrained from chemo- and radiotherapy. In cases of rectal cancer with advanced staging T3N1M0 or T4N2M1 we applied radiotherapy, whereas in cases of cancer localized on other parts of large bowel we had recourse to chemotherapy as combined treatment. Radiotherapy was applied irrespective of grading, whereas chemotherapy was applied in cases of cancer with grading G2 and G3. Radiative energy was used in 65 cases and chemotherapy in 120 cases. Postoperational mortality amounted to 6%. Five years survival after radical surgery achieved 57.4%. Local recurrence took place in 21% of the cases. The control group consisted of the cases subjected to surgery in the years 1984-1989 when only surgery was performed and 5-years survival after radical surgery amounted to 48% and local recurrence was found in 30% of the cases.

Adult↗
PubMed1996

A change of ploidy can modify epigenetic silencing.

A silent transgene in Arabidopsis thaliana was reactivated in an outcross but not upon selfing of hemizygous plants. This result could only be explained by assuming a genetic difference between the transgene-free gametes of the wild-type and hemizygous transgenic plants, respectively, and led to the discovery of ploidy differences between the parental plants. To investigate whether a change of ploidy by itself can indeed influence gene expression, we performed crosses of diploid or tetraploid plants with a strain containing a single copy of a transgenic resistance gene in an active state. We observed reduced gene expression of the transgene in triploid compared with diploid hybrids. This led to loss of the resistant phenotype at various stages of seedling development in part of the population. The gene inactivation was reversible. Thus, an increased number of chromosomes can result in a new type of epigenetic gene inactivation, creating differences in gene expression patterns. We discuss the possible impact of this finding for genetic diploidization in the light of widespread, naturally occurring polyploidy and polysomaty in plants.

Arabidopsis↗
PubMed1994

Gene inactivation in Arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations.

Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.

Arabidopsis↗
PubMed1994

High fidelity extrachromosomal recombination and gene targeting in plants.

The precision of extrachromosomal homologous recombination and gene targeting in plant cells was investigated. Recombination was directed to introns of selectable marker genes where potential changes could persist without affecting the function and therefore the selectability of the genes. Approximately 9 kb of crossover regions was rescued and sequenced. Changes were detected at a frequency below one point mutation per 1000 bp, indicating that extrachromosomal recombination and gene targeting both appear to occur with high fidelity.

Crossing Over, Genetic↗
PubMed1993

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells.

Cytosine↗
PubMed1993

Stress-induced intrachromosomal recombination in plant somatic cells.

Levels of induced homologous recombination between chromosomal repeats in plant somatic cells were examined. Transgenic plants of Nicotiana tabacum hemi- or homozygous for pairs of deletion derivatives of the neomycin phosphotransferase (nptII) marker gene integrated at a single genomic locus were produced. Homologous recombination within the overlapping parts of the nptII gene restored the function and the resulting kanamycin resistance was used for scoring recombination frequency. The recombination events were confirmed by the appearance of a characteristic 1245-base-pair EcoRV fragment detected in all kanamycin-resistant clones tested. The rate of spontaneous recombination was found to be related to the copy number of recombination substrates and was 9 x 10(-5) and 19 x 10(-5) for hemi- and homozygote strains, respectively. Ionizing radiation, mitomycin C, and heat shock markedly increased the frequency of intrachromosomal recombination. Low doses of x-rays (1.25 Gy) enhanced the relative recombination frequency to approximately twice the spontaneous value. The presence of mitomycin C increased the frequency of recombination 9-fold and exposure to an elevated temperature (50 degrees C) increased it 6.5-fold. The x-ray and heat shock treatments reduced cell viability to 53% and 8%, respectively. Mitomycin C treatment had no effect on cell survival.

Journal Article↗
PubMed1993

Replication of the DNA A component of African cassava mosaic virus in a heterologous system.

The capacity for autonomous replication of the DNA A of African cassava mosaic virus (ACMV), a member of the bipartite geminiviruses infecting dicotyledonous plants, has been compared in host and non-host cells. A derivative of the ACMV DNA A was transfected into tobacco and maize protoplasts. Although ACMV is not able to infect maize, replication of the DNA A in maize protoplasts was observed to occur. The efficiency of replication was 10 to 20% of that seen in tobacco protoplasts. In both plant systems, replication was detected after the onset of cell division. ACMV replication in maize cells was compared to that of wheat dwarf virus and found to be 10 to 20% of that observed with the monocotyledon-specific virus. Insertion of 1165 bp of non-viral DNA into the ACMV DNA A prevented replication in maize but not in tobacco.

Blotting, Southern↗
PubMed1992

Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes.

A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the protein-coding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.

Base Sequence↗
PubMed1992

Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays.

Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.

Base Sequence↗
PubMed1991

Reversible inactivation of a transgene in Arabidopsis thaliana.

Fifty percent of Arabidopsis thaliana plants transgenic for a hygromycin resistance gene failed to transmit the resistance phenotype to the progeny. The complete transgene was, however, inherited in all cases according to Mendelian laws as observed by Southern analysis. This discrepancy between genotype and phenotype was the result of a reduced level of transcript in the sensitive transformants. The gene inactivation occurred in plants with multicopy integration of the foreign DNA. No definite correlation was found between gene inactivity and methylation of cytidine residues in the transgene sequence. Explants from several sensitive transformed plants regained a low level of hygromycin resistance on callus induction medium. Subsequent generations obtained by self-pollination were sensitive. In contrast, spontaneous restoration of hygromycin tolerance was observed in seedlings originating from out-crosses with wild-type plants or a different sensitive transformant. A reduction of the copy number was not a prerequisite for spontaneous reactivation. The resistance was often lost again in the next generation. Inactivation and reactivation of the transgene are therefore reversible.

Blotting, Northern↗
PubMed1991

The 3'-terminal region of the hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotiana tabacum.

We have modified the 3'-coding region of the hygromycin B (Hy) phosphotransferase-encoding gene (hph). The level of Hy resistance conferred by the modified hph genes was determined in Escherichia coli and Nicotiana tabacum. Polypeptides up to 12 amino acids shorter or longer than the wild-type (wt) protein remained active in both pro- and eukaryotic cells. A gene construct of wt length, but with a modified 3' sequence, was less active as measured by the level of Hy resistance. Therefore, the nature of the C terminus of the hph gene product, rather than the length of the polypeptide, influences the ability to confer resistance to Hy.

Amino Acid Sequence↗
PubMed1990

Intrachromosomal recombination in plants.

Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin selection did not increase the number of recombinant kanamycin-resistant (KanR) cell clones. DNA analysis of KanR clones derived from cells carrying multiple tandem recombination units suggested that these units have a tendency to undergo concerted recombination. Recovery and analysis of kanamycin-sensitive seedlings with patches of KanR cells provided direct evidence for mitotic recombination in plant tissue.

Base Sequence↗