Cloning of methyltransferase cDNAs from Pisum sativum

Cummings, Michele (1995) Cloning of methyltransferase cDNAs from Pisum sativum. PhD thesis, University of Glasgow.

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Abstract

A number of approaches were adopted to clone pea MTase cDNAs. The only successful approach, however, was PCR amplification of pea MTase-specific segments using degenerate primers, that were designed mainly according to the conserved motifs IV and VI of mouse MTase. A downstream antisense primer based on motif VI was used to prime first-strand cDNA synthesis from pea total RNA. This was then amplified using the same primer in combination with a sense primer designed according to motif IV. Several of the PCR products which were amplified were cloned into pBluescript and then sequenced. Only one type of PCR product was found to be MTase-specific upon translation of the sequences. On comparison with the database, this PCR product was found to share the highest homology with Arabidopsis MTase, followed by mouse and human MTases. No other MTase- specific PCR product was identified out of a total of 18 independent clones that were sequenced. Only one MTase mRNA (of 5.0 kb in length) was identified upon hybridisation of the cloned pea MTase-specific PCR product to a northern blot of pea poly(A)+ RNA. The 3' end of the pea MTase cDNA (corresponding to most of the catalytic domain) was cloned using a 3' RACE strategy and Pfu polymerase. Seven independent clones were mapped, and three were sequenced. The three clones were found to be absolutely identical in sequence, except for at the extreme 3' end, where the sites used for polyadenylation were found to be different for each clone. The nucleotide sequence of the 3' RACE products was also found to match that of the original MTase-specific PCR products in the region between motifs IV and VI. Again, the translated sequence of the 3' RACE product shared the highest homology with Arabidopsis MTase. No evidence for alternative splicing in the 3' end of the pea MTase transcript was found. Developmental changes in the steady state levels of pea MTase mRNA were investigated by hybridisation of one of the cloned PCR products to a northern blot of total RNA extracted from different tissues from pea seedlings, taken at different stages of development. The strength of the hybridisation signal was found to parallel the changes in MTase specific activity (Yesufu et ai, 1991), being highest in rapidly dividing tissues. To determine whether a family of related pea MTase genes exists in pea, the 3' RACE product was hybridised at moderate stringency to a Southern blot of pea genomic DNA digested with various restriction enzymes insensitive to methylation at CpG and CpNpG sites. The results were consistent with the presence of only one type of MTase gene in pea.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Roger I P Adams
Keywords: Plant sciences
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-71520
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:25
Last Modified: 10 May 2019 14:25
URI: http://theses.gla.ac.uk/id/eprint/71520

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