Isolation, characterization and expression ofcDNA and genomic sequences encoding ribulosebisphosphate carboxylase/oxygenase activase from barley
Rundle, Sabine Jos
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Permalink
https://hdl.handle.net/2142/23818
Description
Title
Isolation, characterization and expression ofcDNA and genomic sequences encoding ribulosebisphosphate carboxylase/oxygenase activase from barley
Author(s)
Rundle, Sabine Jos
Issue Date
1991
Doctoral Committee Chair(s)
Zielinski, Raymond E.
Department of Study
Biology, Molecular
Biology, Plant Physiology
Discipline
Biology, Molecular
Biology, Plant Physiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Plant Physiology
Language
eng
Abstract
Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39, rubisco) catalyzes either the carboxylation or oxygenation of ribulose-1,5-bisphosphate (RuBP) to initiate either the Calvin cycle or the photorespiratory pathway. Rubisco needs to be activated in order to catalyze these reactions. Rubisco activation involves the binding of an activator CO$\sb2$ to a lysine residue near but not at the active site of the enzyme, followed by binding of Mg$\sp{2+}$. Rubisco activase is a nuclear-encoded chloroplast protein involved in the activation of rubisco.
The study describes the isolation and structural characterization of genomic and cDNA sequences encoding rubisco activase from the monocot plant barley (Hordeum vulgare L.). Three rubisco activase polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rca polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism. RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire rubisco activase gene family in barley. The genes share 80% nucleotide sequence identity within their coding regions, and the 42 kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kb of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5$\sp\prime$ end of both barley Rca genes.
Probes specific to the three activase mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool. These experiments were performed on mRNA isolated from whole leaves, from leaf sections along the acropetal barley leaf developmental gradient, and from plants grown under different irradiance levels. In addition, Rca mRNA expression was compared with that of other genes encoding proteins involved in photosynthesis.
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