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Biomedical subjects

G C Machray

Publications and source records attributed to G C Machray.

13 recordsLinked to original sources
PubMed2003

Exploiting plant somatic radiation hybrids for physical mapping of expressed sequence tags.

Methods are described for the optimisation of the generation of radiation hybrids suitable for physical mapping of a plant (barley) genome. A combination of PCR-based technologies, involving the use of whole genome, mixed primer and hemi-nested primer amplifications, can greatly extend their utility for the physical mapping of expressed sequence tags (ESTs). Using panels of hybrids and ESTs, donor DNA retention and individual marker retention frequencies for the expressed portion of the barley genome in the hybrids were estimated.

Expressed Sequence Tags↗
PubMed2001

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading.

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.

Carbohydrate Metabolism↗
PubMed2000

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.

Base Sequence↗
PubMed1999

Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the beta-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

Base Sequence↗
PubMed1996

Exon skipping induced by cold stress in a potato invertase gene transcript.

We show that two invertase genes in potato, like most other plant invertase genes, include a very short second exon of 9 bp which encodes the central three amino acids of a motif highly conserved in invertases of diverse origin. This mini-exon is one of the smallest known in plants and pre-mRNA from these genes may be susceptible to alternative splicing, because of a potential requirement for specialized interaction with the splicing machinery to ensure correct processing for the production of a mature mRNA. No evidence of aberrant post-transcriptional processing was observed during normal invertase gene expression in potato. The fidelity of post-transcriptional processing of the pre-mRNA from one of the genes was perturbed by cold stress, resulting in the deletion of the mini-exon from some transcripts. This alternative splicing event occurred under cold stress in both leaf and stem, but was not induced by wounding. This adds an example of exon skipping and the induction of alternative processing by cold stress to the small number of transcripts which have been shown to exhibit alternative splicing in plants. The differential sensitivity of post-transcriptional processing to cold stress observed for the two transcripts examined will permit further dissection of the nucleotide sequence requirements for their accurate splicing.

Alternative Splicing↗
PubMed1995

Hypervariable microsatellites provide a general source of polymorphic DNA markers for the chloroplast genome.

BACKGROUND: The study of plant populations is greatly facilitated by the deployment of chloroplast DNA markers. Asymmetric inheritance, lower effective population sizes and perceived lower mutation rates indicate that the chloroplast genome may have different patterns of genetic diversity compared to nuclear genomes. Convenient assays that would allow intraspecific chloroplast variability to be detected are required. RESULTS: Eukaryote nuclear genomes contain ubiquitous simple sequence repeat (microsatellite) loci that are highly polymorphic in length; these polymorphisms can be rapidly typed by the polymerase chain reaction (PCR). Using primers flanking simple mononucleotide repeat motifs in the chloroplast DNA of annual and perennial soybean species, we demonstrate that microsatellites in the chloroplast genome also exhibit length variation, and that this polymorphism is due to changes in the repeat region. Furthermore, we have observed a nonrandom geographic distribution of variations at these loci, and have examined the number and location of such repeats within the chloroplast genomes of other species. CONCLUSIONS: PCR-based analysis of mononucleotide repeats may be used to detect both intraspecific and interspecific variability in the chloroplast genomes of seed plants. The analysis of polymorphic microsatellites thus provides an important experimental tool to examine a range of issues in plant genetics.

Base Sequence↗
PubMed1994

Characterisation of a complementary DNA encoding a novel plant enzyme with sucrolytic activity.

The cloning of a 1332 bp cDNA from a potato (Solanum tuberosum L.) cv. Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described. The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum. Expression of the cDNA in E. coli yields a fusion protein with sucrolytic activity.

Amino Acid Sequence↗
PubMed1994

Potato (Solanum tuberosum) invertase-encoding cDNAs and their differential expression.

A full-length cDNA clone encoding a potato invertase (Inv) has been isolated. It is highly related (77% nucleotide identity) to a previously characterised potato cDNA clone encoding a putative extracellular Inv. These Inv genes encode a subfamily of apoplastic enzymes which are shown to be distinct, on the basis of sequence similarity, from the related subfamily of vacuolar enzymes. In order to differentiate between the expression of the two potato genes encoding apoplastic Inv, a single-stranded conformational polymorphism (SSCP) assay was developed for products generated by reverse transcription-polymerase chain reaction (RT-PCR) utilising primers designed to amplify both potato sequences. Using this approach, we have shown that these two identified Inv from potato are expressed in a tissue-specific and developmentally regulated manner.

Amino Acid Sequence↗
PubMed1993

cDNA cloning and expression of a potato (Solanum tuberosum) invertase.

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.

Amino Acid Sequence↗
PubMed1988

Insertion sequence IS2 in the cyanobacterium Chlorogloeopsis fritschii.

A cloned DNA fragment, previously demonstrated to encode ribulose bisphosphate carboxylase/oxygenase (RuBisCO) of Chlorogloeopsis fritschii strain CCAP1411/1b, is shown also to include the entire transposable element, IS2, normally a resident in the Escherichia coli genome. Southern-blot hybridisation experiments confirm the presence of IS2 in the C. fritschii genome. This finding adds a new and unrelated species to the known host range of this element and provides evidence of genetic transfer between the Gram-negative E. coli and cyanobacteria. This may also have significance in relation to the nucleotide sequence rearrangements known to occur adjacent to RuBisCO and nif genes in other nitrogen-fixing cyanobacteria.

Base Sequence↗
PubMed1981

Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin.

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.

Animals↗
PubMed1975

The binding sites for tRNA on eukaryotic ribosomes.

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.

Animals↗