Biomedical subjects

D C Baulcombe

Publications and source records attributed to D C Baulcombe.

62 records · Page 4Linked to original sources

PubMed1987

A gibberellin responsive wheat gene has homology to yeast carboxypeptidase Y.

An earlier report (Baulcombe, D. C., and Buffard, D. (1983) Planta 157, 493-501) described the isolation of cDNA clones from mRNAs which are produced in increased amounts when aleurone layers of wheat are treated with gibberellic acid. It is shown here that for one of those cDNAs (2473) the change in level of mRNA in aleurone parallels the change in level of alpha-amylase mRNA. This result was obtained in experiments where the level of gibberellic acid was varied and also when the mRNA was isolated from wheat genotypes which varied in ability to respond to gibberellic acid. In contrast to this, the pattern of 2437 mRNA accumulation in immature grains and in leaf tissue was quite distinct from the pattern of alpha-amylase mRNA accumulation. Analysis of wheat DNA showed that the 2437 mRNA is encoded by a small family of genes located on the short arm of the group 6 chromosomes. One member of this gene family was cloned and sequenced. The coding sequence is interrupted by eight introns and encodes a protein of Mr 55,433. By using hybridization probes from the 5' exon in an S1 nuclease protection assay it was shown that the 2437 mRNA was produced in aleurones (coordinately with alpha-amylase) and in immature grains (not coordinately with alpha-amylase). However, sequence comparison of 1 kilobase of the 5'-flanking region with the sequence of alpha-amylase genes provided no indication of the regulatory elements which would be active in aleurone cells. The protein sequence deduced from the gene sequence has extensive homology with the yeast carboxypeptidase Y, especially in the active site and substrate binding regions. This homology is greater than with the carboxypeptidase I from barley. It is suggested therefore that there are several types of carboxypeptidase encoded in the cereal genome. The sequence of the 2437 protein would represent one of these types and the barley carboxypeptidase I, another.

Amino Acid Sequence↗

PubMed1987

A novel wheat alpha-amylase gene (alpha-Amy3).

A genomic clone of a wheat alpha-amylase gene (lambdaAmy3/33) was identified, on the basis of hybridisation properties, as different from alpha-Amy1 and alpha-Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of alpha-amylase from the alpha-Amy1 and alpha-Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca++ binding region. In addition, the introns are at positions equivalent to the position of introns in the alpha-Amy1 and alpha-Amy2 genes. However, the sequence was less similar to alpha-Amy1 and alpha-Amy2 than these are to each other. Southern blot analysis showed that the lambdaAmy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the alpha-Amy1 or alpha-Amy2 genes. A further difference from the alpha-Amy1 and alpha-Amy2 genese was the pattern of expression. lambdaAmy3/33 was expreseed only in immature grains and, unlike the alpha-Amy1 and alpha-Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of alpha-amylase gene, not described before, which shares a common evolutionary ancestor with the alpha-Amy1 and alpha-Amy2 genes.

Amino Acid Sequence↗

PubMed1987

Synthesis and secretion of wheat alpha-amylase in Saccharomyces cerevisiae.

A wheat alpha-amylase cDNA clone has been fused to the phosphoglycerate kinase initiator methionine to enable synthesis in the yeast Saccharomyces cerevisiae of an alpha-amylase enzyme that is identical in size to the wild-type alpha-amylase. The alpha-amylase is synthesized with an N-terminal plant signal peptide which is recognized in the yeast host, leading to efficient processing and secretion into the medium. The secretion of alpha-amylase into the medium is quite efficient in rich medium, but barely detectable in a minimal medium.

Culture Media↗

PubMed1987

The complete nucleotide sequence of tobacco rattle virus RNA-1.

The complete nucleotide sequence of tobacco rattle virus (TRV) strain SYM RNA-1 was determined from a series of overlapping cDNA clones. cDNA prepared by primer extension was used to determine the exact 5' terminus. The RNA sequence was 6791 nucleotides in length and contained four open reading frames (ORFs). The ORF nearest the 5' terminus coded for a polypeptide of predicted mol. wt. 134,000 (134K) and terminated at an opal (UGA) stop codon. Readthrough of this stop codon would result in the production of a protein of 194K. The gene for a 29K polypeptide started one base beyond the 194K stop codon and, in turn, was followed by the gene for a 16K protein at the 3' end of RNA-1. Amino acid comparisons of the 194K protein with the putative replicase of tobacco mosaic virus showed three regions of strong homology, suggesting that the 134K and 194K proteins were similarly involved in virus replication. The 5' terminal sequences of both genome RNA species of TRV strains ORY, N5 and PRN together with that of SYM RNA-2 were also determined. Alignments of these sequences showed that there was a 22 base repeated sequence close to the 5' terminus in all these RNA species. It was also shown that the 5' terminus of RNA-1 was homologous with the same region in RNA-2.

Amino Acid Sequence↗

PubMed1986

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1.

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.

Journal Article↗

PubMed1986

Expression of a wheat alpha-amylase gene in Escherichia coli: recognition of the translational initiation site and the signal peptide.

Transcription of a full-length cDNA clone of wheat alpha-amylase using a lac promoter in Escherichia coli results in synthesis of a precursor alpha-amylase polypeptide of the correct size, indicating that translation initiates correctly. Recognition of the plant translational initiation site by E. coli ribosomes is 15-20% as efficient as the ribosome-binding site of the beta-lactamase gene when it is fused to alpha-amylase. The alpha-amylase signal peptide is recognised in E. coli resulting in secretion of the enzyme into the periplasmic space; deletion of the signal peptide prevents secretion. Replacement of the alpha-amylase signal peptide with a beta-lactamase signal peptide also enables the bacterial cell to secrete the enzyme. The presence of the beta-lactamase and the alpha-amylase signal peptides in tandem results in secretion of the enzyme and removal of both signal peptides.

DNA↗

PubMed1980

Polyadenylated RNA sequences which are reduced in concentration following auxin treatment of soybean hypocotyls.

Previous work has shown that any effect of exogenous auxin on gene expression in soybean hypocotyl tissue must be restricted to a relatively small fraction of the polyadenylated RNA. However, kinetic hybridization analysis with cDNA probes revealed that a minor abundant class of sequences is markedly reduced in concentrations in the auxin-treated polyadenylated RNA. Recombinant plasmids containing copies of polyadenylated RNA species were constructed using the G-C tailing procedure and clones of auxin-regulated sequences were detected by differential in situ hybridization with cDNA of polyadenylated RNA from auxin-treated or untreated hypocotyls. Although the 12 clones which were selected all contained different size inserts, and were therefore independent, 11 of these apparently hybridized to just two different RNA species. The rate constant of the auxin-sensitive abundant component of the untreated polyadenylated RNA/DNA hybridization was similar to that of the reaction between the two major groups of clones and untreated polyadenylated RNA. This indicates that these cloned sequences are homologous with that cDNA fraction. The twelfth clone is thought to be representative of a group of less abundnt auxin-regulated polyadenylated mRNA species which had been detected in an earlier analysis of the in vitro translation products of soybean hypocotyl RNA. Both the timing and the extent of the influence of auxin on the relative concentration of these cloned sequences are quite consistent with a close relationship between growth regulation by auxin and its effects on gene expression.

Base Sequence↗

PubMedDate not supplied

Molecular analysis of potato virus X isolates in relation to the potato hypersensitivity gene Nx.

The coat protein gene of potato virus X is known to affect the outcome of interactions between different strains of the virus and potato plants carrying the Nx resistance gene. To analyze the role of the coat protein in interactions with Nx hosts, we used the potato virus X strain PVXDX, which induces a hypersensitive response on potato cultivars carrying the Nx resistance gene and the strain PVXDX4, which was originally derived from PVXDX and which overcomes Nx-mediated resistance. Sequencing of cloned coat protein genes representing the strains PVXDX and PVXDX4 showed that they differed at a single nucleotide. This change results in the substitution of glutamine-78 in the PVXDX coat protein for proline in PVXDX4. We constructed hybrid viral genomes by replacing the coat protein gene of a full-length clone of isolate PVXUK3 with the corresponding sequence from either PVXDX or PVXDX4. Progeny virus, derived from in vitro transcripts of these hybrid clones, showed that the single nucleotide difference between the coat protein genes of isolates PVXDX and PVXDX4 was sufficient to alter the outcome of the interaction between the virus and potato plants carrying the resistance gene Nx. Additional coat protein mutants generated in planta from transcript-derived inocula induced an intermediate host response on Nx potato cultivars which is influenced by the presence of a second, PVX-specific, resistance gene in the host plant genome.

Base Sequence↗