Abstract:
Taught is how to make a growth hormone transcription factor that is useful for the control of gene expression and growth rate in organisms.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    The invention described in the current application was also disclosed in provisional application 60/258,237, filed Dec. 25, 2000, from which priority is claimed. 
     
    
     
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
         [0002]    Research support which led to the making of the present invention was provided in part by funding from the National Science Foundation under Grant No. IBN-9600805. Accordingly, the federal government may possess certain statutory rights to the invention.  
         FIELD OF THE INVENTION  
         [0003]    The invention relates to a growth hormone transcription factor.  
         DESCRIPTION OF RELATED ART  
         [0004]    Cell-specific gene expression leads to the exclusive production of secreted hormones by selected cell types in the anterior pituitary. These hormones are produced in differentiated cells through selective processes of transcriptional and translational control. In addition, each cell type displays a distinctive pattern of differentiation during development, and specific neuroendocrine regulation.  
           [0005]    Transcriptional control of the growth hormone (GH) gene resides primarily in the promoter region that contains multiple transcription factor response elements. Pit-1, also known as GHF-1, is a prototypical POU-domain protein which was isolated by virtue of its ability to activate transcription of the GH gene by binding to the GH promoter at two sites. Pit-1 was also found to regulate transcription of the prolactin and TSH beta-subunit genes. Expression of all these genes is pituitary-specific, implying a central role of Pit-1 as a transcription factor controlling pituitary development and differentiation. The important role of Pit-1 in the regulation of anterior pituitary differentiation was confirmed by the identification of Pit-1 mutations as the causes of congenital hypopituitary dwarfism in the Snell dwarf mouse and in humans.  
           [0006]    Because Pit-1 promotes transcription of three distinct hormones, other transcription factors must establish the specificity of GH production in somatotrophs. One such protein was isolated by virtue of its binding to an 18 base pair (bp) Z-box response element (ZRE) conserved among mammals in the GH promoter. The protein that bound to the ZRE contained 15 consensus sequences for DNA-binding zinc fingers, and was named Zn-15. It was also shown that in synergy with Pit-1, Zn-15 activates GH transcription 100-fold above basal levels. The importance of Zn-15 in the physiological regulation of GH gene expression was shown when mutations in the ZRE in the GH promoter abrogated pituitary expression of a reporter gene in transgenic animals. See Lipkin et al., Genes Devel. 7, 1674.  
           [0007]    Although Zn-15 plays an important synergistic role in GH transcriptional control, only the C-terminal portion appears to be necessary for this synergism. Zinc fingers IX, X and XI have been shown to bind to the ZRE in the GH promoter, but DNA binding elements recognized by the remaining 12 zinc fingers of rat Zn-15 have not been found. Some of these fingers in the rat are separated by long linker regions of 20 or more amino acids, a structural feature evidently determinative of DNA binding or transcriptional activation. Zn-15 protein also can bind to a subset of thyroid response elements using fingers IX-XI, and may bind to RNA as well as DNA using the zinc fingers in the N-terminus of the protein. In order to effect cell type-specific regulation of GH, appropriate use must be made of the linkers and zinc fingers which do not bind the ZRE. See Tuggle and Trenkle, Domes. Anim. Endocrinol. 13, 1.  
         BRIEF SUMMARY OF THE INVENTION  
         [0008]    The invention relates to a growth hormone transcription factor that is useful for the control of gene expression and growth rate in organisms. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0009]    The present invention is useful, inter alia, for the control of cell-type specific regulation of GH in mammals, including, importantly, the mouse. Pituitary differentiation has been extensively studied in the mouse, where both transgenic and spontaneous GH-deficient mutants are available. To make a representative embodiment of the present invention, a mouse cDNA, hereinafter called mouse Zn-16 or mZn-16, was isolated. Expression of mouse Zn-16 mRNA was detected in 1 day old normal and GH-deficient Ames dwarf (Prop-1&lt;df−/−&gt;) mouse pituitary samples, as well as in the murine pre-somatotrophic GHFT1-5 cell line, see Lew et al., Genes Development 7, 683, consistent with a developmental role for Zn-16.  
         [0010]    To demonstrate an aspect of the invention, comparison between the GH-altered Ames dwarf (Prop-1&lt;df−/−&gt;), Snell dwarf (Pit-1&lt;dw J −/−), little (GHRHR, &lt;lit−/−&gt;) transgenic GHRH excess (MT, GHRF, Bri 11) and normal littermate mouse pituitaries showed predicted changes in GH mRNA, as well as other changes in hormone products that had not been previously evaluated or reported. Such quantitative analysis suggests that changes in the regulation of factors functioning in the GH homeostatic regulatory system are consistent with a role for Zn-16 in GH transcriptional control.  
         [0011]    Animals, Cell Culture and Isolation of RNA. Normal and Ames dwarf mice maintained in a breeding colony were sacrificed by carbon dioxide euthanasia. The day of birth (by 1400 hrs) was termed day 1. Adult rat pituitaries were obtained by the same procedure. Pituitaries were collected on dry ice, then stored at −70° C. until extracted. GHFT1-5 cells were obtained from Dr. Pamela Mellon (Univ. of California, San Diego), and cultured as described, see Lew et al., supra. Pituitaries and cells were first homogenized (Tissue Tearor, BioSpec, Bartlesville Okla.) in extraction buffer. RNA extraction was performed using the guanidinium-isothiocyanate:phenol:chloroform method as modified by the manufacturer (RNAzol B or Ultraspec, Biotecx, Houston, Tex.). Purity and concentration were checked by UV spectrophotometry (GeneQuant, Pharmacia, Piscataway N.J.).  
         [0012]    Reverse Transcription. The RNA pellet was briefly dried before resuspension in 0.1 μM oligo-dT for subsequent reverse transcription of poly-A RNA using the SuperScript II Preamplification System (Life Technologies, Gaithersburg, Md.) according to the manufacturer&#39;s instructions. The reaction was carried out in a 20 μl volume containing 200 units of MMLV reverse transcriptase at 45° C. for 60 minutes, then the enzyme was denatured by heating at 65° C. for 15 minutes. Hybridized RNA was removed by digestion with 2 units of  E. coli  RNase H. The volume was then brought to 50 μl with diethylpyrocarbonate-treated water and stored at −20° C. UV absorbance at 260 nm was measured on a GeneQuant spectrophotometer (Pharmacia, Piscataway, N.J.) in a 50 μl cuvette.  
         [0013]    Primer Design. The sequence of rat Zn-15 (accession number L23077) was obtained from GenBank. Prospective primer pairs were computer designed (Right Primer 1.2, BioDisk, San Francisco Calif. or Oligo 4.0, National Biosciences, Plymouth Minn.) using the following criteria: location with respect to zinc fingers, match of T m  between the primers, possible secondary structure within the primers, self-hybridization, and hybridization between primers. Primers were synthesized, quantified by UV spectrophotometry (Pharmacia GeneQuant), and stored desiccated at −70° C.  
         [0014]    PCR Amplification. Standard PCR conditions for cDNA amplification included each dNTP at a concentration of 0.2 mM and 1.25 U of Taq polymerase in a final volume of 50 μl. Amplifications were performed in a Model TC-1 thermal cycler (Perkin Elmer-Applied Biosystems, Foster City, Calif.). Amplification reactions were initiated with a hot-start using wax beads (Perkin Elmer) to separate the primers, MgCl 2 , and dNTPs (Lower master mix) from the DNA template and Taq polymerase (Upper master mix). Thermal cycling conditions consisted of initial denaturation for 60 s at 94° C.; followed by a 3-step profile (94° C. for 60 s; 54° C. for 45 s; and 72° C. for 90 s) for the desired number of cycles, and a terminal extension step of 72° C. for 5 min. For some reactions, a final, non-template dependent extension was carried out at 60° C. for 30 min.  
         [0015]    Sequencing Methods, Strategy and Analysis. PCR products from amplification reactions were cloned according to the manufacturer&#39;s instructions (TA Cloning Kit, InVitrogen, San Diego Calif.). Ligation reactions were incubated at 14° C. overnight, and then transformed into  E. coli  INValphaF cells (InVitrogen). Colonies with putative inserts were cultured overnight in 2×YT or TB containing 100 μg/ml ampicillin (Sigma). Bacteria were lysed and plasmid DNA was isolated with a commercial DNA binding matrix (PERFECTprep Plasmid DNA kit, 5 Prime-&gt;3 Prime, Boulder Colo.). Restriction digestion with EcoRI followed by agarose gel electrophoresis was used to confirm the presence of inserts prior to sequencing. DNA sequencing reactions were performed using 400 ng of plasmid template and fluorescent dye-labeled dideoxy terminators with AmpliTaq FS DNA polymerase according to the manufacturer&#39;s protocol (PRISM Ready Reaction DyeDeoxy Terminator kit, Applied Biosystems, Inc., Foster City Calif.). Thermal cycler conditions were 30 sec. at 96° C., 15 sec. at 50° C., and 4 min. at 60° C. for a total of 25 cycles. Unreacted fluorescent dye-labeled dideoxy terminators were removed from the sequencing reactions using size exclusion gel columns (CentriSep, Princeton Separations, Inc., Adelphia N.J.). Sequencing reactions were electrophoresed on an automated sequencer (Applied Biosystems Model 373A) using a 36 cm well-to-read 6% acrylamide gel (Sooner Scientific, Garvin Okla.) at 28 watts constant power for 10 hours. Data collected on a Macintosh computer were evaluated for ambiguities and “clear” sequence length using Factura 1.2.Or6 software (Applied Biosystems, Inc.). Sequences were aligned using GeneWorks 2.45 software (Oxford BioMolecular, San Diego Calif.) and remaining ambiguities in the electropherograms were resolved from overlapping information, manual inspection, or resequencing. Nucleotide and amino acid sequence data were analyzed, aligned and compared with reported sequences using GeneWorks and Statistical Analysis of Protein Sequences. See Brendel et al., Proc. Natl. Acad. Sci. USA 89, 2002.  
         [0016]    Ribonuclease Protection Assay. Total RNA extracted from pituitaries or cells was subjected to ribonuclease protection assay using non-radioactive probe synthesis and detection according to the manufacturer&#39;s protocol (RPA II/BrightStar systems, Ambion, Austin Tex.). A 651 nt probe from positions 840 to 1490 of mZn-15 was employed, and a 250 nt probe for mouse beta-actin was used as a control. Chemiluminescence after substrate treatment was visualized on x-ray film (Fuji RX) and then quantified using constant intensity illumination (FotoDyne, Madison Wis.), a CCD video camera (Hamamatsu C2400) or scanner (Nikon) and computer assisted image analysis (Gel-Pro Analyzer, Media Cybernetics).  
         [0017]    Mouse Zn-16 cDNA Isolation and Characterization. In order to obtain a probe for mouse (m) Zn-16, mouse pituitary RNA was reverse transcribed using an oligo-dT primer and then amplified using gene-specific oligonucleotide primers which annealed to zinc fingers 1× and XI of rat (r) Zn-15, the region that binds to the GH promoter. Cloned products near the expected size of 506 bp in the rat were found to have a similar DNA sequence, and were used as probes in hybridization screens of a mouse pituitary cDNA library previously constructed in the isolation of mLIM-3. See Seidah et al., DNA 13, 1163. However, no positive colonies were found through several rounds of hybridization. Therefore, the majority of the coding region of mZn-16 cDNA was isolated by amplification using primers specific for different zinc fingers of rZn-15, and the 5′ and 3′ ends were isolated using rapid amplification of cDNA ends.  
         [0018]    Six portions of mZn-16 of at least 1500 bp were independently amplified, cloned and sequenced. Sequences were obtained in both directions from multiple clones of each fragment, and then aligned to assemble the entire cDNA sequence of 6879 nucleotides. The full-length mZn-16 cDNA sequence was assembled from overlapping regions of at least 1500 bp.  
         [0019]    The open reading frame in the mZn-16 cDNA as shown in SEQ ID:NO 1 encodes a polypeptide as shown in SEQ ID:NO 2. The mZn-16 amino acid sequence of 2292 amino acids has several additional aa that are not present in rZn-15, particularly in the N-terminus. There are four in-frame methionine residues upstream from the initial methionine in the rat, for additional translational start sites not reported in the rat. A feature of the protein that has been identified based on computer analysis are several regions (aa 830-845, 1550-1567, 1999-2016) encoding consensus eukaryotic nuclear localization signals, see Robbins et al., Cell 64, 615.  
         [0020]    Importantly, multiple zinc fingers of the Cys2His2-type, see Berg and Shi, Science 271, 1081, similar to those found in rZn-15, are present in mZn-16. There is 97% amino acid identity between the zinc fingers of rZn-15 and mZn-16. However, the four differences in mZn-16 in finger V change three proline residues. This region of mZn-16 is now predicted to contain two consensus zinc fingers, designated as Va and Vb in FIG. 2B, and finger Va now agrees at all positions with the zinc finger consensus sequence. Changed residues in fingers II, IX, X and XIII are conservative substitutions, and substitutions in regions outside the fingers would not be predicted to have any impact on zinc coordination or DNA binding, see Berg and Shi, Science 271, 1081. As found in rZn-15, there are extended linker regions between the zinc fingers in mZn-16, particularly in the C-terminal half of the protein. The largest region of consecutive difference between mouse Zn-16 and rat Zn-15 is found in the linker between fingers VII and VIII (aa 845 to 860 in mZn-16), where only two of the 14 consecutive residues are similar.  
         [0021]    Mouse Zn-16 mRNA expression in Normal and Ames dwarf pituitaries. Zn-16 expression in normal and GH-deficient Ames dwarf (Prop-1&lt;df−/−&gt;) mouse pituitaries was studied at the day of birth (postnatal day one). Total RNA was isolated, reverse transcribed using oligo-dT priming, then amplified using primers in fingers IX-XI for 30 cycles. The amount of 503 bp product of these amplifications was then determined using image analysis of ethidium bromide-stained gels. Samples in which no pituitary cDNA was added as control showed no amplification. The average relative image intensity of the bands was determined for normal and Ames dwarf mice (n=3). At one day of age, there was no significant difference in the expression of Zn-16 between normal and Ames dwarf pituitary.  
         [0022]    Mouse Zn-16 mRNA Expression in GHFT1-5 pre-somatotroph cells. The expression of Zn-16 mRNA in Ames dwarf pituitaries suggested that Zn-16 might be expressed in the mouse cell line GHFT1-5, which is derived by immortalization of pituitary cells with a Pit-1 promoter-driven large T antigen construct. These cells have been characterized as pre-somatotrophs, but they do not express Pit-1 protein or GH mRNA. Probes were produced to measure the expression of mZn-15 mRNA in total RNA using ribonuclease protection assays (RPA). Total RNA was isolated either from ca. 30 pooled mouse pituitaries or 107 GHFT1-5 cells, and used for RPA for either Zn-16 or actin mRNA in each sample. Actin mRNA was assayed to serve as a control for each sample in a 5 μg RNA aliquot. The size of the protected fragment for Zn-16 was the same in both pituitary and GHFT1-5 samples, and was of the size predicted from the length of homologous probe sequences (651 nt). In comparing the pituitary and GHFT1-5 samples, the detectable amounts of mZn-16 were different, suggesting that Zn-16 expression levels varied between normal pituitary and GHFT1-5 samples.  
         [0023]    The further description found immediately below shows Zn-16 function in the transcriptional regulatory control of pituitary GH expression.  
         [0024]    Animal care and use. Male mice 3-4 months old were used in this study. Mice were euthanized with carbon dioxide anesthesia, then individual pituitaries were removed using washed instruments and stored in single tubes at −70° C.  
         [0025]    RNA preparation. Total RNA was extracted from individual mouse pituitaries using a modified phenol/chloroform/guanidinium protocol (Ultraspec; Biotecx, Houston, Tex.). The RNA pellet was briefly dried before resuspension in 0.1 μM oligo-dT for subsequent reverse transcription of poly-A RNA using the SuperScript II Preamplification System (Life Technologies, Gaithersburg, Md.) according to the manufacturer&#39;s instructions. The volume was brought to 50 μl with diethylpyrocarbonate-treated water for storage at −20° C. UV absorbance at 260 nm was measured on a GeneQuant spectrophotometer (Pharmacia, Piscataway, N.J.) in a 50 μl cuvette.  
         [0026]    PCR. Primers were selected for specificity for mouse mRNAs, high annealing temperature, and absence of secondary structure using the computer programs Right Primer (BioDisk, San Francisco, Calif.) and Oligo 5.0 (National Biosciences, Plymouth, Minn.). Standard cDNA amplification reactions included each dNTP at a concentration of 0.2 mM and 1.25 U of Taq polymerase in a final volume of 50 μl. The fluorescent dye-labeled dUTPs ([F]dUTP) used for labeling were [R110], [R6G], or [TAMRA] (ABI; Foster City, Calif.). [F]dUTPs were diluted for a constant addition volume of 0.1 μl per reaction. Amplifications were performed in a Model TC-1 thermal cycler (ABI). Amplification reactions were initiated with a hot-start using AmpliWax PCR Gem 50 beads (ABI). Thermal cycling conditions consisted of initial denaturation for 60 s at 94° C.; followed by a 3-step profile (94° C. for 60 s; 54° C. for 45 s; and 72° C. for 90 s) for the desired number of cycles, and a terminal extension step of 72° C. for 5 min. For some reactions, a final, non-template dependent extension was carried out at 60° C. for 30 min. After amplification, unincorporated primers and dNTPs were removed by centrifugation through Centricon-50 or Microcon-30 filters (Amicon, Beverly, Mass.) filters. Products were analyzed on 12 cm well-to-read (WTR) gels composed of 10% Long Ranger (FMC BioProducts, Rockland, Me.) with a 373A instrument using GeneScan 1.2 software. Electrophoresis was performed with power limiting at 12W in 0.5×TBE buffer. Samples were mixed with a ROX-dye labeled size marker (ROX-500, ABI) and a sucrose- or Ficoll-bromophenol blue dye solution before loading on the gel. Results were statistically analyzed using the computer programs Excel (Microsoft, Redmond, Wash.) and SuperANOVA (Abacus Concepts, Berkeley, Calif.).  
         [0027]    Comparison of transcript abundance. In order to compare product intensities among a large number of amplification reactions, the fluorescence detection and sizing capacity of an automated sequencing instrument was used. Amplifications were performed in the presence of fluorescence-labeled dUTP, which was detectable during electrophoresis. Fluorescence labeled products were examined for molecular weight compared to the standards present in each lane as an internal control, and for peak intensity from the processed electropherograms. From peak heights and the calculated efficiency, the transcript abundance was determined as related by the equation in Gilliland et al., Proc. Natl. Acad. Sci. USA 87, 2725. For normal littermates and GH-affected mice, pituitaries were amplified for GH, Zn-16 and Pit-1 abundance. The results, expressed as percentage of normal littermate expression, were:  
                                                                                 mouse type   GH   Zn-16   Pit-1                                        Ames dwarf   0   4.5   0           Snell dwarf   0   5.9   0.2           Little   1.3   5.9   2.9           GHRH giant   242.4   198.2   199.8                      
 
         [0028]    Simple regression correlation tests showed that there was a significant correlation (p&lt;0.001) for Pit-1 vs. GH (F 1.17 , 26.82) and Zn-16 vs. GH (F 1.17 =12.48). This correlation from an in vivo model implicates Zn-16 function in the transcriptional regulatory control of pituitary GH expression.  
         [0029]    Further embodiments of aspects of the invention are described below.  
         [0030]    In an embodiment of an aspect of the invention, a Zn-16-expressing construct with inducible control by exogenous factors (e.g., tetracycline) is stably transfected into mammalian cells (e.g., GC cells). The transfected cells are placed within a permeable membrane for immunological protection (“hollow fiber”). The hollow fiber containing the transfected cells is implanted into the kidney capsule of a patient who has undergone surgery. The cells within the hollow fiber express GH regulated by the Zn-16 which is induced by the physician (e.g., with tetracycline) as needed to promote healing. In a preferred embodiment, localized administration of GH expression is provided by implantation of the hollow fiber unit during the surgery, where compatible with needed removal processes. Such an embodiment of the invention is useful for enhancing GH production, desired in particular during the healing process, promoting tissue regeneration and lessening the need for invasive repetitive injections.  
         [0031]    In another embodiment of an aspect of the invention, a Zn-16-expressing construct with inducible control by exogenous factors (e.g., tetracycline) is stably transfected into mammalian IQ cells (e.g., GC cells). The transfected cells are placed within a permeable membrane for immunological protection (“hollow fiber”). The hollow fiber containing the transfected cells is implanted into the kidney capsule of a patient who has experienced severe burns. The cells within the hollow fiber express GH regulated by the Zn-16 which is de-induced by the physician (e.g., with tetracycline) after promoting healing. Such an embodiment of the invention is useful for enhancing GH production, desired in particular during the healing process, promoting tissue regeneration and lessening the need for invasive repetitive injections.  
         [0032]    In yet another embodiment of an aspect of the invention, a Zn-16-expressing construct with inducible control by exogenous factors (e.g., tetracycline) is stably transfected into mammalian cells (e.g., GC cells). The transfected cells are placed within a permeable membrane for immunological protection (“hollow fiber”). The hollow fiber containing the transfected cells is implanted into the kidney capsule of a patient who has experienced muscle wasting, as is observed, for example, in later-stage HIV-infected patients. The cells within the hollow fiber express GH regulated by the Zn-16 which is subject to control by the physician (e.g., with tetracycline) for the optimal control of healing and the lessening of the pace of muscle wasting. Such an embodiment of the invention is useful for enhancing GH production, desired in particular during the healing process, promoting tissue regeneration and lessening the need for invasive repetitive injections.  
         [0033]    In certain embodiments of the invention, Zn-16 is administered to a patient. As used in this application, “administration” includes the implantation of a construct or a host cell containing a Zn-16 nucleic acid sequence or, more generally, encoding the Zn-16 polypeptide, for instance as described above. That is, administration of Zn-16 is effected also by the administration to a patient, or the implantation into a patient, of a construct encoding the Zn-16 polypeptide.  
         [0034]    In an embodiment of an aspect of the invention, Zn-16 is used to alter gene expression in a patient who has a pituitary tumor. In a preferred embodiment, Zn-16 and somatostatin are co-administered to the patient. Upon administration, the somatostatin binds its receptor, is internalized, and the Zn-16 alters gene expression in such a way as to ameliorate the effects of the tumor.  
         [0035]    In another embodiment of an aspect of the invention, Zn-16 is used to control expression of proteins whose expression is otherwise driven by other zinc finger proteins. In a particular embodiment, Zn-16 is administered to a patient suffering from a cancer that is characterized by the overexpression of proteins whose expression is driven by zinc finger proteins other than Zn-16. The administered Zn-16 blocks the overexpression driven by other zinc finger proteins and slows tumor growth.  
         [0036]    In another embodiment, Zn-16 is affixed to a surface. The presence of heavy metals is detected by their binding to the affixed Zn-16. Methods of the detection of subtle changes in binding, such as surface plasmon resonance or methods capable of detection of small changes in potential difference across a very limited space, enable the detection of very low concentrations of heavy metals when the metals bind Zn-16. In a particular embodiment, Zn-16 is preloaded with one heavy metal, and displacement of that heavy metal is measured.  
         [0037]    In another embodiment, Zn-16 is used as a heavy-metal responsive factor that indicates contamination levels. In such an embodiment, Zn-16-driven expression of a reporter gene changes when there is a change in the heavy metal concentration. Changes in the concentration of not only zinc but also other heavy metals such as mercury and lead are detected.  
         [0038]    In yet another embodiment, the heavy-metal-binding property of Zn-16 is exploited to ameliorate the adverse effect on a patient of heavy metal exposure. By virtue of its heavy-metal-binding property, Zn-16 serves as a sink that binds excess heavy metal that is toxic to key tissues in the patient, such as liver and kidney.  
         [0039]    In another embodiment, Zn-16 is pre-administered to a human to prevent the adverse effect on the human of anticipated heavy metal exposure. A human anticipating participation in cleanup of heavy metal environmental contamination, for example from a spill of stored liquids or from radioactive fallout, self-pre-administers Zn-16, which binds environmental heavy metals to lessen harm to the human. The Zn-16 is a reservoir for the metals which otherwise pose a danger to the human.  
         [0040]    In another embodiment of an aspect of the invention, Zn-16 is administered to a human patient who has diabetes or, more generally, experiences dysregulation of expression or availability of insulin or insulin-like growth factor. The administered Zn-16 regulates the expression of GH to alter levels of insulin and insulin-like growth factor and ameliorates the patient&#39;s condition.  
         [0041]    In an embodiment of an aspect of the invention, Zn-16 is used in a method of controlling the expression of gene products at a multiplicity of loci in the genome of an organism, said method comprising: the site-directed or random mutagenesis of Zn-16 to encode an altered polypeptide, said altered polypeptide possessing an affinity for a transcriptional regulatory factor different from the affinity of the polypeptide of Zn-16 for said transcriptional regulatory factor; the formation of an assemblage of said transcriptional regulatory factor with said altered polypeptide; and the direction of said expression at said loci by said assemblage. In a particular embodiment, the altered polypeptide binds a multiplicity of transcriptional regulatory factors to form the assemblage. In a particularly preferred embodiment, the method controls the expression of gene products throughout the entire genome of the organism, allows specific targeting of gene promoters, and creates a controllable platform to assemble other transcriptional regulatory factors.  
         [0042]    In another embodiment, Zn-16 is modified to have temperature-responsive zinc binding. This modified Zn-16 is useful as a temperature-dependent regulator of gene expression.  
         [0043]    In another embodiment, a cell or tissue sample is taken from a patient. Nucleic acids from the patient are extracted from the sample. Hybridization of the patient&#39;s nucleic acids to the nucleic acid encoding Zn-16 is measured. Detection of a difference between the hybridization of the patient&#39;s sample and the hybridization of a control sample is useful in the diagnosis of dwarfism, gigantism, hypothyroidism, and other disorders of metabolism.  
         [0044]    The invention is not limited to the exact details of operation, or to the exact compositions, methods, procedures, or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art. As the invention would therefore be limited only by the full scope which could reasonably, legally and equitably be accorded any of the appended claims, the foregoing examples are provided merely by way of illustration of the breadth of the present invention, which exceeds any and all of these examples.  
     
       
       
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             6879  
             DNA  
             Mus musculus  
           
            1 

atgttataca accagccaga ccagaaatat gatgaagaga atcttccaat accaaattct     60 

ctacgttgtg agctcttact tgttttgaaa actcagtggc cctttgatcc agaattttgg    120 

gattggaaaa ctttgaagcg ccagtgtctt gctctcatgg gagaagaggc gtctattgtg    180 

tcctcaattg atgaactgaa tgacagtgaa gtctatgaga aagtagacta ccagggtgaa    240 

aggggagaca catctgtgaa tggcctttct gctgctggac ttggtactga ttctggcctg    300 

ctgatggata ctggtgatga aaagcagaag aagaaagaga taaaagaatt aaaagatagg    360 

gggtttatat ctgctaggtt taggaattgg caagcctaca tgcagtattg tttgctctgt    420 

gacaaagaat tccttggaca cagaatagta cggcatgctc aaaaacatta caaagatggg    480 

atttacagct gtcccatatg tgcaaagaat tttaattcta aagactcgtt tgtccctcat    540 

gttaccctgc atgttaaaca gtctagtaaa gagagactag cagctatgaa gccattaaga    600 

agattgggaa ggcctcctaa aatcacagcc acccatgaaa atcaaaagac taatattaat    660 

actgtggcta aacaggaaca gcgacccata aaaaagaata gtctttattc aacagatttc    720 

atagtgttta atgacaacga tggttcagat gatgaaaatg acgacaagga caagtcttat    780 

gagcccgagg tgatccctgt ccagaaacca gtacctgtta atgagtttaa ttgtcctgtg    840 

accttttgta aaaagggctt taagtacttc aaaaatttaa ttgctcatgt gaaaggccat    900 

aaggatagtg aagatgccaa acgctttctt gaaatgcaaa gcaagaaagt catttgccag    960 

tactgtagaa ggcactttgt aagcgtcact catctcaatg atcacttaca aatgcactgt   1020 

ggcagtaagc catatatatg tatacagatg aaatgtaagg ctggttttaa tagttacgca   1080 

gagctgttag cccaccgaaa ggagcatcaa gtctttagag caaagtgctt atttccaaaa   1140 

tgtggcagaa ttttttccca agcttattta ctgtatgatc atgaggccca acattataat   1200 

acgtacacgt gcaagttcac aggttgtggt aaggtgtacc gttctcagag cgagatggag   1260 

aagcaccagg atggccacag tcatcctgaa acagggctgc ctcctgaaga ccagcttcag   1320 

ccatctggaa atgatgtgaa tccggactca ggagcgacgg ctgcaggagg aaggtccgag   1380 

aacagcattg acaagaacct gggttcaaac agaagtgcag attgggagaa aaacagagca   1440 

gagccagctg tgactaaaca cggccagatc tctgccgctg aactcaggca agctaacata   1500 

ccattgtcaa atggtctgga aacccgtgat aatactactg ttcttcggac caatgaagta   1560 

gctgtgtcca tcaaggtgtc tgtcaaccat ggggtagagg gtgactttgg aaagcaagaa   1620 

aacctaacca tggaaggcac tggtgagccg ctgatcacag atgtgcataa accaggtata   1680 

ggtgctgggg tccagttatg tcatccaggt ttccaagaaa agaaaggtca cgagtgcctg   1740 

aacgaagccc agaattcttt atcaaactca gaatcactga agatggatga ccttaaccca   1800 

caaagcttag aaagacaggt gaacactctg atgacctttt ctgtacaaaa tgaggcagga   1860 

cttgaagaca attcacaaat ttgcaagttt gaatgtggag gtgatgttaa aacctcatcc   1920 

agcctttatg atttacctct taagacacta gaaagtatca catttgttca gtcacagccc   1980 

gacctaagca gtccgttggg atctccatca gtacctccaa aagctccagg tcagaagttc   2040 

agctgccagg ttgagggatg cactcgaaca tataactctt cacagagtat tggaaaacac   2100 

atgaagacag cacaccctga ccaatatgct gcttttaaac tgcagcgcaa gacgaaaaaa   2160 

ggtcagaaat ctaacaactt aaatacacca aatcatggaa agtgtgttta ttttttgcca   2220 

tcacaagtga gcagctctaa tcatgctttt tttacaccac agaccaaagc caatgggaac   2280 

cctgcctgtt cagcccaggt gcagcatgtc tcgccttcca ttttcccagc tcatttagca   2340 

agtgtatcag ctccattgtt accctcagtg gaaagtgtcc taagtccaaa tataccttct   2400 

caggataaac atggacaaga tggcatatta tgttcacaaa tggaaaattt gtcttatgct   2460 

cccttgccag cacaaatgga agatctaacc aagacagttt tgcctttgaa tattgacagc   2520 

ggctcagatc cgtttcttcc tttacccaca gaaaatagct ctctcttctc ttcaccagca   2580 

gacagtgaga ataattctgt tttttcccaa ctggaaaata gtacaaatca ttatccctcc   2640 

cagacggatg gaaacataaa ttcctctttt ctgaaaggag gcagcagtga aaatggagtt   2700 

tttccttccc aagtaagttc tgcagatgac ttcagtagca ccagtgccca accgtctaca   2760 

cctaagaaag tgaaaaaaga ccgtggtcga ggcccaaatg ggaaggaaag aaaacccaag   2820 

cacaacaaaa gggctaaatg gcctgcgatt atcagggatg ggaaattcat ctgtagcagg   2880 

tgttacaggg ctttcaccaa ccccaggtcc ctgggtggac acctgtctaa aaggtcttac   2940 

tgcaaaccac tggatggagc agaaatagca caggaacttc tacagaccaa cagacagcct   3000 

tccctcctag ctagcatgat tctctccaca agtgcagtaa atatgcaaca gccgcaacag   3060 

tctaacttta atccagaaac atgctttaaa gacccatcat tcctgcaact tcttaatgtg   3120 

gaaaatcgtc caaccttttt accaagtaca tttccaagat gtgacgtgag taactttaat   3180 

gccagtgtta gtcaggaagg cagtgaaatt attaagcagg ctttagaaac tgctggcatt   3240 

cccagcacgt ttgagagtgc cgaaatgctt tctcaggttg ttccaatagg cagtgtctcc   3300 

gatgcagcac aagtcagtgc agcggggatg ccagggccac ctgtgacacc cttgttacag   3360 

actgtttgcc acccaaacac ctcaccatca aaccagaatc aaacgccaaa ttccaaaacc   3420 

ctcaaagaat gtaacagttt gcctctcttt acaacaaatg atttactgct aaagactatt   3480 

gaaaatggct tgtgctccaa ttcattcagt agttctactg aaccaccaca aaattttacc   3540 

aataatagtg cacatgtttc tgttataagt gggcctcaga atacaagatc cagtcatttg   3600 

aataaaaaag gaaatagtgc atctaagaag agaaaaaaag ttgctcctgc agtaagtgta   3660 

tctaatactt cccaaaatgt gctaccaact gatttaccag tgggccttcc atcgaagaat   3720 

cttacagtcc ctgataccaa cacacggtca gacatgaccc cagattgtga acctcgggct   3780 

ttggtggaaa atctcacaca gaaattaaat aacattgaca atcatttgtt tataactgat   3840 

gtaaaagaga actgtaaagc cagtcttgag ccccatacaa tgttaacccc tttaacatta   3900 

aaaacggaaa acggcgattc ccgaatgatg cctttgagtt catgcacacc agtgaattct   3960 

gatttgcaga tttctgaaga taatgttatt cagaactttg agaagactct tgaaattatt   4020 

aaaactgcta tgaattctca aatacttgag gtaaaaagtg gatctcaggg tactggtgag   4080 

acaacacaga atgctcagat aaattacagc atgcaacttc cctcagtaaa ctctatccca   4140 

gatagcaagc tgcctgatgc ttctcagtgc tcctctttcc taactgtaat gccaacaaag   4200 

tctgaagcat tacataagga ggatcaaata caggacattt tagagggttt gcaaaactta   4260 

aaactagaaa atgacacttc tgctccagct tcccagagta tgctaatgaa caaatcagta   4320 

gcactgtccc ctactcctac taaatcaact ccaaatattg tagtccagcc agtacccgaa   4380 

gtgatacatg ttcagcttaa tgacagagtt aataagccct ttgtgtgtca aaaccaaggc   4440 

tgtaactaca gtgctatgac aaaggatgcc ctgtttaaac actatggtaa aatccatcag   4500 

tatactccag agatgattct tgaaattaag aagaatcaat taaaatttgc tccatttaaa   4560 

tgtgtagtac cttcatgtac caaaacattt acaagaaatt ctaatctccg ggcacactgt   4620 

cagttggtgc atcattttac aatagaagaa atggtaaagc taaaaataaa aaggccctat   4680 

ggaagaaaat ctcagagtga aaatttgtca tctccacaga ataatcaagt gaagaaacag   4740 

ccatccatgg ccgaggaaac aaaaactgag tcacaaccag ccttcaaggt accagcagca   4800 

acaggtgatg ctgcacttgc taatgcaaca gtaatcccag aaaaacaact tgcagaaaaa   4860 

aaaagtcctg agaaaccaga aagttcttca cagcctgtca catcttctgc tgaacaatat   4920 

aatgcaaatc ttgcaaacct aaaaaccaaa ggaaggaaaa ataagaggca tagaaaagaa   4980 

aaggaagaaa aacgggaaaa gaatccagtt tcccaggcct ttgaacttcc aacaaaatac   5040 

agttcgtaca gaccttactg ctgtgtccac cagggatgct ttgctgcttt tacaatacag   5100 

caaaacttga ttcttcatta ccaggctgta cataaatcaa atcttcctac attttctgca   5160 

gaggttcaag aggaaagtga agctgttaaa gaaagtgaag aaactgaacc gaaacaatca   5220 

atgaaagaat ttaggtgtca ggtgagtgac tgttctagga ttttccaagc aattactggc   5280 

ctaatacagc actacatgaa acttcatgaa atgacccccg aggaaattga aagcatgact   5340 

gctgctgtgg atgttggcaa atttccatgt gatcagttgg agtgtaagtt gtcttttaca   5400 

acatacctga gctatgttgt tcatcttgag gtagaccatg gaattggaac aaggacaagt   5460 

aaggcagaag aagatggcat atacaagtgt gactgtgagg gctgtgacag gatatatgcc   5520 

actcggtcta atcttctccg acacatcttt aataaacata atgacaagca taaagcccat   5580 

ctgattcggc caagaaaatt aactggccag gaaaatatat caagtaaggc aaaccaagaa   5640 

aaatcaaagt ctaaacatcg gacaacaaaa cccaacagat ccgggaaaga cggaatgaaa   5700 

atgccaaaga caaagcgaaa gaaaaaaagt aatttagaaa acaagagcgc aaaagtagtg   5760 

cagattgagg aaaataagcc ttattctcta aagcgtggga agcacgtgta ttccataaag   5820 

gctaggaatg atgccttggc agagtgtaca agcaaatttg tgacacagta tccatgtatg   5880 

ataaaagggt gtacttcagt cgttacaagt gaaagcaata tcatcagaca ttataagtgt   5940 

cataagttgt ccagggcatt tacatcacaa caccgcaaca ttcttattgt ctttaagcga   6000 

tatggcaacc cacaaggaag ggaaatctct gagcaagaag atgaaaagaa tgataagaaa   6060 

ggtcctgatt catctgtttt agagaaaaat gataactcgg aaccagctgc tgctccacag   6120 

gaagaaggta gaaaaggtga aaaggatgag atggatgagt taacagaatt atttattaca   6180 

aagttaataa atgaagacag cacaaatgca gaaaaccaag gcaataccac tttaaaggga   6240 

aataacgaat ttcaggagca tgattcctgc acatcagaaa gacaaaagcc tggtaatttg   6300 

aagagagttt ataaagaaaa aaacactgtg cagagtaaga aacggaagat tgataaaact   6360 

gagccagaag tatccttggt ggtaaataat acacggaaag aggaagagcc tgccgtagca   6420 

gttcagacca ctgaggagca tcctgcatcc tttgactgga gctccttcaa gcctatggga   6480 

tttgaagcat cctttctgaa gtttcttgaa gagtctgcag tgaagcagaa gaaaaatagt   6540 

gacagagacc attcaaacag tggaagtaaa agaggatccc attccagctc cagaagacat   6600 

gttgataagg ctgctgtggc tggtagcagt catgtgtgtt cctgtaaaga cagtgaaatc   6660 

tttgtacagt ttgccaaccc ctcaaagctt cagtgcagtg agaatgtaaa aattgtttta   6720 

gacaagactc ttaaagatcg ctctgagctt gtcctaaaac agcttcagga aatgaaacct   6780 

actgtcagtc taaaaaaact tgaagtacta tccaatagtc cagataggac tgttttaaaa   6840 

gaaatcagta taggtaaagc cacgggcaga gggcagtac                          6879 

 
           
             2  
             2293  
             PRT  
             Mus musculus  
           
            2 

Met Leu Tyr Asn Gln Pro Asp Gln Lys Tyr Asp Glu Glu Asn Leu Pro 
1               5                   10                  15 

Ile Pro Asn Ser Leu Arg Cys Glu Leu Leu Leu Val Leu Lys Thr Gln 
            20                  25                  30 

Trp Pro Phe Asp Pro Glu Phe Trp Asp Trp Lys Thr Leu Lys Arg Gln 
        35                  40                  45 

Cys Leu Ala Leu Met Gly Glu Glu Ala Ser Ile Val Ser Ser Ile Asp 
    50                  55                  60 

Glu Leu Asn Asp Ser Glu Val Tyr Glu Lys Val Asp Tyr Gln Gly Glu 
65                  70                  75                  80 

Arg Gly Asp Thr Ser Val Asn Gly Leu Ser Ala Ala Gly Leu Gly Thr 
                85                  90                  95 

Asp Ser Gly Leu Leu Met Asp Thr Gly Asp Glu Lys Gln Lys Lys Lys 
            100                 105                 110 

Glu Ile Lys Glu Leu Lys Asp Arg Gly Phe Ile Ser Ala Arg Phe Arg 
        115                 120                 125 

Asn Trp Gln Ala Tyr Met Gln Tyr Cys Leu Leu Cys Asp Lys Glu Phe 
    130                 135                 140 

Leu Gly His Arg Ile Val Arg His Ala Gln Lys His Tyr Lys Asp Gly 
145                 150                 155                 160 

Ile Tyr Ser Cys Pro Ile Cys Ala Lys Asn Phe Asn Ser Lys Asp Ser 
                165                 170                 175 

Phe Val Pro His Val Thr Leu His Val Lys Gln Ser Ser Lys Glu Arg 
            180                 185                 190 

Leu Ala Ala Met Lys Pro Leu Arg Arg Leu Gly Arg Pro Pro Lys Ile 
        195                 200                 205 

Thr Ala Thr His Glu Asn Gln Lys Thr Asn Ile Asn Thr Val Ala Lys 
    210                 215                 220 

Gln Glu Gln Arg Pro Ile Lys Lys Asn Ser Leu Tyr Ser Thr Asp Phe 
225                 230                 235                 240 

Ile Val Phe Asn Asp Asn Asp Gly Ser Asp Asp Glu Asn Asp Asp Lys 
                245                 250                 255 

Asp Lys Ser Tyr Glu Pro Glu Val Ile Pro Val Gln Lys Pro Val Pro 
            260                 265                 270 

Val Asn Glu Phe Asn Cys Pro Val Thr Phe Cys Lys Lys Gly Phe Lys 
        275                 280                 285 

Tyr Phe Lys Asn Leu Ile Ala His Val Lys Gly His Lys Asp Ser Glu 
    290                 295                 300 

Asp Ala Lys Arg Phe Leu Glu Met Gln Ser Lys Lys Val Ile Cys Gln 
305                 310                 315                 320 

Tyr Cys Arg Arg His Phe Val Ser Val Thr His Leu Asn Asp His Leu 
                325                 330                 335 

Gln Met His Cys Gly Ser Lys Pro Tyr Ile Cys Ile Gln Met Lys Cys 
            340                 345                 350 

Lys Ala Gly Phe Asn Ser Tyr Ala Glu Leu Leu Ala His Arg Lys Glu 
        355                 360                 365 

His Gln Val Phe Arg Ala Lys Cys Leu Phe Pro Lys Cys Gly Arg Ile 
    370                 375                 380 

Phe Ser Gln Ala Tyr Leu Leu Tyr Asp His Glu Ala Gln His Tyr Asn 
385                 390                 395                 400 

Thr Tyr Thr Cys Lys Phe Thr Gly Cys Gly Lys Val Tyr Arg Ser Gln 
                405                 410                 415 

Ser Glu Met Glu Lys His Gln Asp Gly His Ser His Pro Glu Thr Gly 
            420                 425                 430 

Leu Pro Pro Glu Asp Gln Leu Gln Pro Ser Gly Asn Asp Val Asn Pro 
        435                 440                 445 

Asp Ser Gly Ala Thr Ala Ala Gly Gly Arg Ser Glu Asn Ser Ile Asp 
    450                 455                 460 

Lys Asn Leu Gly Ser Asn Arg Ser Ala Asp Trp Glu Lys Asn Arg Ala 
465                 470                 475                 480 

Glu Pro Ala Val Thr Lys His Gly Gln Ile Ser Ala Ala Glu Leu Arg 
                485                 490                 495 

Gln Ala Asn Ile Pro Leu Ser Asn Gly Leu Glu Thr Arg Asp Asn Thr 
            500                 505                 510 

Thr Val Leu Arg Thr Asn Glu Val Ala Val Ser Ile Lys Val Ser Val 
        515                 520                 525 

Asn His Gly Val Glu Gly Asp Phe Gly Lys Gln Glu Asn Leu Thr Met 
    530                 535                 540 

Glu Gly Thr Gly Glu Pro Leu Ile Thr Asp Val His Lys Pro Gly Ile 
545                 550                 555                 560 

Gly Ala Gly Val Gln Leu Cys His Pro Gly Phe Gln Glu Lys Lys Gly 
                565                 570                 575 

His Glu Cys Leu Asn Glu Ala Gln Asn Ser Leu Ser Asn Ser Glu Ser 
            580                 585                 590 

Leu Lys Met Asp Asp Leu Asn Pro Gln Ser Leu Glu Arg Gln Val Asn 
        595                 600                 605 

Thr Leu Met Thr Phe Ser Val Gln Asn Glu Ala Gly Leu Glu Asp Asn 
    610                 615                 620 

Ser Gln Ile Cys Lys Phe Glu Cys Gly Gly Asp Val Lys Thr Ser Ser 
625                 630                 635                 640 

Ser Leu Tyr Asp Leu Pro Leu Lys Thr Leu Glu Ser Ile Thr Phe Val 
                645                 650                 655 

Gln Ser Gln Pro Asp Leu Ser Ser Pro Leu Gly Ser Pro Ser Val Pro 
            660                 665                 670 

Pro Lys Ala Pro Gly Gln Lys Phe Ser Cys Gln Val Glu Gly Cys Thr 
        675                 680                 685 

Arg Thr Tyr Asn Ser Ser Gln Ser Ile Gly Lys His Met Lys Thr Ala 
    690                 695                 700 

His Pro Asp Gln Tyr Ala Ala Phe Lys Leu Gln Arg Lys Thr Lys Lys 
705                 710                 715                 720 

Gly Gln Lys Ser Asn Asn Leu Asn Thr Pro Asn His Gly Lys Cys Val 
                725                 730                 735 

Tyr Phe Leu Pro Ser Gln Val Ser Ser Ser Asn His Ala Phe Phe Thr 
            740                 745                 750 

Pro Gln Thr Lys Ala Asn Gly Asn Pro Ala Cys Ser Ala Gln Val Gln 
        755                 760                 765 

His Val Ser Pro Ser Ile Phe Pro Ala His Leu Ala Ser Val Ser Ala 
    770                 775                 780 

Pro Leu Leu Pro Ser Val Glu Ser Val Leu Ser Pro Asn Ile Pro Ser 
785                 790                 795                 800 

Gln Asp Lys His Gly Gln Asp Gly Ile Leu Cys Ser Gln Met Glu Asn 
                805                 810                 815 

Leu Ser Tyr Ala Pro Leu Pro Ala Gln Met Glu Asp Leu Thr Lys Thr 
            820                 825                 830 

Val Leu Pro Leu Asn Ile Asp Ser Gly Ser Asp Pro Phe Leu Pro Leu 
        835                 840                 845 

Pro Thr Glu Asn Ser Ser Leu Phe Ser Ser Pro Ala Asp Ser Glu Asn 
    850                 855                 860 

Asn Ser Val Phe Ser Gln Leu Glu Asn Ser Thr Asn His Tyr Pro Ser 
865                 870                 875                 880 

Gln Thr Asp Gly Asn Ile Asn Ser Ser Phe Leu Lys Gly Gly Ser Ser 
                885                 890                 895 

Glu Asn Gly Val Phe Pro Ser Gln Val Ser Ser Ala Asp Asp Phe Ser 
            900                 905                 910 

Ser Thr Ser Ala Gln Pro Ser Thr Pro Lys Lys Val Lys Lys Asp Arg 
        915                 920                 925 

Gly Arg Gly Pro Asn Gly Lys Glu Arg Lys Pro Lys His Asn Lys Arg 
    930                 935                 940 

Ala Lys Trp Pro Ala Ile Ile Arg Asp Gly Lys Phe Ile Cys Ser Arg 
945                 950                 955                 960 

Cys Tyr Arg Ala Phe Thr Asn Pro Arg Ser Leu Gly Gly His Leu Ser 
                965                 970                 975 

Lys Arg Ser Tyr Cys Lys Pro Leu Asp Gly Ala Glu Ile Ala Gln Glu 
            980                 985                 990 

Leu Leu Gln Thr Asn Arg Gln Pro  Ser Leu Leu Ala Ser  Met Ile Leu 
        995                 1000                 1005 

Ser Thr  Ser Ala Val Asn Met  Gln Gln Pro Gln Gln  Ser Asn Phe 
    1010                 1015                 1020 

Asn Pro  Glu Thr Cys Phe Lys  Asp Pro Ser Phe Leu  Gln Leu Leu 
    1025                 1030                 1035 

Asn Val  Glu Asn Arg Pro Thr  Phe Leu Pro Ser Thr  Phe Pro Arg 
    1040                 1045                 1050 

Cys Asp  Val Ser Asn Phe Asn  Ala Ser Val Ser Gln  Glu Gly Ser 
    1055                 1060                 1065 

Glu Ile  Ile Lys Gln Ala Leu  Glu Thr Ala Gly Ile  Pro Ser Thr 
    1070                 1075                 1080 

Phe Glu  Ser Ala Glu Met Leu  Ser Gln Val Val Pro  Ile Gly Ser 
    1085                 1090                 1095 

Val Ser  Asp Ala Ala Gln Val  Ser Ala Ala Gly Met  Pro Gly Pro 
    1100                 1105                 1110 

Pro Val  Thr Pro Leu Leu Gln  Thr Val Cys His Pro  Asn Thr Ser 
    1115                 1120                 1125 

Pro Ser  Asn Gln Asn Gln Thr  Pro Asn Ser Lys Thr  Leu Lys Glu 
    1130                 1135                 1140 

Cys Asn  Ser Leu Pro Leu Phe  Thr Thr Asn Asp Leu  Leu Leu Lys 
    1145                 1150                 1155 

Thr Ile  Glu Asn Gly Leu Cys  Ser Asn Ser Phe Ser  Ser Ser Thr 
    1160                 1165                 1170 

Glu Pro  Pro Gln Asn Phe Thr  Asn Asn Ser Ala His  Val Ser Val 
    1175                 1180                 1185 

Ile Ser  Gly Pro Gln Asn Thr  Arg Ser Ser His Leu  Asn Lys Lys 
    1190                 1195                 1200 

Gly Asn  Ser Ala Ser Lys Lys  Arg Lys Lys Val Ala  Pro Ala Val 
    1205                 1210                 1215 

Ser Val  Ser Asn Thr Ser Gln  Asn Val Leu Pro Thr  Asp Leu Pro 
    1220                 1225                 1230 

Val Gly  Leu Pro Ser Lys Asn  Leu Thr Val Pro Asp  Thr Asn Thr 
    1235                 1240                 1245 

Arg Ser  Asp Met Thr Pro Asp  Cys Glu Pro Arg Ala  Leu Val Glu 
    1250                 1255                 1260 

Asn Leu  Thr Gln Lys Leu Asn  Asn Ile Asp Asn His  Leu Phe Ile 
    1265                 1270                 1275 

Thr Asp  Val Lys Glu Asn Cys  Lys Ala Ser Leu Glu  Pro His Thr 
    1280                 1285                 1290 

Met Leu  Thr Pro Leu Thr Leu  Lys Thr Glu Asn Gly  Asp Ser Arg 
    1295                 1300                 1305 

Met Met  Pro Leu Ser Ser Cys  Thr Pro Val Asn Ser  Asp Leu Gln 
    1310                 1315                 1320 

Ile Ser  Glu Asp Asn Val Ile  Gln Asn Phe Glu Lys  Thr Leu Glu 
    1325                 1330                 1335 

Ile Ile  Lys Thr Ala Met Asn  Ser Gln Ile Leu Glu  Val Lys Ser 
    1340                 1345                 1350 

Gly Ser  Gln Gly Thr Gly Glu  Thr Thr Gln Asn Ala  Gln Ile Asn 
    1355                 1360                 1365 

Tyr Ser  Met Gln Leu Pro Ser  Val Asn Ser Ile Pro  Asp Ser Lys 
    1370                 1375                 1380 

Leu Pro  Asp Ala Ser Gln Cys  Ser Ser Phe Leu Thr  Val Met Pro 
    1385                 1390                 1395 

Thr Lys  Ser Glu Ala Leu His  Lys Glu Asp Gln Ile  Gln Asp Ile 
    1400                 1405                 1410 

Leu Glu  Gly Leu Gln Asn Leu  Lys Leu Glu Asn Asp  Thr Ser Ala 
    1415                 1420                 1425 

Pro Ala  Ser Gln Ser Met Leu  Met Asn Lys Ser Val  Ala Leu Ser 
    1430                 1435                 1440 

Pro Thr  Pro Thr Lys Ser Thr  Pro Asn Ile Val Val  Gln Pro Val 
    1445                 1450                 1455 

Pro Glu  Val Ile His Val Gln  Leu Asn Asp Arg Val  Asn Lys Pro 
    1460                 1465                 1470 

Phe Val  Cys Gln Asn Gln Gly  Cys Asn Tyr Ser Ala  Met Thr Lys 
    1475                 1480                 1485 

Asp Ala  Leu Phe Lys His Tyr  Gly Lys Ile His Gln  Tyr Thr Pro 
    1490                 1495                 1500 

Glu Met  Ile Leu Glu Ile Lys  Lys Asn Gln Leu Lys  Phe Ala Pro 
    1505                 1510                 1515 

Phe Lys  Cys Val Val Pro Ser  Cys Thr Lys Thr Phe  Thr Arg Asn 
    1520                 1525                 1530 

Ser Asn  Leu Arg Ala His Cys  Gln Leu Val His His  Phe Thr Ile 
    1535                 1540                 1545 

Glu Glu  Met Val Lys Leu Lys  Ile Lys Arg Pro Tyr  Gly Arg Lys 
    1550                 1555                 1560 

Ser Gln  Ser Glu Asn Leu Ser  Ser Pro Gln Asn Asn  Gln Val Lys 
    1565                 1570                 1575 

Lys Gln  Pro Ser Met Ala Glu  Glu Thr Lys Thr Glu  Ser Gln Pro 
    1580                 1585                 1590 

Ala Phe  Lys Val Pro Ala Ala  Thr Gly Asp Ala Ala  Leu Ala Asn 
    1595                 1600                 1605 

Ala Thr  Val Ile Pro Glu Lys  Gln Leu Ala Glu Lys  Lys Ser Pro 
    1610                 1615                 1620 

Glu Lys  Pro Glu Ser Ser Ser  Gln Pro Val Thr Ser  Ser Ala Glu 
    1625                 1630                 1635 

Gln Tyr  Asn Ala Asn Leu Ala  Asn Leu Lys Thr Lys  Gly Arg Lys 
    1640                 1645                 1650 

Asn Lys  Arg His Arg Lys Glu  Lys Glu Glu Lys Arg  Glu Lys Asn 
    1655                 1660                 1665 

Pro Val  Ser Gln Ala Phe Glu  Leu Pro Thr Lys Tyr  Ser Ser Tyr 
    1670                 1675                 1680 

Arg Pro  Tyr Cys Cys Val His  Gln Gly Cys Phe Ala  Ala Phe Thr 
    1685                 1690                 1695 

Ile Gln  Gln Asn Leu Ile Leu  His Tyr Gln Ala Val  His Lys Ser 
    1700                 1705                 1710 

Asn Leu  Pro Thr Phe Ser Ala  Glu Val Gln Glu Glu  Ser Glu Ala 
    1715                 1720                 1725 

Val Lys  Glu Ser Glu Glu Thr  Glu Pro Lys Gln Ser  Met Lys Glu 
    1730                 1735                 1740 

Phe Arg  Cys Gln Val Ser Asp  Cys Ser Arg Ile Phe  Gln Ala Ile 
    1745                 1750                 1755 

Thr Gly  Leu Ile Gln His Tyr  Met Lys Leu His Glu  Met Thr Pro 
    1760                 1765                 1770 

Glu Glu  Ile Glu Ser Met Thr  Ala Ala Val Asp Val  Gly Lys Phe 
    1775                 1780                 1785 

Pro Cys  Asp Gln Leu Glu Cys  Lys Leu Ser Phe Thr  Thr Tyr Leu 
    1790                 1795                 1800 

Ser Tyr  Val Val His Leu Glu  Val Asp His Gly Ile  Gly Thr Arg 
    1805                 1810                 1815 

Thr Ser  Lys Ala Glu Glu Asp  Gly Ile Tyr Lys Cys  Asp Cys Glu 
    1820                 1825                 1830 

Gly Cys  Asp Arg Ile Tyr Ala  Thr Arg Ser Asn Leu  Leu Arg His 
    1835                 1840                 1845 

Ile Phe  Asn Lys His Asn Asp  Lys His Lys Ala His  Leu Ile Arg 
    1850                 1855                 1860 

Pro Arg  Lys Leu Thr Gly Gln  Glu Asn Ile Ser Ser  Lys Ala Asn 
    1865                 1870                 1875 

Gln Glu  Lys Ser Lys Ser Lys  His Arg Thr Thr Lys  Pro Asn Arg 
    1880                 1885                 1890 

Ser Gly  Lys Asp Gly Met Lys  Met Pro Lys Thr Lys  Arg Lys Lys 
    1895                 1900                 1905 

Lys Ser  Asn Leu Glu Asn Lys  Ser Ala Lys Val Val  Gln Ile Glu 
    1910                 1915                 1920 

Glu Asn  Lys Pro Tyr Ser Leu  Lys Arg Gly Lys His  Val Tyr Ser 
    1925                 1930                 1935 

Ile Lys  Ala Arg Asn Asp Ala  Leu Ala Glu Cys Thr  Ser Lys Phe 
    1940                 1945                 1950 

Val Thr  Gln Tyr Pro Cys Met  Ile Lys Gly Cys Thr  Ser Val Val 
    1955                 1960                 1965 

Thr Ser  Glu Ser Asn Ile Ile  Arg His Tyr Lys Cys  His Lys Leu 
    1970                 1975                 1980 

Ser Arg  Ala Phe Thr Ser Gln  His Arg Asn Ile Leu  Ile Val Phe 
    1985                 1990                 1995 

Lys Arg  Tyr Gly Asn Pro Gln  Gly Arg Glu Ile Ser  Glu Gln Glu 
    2000                 2005                 2010 

Asp Glu  Lys Asn Asp Lys Lys  Gly Pro Asp Ser Ser  Val Leu Glu 
    2015                 2020                 2025 

Lys Asn  Asp Asn Ser Glu Pro  Ala Ala Ala Pro Gln  Glu Glu Gly 
    2030                 2035                 2040 

Arg Lys  Gly Glu Lys Asp Glu  Met Asp Glu Leu Thr  Glu Leu Phe 
    2045                 2050                 2055 

Ile Thr  Lys Leu Ile Asn Glu  Asp Ser Thr Asn Ala  Glu Asn Gln 
    2060                 2065                 2070 

Gly Asn  Thr Thr Leu Lys Gly  Asn Asn Glu Phe Gln  Glu His Asp 
    2075                 2080                 2085 

Ser Cys  Thr Ser Glu Arg Gln  Lys Pro Gly Asn Leu  Lys Arg Val 
    2090                 2095                 2100 

Tyr Lys  Glu Lys Asn Thr Val  Gln Ser Lys Lys Arg  Lys Ile Asp 
    2105                 2110                 2115 

Lys Thr  Glu Pro Glu Val Ser  Leu Val Val Asn Asn  Thr Arg Lys 
    2120                 2125                 2130 

Glu Glu  Glu Pro Ala Val Ala  Val Gln Thr Thr Glu  Glu His Pro 
    2135                 2140                 2145 

Ala Ser  Phe Asp Trp Ser Ser  Phe Lys Pro Met Gly  Phe Glu Ala 
    2150                 2155                 2160 

Ser Phe  Leu Lys Phe Leu Glu  Glu Ser Ala Val Lys  Gln Lys Lys 
    2165                 2170                 2175 

Asn Ser  Asp Arg Asp His Ser  Asn Ser Gly Ser Lys  Arg Gly Ser 
    2180                 2185                 2190 

His Ser  Ser Ser Arg Arg His  Val Asp Lys Ala Ala  Val Ala Gly 
    2195                 2200                 2205 

Ser Ser  His Val Cys Ser Cys  Lys Asp Ser Glu Ile  Phe Val Gln 
    2210                 2215                 2220 

Phe Ala  Asn Pro Ser Lys Leu  Gln Cys Ser Glu Asn  Val Lys Ile 
    2225                 2230                 2235 

Val Leu  Asp Lys Thr Leu Lys  Asp Arg Ser Glu Leu  Val Leu Lys 
    2240                 2245                 2250 

Gln Leu  Gln Glu Met Lys Pro  Thr Val Ser Leu Lys  Lys Leu Glu 
    2255                 2260                 2265 

Val Leu  Ser Asn Ser Pro Asp  Arg Thr Val Leu Lys  Glu Ile Ser 
    2270                 2275                 2280 

Ile Gly  Lys Ala Thr Gly Arg  Gly Gln Tyr 
    2285                 2290