Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Patent
1993-10-25
1996-12-17
Chereskin, Che S.
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
800DIG44, 536 232, 4353201, 530370, 530379, A01H 400, C07K 14415, C12N 1529, C12N 1582
Patent
active
055855423
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a DNA sequence which encodes at least part of the tomato enzyme endopolygalacturonase PG1 .beta.-subunit. The present invention further relates to the use of this DNA sequence in modulating endopolygalacturonase expression in tomatoes.
BACKGROUND OF THE INVENTION
The structure of tomato fruit endopolygalacturonase (EC 3.2.1.15, poly (1,4 .alpha.-D-galacturonide) glycanohydrolase or PG) resides in three proteins termed PG1, PG2A and PG2B (Ali and Brady 1982). The three proteins are immunologically related (Ali and Brady 1982) and appear to be, in part, the products of a single copy gene that is developmentally regulated (Bird et al. 1988). PG1 apparently also contains another polypeptide (Moshrefi and Luh 1983). The gene, DNA copies of the mRNA encoding PG2A, and the PG2A form of the protein have been sequenced (Bird et al. 1988; Grierson et al. 1986; Sheehy et al. 1987).
As ripening is induced in tomato fruit the following increase: transcription of the PG gene (DellaPenna et al. 1989), the steady state level of the PG mRNA (DellaPenna et al. 1986; Maunders et al. 1987), PG activity and the amount of PG protein (Brady et al. 1982; Tucker and Grierson 1982). Concomitantly, during ripening there is an increase in the ease of pectin extraction and in the compressibility (`softness`) of the fruit pericarp (Sawamura et al. 1978; Brady et al. 1982; Seymour et al. 1987). These correlations led to the hypothesis that PG activity is primarily responsible for the increase in softness during ripening (Grierson 1985).
Several aspects of the function and regulation of PG activity in tomato fruit remain unclear. The occurrence and roles of the three isoforms of the enzyme are undefined; the details of their movement to, and interaction with, the cell wall are unknown.
It may be significant that PG1 is the predominant isoform when the total amount of enzyme is small. This is the case in the slow ripening (Nr) mutant, in hetarozygotes of the non-ripening (nor) and ripenings inhibited (rin) mutants and in the early stages of ripening of normal lines (Tucker et al. 1980; Brady et al. 1983; Knegt et al. 1988). Under denaturing conditions PG1 contains polypeptides that appear to be identical to PG2A and PG2B and a smaller polypeptide (Moshrefi and Luh 1983). There are suggestions that PG1 is formed from PG2 by interaction with either a specific glycopeptide (Pressey 1984; Knegt et al. 1988) or carbohydrate (Tucker et al. 1981). Alternative theses on PG1's role include: that it is the active in vivo isoform (Tucker et al. 1981; Knegt et al. 1988), that the second polypeptide in PG1 may bind and site PG onto the cell wall (Brady et al. 1987) and that PG1 does not exist in vivo but is formed when PG2 is co-extracted with a nonspecific `convertor` glycoprotein (Pressey 1986, 1988).
PG1 is notably thermostable (Tucker et al. 1981; Moshrefi and Luh, 1983). The thermostability makes it important as a catalyst of polymer changes when tomato fruit are processed and it is an important determinant of the heat input required for processing. For this reason, and because of the possibility that PG1 is the form of the enzyme active in vivo there is commercial interest in the genetic regulation of the content of PG1 in tomato fruit. Knowledge of the structure and in particular, the amino acid sequence of the subunit could allow the gene sequence to be sought. Genetic manipulation using antisense or transgene technology could follow.
The present inventors have, for the first time, developed practical methods for isolating the protein PG1 and each of its subunits. The chemistry of the two subunits is described and N-terminal and some internal sequence of the second or .beta.-subunit of PG1 is described herein for the first time.
SUMMARY OF THE PRESENT INVENTION
In a first aspect the present invention consists in an isolated DNA sequence which encodes at least part of the tomato enzyme endopolygalacturonase PG1 .beta.-subunit.
In a second aspect the present invention consists in a D
REFERENCES:
Compton, T. "Degenerate Primers for DNA Amplification" PCR Protocols: A Guide to Methods and Applications, Academic Press, pp. 39-45. 1990.
Lee, C. C. et al. "cDNA cloning using . . . " PCR Protocols: A Guide to Methods and Applications, 1990, Academic Press, pp. 47-53.
Frohman, M. A., et al. "Rapid production of full-length . . . " Proc. Natl Acad. Sci., vol. 85, Dec. 1988, pp. 8998-9002.
Frohman, M. A. "Race: Rapid amplification . . . " PCR Protocols: A Guide to Methods and Applications, Academic Press, pp. 27-38. 1990.
Smith, C. J. S. et al. "Antisense RNA inhibition . . . " Nature, vol. 334, 25 Aug. 1988, pp. 724-726.
Fillatti et al. "Efficient transfer of a gluphosate . . . " Biotechnology, vol. 5, Jul. 1987, pp. 726-730.
Hamilton et al. Nature, vol. 346, 19 Jul. 1990, pp. 284-286.
Shahin, E. A. et al. "Transformation of cultivated . . . " Theor Appl Genet (1986), 72, pp. 720-777.
Sheehy, R. E. et al. "Reduction of polygalacturonase . . . " Proc. Natl Acad. Sci. USA, vol. 85 (Dec. 1988), pp. 8805-8809.
Pogson, B. J. et al. "On the occurrence and structure of subunits . . . " Aust. J. Plant Physiol. 1991, 18, pp. 65-79.
J. E. Lincoln et al. "Diverse mechanisms for the regulation . . . " Mol. Gen.-Genet (1988) 212, pp. 71-75.
Moshrefi, M. et al "Carbohydrate composition and electrophoretic . . . " European Journal of Biochemistry, vol. 135 No. 3, Oct. 1983, pp. 511-514.
Osteryoung, K. W. et al. "Analysis of tomato polygalaturonase . . . " The Plant Cell, vol. 2 No. 12, Dec. 1990, Rockville, MD, USA, pp. 1239-1248.
Pressey, R. "Purification and characterization of tomato . . . " European Journal of Biochemistry, vol. 144 No. 2, Oct. 1984, pp. 217-221.
Smith, et al (Aug. 1988) Nature 334: 724-726.
Zheng, et al (Sep. 1992) The Plant Cell 4: 1147-1156.
Moshrefi, et al (1983) European Journal of Biochemistry 135 (3): 511-514.
Pressey (Oct. 1984) European Journal of Biochemistry 144(2): 217-221.
Brady Colin
Lee Elizabeth
Orr Glenda R.
Pogson Barry J.
Speirs James
Chereskin Che S.
Commonwealth Scientific and Industrial Research Organisation
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