Comparative studies of the nucleotide sequences of metallothionein-like plant genes

Abstract

Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal binding proteins, whose proposed functions include roles in essential trace metal homeostasis and the detoxification of certain non-essential metals. Genes encoding MTs have been isolated from a wide range of organisms and "MT-like" genes have also been identified from several plant species. At present the function(s) of these plant genes is/are unknown. It appears that plants possess a complement of related genes which may perform different functions, possibly in different organs, and hence appear to show different patterns of expression. Unlike the E(_c) protein from wheatgerm, the translational products of other plant MT-like genes remain to be purified from plant tissue. Previous to this study, there were only two reports which presented 5' flanking sequences of MT-like genes from plants (maize and pea). The aim of this research was to determine the sequences of the 5' flanking regions of several different MT-like genes from several plants and to identify any consensus sequences which could be regulatory elements that control the expression of these genes. PGR was used to amplify the coding regions of two additional MT-like genes from pea designated PsMTb and PsMTq . A partial cDNA from N. tabacum , previously isolated via heterologous probing with PsMT/n, was used to screen a genomic library. Three genomic clones were identified (clones 1, 2, and 13) but subsequent sequencing failed to identify any cysteine-rich motifs. However, clone 13 showed significant sequence similarity to a gene from A. thaliana (meri 5 ) which is known to be expressed in apical mens terns. Two MT-like genes, designated AtMT-1 and AtMT-2 with consideration to the location of the encoded cysteine residues, were isolated by screening an A. thaliana genomic library with cDNA probes generated by PGR based upon known cDNA sequences (MT-1 and MT-2 ). The 5' flanking sequences of both genes were characterised, AtMT-1 via sub- cloning and "gene walking" and AtMT-2 via direct sequencing of cosmid DNA. A PGR product was also amplified using primers designed to a third MT-like cDNA from A. thaliana (which shows some sequence similarity to from wheat) although corresponding genomic clones remain to be isolated. The 5' flanking sequences of MT-like genes from maize, pea and a class 1 MT from N.crassa were compared with the novel 5' flanking sequences obtained for the 2 new genes from A. thaliana. The motif TCGCCA(N)2-4 AATTTG has been identified, at a similar distance from the putative TATA box within the 5' flanking sequences of the MT-like genes from pea and A. thaliana (PsMT(_A) and AtMT-1). This motif has been identified in two plant species which have been catergorised as type 1 MT-like genes, are both from dicotyledenous species and are thought to be expressed in roots in response to copper ions. Several other (short) conserved motifs have also been identified following a series of comparisons of the first 300 nucleotides of 5' flanking sequence adjacent to the putative TATA boxes of these genes. In the course of these studies automated DNA sequence analyses have been optimised with respect to DNA template preparation and the direct sequencing of PGR products and cosmid DNA.