Tissues expressing mRNAs of three cold-induced genes, blt101, blt14, and blt4.9, and a control gene, elongation factor 1␣, were identified in the crown and immature leaves of cultivated barley (Hordeum vulgare L. cv Igri). Hardiness and tissue damage were assessed. blt101 and blt4.9 mRNAs were not detected in control plants; blt14 was expressed in control plants but only in the inner layers of the crown cortex. blt101 was expressed in many tissues of cold-acclimated plants but most strongly in the vascular-transition zone of the crown; blt14 was expressed only in the inner layers of the cortex and in cell layers partly surrounding vascular bundles in the vascular-transition zone; expression of blt4.9, which codes for a nonspecific lipid-transfer protein, was confined to the epidermis of the leaf and to the epidermis of the older parts of the crown. None of the cold-induced genes was expressed in the tunica, although the control gene was most strongly expressed there. Thus, the molecular aspects of acclimation differed markedly between tissues. Damage in the vascular-transition zone of the crown correlated closely with plant survival. Therefore, the strong expression of blt101 and blt14 in this zone may indicate a direct role in freezing tolerance of the crown.

No abstract

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.

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The methylation of DNA is thought to have several biological roles including a role in the control of gene expression. In animals DNA methyltransferase or methylase has been found to be a large single subunit enzyme that catalyses the transfer of methyl groups from S-adenosyl methionine (SAM) to specific cytosines in DNA. In mammals 3 6 % of cytosines are methylated in the dinucleotide CpG but in plants as many as 30% of cytosines can be methylated in CpG and also in the trinucleotide CpNpG. catalysed by the same enzyme. Maintenance methylation requires hemimethylated DNA as substrate and ensures the pattern of DNA methylation is preserved from one generation to the next: while de mvo methylation requires unmethylated DNA and enables new methylation patterns to be generated. Iittle is known about the DNA methylase(s) of plants. Although the rice [ 11 and wheat [2] methylases have been found to be 54 kDa and 55 kDa respectively, we have isolated a higher molecular weight enzyme than these from pea shoots.DNA methylase is purified from a crude nuclear extract from 5 7 day old pea shoots. Initial purification of enzyme from this extract involved gel filtration but sequential use of affinity and ion exchange columns has proved more successful. The nuclear extract is applied to a heparin Sepharose column and the enzyme eluted using a NaCl gradient (0.2 M-0.7 M). The active fractions are pooled, diluted and applied to a Q Sepharose column in 0.15 M NaCI. The enzyme is eluted with a NaCl gradient (0.15 M-0.4 M) and the fractions are dialysed to remove the salt and assayed using synthetic olige nucleotides. A 1W180 kDa band was obtained after SDS polyacrylamide gel electrophoresis of the purified enzyme fraction. Further smaller molecular weight bands are thought to be possible breakdown products. The 160-180 kDa band cross mcts with an antibody to peptides obtained from the active site of the mouse methylase.investigate the sequence specificity of the methylase enzyme. Sequence specificity experiments were carried out in conditions where the enzyme concentration was limiting, or with the DNA present in limiting amounts. Higher levels of enzymic activity were observed with hemi-methylated oligonucleotide substrates than with the unmethylated forms. Overall the highest level of In eukaryotes two processes of methylation occur, apparentlyOligonucleotides have been used as substrate DNA to enzymic activity was achieved with the hemimethylated CpNpG oligonucleotide duplex, (CAG)7 (mCTGh. 1Poly dl-dC].[poly dl-dC], an analogue of [ poly dGdC] and single stranded (CAGh were found to be poor substrates compared with native pea DNA.Addition of 0.1 M NaCl inhibits the methylation reaction if added before a DNA-Enzyme complex has formed. Addition of 0.1 M NaCl 10 min after the start of the incubation rapidly inhibits methylation of unmethylated CpNpG and CpG target sites on DNA. Methylation of hemimethylated CpG target sites is inhibited less quickly but there is no inhibition of methylation of hemimethylated CpNpG'target sites.DN...