Rapid analysis of plant gene expression by a novel reverse transcriptase‐PCR method

Simpson, Craig G.; Sinibaldi, Ralph M.; Brown, John W.

doi:10.1111/j.1365-313x.1992.tb00154.x

Cited by 28 publications

(18 citation statements)

References 6 publications

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“…Transcript size was determined by comparison of electrophoretic mobility with RNA markers of known sizes (Life Technologies). RT-PCR was carried out as described by Simpson et al (1992). Control reactions were carried out in the absence of reverse transcriptase.…”

Section: Analysis Of Gene Expressionmentioning

confidence: 99%

cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)

Taylor¹,

George²,

Ross³

et al. 1998

The Plant Journal

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SummaryUsing an Arabidopsis thaliana expressed sequence tag with sequence similarity to human lysosomal α-glucosidase as a probe, a potato cDNA was isolated. The cDNA encodes a polypeptide with an Mr value of 105,400 and the most significant matches of the deduced amino acid sequence are with members of family 31 of glucosyl transferase. The potato cDNA was expressed in a strain of Saccharomyces cerevisiae that is deficient in maltase activity and unable to grow using maltose as a carbon source (ABYSMAL81). Expression of the potato cDNA in the mutant yeast strain restores its ability to use maltose as a carbon source for growth. Additionally, α-glucosidase activity could be measured in extracts of the yeast cells following complementation. A range of maltodextrins were substrates for this activity. The steady-state expression level of the potato α-glucosidase gene was low in most tissues examined, the highest levels occurring in sprouting tubers and source leaves.

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Section: Analysis Of Gene Expressionmentioning

confidence: 99%

cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)

Taylor¹,

George²,

Ross³

et al. 1998

The Plant Journal

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“…Aliquots of mRNAs were treated with Phosphow~ation RNase-free DNase (5 units per pg of mRNA). Treated mRNAs were reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Gibco BRL) by using the primers GMC34 (5'-CCA-TATACAACATGTGAAACACTG-3' for Fenlfen) and GMC35 (5'-TGA-AAGAAGGATCCACAGGTATAG-3' for P tolpto) to make the cDNAs (Simpson et al, 1992). cDNA templates were amplified by PCR using gene-specific primers RI (5'-ATGGGAAGCAAGTATCCAA-3') and GMC35 (Ptolpto) or GMC2 (5'-GCTACATTGAAGTlTGCCC-3') and GMC34 (Fenlfen).…”

Section: Expression and Purification Of Fusion Proteinsmentioning

confidence: 99%

Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.

et al. 1997

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The Pto gene was derived originally from the wíld tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. The Fen gene is also derived from L. pimpinellifolium and confers sensitivity to the insecticide fenthion. We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L. esculentum, and designated them pfo and fen. High conservation of genome organization between the two tomato species allowed us to ídentífy the pto and fen alleles from among the cluster of closely related Pfo gene family members. The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively. In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases. In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Ptil serine/ threonine kinase. However, the pto kinase shows impaired interaction with Ptil and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system. The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto-and fen-mediated signal transduction pathways.

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“…RNA samples were treated twice with DNase I to deplete contaminating genomic DNA (Simpson et al, 1992).…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Cis-Acting Elements Essential for Light Regulation of the Nuclear Gene Encoding the A Subunit of Chloroplast Glyceraldehyde 3-Phosphate Dehydrogenase in Arabidopsis thaliana

1996

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Rapid analysis of plant gene expression by a novel reverse transcriptase‐PCR method

Cited by 28 publications

References 6 publications

cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)

cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)

Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.

Cis-Acting Elements Essential for Light Regulation of the Nuclear Gene Encoding the A Subunit of Chloroplast Glyceraldehyde 3-Phosphate Dehydrogenase in Arabidopsis thaliana

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