title: Cytosine DNA methyltransferases and CWG islands in Pisum sativum
Creators: name: Pradhan, Sriharsa
Publisher: name: University of Glasgow
Year: 1995
Subjects: subject: Plant sciences.

description: The aim of this project was to characterise the DNA methyltransferases in plants using Pisum sativum as a model system. In order to purify and assess the target specificity of various forms, I applied the partially purified methyltransferase to a MonoQ column, and fractionated two different forms of the DNA methyltransferase. One, a CG methyltransferase, was 140 kDa in size and appeared to be unstable. This protein only methylates CG and Cl oligonucleotide duplexes. The other enzyme, a 110 kDa protein, methylates 5'-CAG-3' and 5'-CTG-3'trinucleotide targets but not 5'- CCG-3' or 5'-CGG-3' sequences, as revealed by in vitro methylation followed by Maxam-Gilbert chemical sequencing of the methylated strand in the model substrates. I fully purified this second methyltransferase using a CNG hemimethylated DNA affinity chromatography column. The purified protein gave two distinct bands on acrylamide gel, one at 110 and the other 100 kDa. This exciting observation opened up two different avenues in my research project, one to understand more about the targets of CWG methylation and the other to understand more about the enzyme itself. CsCl purified nuclear DNA was digested with several restriction endonuclases, which were sensitive to cytosine methylation such as Pstl, Pvull and EcoRll. After end labelling two to three size range of fragments were produced. I gel-purified and cloned these fragments, which arose from the unmethylated region of the genome. The sequence analysis of fifty such Pstl tiny fragment (PTF) clones shows that these DNAs are rich in unmethylated CWGs and are deficient in CG dinucleotides indicating the occurrence of CWG islands in pea. Occasional unmethylated CGsare found in CWG islands Analysis of the 5' region of 40 published plant gene sequences from the GenEMBL database does not shows any particular trend between CG and CWG content of the plant promoters. It is estimated that 5% of the plant genes are associated with CWG islands, in contrast to most of them being associated with CG islands. To address the biological role of CG vs CWG methylation, I performed transient gene expression studies in collaboration with Dr. Nigel Urwin. Plasmid constructs containing promoters of either CAMV35S or rbcs2 fused to a CAT reporter gene were in vitro methylated and electroporated in to bean protoplasts. Methylation of CWG sequences in the rbcs2 promoter leads to a dramatic inhibition of CAT gene expression suggesting the possible involvement of CNG methyltransferase (MTase) in gene regulation. In order to understand the structure, function and pattern of methyltransferase gene expression during plant development, I have isolated a series of overlapping clones from pea cDNA libraries. The assembled nucleic acid sequence is 5 kb. The deduced amino acid sequence has an open reading frame of 1560 amino acids with a predicted protein of 180 kDa. Like other higher eukaryotic DNA MTase this protein has an N terminal domain fused with a catalytic domain with a short linker which, in case of pea, is RKKKG. The pea enzyme has eight of the ten conserved motifs found in prokaryotic enzymes. The C terminal (catalytic domain) of the protein is homologous to both vertebrate and plant enzymes. However, the N-terminal has poor homology with the vertebrate enzymes. The unique feature of the N terminal region is the presence of several, putative nuclear localisation signals that may also be the sites of action for proteases. The presence of a Zn binding domain on this protein makes it different from the Arabidopsis protein. An uncharacterised acidic domain is also present in the protein. By Southern analysis I concluded that the MTase gene is a single copy gene and Northern analysis shows it to be mostly expressed in the meristematic tissue. Amino acid sequence alignment with the N terminal sequence of the CWG MTase suggests that this protein could be a processed product of the 180 kDa protein.