MXPA99003284A - Enhanced transgene expression in a population of monocot cells employing scaffold attachment regions - Google Patents

Abstract

A method of increasing transgene expression in a population of monocot plant cells is described which involves the use of a DNA construct comprising, inter alia, at least one chicken lysozyme gene locus scaffold attachment region.

Description

EXPRESSION TRANSGEN IMPROVED IN A POPULATION OF MONOCOTILEDONE CELLS THAT USE FIXING REGIONS IN ITS STRUCTURE FIELD OF THE INVENTION The present invention pertains to a method of increasing transgene expression and, in particular, to a method for increasing transgene expression in a population of monocotyledonous cells using DNA constructs having at least one attachment region in their structure.

BACKGROUND OF THE INVENTION The improvement of plant crops for a variety of characteristics, including resistance to disease and peste, and improvement of grain quality such as oil, starch or protein composition, can be achieved by introduction of new or modified genes. in the genome of the plant. However, characteristics that require relatively high expression of an introduced gene ("transgen") may not be achieved, or may be achieved in very low frequency in a large population of transformants. REF. 29773 Accordingly, it is necessary to prepare and analyze a large number of independent transformants in order to identify a plant with a level of expression that is adequate to produce the desired characteristic because there is no reproducible method to prepare a population of stable, independent transformed cells that they are characterized because they increase the average expression levels.

Fixation regions in the structure (SARs), also known in the medium as base fixation regions (MARSs), cause various effects on the expression of transgenes. SARs are fragments of DNA that comprise specific nucleotide sequences that are capable of binding to base nuclear preparations derived from eukaryotic cells. The SARs can be either constitutive or transient (Getzenberg et al. (1994) J. Cell. Biol. 55:22). Transient SARs are in spite of temporarily fixing a promoter region to the nuclear base. It is speculated that the fixation is influenced by type cells, stage of development, or gene expression conditions. The constitutive SARs are in spite of being in the bonds of DNA ring regions that are transcriptionally independent. In animals, the effects of SARs on expressions of an associated transgene have been shown to include one or more of the following: position, independence, dependence on the number of copies, increased expression level, and reduced expression variation (Stief et al. 1989) Nature 341: 343, Bonifer et al (1990) EMBO J 9: 2843, Klews et al (1991) Biochemistry 30: 1264, McKnight et al. (1992) PNAS 89: 6943).

The PCT application having International Publication Number WO 94/07902 and published on April 14, 1994 describes a method for increasing the expression and reducing the variation of foreign gene expression in plant cells using a DNA construct comprising , "inter alia", a region of fixation in its structure, positioned either in 5 'in a region of transcription initiation or in 31 in a structural gene.

Avra Ova et al., JCB Suplement D (21B), page 129 (1995) presented at the Keystone Assembly on April 4-10 (1995), discusses the use of a MAR yeast to regulate gene expression in maize cell lines. In the same way it was exposed that it is an Adhl MAR maize that worked and a Mhal MAR maize that did not work in regulation of gene expression in maize cell lines.

In plants, the reported effects of SARs on transgene expression has been completely variable. Breyne et al. ((1992) The Plant Cell 4: 463) reported that a tobacco SAR reduced the variation level of transgene expression among a population of transformant tobacco produced by transformation by Agrobacterium. In this study, the reduction in the variation was due to the collation of expression levels at the lower end of the observed range of expression level and complete elimination of upper range lines. Thus, the average expression level for the population of transformants was decreased. In addition, the presence of a SAR derived from the ß-globin human locus had no effect on the variation or range of the transgene expression.

A SAR derived from a soybean locus gene by heat of shock was shown to confer 5 to 9 fold increase in transgene expression in tobacco plants transformed by Agrobacterium (Schoffl et al. (1993) Transgenic Research (2:93) The level of transgene expression correlated with the number of copies transgen, however, the levels of transgene expression that crosses the population of transformants examined was highly variable.

A SAR yeast was able to confer 12 times higher average expression in a reporter gene introduced into tobacco cells by transformation mediated by particle bombardment (Alien et al. (1993) The Plant Cell 5: 603). Slight reduction in the variation in the number of independent lines that were observed. Preferably, in the observation of the dependence on the number of copies, the lines with the highest number of copies actually had lower levels of expression.

Alien et al. ((1996) The Plant Cell 8: 899), using a tobacco SAR, showed a 60-fold higher average transgene expression in tobacco cells transformed by particle bombardment, with variation in the number of independent lines.

A SAR located in the upper region of the phase or line of the grain promoter was shown to confer reduced variability and slightly increase the expression levels of the transgene reporter in the number of independent transformants of Agrobacterium-transformed tobacco plants (Van der Grrst et al. (1994) The Plant Journal 8 (3): 413).

A SAR derived from the locus gene gene of chicken lizozyme greatly reduced the variability of transgene expression in independent number, Agrobacterium-transformed tobacco plants (Mlyrova et al. (1994) The Plant Cell 6: 417). The average level of expression was increased approximately 4 times, but the maximum level of expression in any transformant alone was not greater than that of the transformations of plants with constructions that did not contain SARs.

Thus, it is clear from the above discussion that the inclusion of nucleic acid fragments encoding SARs in DNA constructs that were transformed into plant cells affect the expression of associated transgenes. However, the effect on expression is variable, and may be dependent on the nature of the host cell, the source of the SAR, and the means of introducing transgenes into the host cell. In addition none so far has demonstrated the effect of SAR of the locus chicken gene locus on the expression of transgenes in a population of monocotyledonous cells of plants.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, this invention concerns a method for increasing the level of expression of a transgene in a population of monocotyledonous cells comprising: (a) transform the population with a DNA construct comprising: (1) a transgene comprising, in the 5 'to 3' direction: (i) a promoter (ii) a coding sequence operably linked to the promoter; and (iii) a polyadenylation indicating sequence operably linked to the coding sequence; Y (2) at least one locus chicken gene locus that has a binding region which is characterized in that the fixation region in the structure is in position 5 ', 3', or 5 'and 3' of the transgene; and (b) incubate the transformed population under conditions suitable for cell development.

In another embodiment this invention concerns a population of monocotyledonous cells containing a DNA construct comprising: (1) a transgene comprising, in the 5 'to 3' direction: (i) a promoter; (ii) a coding sequence operably linked to the promoter; and (iii) a polyadenylation indicating sequence operably linked to the coding sequence; Y (2) at least one chicken lozozyme locus gene having a binding region which is characterized in that the fixation region in the structure is 5 ', 3', or 5 'and 3' of the transgene.

BRIEF DESCRIPTION OF THE FIGURES AND LIST OF SEQUENCES.

Figure 1 is a diagram of the plasmid pMH40. The chimeric gene fragments described in this figure are defined as follows: 35S / P-cabL represents a BamHI fragment up to Ncol, GUS represents a Ncol fragment up to Kpnl, and NOS 3 'represents a Kpnl fragment up to Salí. The vector sequences are derived from pGEM9Zf and contain the ampicillin resistance gene.

Figure 2 is a diagram of plasmid p40A53. A chimeric gene consisting of the 35S / P-cabL, the GUS coding region, and the 3 'NOS region, is linked to the SAR locus of the chicken lizsozyme ("element A"). The element 5'A is located between the sites BamHI and Ba / Bgl, and the element 3 'A is located between Salí and Spe / Xbal. "J" refers to the point of intersection between the BamHI and BglII sites in one case, and between the Xbal and Spl sites in the other case.

Figure 3 is a graphical representation of activities of the GUS enzyme in the BMS SAR (+) and SAR (-) lines and SAR (+) n = 8 lines.

SEQ ID Nos: 1 and 2 are the pair of oligonucleotides encoding the poly binding fragment that were used to modify the 3 'end of element A in order to facilitate the construction of p40A53.

SEQ ID Nos: 3 and 4 are the pair of oligonucleotides encoding the poly binding fragment that was used to modify the 5 'end of element A in order to facilitate the construction of p40A53.

BIOLOGICAL DEPOSIT The following plasmids have been deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Pakla n Drive, Rockville, MD 20852, and have the following access numbers: Plasmid Accession No.: Deposit Date p40A53 ATCC 97725 September 20, 1996 DETAILED DESCRIPTION OF THE INVENTION This invention provides a method for increasing transgene expression in a population of cells of monocotyledonous plants by using DNA constructs comprising, inter alia, at least one SAR of the chicken lysozyme locus gene. The term "population" as used herein refers to a group of monocotyledonous cells in culture or as part of a plant or seed thereof. Specifically, the inclusion of a SAR gene locus chicken lysozyme in DNA constructs used to transform cells of monocotyledonous plants that have a double effect: (1) the expression transgene as a measure on the entire population of individual transformants is increased, and (2) the highest levels of transgene expression by SAR containing individual transformants is increased on SARs containing non-individual transformants, eg, transformants with trangenes of identical SAR characters.

Monocotyledonous cells that can be used to practice the present invention include a group of examples of monocotyledonous plants which are corn, wheat and rice.

DNA constructs used to transform a population of cells of monocotyledonous plants comprise: (1) a transgene comprising in the 5 'to 3' directions: (a) a promoter; (b) a coding sequence operably linked to the promoter; and (c) a polyadenylation indicating sequence operably linked to the coding sequence; and (2) at least one SAR of loci chicken lysozyme gene in position 5 ', 3' or 5 'and 3' of the frangen.

The SAR gene locus of chicken lysozyme-construction transgene can be introduced into a monocotyledonous genome using techniques well known to those skilled in the art. These methods include, but are not limited to, (1) DNA directed by uptake, such as particle bombardment or electrophoresis, and (2) Agrobacterium-mediated transformation.

The improvement of transgene expression by such SARs can be practiced with any transgene that is regulated by a constitutive, tissue-specific or developmentally regulated promoter. The transgene can encode a protein product or can produce a functional RNA that can, in turn, mediate the control of gene expression by counter-sense, co-suppression or other gene expression technology.

"Gene" refers to a nucleic acid fragment that expresses a specific protein or specifies the production of a functional RNA, which includes the preceding regulatory sequences (5 'non-coding sequences), following (3 'non-coding sequences) and in the coding sequence. "Native gene" refers to a gene such as ses found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native, comprising regulatory and coding sequences that are not found together in nature.

Accordingly, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a different way from that found in nature. "Endogenous gene" refers to a gene. native in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is not normally found in the host organism, but that is introduced into the host organism by transformation. Foreign genes may include native genes inserted into a non-native organism, or chimeric genes. A "transgene" is any gene that is introduced into the genome of an organism through a transformation process.

"Coding sequence" refers to a DNA sequence that encodes a specific amino acid sequence or a functional RNA. "Regulatory sequences" refers to localized nucleotide sequences. upwards (5 'non-coding sequences), in, or down (3 'non-coding sequences) of a coding sequence, and with influence on the transcription, process, stability and subsequent translation of the transcribed RNA. Regulatory sequences include promoters, enhancers, introns, leader translational sequences and polyadenylation reporter sequences.

"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence. In general, a coding sequence is located 3 'of a promoter sequence. The promoter sequence consists of elements near or more distant from the center upwards, the late elements are often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and can be an innate element of the promoter or a heterologous element inserted to improve the level or tissue specificity of a promoter. The promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or equally comprise segments of synthetic DNA. It is over-understood by those skilled in the art that different promoters can direct the expression of a gene in different types of tissues or cells ("tissue-specific"), or in different stages of development ("regulated development"), or in response to different environmental conditions (see Okamuro, JK and Goldberg, RB in The Biochemistry of Plants, Academic Press: New York, 1989, Vol 2, pp 1-82, and Goldberg, RB and collaborators (1989) Cell 56: 149 and the references cited here). The promoters that cause a gene to be expressed mostly in cell types in which most of the time are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact linkages of regulatory sequences has not been fully defined, DNA fragments of different lengths may have identical promoter activity.

The "leader translation sequence" refers to a DNA sequence located between the start site of transcription of a gene and the coding sequence. The leader translational sequence is present in all mRNA processed upstream of the translation start sequence, and may affect one or more of the following: primary mRNA transcript processing, mRNA stability and translational efficiency. Turner, R. and Foster, G. D. '1995) Molecular Biotechnology 3: 225.

The "3 'non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation signal sequences and other indicator regulatory coding sequences capable of affecting the mRNA process or gene expression. Ingelbrecht, Y. L. W. et al. (1989) Plant Cell 1: 671.

The term "operably linked" refers to nucleic acid sequences of a single nucleic acid fragment that are associated such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (for example that the coding sequence is under the transcriptional control of a promoter).

The coding sequences can be operably linked to regulatory sequences oriented in the sense or in contradiction. "Orientation in the sense" refers to the arrangement of regulatory sequences and coding sequences characterized in that the transcription will result from the production of a transcribed RNA that can be translated to the polypeptide encoded by the coding sequence. "Counter-sense orientation" refers to the arrangement of regulatory and coding sequences characterized in that the transcription will be re-ligated from the production of a transcribed RNA that is totally or partially complementary to a target primary transcript or mRNA and that blocks the expression of a target gene.

The term "expression", as used herein, refers to transcription in sense (mRNA), or in contradiction to RNA derived from the nucleic acid fragment of the invention. The term "expression" may also include subsequent translation of mRNA into a polypeptide. "Inconsensus inhibition" refers to the production in contradict of transcribed RNA and the resulting deletion of the expression of identical or foreign essentially similar or endogenous genes (US Patent No. 5,107,065 the presentation of which is incorporated herein by reference) .

"Overexpression" refers to the production of a gene product of transgenic organisms that exceed production levels in normal or non-transformed organisms. "Coexpression" refers to the production of RNA transcripts in sense and the resulting deletion of the expression of identical or foreign essentially similar or endogenous genes (U.S. Patent No. 5,231,020 the presentation of which is incorporated herein by reference).

"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing transformed nucleic acid fragments are referred to as "transgenic cells, and organisms containing transgenic cells are referred to as" transgenic organisms. "Examples of plant and plant cell transformation methods include Agrobacterium-mediated transformation (De Blaere et al. collaborators (1987) Meth. Enzymol 143: 277) and particle bombardment technology (Klein et al. (1987) Nature (London) 327: 70-73; US Patent No. 4,945,050). Complete plants can be regenerated from transgenic cells by methods well known to the experts (see, for example, Fromm et al. (1990) Bio / Technology 8: 833).

By the method set forth herein, the expression "transgene" in a transformed monocotyledonous population can be enhanced by over-expression in monocotyledons that have been transformed with transgenes lacking at least one SAR of the chicken lysozyme locus gene. Accordingly, the method is useful for increasing the levels of expression of desirable polypeptides. In addition, more effective control of gene expression by counter-sense or co-suppression technologies can be achieved by allowing higher levels of expression of functional RNA transcripts (e.g. in contradiction or in sense).

EXAMPLES The present invention is further defined in the following examples. It will be understood that the examples are illustrative only and the present invention is not limited to the uses described in the examples. From the foregoing discussion and the following examples, a person skilled in the art can investigate, and without departing from the spirit and scope thereof, he can make various changes and modifications to the invention to adapt it to various uses and conditions. All the modifications can have the purpose of falling within the scope of the claims.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., frisch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (from here "Maniatis").

EXAMPLE 1 Construction of Transgen Expression SAR Vectors The plasmid pMH40 (Figure 1) comprises the following genetic elements: a CaMV 35S promoter, cab leader, the uidA coding region, and the NOS polyadenylation reporter sequence. the CaMV 35S promoter is a 1.3 kb DNA fragment that extends beyond 8 bp (for example 3 'of) the transcription start site. The leader cab is a 60 bp untranslated leader DNA fragment derived from the chlorophenyl (a / b "cab") 22L gene binding protein (Harpster et al. (1988) Mol, Gen. Genet, 212: 182). The leader cab was operably linked to the 3 'end of the 35S promoter fragment. The uidA coding region, which encodes the β-glucuronidase protein ("GUS"; Jefferson et al. (1987) EMBO J. 6: 3901) was operably linked to the 3 'end of the cab leader. Finally, an 800 bp DNA fragment containing the region of the polyadenylation report sequence of the nopaline synthesi gene ("NOS"); Depicker et al. (1982) J. Mol. Appl. Genet 1: 561) was operably linked to the uidA coding region. These DNA fragments, together comprising a 35S-GUS chimeric gene, were inserted by standard cloning techniques in the vector pGEM9Zf (Promega, Madison Wl) to produce the plasmid pMH40,. PMH40, which represents a SAR (-) construct, was used in control experiments in order to establish expression reference values in the absence of structure of the binding regions.

The SAR of the chicken lysozyme gene locus is contained in a 3 kb DNA fragment that is located between 8.7 kb and 11.7 kb upstream (for example 5 ') of the coding region of the chicken lysozyme gene (Phi). -Van, L. and Stratling, WH (1988) EMBO J. 7: 655). This SAR also called "element A", is present in the plasmid pUC-B-1-XI (Phi-Van, L. and Stratling, WH, supra) as a BamHI-Xbal fragment, and is flanked by the following sites of restriction: Kpnl and Smal on the 5 'side; and I left, PstI and Sphl on the 3 'side.

For insertion of an A element on the 5 'side of a 35S-GUS chimeric gene that is identical to another in pMH40 except for a 3-NOS fragment cut from 300 bps, the next double-stranded poly-linked oligonucleotide was inserted into pUC-B- lXl which has been previously digested with Xbal and Pstl: *. - -AGWAA7TCAGATCTCTSCA - 3 '(SECIDNO: l) ¡i!! S! L! L! Ll,. _ t tr.-_-.-: tcrAG? G - 5 '(SEC IDNOS).

This manipulation is a result of the deletion of the SalI site and introduction of EcoRI and BglII sites of restriction of enzymes between the Xbal and PstI sites present at the 3 'end of element A. Element A was cut as a Ba HI-BglII fragment. inserted in the Ba HI site located at the 5 'end of the 35S promoter of the 35S-GUS chimeric gene.

For insertion of an A element on the 3 'side of the 35S-GUS chimeric gene, the following double-stranded poly-linked oligonucleotide was inserted into pUC-B-1-XI which has been previously digested with Kpnl and BamHI: (SECIDN0: 3) ÜüllM "GCTT? .-. GCCTAG t '(SECID O: 4).

This manipulation is the result of the deletion of the Smal site and introduction of SalI and EcoRI sites of restriction of enzymes between the Kpnl and BamHI sites present in the 5 'end of element A. Element A was then cut as a Sall-Xbal fragment and inserted in Salí and Spel sites located in 3 'of the 35S-GUS chimeric gene.

The manipulations described above are a result of the insertion of the elements A both 5 'and 3' of the chimeric 35S-GUS gene (see figure 2).

EXAMPLE 2 Transformation of Monocotyledonous Cells with Transgene Expression SAR Vectors "Black Mexican Sweet" (BMS), monocotyledonous cell line, derived from corn, commonly used. BMS cells were maintained as suspension cultures in the following media ("MS +"): MS salts (GIBCO Laboratories, Grand Island NY), 0.5 mg./L. of thiamine, 150 mg./L. of L-aspargine, 20 g./L. of sucrose, and 2 mg./L of 2,4-dichlorophenoxyacetic acid. The pH of this medium was adjusted to 5.8 using 1 N KOH. The cells were subcultured 2 to 3 times per week by adding 25 mL of cells to 25 mL of fresh medium in a 250 mL flask. The flask was incubated with shaking (125 rpm) and cultured at 26 ° C in the dark.

Suspension cultures of BMS cells were transformed by the particle bombardment trigger method (Klein et al. (1987) Nature 327: 70). An instrument of Du Pont Biolistic ™ PDS 1000 / He was used for the transformations. Seven of 10 mi. of the BMS culture in suspension, obtained 2-4 days after subculturing, was also distributed on top of a whatman filter disc no. 1 installed in a buchner funnel under light vacuum. The filtrates were transferred onto plates of MS + solid medium (MS + containing 6 g./L of agar) and stored at 26 ° C overnight.

DNA plasmid was precipitated on golden particles as follows. The following components were added to 50μL of 60 mg./mL of a suspension of 1 mm of gold particles in the order listed: DNA plasmid (5 μg of pMH40 or 9 μg of p40A53, each mixed with 5 μg of pDETRIC, a plasmid containing the bar gene of Streptomyces hygroscopicus which confers resistance to the herbicide glufosinate (Thompson et al. (1987) EMBO J 6: 2519) (the bar gene had its codon translation changed from GTG to ATG for appropriate initiation of translation in plants (De Block et al. (1987) EMBO J 6: 2513), is driven by the 35S promoter of the "Cauliflower Mosaic Virus, and used the polyadenylation signal of the octopine synthesis gene of Agrobacterium tumefaciens, 50 μL of Ca2 CI 2.5 M and 20 μL of 0.1 M spermidine Equimolar amounts of SAR (-) and SAR (+) plasmids were used in the bombardment This particle preparation was shaken for 3 minutes, rotating in a microfuge for 10 seconds, and the supernatant removed The coated DNA-particles were then washed once with 400 μL of 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The coated DNA-particle suspension was sonicated 3 times for 1 second each. Seven and a half microliters of the DNA-coated particle suspension were then loaded onto each macro transporter disc.

The disk filter containing BMS cells was placed about 3.5 inches away from the retainer screen and bombarded 2 times. The membrane rupture pressure was set at 1000 psi and at least 28 inches of mercury was removed from the chamber. Two dishes were bombed "per" construction "per" experiment, The bombed dishes were incubated for 7 days at 26 ° C in the dark. After 7 days, the bombarded tissues were scraped from the filter, resuspended in MS + liquid and placed on avocado on MS + solid medium supplemented with 3 mg / L of Bialophos. Over a period of 3 to 7 weeks, rapidly growing tissue groups, representing the transformed lines, were transferred by spraying onto MS + fresh solid medium supplemented with 3 mg./L of Bialaphos. The transformed tissue derived from the bombardment of p40A53 was generally slow in appearance. The developed lines were chopped in the course of time until a population of 54 lines was obtained for each DNA construct. The lines were subcultured after 2-3 weeks on a solid MS + medium supplemented with 2 mg./L. of Bialaphos.

EXAMPLE 3 Transgene Expression Assay Fifty-four lines transformed with DNA constructs containing SARs (+) and 54 lines transformed with DNA constructs without SARs (-) were compared by reporter expression expression by histochemical staining. the following solution for histochemical staining was prepared: GüS Histochemical Test Solution 0.1 M NaP04 regulator, pH 7.0 50 mL K3 (Fe (CN) 6) 0.1 M 0.50 mL K4 (Fe (CN) 6) .3H20 0.1 M 0.50 mL Na2EDTA 0.5 M 0.50 mL Deionized H20 48.5 ml. * X-gluc 100 mg. * X-gluc = 5-bromo-4-chloro-3-indoyl-β-glucuronide A small amount of tissue from each line was placed in each of 96 deep plates containing 0.25 ml. of GUS test solution. Following incubation overnight at 37 ° C, each line was marked on a scale of 0 to 5 based on a visual measurement of the intensity of the blue spot, indicating the amount of GUS enzymatic activity. The results are presented in Table 1.

Table 1 Visual Activity Test GUS Lines examined 0 (Negative) Activity GÜS 4 or 5 (High) 1. 2 or 3 (low) ? AR (-) (n = 54) 35 12 7 SAR (+) (n = 54) 25 '5 24 These results indicate that the presence of SAR reduces the percentage of negative lines in a population, and increases the percentage of high activity lines.

Quantitative values for GUS activity in transformants were determined for high and low activity lines, as well as a portion of negative lines. They prepared sample extracts for each line as follows. 200-300 mg. of fresh BMS tissue was suspended in 500 μL of regulatory extraction (50 mM NaP04, 10 mM EDTA, 0.1% TritonX-100 and 0.1% Sarkosyl) and macerated with a mortar. A small amount of sand was included in the cell suspension in order to help in the breakdown of the cells.

Following a brief centrifugation, the supernatant was collected and stored at -70 ° C until assayed.

Extracts from the sample were preheated to 37 ° C prior to the test. 120 microliters of each sample extract was placed in an individual tank of a 96-well microtiter plate. 30 microliters of MUG regulating substrate (10 mM 4-methylumbelliferyl-β-D glucuronide (Sigma) in regulatory extraction) preheated (37 ° C), freshly prepared, was then added to each tank. Aliquots of 20 microliters were then removed at the times of 0, 10, 15, 30, 60 and 120 minutes after the addition of the MUG regulatory substrate and placed in individual tanks of a fluorometric microtiter plate (Titretek Fluoroplate, ICN Biomedical), each deposit containing 1809 μL of Na2C03 0.2 M in order to stop the reaction. Fluorescence was detected and quantified using Perkin-Elmer LS-3B spectrophotometer. The sampled activities were interpolated from a standard curve constructed by standard concentration graph of 4-MU (4-methylumbelliferone) (Sigma) against its fluorescence intensity measurement. This curve was used to convert fluorescence intensity of the sample extracts to uM 4-MU.

Protein assays were performed on the same samples samples using the "BCA Protein Assay Reagent" (Pierce Chemical, Rockford, IL) following the manufacturer's instructions for the Microtiter Plate Protocol. The enzymatic activities GUS were then calculated as pmoles 4-MU / mg protein. Punctual times of intake over the course of the trial were evaluated and the data converted to pmoles 4-MU / mg. of protein / min. For purposes of data presentation, the activities were then multiplied by 1000.

Fourteen of the lines that were rated between 0 or 1 in the visual test were examined in the quantitative assay; these lines had no detectable enzymatic activity. The visual ratings of 1 were based on microscopic detection of activity spots GUS in negative tissues generally. This amount of activity was not detected enough in the quantitative assay (MUG). The remainder of the lines with values of 0 or 1 were assigned enzymatic activity results of zero. But all the 2 SAR (-) lines with visual assessment of 2 and more showed levels of enzymatic activity that varied between 15.8 and 215.9 pmoles 4-MU / mg protein / ín. X 1000 as shown in Table 2. The 2 exceptions have non-detectable activities, and more similarly results from unequal sampling of chimeric tissue.

Table 2 Quantitative Essay of GUS Activity : "7.0 38 317.2 € * > - ~.". < -, 39 378.0 5? 16).: 40 403.2 2! 5.9 41 576.5 42 805.1 43 830.6 44 1123.2 45 1135.2 46 1363.1 47 1536.3 48 1707.6 49 2036.8 50 2590.5 51 2915.3 52 4022.4 5? 6603.6 54 8596.0 Based on these quantitative tests, the population of 54 SAR (-) lines had an average enzyme activity level of 20.0. The population of 54 SAR lines (+) had an average enzymatic activity of 718.8.

This result demonstrates a SAR-dependent increase in expression 36 fold between the two populations of transformants. When only lines with measurable enzymatic activity levels are compared, SAR lines (+) again show an increase in expression. The SAR (-) lines have an average enzyme activity level of 72.0 while the SAR (+) lines have an average enzyme activity level of 1386.2, an increase of 19 times. These results are graphically presented in Figure 3.

The data in Table 2 and Figure 3 also indicate that 19 of 54 (35%) of the SAR (+) lines have GUS activities that are higher than any of the SAR (-) lines. The maximum level of expression achieved by an individual line was increased by the presence of SAR. The enzymatic activity of the SAR (-) line with the highest expression was 215.9, while the activity of the highest SAR (+) line was 8596.0, an increase of 39.8 times.

The variation in expression between transformants of the SAR (+) line increased the population over the range of activities presented by the SAR (-) lines. The range of enzymatic activities between 35.0 and 8596.0, the major expressor being 245.6 times greater than the lowest expressor. The range of activities for the SAR (-) population was between 15.8 and 215.9, the highest being 13.7 times higher than the lowest.

LIST OF SEQUENCES (1. GENERAL INFORMATION (i) APPLICANT: (A) NAME: E.I. DUPONT DE NEMOURS AND COMPANY (B) STREET: 1007 MARKET STREET (C) CITY: ILMINGTON (D) STATE: DELAWARE (E) COUNTRY: U.S.A. (F) POSTAL CODE (ZIP): 19898 (G) TELEPHONE: 302 - 992 - 5481 (H) TELEFAX: 302 - 773 - "0164 (I) TELEX: 6717325 (ii) TITLE OF THE INVENTION: EXPRESSION TRANSGEN IMPROVED OCOTILEDONEAS (iii) SEQUENCE NUMBER: 4 (iv) READING COMPUTER FORM: (A) MIDDLE TYPE: 3.5 INCH DISKET (B) COMPUTER: PC COMPATIBLE WITH IBM (c) OPERATING SYSTEM: MICROSOFT WORD FOR WINDOWS 95 (D) SOFTWARE: MICROSOFT WORD VERSION 7.0 A (v) BASIC DATA OF THE APPLICATION: (A) APPLICATION NUMBER: (B) REGISTRATION DATE: (C) CLASSIFICATION: (vi) PRIORITY DATA OF THE APPLICATION (A) APPLICATION NUMBER: 60 / 028,165 (B) REGISTRATION DATE: OCTOBER 17, 1996 (vii) OFFICIAL / APPLICANT INFORMATION: (A) NAME: CHRISTENBURY, LYNNE M. (B) REGISTRATION NUMBER: 30,971 (C) REFERENCE / NO. OF CÉDULA: BB-1072 [2) INFORMATION OF SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) TYPE OF FILAMENT: ONE ONLY (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: DNA (GENOMIC) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 CTAGAGAATT CAGATCTCTG CA (2) INFORMATION OF SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) TYPE OF FILAMENT: ONE ONLY (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: DNA (GENOMIC) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 TCTTAAGTCT AGAG (2) INFORMATION OF SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) TYPE OF FILAMENT: ONE ONLY (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: DNA (GENOMIC) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: GTACCGTCGA CGAATTCG (2) INFORMATION OF SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) TYPE OF FILAMENT: ONE ONLY (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: DNA (GENOMIC) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4: GCAGCTGCTT AAGCCTAG INDICATIONS IN RELATION TO DEPOSIT OF MICROORGANISMS (PCT RULE 13 BIS) A. The indications made below in relation to the microorganism referred to in the description on page 4, line 32.

B. IDENTIFICATION OF THE DEPOSIT Institution name depositary: AMERICAN TYPE CULTURE COLLECTION Address of depository institution (including zip code and country: 12301 Parkla n Drive Rockville, Maryland 20852 USA Date of deposit: Access Number: September 20, 1996 (20.09.96) 97725 C. ADDITIONAL INDICATIONS: With respect to these designations in which a European patent is procured, a sample of the deposited microorganisms will be available until the publication of the mention of the grant of the European patent or until the date on which the application has been rejected or withdrawn or consider that it be withdrawn, only by the result of such a sample from an expert named by the person requesting the sample. (Rule 28 (4) EPC) D. DESIGNATED COUNTRIES FOR WHICH THE INDICATIONS ARE MADE.

E. ANNEX OF INDICATIONS IS PROVIDED. (x) This sheet was received with the international application. Authorized Official: (Illegible Signature) It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following,

Claims (9)

1. A method for increasing the level of expression of a transgene in a population of monocotyledonous cells which is characterized in that it comprises: (a) transform the population with a DNA construct that is characterized in that it comprises: (1) a transgene comprising, in the 5 'to 3' direction: (i) a promoter; (ii) a coding sequence operably linked to the promoter; and (iii) a polyadenylation indicating sequence operably linked to the coding sequence; Y (2) at least one locus gene of chicken lysozyme in the structure of the binding region which is characterized in that it is in position 5 ', 3', or 5 'and 3' of the transgene; Y (b) incubating the transformed population under conditions suitable for cell growth.

2. The method of claim 1 which is characterized in that the population of monocotyledonous cells are maize cells.

3. The method of claim 1 which is characterized in that it further comprises regenerating the whole plants of the transformed cells.

. The method of claim 1 which is characterized in that the promoter is a tissue-specific promoter.

5. A population of monocotyledonous cells that is characterized in that they contain a DNA construct comprising: (1) a transgene that is characterized in that it comprises, in the direction 5 'to 3': (i) a promoter; (ii) a coding sequence operably linked to the promoter; and (iii) a polyadenylation indicating sequence operably linked to the coding sequence; and (2) at least one chicken lysozyme locus gene in the structure of the binding region which is characterized in that the structure of the binding region is 5 ', 3', or 5 'and 3' of the transgene.

6. The population of the monocotyledonous cells of claim 5 is characterized in that the monocotyledonous cells are maize cells.

7. Regenerated plants of the population of claim 5 or 6.

8. Seeds obtained from the plants of claim 7.

9. The population of monocotyledonous cells of claim 5 which is characterized in that it is a tissue-specific promoter.