Many attempts have been made to obtain improved plants for cultivation through breeding programs. A conventional plant breeding program requires as much as ten years to develop a new variety. In addition to the initial hybridization step, several years are typically spent replanting successive generations in order to obtain homozygous plants. An alternative to a conventional plant breeding program is anther culturing in which anthers from one plant are used to pollinate the ovaries of another plant. However, many traits cannot be successfully introduced via such hybridization techniques since the genes for such traits are not found in breeds available for hybridization.
An alternative to hybridization is somaclonal variation. This technique involves the use of vegetative plant parts, such as callus tissue, as explant material. For example, callus that develops from vegetative explants of rice frequently regenerates plants which have genetic characteristics not found in the variety from which the explant was originally obtained. These somatic mutants occur at high frequencies, and the percentage of regenerated plants which differ from the starting variety exceeds, for example, 75 percent in rice. This technique is therefore useful for producing genetic variability. Again, however, there are limits to the extent of variation which can be obtained.
Development of plant genetic engineering began in the early 1940s when experiments were being carried out to determine the biological principle causing formation of crown gall tumors. The tumor-inducing principle was shown to be a bacterial plasmid from the infective organism Agrobacterium tumefaciens. This plasmid has since been characterized in much detail utilizing the currently available techniques of recombinant DNA technology. The bacterium elicits its response by inserting a small fragment of bacterial plasmid into the plant nucleus where it becomes incorporated and functions as a plant gene. This discovery opened the door to using Agrobacterium and its plasmids as vehicles to carry foreign DNA to the plant nucleus. There are, however, limitations to the application of this technique which include: (1) susceptibility to infection with the Agrobacterium plasmid and (2) available tissue culture technology for regeneration of the transformed plants. Thus, there are no successful reports on genetic engineering of monocots such as cereals with Agrobacterium plasmid vectors because of the general inability of Agrobacterium to infect monocots.
More recently, other techniques have been used to genetically transform monocots. For example, electroporation of protoplasts of rice, wheat and sorghum to obtain expression of a foreign gene was reported in Ou-Lee et al, Botany, vol. 83, pp. 6815-6819 (1986). Similar plant protoplast electroporation and electroinjection through cell walls and membranes have also been reported for other monocots, and dicots as well. See, Fromm et al, Proc. Natl. Acad Sci USA, vol. 82, pp. 5824-5828 (1985); Hibi et al, J. Gen. Virol., vol. 67, pp. 2037-2042 (1986); Langridge et al, Plant Cell Reports, vol. 4, pp. 355-359 (1985); Fromm et al, Nature, vol. 319, pp 791-793; Shillito et al, Bio/Technology, vol. 3, pp. 1099-1103 (1985); and Okada et al, Plant Cell Physiol., vol. 27, pp. 619-626 (1986). Similarly, direct and chemical-induced introduction of DNA into monocot and dicot cells has been disclosed. See, Lorz et al, Mol. Gen. Genet., vol. 199, pp. 178-182 (1985); Potrykus et al, Mol. Gen. Genet., vol. 199, pp 183-188 (1986); Uchimaya et al, Mol. Gen. Genet., vol. 204, pp 204-207 (1986); Freeman et al, Plant Cell Physiol., vol. 25, no. 8, pp. 1353-1365 (1984); and Krens et al, Nature, vol. 296, pp. 72-74 (1986). Another technique of interest is the injection of DNA into young floral tillers of rye plants reported in de la Pena et al, Nature, vol. 325, pp. 274-276 (1987).
The agricultural production of major crops has long been significantly affected by insects and plant pathogens. For example, blight and blast are major diseases of rice plants which can decimate a crop. Some plants cannot be cultivated in certain parts of the world because of the presence of diseases in such locations to which the plants are susceptible. For example, the main diseases in potato are bacterial soft rot and bacterial wilt caused by Erwinia carotovora and Pseudomonas solanacearum, respectively. These diseases are primarily responsible for limiting the growth of potatoes in many areas of Asia, Africa, South and Central America. Moreover, pesticides are becoming increasingly difficult to use in an effective, and yet environmentally acceptable manner. Therefore, it would be desirable to have available for cultivation plants which are resistant to insects and other pathogens.
It is well known that the pupae of Hyalophora (a type of silk moth) respond to bacterial infection by the synthesis of mRNAs which culminate in the production of about 15 to 20 new proteins. Lysozyme, the antibacterial protein found in egg white and human tears, and two other classes of antibacterial peptides, called cecropins and attacins, have been purified from Hyalophora humor. These proteins have a rather broad spectrum of activity in that they are effective on many different types of bacteria. Thus, the insects have evolved a rather successful and novel means to fight bacterial infections. Although a traditional immunologist would think this system lacks specificity, the insect has a rather potent arsenal of at least three different antibacterial proteins which may work in different ways to destroy bacterial pathogens. Thus, the invading bacteria is presented with a formidable challenge which is very difficult to circumvent. While a bacterial pathogen may be naturally resistant to one, it is highly improbable that it would be resistant to all three toxins. Although the exact mode of action of the protein toxins is not fully understood, they are generally procaryote specific and appear to be benign to eucaryotic insect cells.
As mentioned above, the property of certain peptides to induce lysis of procaryotic microorganisms such as bacteria is well known. For example, U.S. Pat. Nos. 4,355,104 and 4,520,016 to Hultmark et al describe the bacteriolytic properties of some cecropins against Gram-negative bacteria. Quite interestingly, the cecropins described in the Hultmark et al patents were not universally effective against all Gram-negative bacteria. For example, the cecropins described therein lysed Serratia marcescens D61108, but not Serratia marcescens D611. Moreover, cecropins have generally been reported to have no lytic activity towards eucaryotic cells such as insect cells, liver cells and sheep erythrocytes, as reported in the Hultmark patents; International Patent Publication WO/8604356; Andreu et al, Biochemistry, vol. 24, pp. 1683-88 (1985); Boman et al, Developmental and Comparative Immunology, vol. 9, pp. 551-558 (1985); and Steiner et al, Nature, vol. 292, pp. 246-248 (1981).
Other lytic peptides heretofore known include, for example, the sarcotoxins and lepidopterans. Such peptides generally occur naturally in the immune system of Sarcophaga peregrina and the silkworm, lepidopteran, respectively, as reported in Nakajima et al, The Journal of Biological Chemistry, vol. 262, pp. 1665-1669 (1987) and Nakai et al, Chem. Abst. 106:214351w (1987).
A number of the antibacterial polypeptides have been found to be useful when the genes encoding therefor are incorporated into various animals. Such transformation of animals with genes encoding therefor are described in U.S. patent application Ser. No. 069,653, filed Jul. 25, 1986, now abandoned by Jesse M. Jaynes, Frederick M. Enright and Kenneth F. White, which is hereby incorporated herein by reference.
Polynucleotide molecules expressible in E. coli and having the sequence araB promoter operably linked to a gene which is heterologous to such host are also known. The heterologous gene codes for a biologically active polypeptide. A genetic construct of a first genetic sequence coding for cecropin operably linked to a second genetic sequence coding for a polypeptide which is capable of suppressing the biological effect of the resulting fusion protein towards an otherwise cecropin-sensitive bacterium is also described in International Publication WO86/04356, Jul. 31, 1986.
The Hultmark et al patents mentioned above also mention that there are no known antibodies to cecropin, indicating a wide acceptability for human and veterinary applications, including one apparently useful application for surface infections because of the high activity against Pseudomonas. Similarly, EPO publication 182,278 (1986) mentions that sarcotoxins may be expected to be effective in pharmaceutical preparations and as foodstuff additives, and that antibacterial activity of sarcotoxin can be recognized in the presence of serum Shiba, Chem. Abstr. 104:230430K (1985) also mentions preparation of an injection containing 500 mg lepidopteran, 250 mg glucose and injection water to 5 ml.
Several analogs of naturally-occurring cecropins, sarcotoxins and lepidopterans have been reported. For example, it is reported in Andreu et al, Proc. Natl. Acad. Sci. USA, vol. 80, pp. 6475-6479 (1983) that changes in either end of the amino acid sequence of cecropin generally result in losses in bactericidal activity in varying degrees against different bacteria. It is reported in Andreu et al (1985) mentioned above that Trp.sup.2 is clearly important for bactericidal activity of cecropin, and that other changes in the 4, 6 or 8 position have different effects on different bacteria. From the data given in Table II at page 1687 of Andreu et al (1985), it appears that almost any change from natural cecropin generally adversely affects its bactericidal activity. Cecropin is defined in International Publication WO86/04356 to include bactericidally active polypeptides from any insect species and analogs, homologs, mutants, isomers and derivatives thereof having bactericidal activity from 1% of the naturally-occurring polypeptides up to 100 times or higher activity of the naturally-occurring cecropin. Other references generally discuss the effects of the .alpha.-helix conformation and the amphiphilic nature of cecropin and other lytic peptides.
It is known that lysozyme and attacins also occur in insect hemolymph. For example, it is reported in Okada et al, Biochem. J., vol. 229, pp. 453-458 (1985) that lysozyme participates with sarcotoxin against bacteria, but that the bactericidal actions are diverse. Steiner et al mentioned above suggests that lysozyme plays no role in the antibacterial activity of insect hemolymph other than to remove debris following lysis of bacteria by cecropin. Merrifield et al, Biochemistry, vol. 21, pp. 5020-5031 (1982) and Andreu et al (1983) mentioned above state that cecropin purified from insect hemolymph may be contaminated with lysozyme, but demonstrate that the synthetically prepared cecropin is as bactericidally active as purified cecropin from insect hemolymph.
The treatment of eucaryotic pathogens and other cells with lytic peptides, and novel lytic peptides, is the subject matter of U.S. Ser. No. 102,175, filed Sep. 29, 1987, now abandoned by Jaynes, Enright, White and Jeffers, which is hereby incorporated herein by reference.
Approximately 70% of the world's human population lives in underdeveloped countries, and have diets nutritionally inadequate in proteins, fats and calories. Malnutrition in these countries is wide-spread and persistent. Protein malnutrition can usually be attributed to a deficiency in the diet of one or more of the essential amino acids. The major food staples of many underdeveloped countries, cereals and tubers, are deficient in most limiting essential amino acids. When a major portion of the diet consists of such staples, the result is limiting essential amino acid deficiency. In children, this condition is particularly debilitating, because of the large requirement for high quality polypeptide needed for normal growth and development.
Protein malnutrition of this type could be alleviated or eliminated by adding supplements to the diet, which supplements contained polypeptides high in these limiting essential amino acids. Such polypeptides would have to be susceptible to digestion by normal human or animal proteases. Further, such polypeptides would be manufactured by cloning and expression of synthetic DNA.
Synthetic DNA of a desired sequence can now be constructed using modern chemical techniques, and the DNA can then be cloned into various microorganisms using recombinant DNA technology. It is known, for example, that a synthetic DNA which codes for poly(1-aspartyl-1-phenylalanine) can be cloned and expressed in E. coli. Doel et al, Nucleic Acids Research, Vol. 8, No. 20, pp. 4575-4592 (1980). Also Tangus et al, Applied and Environmental Microbiology, Vol. 43, No. 3, pp. 629-635 (March, 1982), have obtained expression of a cloned homopolymeric synthetic DNA sequence coding for poly-1-proline.
A number of United States patents have issued concerning the synthesis of polypeptides by conventional polypeptide sequencing, or by using DNA technology. Such patents include U.S. Pat. No. 3,796,631 to Choay et al, U.S. Pat. No. 3,850,749 to Kaufman et al, U.S. Pat. No. 3,299,043 to Schramm et al, U.S. Pat. No. 3,594,278 to Naylor, and U.S. Pat. No. 3,300,469 to Bernardi et al. U.S. Pat. No. 4,338,397, issued Jul. 1982 to Gilbert et al, discloses a method for synthesizing within a bacterial host, and secreting through the membrane of the host, a selected mature protein or polypeptide using these DNA techniques. The polypeptide can be any selected polypeptide such as proinsulin, serum albumin, and the like.
Peptides high in limiting essential amino acids and transformed plants expressing the same are the subject matter of U.S. Ser. No. 837,722, filed Mar. 7, 1986, now abandoned by Jaynes; and U.S. Ser. No. 837,211 filed Mar. 8, 1986, now abandoned by Jaynes, which are hereby incorporated herein by reference.