University of Hawai'i

The United States of America As Represented by the Secretary of

John I Stiles

Kabi Raj Neupane

Istefo Moisyadi

Olin D Anderson

John D Neill

Frank C Greene

James C Litts

The invention establishes that coffee fruit ripening is climacteric. The invention further provides techniques to isolate substantially pure RNA from coffee fruit even though the fruit contains high levels of phenolic compounds and carbohydrate which would otherwise interfere with obtaining clean RNA preparations from this tissue. The invention provides purified proteins, nucleic acid sequences that code on expression therefore and recombinant DNA molecules, including hosts transformed therewith, and methods for transforming coffee plants to suppress the expression of coffee fruit-expressed ACC synthase and/or coffee fruit-expressed ACC oxidase necessary for ethylene biosynthesis and the ripening of coffee fruit. Coffee plants are transformed with vectors containing coffee fruit-expressed ACC synthase and/or with ACC oxidase DNA sequences that code on expression for the respective RNA that is antisense or sense to the mRNA for the respective ACC synthase and/or ACC oxidase. The result is that the expression of the respective enzyme is eliminated and the transformed plants are incapable of synthesizing ethylene during coffee fruit ripening, although other aspects of their metabolism is not affected. The invention further provides methods for controlling the ripening of coffee fruit from the transformed coffee plants by applying exogenous ethylene.

Purified proteins, recombinant DNA sequences and processes for controlling the ripening of coffee plants

The invention provides purified proteins, DNA sequences that code on expression therefore, and recombinant nucleic acid sequences, including hosts transformed with such sequences, for transforming coffee plants to reduce or eliminate the synthesis of caffeine. Coffee plant cells are transformed by means of transforming vectors that comprise a nucleic acid sequence, operably linked to a transcription promoter in an antisense or sense orientation, that codes on transcription for an RNA that has a length sufficient to interfere with the expression of a specific enzyme in the pathway for caffeine biosynthesis. Coffee plants regenerated from transformed coffee plant cells exhibit caffeine production that has been reduced by up to 98% compared with untransformed coffee plants.

Purified proteins, recombinant DNA sequences and processes for producing caffeine-free beverages

Purified proteins, DNA sequences that code on expression therefore and recombinant DNA molecules, including hosts transformed therewith, for transforming coffee plants to suppress the expression of caffeine. The DNA sequences and recombinant DNA molecules are characterized in that they code on expression for an enzyme in the pathway for caffeine synthesis in coffee. Coffee plants transformed with DNA molecules that code on transcription for mRNA that is antisense to mRNA that codes on expression for at least one enzyme in the pathway for caffeine biosynthesis.

Purified proteins, recombinant DNA sequences and processes for producing

The invention provides purified proteins, DNA sequences that code on expression therefore and recombinant DNA molecules, including hosts transformed therewith for transforming coffee plants to suppress the expression of enzymes necessary for ethylene synthesis. The DNA sequences and recombinant DNA molecules are characerized in that they code on expression for the enzymes ACC synthase or ACC oxidase that are elements of the pathway for ethylene biosynthesis in coffee plants. Coffee plants are transformed with vectors containing ACC synthase and/or with ACC oxidase DNA sequences that code on expression for the respective mRNA that is antisense to the mRNA for ACC synthase and/or ACC oxidase. The resulting antisense mRNA binds to the respective ACC synthase and/or ACC oxidase mRNA, thereby inactivating the mRNA encoding one or more enzymes in the pathway for ethylene synthesis. The described DNA sequences can also be used to block synthesis of ACC synthase or ACC oxidase using co-suppression. The result in either event is that the transformed plants are incapable of synthesizing ethylne, though other aspects of their metabolism is not affected.

Purified proteins, recombinant DNA sequences and processes for

The present invention provides 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase) cDNA sequences from papaya fruit. These sequences are useful in regulation of ethylene biosynthesis in papaya cells and in regulating fruit ripening, including delaying of ripening. This is accomplished by co-suppression or by antisense technology. The present invention also provides methods for regulation of fruit ripening.

Nucleic acids encoding a papaya ACC synthase gene

A strain of yeast Saccharomyces cerevisiae has been developed which, when grown under defined culture conditions, will produce protein indistinguishable from wheat gluten protein. This new yeast strain was developed by introducing a specially constructed autonomously replicating extrachromosomal genetic element, gluten plasmid pAY31, into the parent yeast strain. This plasmid is a circular DNA molecule, constructed by enzymic fusion of the following elements: (1) the E. coli plasmid pUC8 wherein the EcoRI site has been removed; (2) the autonomously replicating yeast sequence ARS1; (3) the yeast URA3 gene; (4) a modified yeast iso-1-cytochromic gene retaining the promoter region and transcription termination sequence, and wherein the protein coding sequences have been deleted and replaced with a synthetic EcoRI restriction site, the site at which the wheat gluten protein gene is cloned; and (5) a fragment of a wheat gluten protein gene which includes the amino acid coding region, translation initiation and termination sequence, and short flanking nucleotide sequences, but excludes transcription initiation and termination sequences. Wheat gluten protein synthesized by the new yeast strain can be used to supplement wheat and non-wheat flours for baked products and for use in diagnosis and treatment of illness in humans caused by wheat gluten proteins.

Growing community of inventors

Kaneohe, HI, United States of America

John I Stiles