Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1995-05-04
2001-01-16
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C435S069100, C435S070100, C435S252200, C435S252300, C435S252330, C435S418000, C435S469000, C536S023200, C536S023700, C536S024100, C536S023600, C800S288000, C800S294000
Reexamination Certificate
active
06174724
ABSTRACT:
TECHNICAL FIELD
This invention is in the fields of genetic engineering, plant biology, and bacteriology.
BACKGROUND ART
In the past decade, the science of genetic engineering has developed rapidly. A variety of processes are known for inserting a heterologous gene into bacteria, whereby the bacteria become capable of efficient expression of the inserted genes. Such processes normally involve the use of plasmids which may be cleaved at one or more selected cleavage sites by restriction endonucleases, discussed below. Typically, a gene of interest is obtained by cleaving one piece of DNA and the resulting DNA fragment is mixed with a fragment obtained by cleaving a vector such as a plasmid. The different strands of DNA are then connected (“ligated”) to each other to form a reconstituted plasmid. See, for example, U.S. Pat. No. 4,237,224 (Cohen and Boyer, 1980); U.S. Pat. No. 4,264,731 (Shine, 1981); U.S. Pat. No. 4,273,875 (Manis, 1981); U.S. Pat. No. 4,322,499 (Baxter et al., 1982), and U.S. Pat. No. 4,336,336 (Silhavy et al., 1982). A variety of other reference works are also available. Some of these works describe the natural processes whereby DNA is transcribed into messenger (mRNA) and mRNA is translated into protein; see, e.g., Stryer, 1981 (note: all references cited herein, other than patents, are listed with citations after the Examples); Lehninger, 1975. Other works describe methods and products of genetic manipulation; see, e.g., Maniatis et al., 1982; Setlow and Hollaender, 1979.
Most of the genetic engineering work performed to date involves the insertion of genes into various types of cells primarily bacteria such as
E. coli
, various other types of microorganisms such as yeast, and mammalian cells. However, many of the techniques and substances used for genetic engineering of animal cells and microorganisms are not directly applicable to genetic engineering involving plants.
As used herein, the term “plant” refers to a multicellular differentiated organism that is capable of photosynthesis, such as angiosperms and multicellular algae. This does not include microorganisms, such as bacteria, yeast, and fungi. However, the term “plant cells” includes any cell derived from a plant; this includes undifferentiated tissue such as callus or crown gall tumor, as well as plant seeds, propagules, pollen, and plant embryos.
A variety of plant genes have been isolated, some of which have been published and/or are publicly available. Such genes include the soybean actin gene (Shah et al., 1982), corn zein (Pederson et al., 1982) soybean leghemoglobin (Hyldig-Nielsen et al., 1982), and soybean storage proteins (Fischer and Goldberg, 1982).
The Reigons of a Gene
The expression of a gene involves the creation of a polypeptide which is coded for by the gene. This process involves at least two steps: part of the gene is transcribed to form messenger RNA, and part of the mRNA is translated into a polypeptide. Although the processes of transcription and translation are not fully understood, it is believed that the transcription of a DNA sequence into mRNA is controlled by several regions of DNA. Each region is a series of bases (i.e., a series of nucleotide residues comprising adenosine (A), thymidine (T), cytidine (C), and guanidine (G)) which are in a desired sequence. Regions which are usually present in a eucaryotic gene are shown on FIG.
1
. These regions have been assigned names for use herein, and are briefly discussed below. It should be noted that a variety of terms are used in the literature, which describes these regions in much more detail.
An association region 2 causes RNA polymerase to associate with the segment of DNA. Transcription does not occur at association region 2; instead, the RNA polymerase normally travels along an intervening region 4 for an appropriate distance, such as about 100-300 bases, after it is activated by association region 2.
A transcription initiation sequence 6 directs the RNA polymerase to begin synthesis of mRNA. After it recognizes the appropriate signal, the RNA polymerase is believed to begin the synthesis of mRNA an appropriate distance, such as about 20 to about 30 bases, beyond the transcription initiation sequence 6. This is represented in
FIG. 1
by intervening region 8.
The foregoing sequences are referred to collectively as the promoter region of the gene.
The next sequence of DNA is transcribed by RNA polymerase into messenger RNA which is not translated into protein. In general, the 5′ end of a strand of mRNA attaches to a ribosome. In bacterial cells, this attachment is facilitated by a sequence of bases called a “ribosome binding site” (RBS). However, in eucaryotic cells, no such RBS sequence is known to exist. Regardless of whether an RBS exists in a strand of mRNA, the mRNA moves through the ribosome until a “start codon” is encountered. The start codon is usually the series of three bases, AUG; rarely, the codon GUG may cause the initiation of translation. The non-translated portion of mRNA located between the 5′ end of the mRNA and the start codon is referred to as the 5′ non-translated region 10 of the mRNA. The corresponding sequence in the DNA is also referred to herein as 5′ non-translated region 12. The specific series of bases in this sequence is not believed to be of great importance to the expression of the gene; however, the presence of a premature start codon might affect the translation of the mRNA (see Kozak, 1978).
A promoter sequence may be significantly more complex than described above; for example, certain promoters present in bacteria contain regulatory sequences that are often referred to as “operators.” Such complex promoters may contain one or more sequences which are involved in induction or repression of the gene. One example is the lac operon, which normally does not promote transcription of certain lactose-utilizing enzymes unless lactose is present in the cell. Another example is the trp operator, which does not promote transcription or translation of certain tryptophan-creating enzymes if an excess of tryptophan is present in the cell. See, e.g., Miller and Reznikoff, 1982.
The next sequence of bases is usually called the coding sequence or the structural sequence 14 (in the DNA molecule) or 16 (in the mRNA molecule). As mentioned above, the translation of a polypeptide begins when the mRNA start codon, usually AUG, reaches the translation mechanism in the ribosome. The start codon directs the ribosome to begin connecting a series of amino acids to each other by peptide bonds to form a polypeptide, starting with methionine, which always forms the amino terminal end of the polypeptide (the methionine residue may be subsequently removed from the polypeptide by other enzymes). The bases which follow the AUG start codon are divided into sets of 3, each of which is a codon. The “reading frame,” which specifies how the bases are grouped together into sets of 3, is determined by the start codon. Each codon codes for the addition of a specific amino acid to the polypeptide being formed. The entire genetic code (there are 64 different codons) has been solved; see, e.g., Lehninger, supra, at p. 962. For example, CUA is the codon for the amino acid leucine; GGU specifies glycine, and UGU specifies cysteine.
Three of the codons (UAA, UAG, and UGA) are “stop” codons; when a stop codon reaches the translation mechanism of a ribosome, the polypeptide that was being formed disengages from the ribosome, and the last preceding amino acid residue becomes the carboxyl terminal end of the polypeptide.
The region of mRNA which is located on the 3′ side of a stop codon in a monocistronic gene is referred to herein as 3′ non-translated region 18. This region is believed to be involved in the processing, stability, and/or transport of the mRNA after it is transcribed. This region 18 is also believed to contain a sequence polyadenylation signal 20, which is recognized by an enzyme in the cell. This enzyme adds a substantial number of adenosine residues to the mRNA molecule, to form po
Fraley Robert T.
Rogers Stephen G.
Fox David T.
Howrey Simon Arnold & White L.L.P.
Lavin, Jr. Lawrence M.
Monsanto Company
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