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D Dudits

Publications and source records attributed to D Dudits.

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

Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells.

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa. Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements. Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases. The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation. The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments. Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities. the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex. Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa. The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants.

Amino Acid Sequence↗
PubMed1997

Factors affecting transient expression of vector constructs in wheat protoplasts.

Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

Gene Expression↗
PubMed1996

Identification of three highly expressed replacement histone H3 genes of alfalfa.

One genomic and six cDNA clones for the replacement histone H3.2 protein of alfalfa (Medicago sativa) were isolated and sequenced. By gene organization they represent 3 distinct genes. PCR methods were used to confirm that only three intron-bearing histone H3.2 genes of this type exist per haploid genome. They co-exist with approximately 56 copies of the previously characterized replication-dependent, intronless histone H3.1 variant gene. Comparison of the relative expression of few constitutive H3.2 genes with the high S phase expression of the abundant cell cycle-dependent H3.1 genes by mRNA levels and protein synthesis measurements revealed that the replacement histone H3.2 genes are very highly expressed. Structural analysis of the genomic replacement H3.2 gene revealed a unique feature. A repeated polypyrimidine sequence motif in the 5' untranslated region of this gene replaces the ubiquitous intron present in all known replacement H3 genes. A hypothesis is presented that this motif and other, non-randomly distributed polypyrimidine sequences in the introns of replacement histone H3 genes of alfalfa and Arabidopsis, may affect nucleosome assembly. Chromatin repression of these replacement genes would be avoided, consistent with the high, constitutive expression of replacement H3 histone genes in plants.

Amino Acid Sequence↗
PubMed1994

Activation of the cell cycle machinery and the isoflavonoid biosynthesis pathway by active Rhizobium meliloti Nod signal molecules in Medicago microcallus suspensions.

We have shown that treatment of Medicago microcallus suspensions with the cognate Rhizobium meliloti Nod signal molecule NodRm-IV(C16:2,S) can modify gene expression both qualitatively and quantitatively. At concentrations of 10(-6) - 10(-9) M, this host specific plant morphogen but not the inactive non-sulfated molecule stimulated cell cycle progression as indicated by the significantly enhanced thymidine incorporation, elevated number of S phase cells, increase in kinase activity of the p34cdc2-related complexes and enhancement of the level of expression of several cell cycle marker genes, the histone H3-1, the cdc2Ms and the cyclin cycMs2. The presented data suggest that at least part of the physiological role of the Nod factor may be linked to molecular events involved in the control of the plant cell division cycle. In situ hybridization experiments with antisense H3-1 RNA probe indicated that only certain cells of the calli were able to respond to the Nod factor. High (10(-6) M) but not low (10(-9) M) concentrations of the active Nod factors induced the expression of the isoflavone reductase gene (IFR), a marker gene of the isoflavonoid biosynthesis pathway in most callus cells. Our results indicate that Medicago cell responses to the Nod signal molecules can be investigated in suspension cultures.

CDC2 Protein Kinase↗
PubMed1993

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize.

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the beta-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the -208 to -46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to -389 bp from ATG) promoter of wheat, alpha-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10-20 resistant calli, or GUS-expressing colonies after treatment of 10(6) protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80-90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.

Aminobutyrates↗
PubMed1992

Differential Expression of Histone H3 Gene Variants during Cell Cycle and Somatic Embryogenesis in Alfalfa.

Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [(3)H]thymidine incorporation. Histone H3-11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4-dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belòngs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription.

Journal Article↗
PubMed1992

Alfalfa cyclins: differential expression during the cell cycle and in plant organs.

Cell division in eukaryotes is mediated by the action of the mitosis promoting factor, which is composed of the CDC2 protein kinase and one of the various mitotic cyclins. We have recently isolated a cdc2 gene from alfalfa. Here, we report the isolation of two cyclin genes, cycMs1 and cycMs2, from alfalfa. The cycMs2 gene shows highest similarity to type B cyclins. In contrast, the predicted amino acid sequence of the cycMs1 gene shows similar homology scores to cyclins of all types (25 to 35%). Both genes are expressed in dividing suspension cultured cells but cease to be expressed when the cells enter stationary phase. In synchronized alfalfa suspension cultured cells, the mRNAs of cycMs1 and cycMs2 show maximal expression in the G2 and M phases. Transcripts of cycMs2 are found only in late G2 and M phase cells, an expression pattern typical for cyclin B genes, whereas cycMs1 appears with the onset of G2. This pattern indicates that alfalfa cycMs1 and cycMs2 belong to different classes of cyclins. In young leaves, expression of both genes is high, whereas in mature leaves no transcripts can be detected, indicating that the two cyclin genes are true cell division markers at the mRNA level. In other organs, a more complex expression pattern of the two cyclin genes was found.

Amino Acid Sequence↗
PubMed1991

Complementation of a yeast cell cycle mutant by an alfalfa cDNA encoding a protein kinase homologous to p34cdc2.

The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.

Amino Acid Sequence↗
PubMed1991

Alfalfa heat shock genes are differentially expressed during somatic embryogenesis.

We have isolated two cDNA clones (Mshsp18-1; Mshsp18-2) from alfalfa (Medicago sativa L.) which encode for small heat shock proteins (HSPs) belonging to the hsp17 subfamily. The predicted amino acid sequences of the two alfalfa proteins are 92% identical and a similar degree of homology (90%) can be detected between Mshsp18-2 and the pea hsp17. In comparison to various members of small HSPs from soybean amino acid sequence similarities of 80-86% were identified. The alfalfa HSPs share a homologous stretch of amino acids in the carboxy terminal region with hsp22, 23, 26 from Drosophila. This region contains the GVLTV motif which is characteristic of several members of small HSPs. At room temperature alfalfa hsp18 mRNAs were not detectable in root and leaf tissues but northern analysis showed a low level of expression in microcallus suspension (MCS). The transcription of Mshsp18 genes is induced by elevated temperature, CdCl2 treatment and osmotic shock in cultured cells. In alfalfa somatic embryos derived from MCS a considerable amount of hsp18 mRNA can be detected during the early embryogenic stages under normal culture conditions. The differential expression of these genes during embryo development suggests a specific functional role for HSPs in plant cells at the time of the developmental switch in vitro.

Amino Acid Sequence↗
PubMed1991

Promoter and genotype dependent transient expression of a reporter gene in plant protoplasts.

For analysis of expression of three different plant promoters such as CaMV 35S, rbc S and mas, compact plasmid vectors were constructed by use of beta-glucuronidase (GUS) gene and nos termination signal. The plasmid molecules were introduced into tobacco and tomato protoplasts by using the Mg2+/PEG transformation protocol described by Negrutiu et al. The transient assays revealed maximum expression two days after DNA uptake. The comparative studies show the following order of promoters mas, CaMV 35S, rbc S as far as the activity is concerned. We also detected genotype-dependent promoter activity in the case of tomato.

DNA↗
PubMed1989

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa.

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.

Amino Acid Sequence↗
PubMed1989

Differentiation of potato (Solanum tuberosum L.) plants from cultured leaf protoplasts.

Through induction of totipotent stage in cultured cells plants were regenerated from protoplast derived callus colonies of potato. Growing of the plants in vitro with optimal aeration and pretreatment of the leaves in dark and cold prior to protoplast isolation has improved the protoplast yield and frequency of cell division. Protoplasts of four potato genotypes have started to divide within 2-5 days after plating them into culture medium containing 2,4-D (0.2 mg/l); ZEA (0.5 mg/l); and NAA (1 mg/l) as growth regulators. Embedding of the cells into agarose proved to be favourable to avoid cell browning and to increase colony formation. The series of hormone treatments based on complex action of NAA and BAP promoting colony growth and greening, ZEA and IAA inducing shoot redifferentiation, and GA3 plus NAA supporting shoot elongation and rooting, finally resulted in high frequency of plant regeneration from microcolonies.

Plant Cells↗
PubMed1987

Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants.

Transfer of methotrexate and 5-methyltryptophan resistance from carrot (Daucus carota) to tobacco (Nicotiana tabacum) was achieved by fusion between leaf mesophyll protoplasts of tobacco and irradiated cell culture protoplasts of carrot. Some of the regenerated somatic hybrids exhibited normal tobacco morphology with coexpression and independent segregation of the transferred resistance markers. Chromosomal instability resulted in aneuploid somatic hybrids with significantly lower chromosome number than predicted by simple addition of parental chromosome number. The methotrexate resistance phenotype was correlated with the expression of carrot-specific dihydrofolate reductase as judged by isozyme and immunological characteristics of the enzyme. The genomic construct of these somatic hybrids made the transmission of the resistance character into the next sexual generation possible.

Journal Article↗
PubMed1982

Isolation of a wheat cell line with altered membrane properties.

A spontaneous dimethylsulfoxide (DMSO)-tolerant cell line was isolated from a cell culture of wheat (Triticum monococcum L.). The tolerant cells were able to grow in the presence of 4% DMSO. Cells formed from protoplasts of the tolerant line required DMSO for division in culture medium of high osmotic value.Fatty acid composition and the molar ratio of phospholipids/sterols suggest a more ordered membrane structure in the tolerant line. Accordingly, a lower K(+) influx rate was detected in the tolerant cells in comparison with the original line. These characteristics were maintained after 6 months' cultivation of the cells in DMSO-free growth medium. This suggested that genetic changes could be responsible for differences between the two cell lines.

Journal Article↗
PubMed1980

DNA synthesis and division in interkingdom heterokaryons.

Drosophila cells and plant protoplasts were fused by polyethylene glycoldimethylsulfoxide treatment. DNA synthesis in plant and animal cells and in their heterokaryons were monitored by [3H]thymidine autoradiography. In appropriate media both the parental cells and the heterokaryons preserved their DNA synthetic activity. Division of the heterokaryons were also observed.

Animals↗