Pea DNA Research Articles

Characterization of a 20 kDa DNase elicitor from Fusarium solani f. sp. phaseoli and its expression at the onset of induced resistance in Pisum sativum

Summary DNase released from Fusarium solani f. sp. phaseoli (Fsph DNase) has previously been reported to induce pathogenesis-related (PR) genes, phytoalexin accumulation and disease resistance against subsequent challenge with the true pea pathogen, Fusarium solani f. sp. pisi (Fspi). This report is a further analysis of DNase production with probes specific for both the gene and protein. N-terminal analysis of the approximately 20 kDa Fsph DNase protein facilitated both the development of anti-Fsph DNase antiserum and the cloning of the Fsph DNase gene. Utilizing the anti-Fsph DNase antiserum to prepare an affinity column, we demonstrated that the retention and recovery of the DNase activity was associated with this protein. Fsph DNase protein was detectable by Western analysis in both the fungi and plant cytoplasm within 6-8 h following inoculation of the pea endocarp surface. Partially purified DNase detected via catalytic activity began accumulating within pea tissue at 3 h post-inoculation. Enhanced fragmentation of pea DNA occurred within 5 h following treatment of pods with Fsph DNase or inoculations with the two fungi. DNA cleavage within the nuclei of endocarp pea cells was detectable via a TUNEL assay at 3 h post-inoculation. As a result of these findings, we propose that the entrance of Fsph DNase into the pea cell and the signalling of plant defence responses is temporally associated with the damage of host DNA.

A pea homologue of human DNA helicase I is localized within the dense fibrillar component of the nucleolus and stimulated by phosphorylation with CK2 and cdc2 protein kinases

SummaryDNA helicases catalyse the transient opening of duplex DNA during nucleic acid transactions. Here we report the isolation of a second nuclear DNA helicase (65 kDa) from Pisum sativum (pea) designated pea DNA helicase 65 (PDH65). The enzyme was immunoaffinity purified using an antihuman DNA helicase I (HDH I) antibody column. The purified PDH65 showed ATP‐ and Mg2+‐dependent DNA and RNA unwinding activities, as well as ssDNA‐dependent ATPase activity. The direction of DNA unwinding was 3′ to 5′ along the bound strand. Antibodies against HDH I recognized the purified PDH65, and immunodepletion with these antibodies removed the DNA and RNA unwinding and ATPase activities from purified preparations of PDH65. The DNA and RNA unwinding activities were upregulated after phosphorylation of PDH65 with CK2 and cdc2 protein kinases. By incorporation of BrUTP into pea root tissue, followed by double immunofluorescence labelling and confocal microscopy, PDH65 was shown to be localized within the dense fibrillar component of pea root nucleoli in the regions around the rDNA transcription sites. These observations suggest that PDH65 may be involved both in rDNA transcription and in the early stages of pre‐rRNA processing.

A pea homologue of human DNA helicase I is localized within the dense fibrillar component of the nucleolus and stimulated by phosphorylation with CK2 and cdc2 protein kinases.

DNA helicases catalyse the transient opening of duplex DNA during nucleic acid transactions. Here we report the isolation of a second nuclear DNA helicase (65 kDa) from Pisum sativum (pea) designated pea DNA helicase 65 (PDH65). The enzyme was immunoaffinity purified using an antihuman DNA helicase I (HDH I) antibody column. The purified PDH65 showed ATP- and Mg(2+)-dependent DNA and RNA unwinding activities, as well as ssDNA-dependent ATPase activity. The direction of DNA unwinding was 3' to 5' along the bound strand. Antibodies against HDH I recognized the purified PDH65, and immunodepletion with these antibodies removed the DNA and RNA unwinding and ATPase activities from purified preparations of PDH65. The DNA and RNA unwinding activities were upregulated after phosphorylation of PDH65 with CK2 and cdc2 protein kinases. By incorporation of BrUTP into pea root tissue, followed by double immunofluorescence labelling and confocal microscopy, PDH65 was shown to be localized within the dense fibrillar component of pea root nucleoli in the regions around the rDNA transcription sites. These observations suggest that PDH65 may be involved both in rDNA transcription and in the early stages of pre-rRNA processing.

Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses.

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m(-2)) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.

Suppression of pea nuclear topoisomerase I enzyme activity by pea PCNA.

Proliferating cell nuclear antigen (PCNA), a highly conserved DNA polymerase accessory protein of eukary- otic kingdom, has not been studied thoroughly in bio- chemical terms in plants. We describe the isolation of the cDNA encoding PCNA from the pea cDNA library using the PCR approach. The cDNA was used for expression of pea PCNA in bacteria as a fusion protein (GST.PCNA) with the GST tag at the amino terminal end. The GST.PCNA stimulated the partially purified pea DNA polymerases approximately 30-fold. The stimulation was due to the oligomeric form of GST.PCNA. The pea PCNA interacted with the recombinant type I pea topoiso- merase as well as the native pea nuclear topoisomerase I and repressed the DNA relaxation activities. However, the DNA binding activity of Topo I remained undisturbed in the presence of high amounts of PCNA, thereby signify- ing that the catalysis of Topo I was probably affected by PCNA.

Plant Methyltransferases and Their Targets in the Plant Genome

In addition to the methylation of cytosine in some CG dinucleotides, plant genomes contain 5-methylcytosine (a cytosine with a methyl group in place of the hydrogen at position 5) in the trinucleotide sequence mCNG, where N is reportedly any of the four common DNA bases (Gruenbaum et al. 1981). 5-Methylcytosine has also been found in non-symmetrical sequences in transgenes (Meyer et al. 1994), but there is no evidence in plants for the N-4 methylcytosine that is found in some prokaryotes. We have shown that CG and CNG methylation are carried out by different methyltransferases (Pradhan and Adams 1995), and one possible consequence of this is that the two reactions could be independently regulated. This would allow the methylation of CG and CNG sequences to have unrelated functions in the plant cell, although to date there are no data to support this suggestion. A typical base composition for angiosperm DNA is 40% G+C. If all CG and CNG sequences were to be methylated, then the percentage of cytosines methylated would be 36% (20% in mCG and 20% in mCNG, of which 4% would also be in mCG). Some angiosperms approach this value, and cleavage of pea DNA with methyl-sensitive restriction enzymes indicates that most CG and CWG sequences are methylated (Pradhan et al. 1995). (W stands for A or T and hence the CWG trinucleotide contains CAG on one strand of DNA and CTG on the other.) Yet in the DNA from Arabidopsis, only 5% of the cytosines are methylated (Leutwiler...

DNA substrate specificity of pea DNA methylase

DNA substrate specificity of pea DNA methylase

Cloning and sequencing of a soybean nuclear gene coding for a chloroplast translation elongation factor EF-G

A plant nuclear gene coding for a chloroplast specific translation elongation factor EF-G (cEF-G) was cloned and sequenced for the first time. We screened two partial soybean genomic libraries with a short PCR amplified pea DNA probe constructed according to the N-terminal peptide sequence of pea chloroplast EF-G. The gene is three times split, codes for a chloroplast type transit peptide and a protein very similar to bacterial translation elongation factor EF-G. The gene is expressed as evidenced by Northern hybridisations.

DNA substrate specificity of pea DNA methylase.

DNA methylase, present in low-salt extracts of nuclei prepared from Pisum sativum shoot tips, methylates model DNA substrates containing CNG trinucleotides or CI dinucleotides only. The binding to the hemimethylated trinucleotide substrates is very much stronger and more persistent than the binding to the unmethylated substrates or to the hemimethylated dinucleotide substrate. When the DNA concentration is limiting, the rate of methyl-group transfer with the hemimethylated CNG substrate is much greater than that with the unmethylated CNG. However, the Vmax. is similar for the two CNG substrates. On fractionation using Q-Sepharose, two peaks of activity are seen with different relative activities using the di- and trinucleotide substrates. The relative activity with these substrates changes during purification, during plant growth and on heating at 35 degrees C as well, indicating that more than one enzyme or more than one form of the enzyme may be present.

Cloning the pea DNA methylase cDNA

Cloning the pea DNA methylase cDNA

DNA methylase from Pisum sativum

DNA methylase activity was detected in nuclei from pea shoots. The enzyme can only be extracted by low-salt treatment if the nuclei are pretreated with micrococcal nuclease. Only a single enzyme was detected, and it was purified to a specific activity of 1620 units/mg of protein. It has an Mr of 160,000 on gel filtration and SDS/PAGE. Pea DNA methylase methylates cytosine in all four dinucleotides, and this is interpreted to show that it acts on CNG trinucleotides. Although it shows a strong preference for hemi-methylated double-stranded DNA, it is also capable of methylation de novo. Homologous DNA is the best natural substrate. In vitro the enzyme interacts with DNA to form a salt-resistant complex with DNA that is stable for at least 4 h.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Plastocyanin is encoded by a single-copy gene in the pea haploid genome

cDNA clones for pea plastocyanin were isolated from a pea leaf cDNA library screened with a (32)P-labelled mixed oligonucleotide probe predicted from part of the N-terminal amino acid sequence of pea plastocyanin. The six cDNA clones isolated were found to be identical in the regions in which they overlapped. A Southern blot of restricted pea DNA probed with one of these cDNA clones showed the pea plastocyanin gene to exist as a single copy in the haploid genome. A pea genomic library in λEMBL3 screened with the same cDNA clone gave three positive plaques which contained identical 16 kbp Bam HI fragments. A single uninterrupted plastocyanin gene was located near the middle of the fragment and was characterised by DNA sequencing. The derived amino acid sequence indicates that the plastocyanin precursor consists of 168 amino acid residues including a presequence of 69 amino acid residues. The transcription initiation site was located by S1 nuclease mapping approximately 50 bp upstream of the translation initiation site. A sequence similar to a consensus light-responsive element found in a large number of phytochrome-dependent light-inducible genes is located just upstream of the TATA box. A cluster of direct repeats containing potential Z-DNA-forming elements occurs 600-750 bp upstream of the transcription initiation site.

Localization and nucleotide sequence of the gene for the 8 kDa subunit of photosystem I in pea and wheat chloroplast DNA

The gene for the 8 kDa subunit of photosystem I has been located in the small single copy region of wheat chloroplast DNA by coupled transcription-translation of cloned fragments of DNA and by DNA sequence analysis. The pea gene for this subunit was located in pea chloroplast DNA by using the wheat gene as a probe. The location was confirmed by immunoprecipitation of the products of coupled transcription-translation of cloned DNA with antiserum raised against the small subunits of pea photosystem I and by DNA sequence analysis. The deduced amino acid sequences of the pea and wheat proteins are identical in seventy-six out of the eighty-one amino acid residues. There are nine conserved cysteine residues, eight of which are arranged in the primary structure in a similar way to those in bacterial ferredoxins containing two 4Fe-4S centres, suggesting that the polypeptide binds iron sulphur centres A and B of photosystem I.

The structure and light-dependent transient expression of a nuclear-encoded chloroplast protein gene from pea (Pisum sativum L.).

A nuclear gene encoding a light-induced transiently expressed protein that is localized in the chloroplast has been isolated from an EMBL3 library of pea DNA. The gene is a member of a multigene family. The sequence of the gene contains the complete reading frame of previously characterized cDNA clones and two introns in the 5' region of the protein coding sequence. Primer extension and S1 nuclease studies have defined the cap site. Two TATA boxes are found 5' to the initiating methionine codon. Only a limited homology is found between the presequence of the gene and transit sequences of other previously sequenced precursors. Isolated nuclei of pea have been labelled and the in vitro synthesized transcripts analysed. The results show that the light-dependent expression of the gene family is regulated at the level of transcription.

Localisation of genes for components of photosystem II in chloroplast DNA from pea and wheat

The genes for three components of photosystem II have been localised in chloroplast DNA from pea and wheat by hybridisation with gene-internal sequences from spinach chloroplast DNA. In both pea and wheat, the gene for the 51 kDa polypeptide is located close to the genes for cytochrome b-563 and the 15 kDa polypeptide of the cytochrome b-f complex. The genes for the D2 and 44 kDa polypeptides are located close together, approximately 55 kbp from the gene for the 51 kDa polypeptide, in both pea and wheat chloroplast DNA. The location and orientation of the genes for the D2 and 44 kDa polypeptides in wheat chloroplast DNA indicate that the rearrangement of the wheat genome with respect to the spinach genome is the result of at least two inversions.

Sequence organization of a pea chloroplast DNA gene coding for a 34,500-dalton protein.

A gene (PGII), which codes for a 34.5-kilodalton protein, has been isolated and cloned from pea chloroplast DNA. The production of its 1.2-kilobase mRNA is photodependent. The direction of transcription has been determined, the site of initiation of transcription has been found, and an in vitro protein product has been produced. The gene, including the 5' and 3'-flanking regions, has been sequenced. It shows ca. 95% homology to the photosystem II thylakoid membrane protein, photogene 32, from spinach and tobacco. There are no intervening sequences. The 5'-flanking region suggests similarities with Escherichia coli promoters. The 5'-flanking region is remarkably conserved among pea, spinach, and tobacco DNA.

Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide

A nuclear gene AB80 has been isolated from a phage lambda Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an uninterrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80. The first methionine codon 3' from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A "TATA" sequence occurs 31 nucleotides 5' from the cap site. A second TATA sequence is found 7 nucleotides on the 5' side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within the constituent polypeptides of the light-harvesting chlorophyll a/b-protein complex. The sequence Arg-Lys-Ser-Ala-Thr-Thr-Lys-Lys occurs at, or near, the NH(2)-terminus of the mature polypeptide encoded by AB80. This basic peptide is of interest because of its apparent involvement in changes in excitation-energy distribution in chloroplast membranes. Some general similarities, but no extensive sequence homology, is found on comparing the transit sequence for the precursor to the chlorophyll a/b-binding polypeptide with the transit sequences previously determined for the precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase.

Repeat sequence interspersion in coding DNA of peas does not reflect that in total pea DNA

The pattern of sequence organization in the regions of the pea genome near sequences coding for mRNA differs significantly from that in total DNA. Interspersion of repeated and single copy sequences is so extensive that 85% of 1300 nucleotide-long fragments contain highly repetitive sequences (about 5000 copies per haploid genome). However, data presented here demonstrate that sequences which code for mRNA are enriched in the small fraction of fragments which do not contain these highly repetitive sequences. Thus, in contrast to the great majority of other sequences in the genome, most mRNA coding sequences are not located within 1300 nucleotides of highly repetitive elements. Moreover, our data indicate that those repeats (if any) which are closely associated with mRNA coding sequences belong to low copy number families characterized by an unusually low degree of sequence divergence.

Reiteration frequency of the gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase

The messenger RNA coding for the precursor of the small subunit of ribulose-1,5-bisphosphate (Ru-P 2) carboxylase has been partially purified from pea leaves. The mRNA has a size of 11.5S, is approximately 800 nucleotides long and, on cell-free translation, directs the synthesis of a single major polypeptide of 20,000 daltons. Antiserum for the small subunit of Ru-P 2 carboxylase immunoprecipitated 52% of the 35S-methionine-labeled cell-free translation products. The RNA hybridizes with pea DNA with monophasic kinetics corresponding to the presence of one, or very few, gene copies per haploid genome.