Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1999-11-19
2002-06-25
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C435S320100, C435S419000, C435S468000, C536S024100, C800S298000
Reexamination Certificate
active
06410828
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to plant molecular biology. In particular, it relates to promoter sequences useful for gene expression in selected plant organs and tissues.
Isolated plant promoters are useful in the genetic engineering of plants to produce transgenic plants with desired phenotypes. To produce such transgenic plants, an isolated plant promoter is inserted into a vector and operably linked to a heterologous DNA sequence. Plant cells are then transformed with the vector such that expression of the heterologous DNA is controlled by the promoter.
Some plant promoters are tissue-specific, while others are constitutive and drive expression in essentially all tissues and organs. Tissue-specific promoters can be identified from genes that are expressed in particular tissues or at particular times during development.
A need exists for a variety of different promoters to be used in the genetic engineering of plants. New tissue-specific promoters are particularly useful for the controlled expression of various nucleic acid sequences in transgenic plants. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides embryo specific maize metallothionein promoters. The promoters can be used to provide embryo specific expression of the heterologous sequences in plants. In particular, the promoters are useful in expression in maize. More specifically, the invention provides an isolated DNA molecule comprising base pairs 50 to 1649 of SEQ ID NO:5, or a fragment, genetic variant or deletion of such a sequence which retains the ability of functioning as an embryo specific promoter in plant cells.
The invention also provides expression cassettes comprising an embryo specific promoter operably linked to a heterologous nucleic acid sequence, wherein the promoter selectively hybridizes to a 20 consecutive base pair portion of the sequence set forth in SEQ ID NO:5.
The invention also provides an expression cassette of wherein the promoter comprises a sequence extending from about nucleotide 50 to nucleotide 1649 of SEQ ID NO:5.
The invention also provides a method of expressing a heterologous nucleic acid sequence in a plant comprising:
a) introducing into plant tissue a vector comprising an embryo specific maize metallothionein promoter operably linked to the heterologous nucleic acid sequence; and
b) regenerating the plant tissue into a whole plant.
The invention also provides an isolated DNA molecule having a 20 base pair nucleotide portion identical in sequence to a 20 consecutive base pair portion of the sequence set forth in SEQ ID NO:5.
The invention also provides transgenic plant comprising the expression cassettes described above. The plant may be any agronomically useful plant.
DEFINITIONS
The term “nucleic acids”, as used herein, refers to either DNA or RNA. “Nucleic acid sequence” or “polynucleotide sequence” refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Sequence regions on a DNA strand that are 5′ to the 5′ end of an RNA transcript encoded by the DNA are referred to as “upstream sequences”. Upstream sequences are usually counted in a negative direction from the transcription start site. Sequence regions on the DNA strand that are 3′ to the 3′ end of the RNA transcript are referred to as “downstream sequences.”
The term “promoter” refers to a region of DNA upstream from the translational start codon and which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. The term maize metallothionein promoter as used herein refers to plant promoters comprising sequences derived from the promoter region of a maize metallothlonein gene. The promoters of the invention contain tissue specific elements that allow embryo specific transcription of operably linked DNA sequences. The promoters are considered to be embryo specific promoters because transcription of the operably linked DNA is higher in embryo tissues than it is in other tissues.
A “tissue-specific” promoter as used herein refers to a promoter that drives expression of an operably linked nucleic acid sequence in a particular tissue in a plant or at a particular stage in the plant life-cycle.
The term “operably linked” as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. It is understood that the promoter sequence aim includes transcribed sequences between the transcriptional start and the translational start and the translational start codon.
The phrase “expression cassette”, refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible wanh such sequences. Such cassettes include at least promoters and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.
A “heterologous” nucleic acid or protein is one that originates from a foreign source (or species) or, if from the same source is modified from its original form. Thus, a heterologous promoter sequence in an expression cassette is from a source different from the source of the coding sequence, or, if from the same source, is modified from its original form. Modification may occur, e.g., by treating the DNA with a restriction enzyme to generate a promoter element that is capable of conferring tissue-specific expression on the expression cassette which includes it.
The phrases “isolated” or “substantially pure” when referring to a polynucleotide or protein, means a chemical composition which is free of other subcellular components of the organism from which it its derived. Typically, a compound is substantially pure when at least about 85% or more of a sample exhibits a single polypeptide backbone, or polynucleotide sequence. Minor variants or chemical modifications may typically share the same polypeptide sequence. Depending on the purification procedure, purity of 85%, and preferably over 95% pure are possible. Nucleic acid and protein purity or homogeneity may be indicated by a number of means well known in the art, such as gel electrophoresis and the like.
The term “plant” includes whole plants, plant organs (e.g., leaves, stems roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
“Percentage of sequence identity” for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection. Sequences are typically compared using GESTFIT or BlastN with default parameters.
Substantial identity of polynucleotide sequences means that a polynucleotide comprises a s
Armstrong Katherine
Folkerts Otto
Hey Timothy D.
Pareddy Dayakar R.
Rubin-Wilson Beth C.
Collins Cynthia
Dow AgroSciences LLC
McElwain Elizabeth F.
Stuart Donald R.
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