Carl I. Webster

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the β-glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, β-glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.

Chromosomal location and expression of the single-copy gene encoding high-mobility-group protein HMG-I/Y in Arabidopsis thaliana.

A cDNA encoding the HMG-I/Y protein from Arabidopsis thaliana has been isolated and characterised by nucleotide sequencing. The 903 bp cDNA contains a 612 bp open reading frame encoding a protein of 204 amino acid residues showing homology to HMG-I/Y proteins from other plant species. The protein contains four copies of the ‘AT-hook’ motif which is involved in binding A/T-rich DNA. Southern blotting showed that the HMG-I/Y gene was present in a single copy in the Arabidopsis genome. The gene was localised to the top of chromosome 1 by RFLP analysis of F8 recombinant inbred lines. Northern blotting showed that the gene was expressed in all organs examined, with the highest expression in flowers and developing siliques.

The single-copy gene encoding high-mobility-group protein HMG-I/Y from pea contains a single intron and is expressed in all organs

The coding and 3′-downstream regions of the gene encoding the high mobility group protein HMG-I/Y from pea have been isolated, sequenced and characterised. A 795 bp pea genomic fragment containing the coding region of the pea HMG-I/Y gene with a single intron of 201 bp was isolated by PCR. The gene encodes a protein of 197 amino acid residues with four copies of the AT-hook DNA-binding motif encoded by exon 2. Southern blot analysis on genomic DNA revealed the presence of a single copy of the HMG-I/Y gene in the haploid genome. The pea HMG-I/Y gene is expressed in all organs of pea including roots, stems, leaves, flowers, tendrils and developing seeds, as determined by northern blot analysis.

Characterisation and promoter analysis of the Arabidopsis gene encoding high-mobility-group protein HMG-I/Y

The single-copy gene encoding the Arabidopsis HMG-I/Y protein was isolated and characterised. The gene encodes a protein of 204 amino acid residues and contains a single intron of 73 bp. Primer extension analysis indicates that transcription starts 115 bp upstream of the translation start and the leader sequence contains a short open reading frame of 13 amino acid residues. The 5′-upstream region of 2117 bp and several 5′ deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium-mediated transformation. Analysis of transgenic tobacco plants containing HMG-I/Y promoter regions of -2117, -1468 and -707 from the translation start detected GUS activity in all organs examined, including roots, stems, leaves and floral organs. Deletion from -707 to -185 resulted in a 20–30-fold reduction in GUS activity in roots and stems, indicating the presence of important quantitative regulatory elements in this region.

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.

The Use of MALDITOF Mass Spectrometry and Amino Acid Sequence Analysis in Characterising Small Amounts of N-Blocked Protein

One hundred picomoles of a high mobility group (HMG) protein were isolated by reverse phase hplc from an extract of pea nuclei. Amino acid sequence and composition analysis of half the sample showed the protein to be blocked at the N-terminus. Of the remaining material, 40pmol was subjected to digestion with proteinases and the peptides from the tryptic digest were separated on reverse phase hplc. Each eluted peak was examined by matrix-assisted laser desorption time-of-flight (MALDITOF) mass spectrometry and amino acid sequence analysis. From the resulting information, it was clear that the target protein sequence correlated with the inferred sequence of a previously isolated pea leaf cDNA encoding an HMG-I-like protein. The expressed protein was smaller than the DNA sequence suggested. From a series of further digests on 1–2pmol of protein, the likely identity of the N-terminal block was established as well as several sites of C-terminal processing. This works illustrates how extensive amounts of data can be derived from a small amount of protein by the combined use of sequence analysis and MALDITOF mass spectrometry.