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Twigg, P. G.  1993.  Isolation of a nodule-specific cDNA encoding a putative glycine-rich protein from Alnus glutinosa.  Ph. D. Dissertation, University of Tennessee, Knoxville.  144 pp.


ABSTRACT

Gene expression specific to symbiotic actinorhizal nodules has until now not been demonstrated due largely to the difficulty in extracting intact and biologically active messenger RNA from actinorhizal host plants.  In the course of this study a dependable protocol was developed for the isolation of RNA from roots and nodules of actinorhizal Alnus glutinosa.  cDNA libraries were constructed from mRNA from roots and nodules and from this a subtraction library enriched in nodule-specific or enhanced sequences was produced.  A total of 197 putative nodule-specific clones was differentially screened with nodule and root first strand cDNA.  A single cDNA clone, pAgNt84, hybridizing specifically to nodule but not to root or leaf RNA was selected for further study.

Sequence analysis of pAgNt84 revealed that it was composed of 681 bp and had a putative open reading frame of 98 amino acids.  The derived amino acid sequence of the open reading frame was found to be rich in glycine and histidine, at 15.3% each.  Alignment of the putative protein product of pAgNt84 with several other glycine-rich proteins revealed high sequence conservation in the N-terminal end in a region known to contain a signal peptide in several members of a plant glycine-rich protein family.  Based on the similarity of the putative signal peptides a possible localization and function of the pAgNt84 cDNA-encoded protein is proposed.

Nucleic acid comparisons revealed a relationship of the pAgNt84 cDNA to a cDNA encoding a glycine-rich protein from Medicago sativa, which lacks a signal peptide.  The similarity of these sequences begins only after the end of the region encoding the putative signal peptide in pAgNt84 indicating a common evolutionary origin, but with the gain or more likely loss of the region encoding the signal peptide.