Molecular cloning of cDNA for Avena phytochrome [Oats]

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)+RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1984-04, Vol.81 (8), p.2332-2336
Main Authors: Hershey, H.P, Colbert, J.T, Lissemore, J.L, Barker, R.F, Quail, P.H
Format: Article
Language:English
Subjects:
Avena
Biological Sciences: Biochemistry
Chromium
Complementary DNA
DNA
DNA probes
Genetic screening
Libraries
Messenger RNA
Plants
Plasmids
RNA
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)+RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)+RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobase-pair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)+RNA hybridized with32P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.81.8.2332