Biomedical subjects

G J Wadsworth

Publications and source records attributed to G J Wadsworth.

7 recordsLinked to original sources

PubMed1998

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase.

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X5-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.

Amino Acid Sequence↗

PubMed1995

Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase, AAT4.

A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency. pSAT2 contained an open reading frame encoding a 47640 Da protein. The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals. It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity. Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide. A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide. Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis. Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria. Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase.

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50,581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide. A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.

Amino Acid Sequence↗

PubMed1990

Molecular characterization of a catalase null allele at the Cat3 locus in maize.

Previous analysis has identified line IDS28 of maize (Zea mays L.) as being homozygous for a Catalase-3 (Cat3) null allele. Catalase-3 (CAT-3) protein-specific antibodies could not detect CAT-3 in extracts of several tissues of IDS28, which in a typical maize line possess CAT-3. The absence of CAT-3 resulted in a significant decrease in total catalase activity in those tissues where CAT-3 is the predominant catalase isozyme. RNA blot analysis indicated that IDS28 does not accumulate Cat3 transcript in any tissues. Genomic DNA blots revealed significant structural alterations in the Cat3 gene in IDS28. The results suggest that the molecular basis for Cat3 null phenotype in IDS28 may be a deletion in the 5' end of the Cat3 gene.

Alleles↗

PubMed1989

Differential expression of the maize catalase genes during kernel development: the role of steady-state mRNA levels.

In maize three isozymic forms of catalase, CAT-1, CAT-2, and CAT-3 are encoded by three distinct and unliked structural genes (Cat1, Cat2 and Cat3). Catalase activity profiles and zymogram analysis were used to examine the spatial and temporal expression of the three genes during kernel maturation. Three developmental stages of catalase expression were observed in the growing kernel. During stage 1 (6-12 days after pollination), both Cat1 and Cat3 were expressed; during stage 2 (15-18 days after pollination) only Cat1 expression was observed; and during stage 3 (21-30 days after pollination), Cat1 and Cat2 were expressed. The major constituent tissues of the kernel were examined to determine their contribution to total kernel catalase expression. Each of the tissues was found to have a unique pattern of catalase gene expression. RNA blot analysis, using catalase gene-specific nucleic acid probes, suggests that the differential expression of the three catalase genes observed in the kernel is regulated by controlling the distribution of steady-state mRNA species for the three genes.

Blotting, Northern↗

PubMed1988

Characterization of catalase transcripts and their differential expression in maize.

In maize, the three unlinked catalase (EC 1.11.1.6) structural genes (Cat1, Cat2 and Cat3) are differentially expressed temporally, spatially and in response to environmental signals in the developing seedling. In order to understand more fully the molecular mechanisms involved in catalase gene expression, full-length cDNA clones representing the maize Cat1, Cat2 and Cat3 transcripts were isolated and characterized. DNA sequence analysis confirmed that each cDNA encodes a unique catalase protein. Gene-specific probes for the three maize catalase cDNAs were isolated and used to probe blots of poly(A)+ RNA isolated from various maize tissues. Cat1 mRNA was found in scutella, milky endosperm of immature kernels, leaves and epicotyls. The Cat2 mRNA was present primarily in post-germinative scutella, with lower levels in leaves and epicotyls. Cat3 mRNA was detected primarily in epicotyls and, to a lesser extent, in leaves and scutella. The gene-specific probes hybridized with maize genomic DNA blots in simple, but unique patterns, indicating that there is one, or a very few copies of each catalase gene. The coding region of the Cat3 cDNA comprised 66% G + C, which led to a strong codon usage bias in this gene. This codon bias was also seen with the Cat2 transcripts, but not with those for Cat1. A high degree of similarity was found between the maize catalase nucleic acid and deduced amino-acid sequences and those of sweet potato and rat liver catalase.

Amino Acid Sequence↗

PubMed1988

A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis.

A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity.

Blotting, Northern↗