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J W Tregear

Publications and source records attributed to J W Tregear.

6 recordsLinked to original sources

Plant MAP kinase kinase kinases structure, classification and evolution.

The increasing number of reports describing plant MAP kinase signalling components reflects the cardinal role that MAP kinase pathways are likely to play during plant growth and development. Relationship and structural analyses of plant MAP kinase kinase kinase related cDNAs and genes established, on one hand, the PMEKKs, which may be distinguished into the alpha, beta, gamma, and zeta groups, and, on the other hand, the PRAFs that consist of the delta, eta and theta groups. Plant MAP3Ks are characterized by different primary structures, but conserved within a single group. A relationship analysis, which included animal, fungal and plant MAP3Ks, revealed a high degree of diversity among this biochemically established set of proteins, thus suggesting a range of biological functions. Four major families emerged, namely the MEKK/STE11, including the PMEKKs, the RAF, including the PRAFs, as well as the MLK and CDC7 families. These four families showed phylum-dependent distributions. Signature sequences characterizing the RAF family and the RAF subfamilies have been evidenced. However, no equivalent sequence motifs were identified for the MEKK/STE11 family, which is highly heterogeneous.

Evolution, Molecular↗

Characterisation of novel plant genes encoding MEKK/STE11 and RAF-related protein kinases.

Various elements of the MAP kinase module have been isolated in plants. We describe here the characterisation of 14 new plant cDNAs and genes encoding putative MAP kinase kinase kinases (MAP3Ks) related to the MEKK/STE11 and RAF protein kinases. Plant MAP3Ks are characterised by a variety of primary structures conserved within closely related proteins. Southern blot analysis suggests that plant MAP3Ks are heterogenous in their genomic structure, existing either as single copy genes or as small gene families. An RT-PCR analysis showed that in Arabidopsis thaliana, all organs studied contain detectable levels of transcripts of each of the MAP3K genes identified; however, signals obtained with mature pollen were weak or non-existent except for AtMAP3Kgamma. None of the reported genes share a cell-cycle or a cold stress regulated expression.

Amino Acid Sequence↗

Functional analysis of linker insertions and point mutations in the alpha-Amy2/54 GA-regulated promoter.

Functional analysis of a gibberellin-regulated wheat alpha-amylase promoter, alpha-Amy2/54, has indicated that three regions were essential for expression. By studying the ability of mutant promoters, containing a randomly inserted 22 bp excision linker, to direct expression in oat aleurone protoplasts we have refined the positions and extents of these three cis elements and also demonstrated the presence of two additional elements. By converting the linker insertions to either single base point mutations or deletions using the class IIS restriction endonuclease BsmI we have shown that nucleotides -119 and -109 within the GARE -121GTAACAGAGTCTGG-108 and nucleotide -152 within the proposed element -156GATTGACTTGACC-144 are essential for high level expression from this promoter.

Base Sequence↗

Localisation of cis elements in the promoter of a wheat alpha-Amy2 gene.

A functional analysis of the promoter from the wheat alpha-amylase gene alpha-Amy2/54 is described. Mutant alpha-Amy2/54 promoters containing replacements or deletions were constructed and their ability to direct expression of the reporter gene beta-glucuronidase (GUS) in gibberellin-responsive oat aleurone protoplasts analysed. Chimaeric promoters using regions of the cauliflower mosaic virus (CaMV) 35S and alpha-Amy2/54 promoters were also analysed. The results suggest that at least three regions within the alpha-Amy2/54 promoter contain cis elements that are necessary for high-level gibberellin-regulated transcription. Fusion of 1.8 kb of promoter sequence upstream from -117 bp to a minimal (-55 CaMV 35S) promoter gave rise to hormone-independent expression implying that the region 3' to -117 bp contains an element which represses transcription in the absence of gibberellin or presence of abscisic acid.

Base Sequence↗

The lectin gene family of Ricinus communis: cloning of a functional ricin gene and three lectin pseudogenes.

Molecular hybridisation using a ricin cDNA probe has revealed that the ricin/Ricinus communis agglutinin (RCA) multigene family is composed of approximately eight members. Several genomic clones containing preproricin and preproricin-like sequences have been isolated. Partial analysis of three different genomic clones by DNA sequencing and ribonuclease protection has indicated that at least three members of the lectin gene family are non-functional. None of the original seventeen positive clones isolated appears to contain a Ricinus communis agglutinin (RCA) gene. One gene member analysed (pCBG3H1) represents a functional ricin gene similar in coding sequence to the published cDNA sequence and possesses typical eukaryotic consensus sequences and seed-specific elements within the flanking sequences. Investigation at the transcriptional level of the expression pattern of this gene revealed that mRNA accumulates during the post-testa stages of seed development. The pattern of accumulation of steady-state transcripts correlates closely with that previously observed at the protein and translatable RNA levels.

Amino Acid Sequence↗

Molecular cloning of ricin.

A variety of strategies have been used to obtain cDNA and genomic clones encoding ricin. Since their isolation these sequences have been manipulated to allow expression of A chain (19) and A chain mutants (15,20,34), B chain (14,21-23) and proricin (24). Utilizing structural information (35), precise changes have been introduced into both A and B chains with the aim of probing catalytic and sugar-binding residues, respectively. In the longer term, such manipulations, coupled with successful expression and purification schemes, will allow the delineation of functional residues and domains, ensuring that ricin remains the prototype plant toxin with which to study cellular intoxication and ribosome inactivation and to utilize in pharmaceutical product development.

Amino Acid Sequence↗