Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4838-4842, July 1989 Biochemistry Rat : Structure and regulation by insulin MARK A. MAGNUSON, TERESA L. ANDREONE*, RICHARD L. PRINTZ, STEVE KOCH, AND DARYL K. GRANNERt Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232 Communicated by Charles R. Park, March 20, 1989

ABSTRACT The glucokinase gene is 15.5-kilobases long, Primer-Extension Analysis. A 36-base oligonucleotide (5'- appears to be present as a single copy, and contains 10 exons ATGTTCCTGACTCCTGAGGCCACCTGTTGCAGGTGA- that range in size from 96 to 977 base pairs. The transcription 3') complementary to sequences near the 5' end of the start site was located 127 upstream from the glucokinase mRNA was synthesized and 5'-end-labeled with translation initiation codon. The 5' flanking DNA contains [_y-32P]ATP (>5000 Ci/mmol; 1 Ci = 37 GBq) and T4 poly- several regions similar to dermed promoter elements. These . The primer (3 x 10s cpm) was annealed in include a probable "TATA box," an Spl , and a total volume of 20 gl to 20 pug to poly(A)+ RNA; the several elements related to liver-specific gene expression. In annealing buffer contained 20 mM Tris-HCl (pH 7.5), 250 mM addition, we determined that transcription of the glucokinase NaCl, and 1 mM EDTA. After hybridization for 1 hr at 60TC gene increased at least 20-fold when diabetic rats were treated the reaction mixtures, containing the annealed primer and with insulin for 2 hr. RNA, were diluted with 130 1A ofa solution containing 50 mM Tris-HCl (pH 7.5), 40 mM KCl, 10 mM dithiothreitol, 3 mM Glucokinase (ATP:D-hexose 6-, EC MgCl2, actinomycin D (75 ,ug/ml), deoxyribonucleotides at 2.7.1.1) plays a key role in the regulation of glucose homeo- 0.5 mM each, and 2 units of avian myeloma virus reverse stasis by catalyzing the first step in glycolysis (1). Expression transcriptase (Promega Biotec) and then incubated at 37TC for ofthe is limited to hepatocytes and pancreatic ,B cells 1 hr. The products of the reactions were size-fractionated on (2, 3), and it is regulated differently in these two tissues. The a 5% polyacrylamide/7 M urea gel and visualized by auto- hepatic enzyme is induced by insulin and repressed by cAMP radiography. (4) whereas in the p cell glucokinase activity is increased by S1 Nuclease Protection Analysis. Single-stranded DNA, glucose (5). The glucokinase gene is, therefore, of interest generated from a 1300-base-pair (bp) (BamHI-BamHI) frag- both because of its tissue-specific expression and because of ment from AGK5, cloned into pEMBL19, was annealed to the the several regulatory processes that can be analyzed. Before same 36-base primer used in the primer-extension analysis the cis-acting DNA elements responsible for the tissue- and extended in the presence of [a-32P]dATP and the Klenow specific expression and hormonal regulation of this enzyme fragment of DNA I. The DNA was digested with can be identified and studied, the structure ofthe glucokinase Bgl I, the strands were separated on an alkaline agarose gel, gene, the transcription unit in each tissue, and the sequence and the desired DNA fragment was isolated. The resulting of its 5' flanking DNA must be determined. 32P-labeled, single-stranded 365-nucleotide DNA fragment Glucokinase is thought to be a member of a family of (1.2 x 105 cpm) was hybridized to 20 gg of poly(A)+ RNA in that have a common evolutionary origin (6). This 30 tkl of a buffer containing 80% (vol/vol) formamide, 40 mM concept was based on indirect evidence because none of the Pipes (pH 7.0), 0.4 M NaCl, and 1 mM EDTA. The mixtures complete structures of the mammalian hexokinases were were heated at 75TC for 15 min and incubated at 500C available. We recently deduced the structure of rat liver overnight. After the hybridization, 300 p.l of a solution glucokinase and found that it shares 33% and 53% amino acid containing 300 mM NaCl, 3 mM ZnSO4, 60 mM sodium sequence identity with yeast and the carboxyl- acetate (pH 4.5), and 200 units of S1 nuclease was added and terminal portion of rat brain hexokinase I, respectively (7). the reaction mixture was incubated at 37°C for 1 hr. After We now have determined the structure of the glucokinase phenol extraction and ethanol precipitation, the DNA was genet as an initial step toward answering how the various size-fractionated on a 5% polyacrylamide/7 M urea gel and hexokinase isozymes are related to each other. an autoradiograph was obtained. Transcription Run-On Analysis. Male Sprague-Dawley EXPERIMENTAL PROCEDURES rats weighing 125-150 g (Harlan Animal Supply, Indianapo- General Techniques. Standard procedures were used for lis) were made diabetic as described (12). Rat liver nuclei screening phage libraries, DNA labeling, restriction enzyme were isolated by the method ofSchibler et al. (13), except that mapping, subcloning, isolation of genomic DNA, and South- the suspension buffer was that of Sasaki et al. (14). The ern transfers (8). RNA was isolated from the livers ofdiabetic elongation of nascent RNA transcripts was quantitated es- rats by the method of Chirgwin et al. (9). Genomic DNA sentially as described by McKnight and Palmiter (15) and fragments were subcloned into pEMBL (10) or Bluescript modified by Sasaki et al. (14). The plasmid bound to M13+ (Stratagene) plasmid vectors for restriction map anal- the filters to measure transcription were, for glucokinase, ysis and DNA sequencing using the dideoxynucleotide pGK.Z1; for the glucokinase control, Bluescript M13+; for method (11). An amplified rat genomic library in EMBL3 was phosphoenolpyruvate carboxykinase (PEPCK), PC116 (16); obtained from G. Scherer (German Cancer Research Center, and for the PEPCK control, pBR322. Heidelberg, F.R.G.). A second, unamplified rat genomic library was constructed in EMBL3 using DNA isolated from Abbreviation: PEPCK, phosphoenolpyruvate carboxykinase. the liver of a male Sprague-Dawley rat. *Present address: Chicago Medical School, North Chicago, IL 60064. tTo whom reprint requests should be addressed at: 607 Light Hall, The publication costs of this article were defrayed in part by page charge Vanderbilt University, Nashville, TN 37232. payment. This article must therefore be hereby marked "advertisement" tThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M24943 to M24952). 4838 Downloaded by guest on October 1, 2021 Biochemistry: Magnuson et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4839 tended further in the 5' direction were isolated (Fig. 2). AGK5 -23 Kilobases extended 13.5 kilobases (kb) farther than AGK9 so it was subjected to additional analysis. Thus genomic DNA was - 9.4 found to contain 10 exons and the entire hepatic transcription unit of the glucokinase gene. -6.6 Intron-Exon Organization of the Gene. The location of individual exons within the glucokinase gene was determined -44 by a combination of Southern blot analysis and DNA se- quencing (Fig. 3). The intron-exon junctions were deter- mined by sequencing the genomic DNA with exon-specific primers and comparing this sequence with the cDNA se- - 2.3 quence. All of the exons of the glucokinase gene were -2.0 sequenced (Fig. 3) and these data were compared to the cDNA sequence (7). Three single-base differences between m CE I C) 0 the cDNAs and genomic DNAs were identified§; however, o g 0 none changed the predicted amino acid sequence of the protein (7). Splice sites, located at points of cDNA and H H genomic DNA homology divergence, were determined by FIG. 1. Southern blot analysis of rat genomic DNA using a obtaining the best fit to the splice consensus sequence (17). glucokinase cDNA fragment. High molecular weight genomic DNA The putative intron-exon splice site sequences are shown in was isolated from a rat liver, digested with EcoRl, HindIII, or Table 1. The exons ranged in size from 96 bp to 977 bp. BamHI, as indicated, size-fractionated on a 1.0% agarose gel, and Identification of the Transcription Initiation Sites. The S1 transferred to a nylon membrane. The hybridization probe was a nuclease protection assay was used to identify the transcrip- 389-bp BstEH-EcoRI 32P-labeled DNA fragment isolated from the GK1 cDNA (7). Size markers were electrophoresed in an adjacent tion initiation site(s) of the glucokinase gene. After hybrid- lane. ization of the S1 probe to poly(A)+ RNA isolated from the livers of diabetic and insulin-treated diabetic rats, the hy- bridization products were treated with S1 nuclease, and then RESULTS the protected DNA fragments were size-fractionated and Southern Analysis of Genomic DNA. A Southern blot hy- visualized by autoradiography. A portion of the autoradio- bridization of rat genomic DNA was performed to determine graph obtained is shown in Fig. 4A. Lanes A-D show DNA whether there were multiple glucokinase or other size markers from a dideoxynucleotide sequencing reaction. closely related genes. A fragment of the liver glucokinase S1 nuclease-resistant fragments were obtained when RNA cDNA was used to probe EcoRI, BamHI, and HindIII from the insulin-treated diabetic rats was used (lane E) but restriction digests of rat genomic DNA. As shown in Fig. 1, not when the RNA was isolated from the untreated diabetic a single DNA species was seen for each restriction digest. rats (lane F). The protected DNA fragments varied in size This result suggests that the glucokinase gene is present in the from 83 to 89 bases but the strongest bands were 87-89 bases rat as a single copy. long, centered around an adenine. Screening for the Rat Glucokinase Gene. A glucokinase A primer-extension assay was also used to locate the genomic DNA fragment, AGK2, was identified by screening transcription initiation site. A portion of the autoradiograph a rat genomic DNA library in EMBL3 with the partial-length obtained from this experiment is shown in Fig. 4B. Lane E glucokinase cDNA GK1 (7). The restriction map ofthe insert shows the DNA products of a primer-extension reaction in AGK2 (Fig. 2) predicted EcoRI, BamHI, and HindIlI using poly(A)+ RNA from the livers of insulin-treated dia- fragments of the lengths seen in the genomic Southern blot betic rats and lane F shows the products from poly(A)+ RNA experiment (Fig. 1), indicating that AGK2 contained some from the livers of diabetic rats. The following results corre- part ofthe glucokinase gene. A DNA fragment from near the spond with the S1 nuclease protection experiment: (i) Prod- 5' end of AGK2 was isolated and used to probe additional ucts derived from glucokinase mRNA were seen only in lane recombinant bacteriophage from a different, unamplified rat genomic library to identify DNA segments that would overlap §The sites at which the glucokinase cDNA and genomic DNA AGK2 but extend farther in the 5' direction. Two additional sequences differ are described in the sequence data submitted to clones (AGK5 and AGK9) that overlapped AGK2 and ex- GenBank.

XGK9 XGK5 XGK2

EcoR I I a I BamH I -- Hind m I I LL- I I I I Others --- H I4 H H FIG. 2. Structure of the rat glucokinase gene. c o a0 P CL a E a The structure of the rat glucokinase gene is illus- y 0n (n cn trated. Shown, from top to bottom, are: (i) the overlapping rat glucokinase genomic DNA clones in the bacteriophage A vector EMBL3; (it) the pGK.HE pGK.E3 pGK.E2 pGK.EI restriction map of the cloned DNA using the EcoRI, HindIlI, BamHI, Bgl II, Kpn I, 1kb Sac I, and Sma I; (iiM) plasmid subclones of the gene; and (iv) the location of the 10 exons as in I I I I 1 * * * _ depicted by the solid boxes. The scales kilo- 2 3 4 56 7 8 9 10 bases is indicated below. Downloaded by guest on October 1, 2021 4840 Biochemistry: Magnuson et al. Proc. Natl. Acad. Sci. USA 86 (1989)

100 bp E F-H obtain it is shown in Fig. 3. As can be seen by examining this CD co _ I sequence, the cap site is located 127 bases upstream from the ATG initiation codon. Eleven additional nucleotides not i\-H i- i -H-al -H zm present in the GK.Z2 cDNA (7) were placed in the first exon Il -- * _ I , of the gene by this analysis. * I Regulation of Glucokinase Gene Transcription by Insulin. insulin exerts H As an initial step in the effort to understand how e Z CL a. positive and negative effects on transcription in the same cell, I CL x -vsmv 36gz~ we quantitated transcription of the PEPCK and glucokinase genes in the same aliquots of nuclei isolated from the livers of diabetic rats. Transcription ofthe glucokinase gene was at the limit of detection in the absence of insulin (Fig. 6) but Ur 0 c 75 r ur CL increased to 15 ppm at 0.5 hr, to 309 ppm at 1 hr, and to 619 w 4 I 5 CID 6 or iffm--G±S.L ppm at 2 hr after insulin treatment. Transcription of the -0 PEPCK gene decreased from 6954 ppm in the untreated diabetic animal to 1666 ppm at 0.5 hr, to 754 ppm at 1 hr, and to 1027 ppm after 2 hr of insulin treatment. A somewhat

_& a different transcription run-on experiment was performed to sDX 7 validate the magnitude of the induction of glucokinase gene transcription by insulin. Nuclei were isolated from untreated diabetic rats and from diabetic rats treated with insulin for 2 hr. In this experiment the hybridization filters were exposed E 'a CL I 8 a. to film for 2 weeks and the induction was quantitated by densitometry. Transcription of the glucokinase gene in- ., I . I creased at least 20-fold (data not shown).

_ w x OL (f) E DISCUSSION a. '0CIDm'P I s- // Structure of the Rat Glucokinase Gene. The 15.5-kb glu- cokinase gene consists of 10 exons separated by 9 introns. It appears to be present as a single copy. The location of the start site of transcription (127 bases upstream from the FIG. 3. Exon locations and sequencing strategy. The strategy translation initiation codon) predicts that the mRNA pro- used to sequence the exons and 5' flanking DNA of the glucokinase duced from this gene in the liver is 2357 bases long, excluding gene is indicated. The sizes of the exons are, for exons 1 to 10, The translation initiation codon is respectively, 172, 163, 155, 120, 96, 100, 184, 156, 234, and 977 bp. the poly(A) tail. putative exon the translation termination signal Each exon is shown as a hatched box and the sequencing strategy is located within 1, and indicated below. is located in exon 10. The portion of the protein homologous with the ATP binding domains in a variety ofproteins [amino E, which contains RNA isolated from the insulin-treated rats. acids 78-102 (7)] is encoded entirely by DNA located within (it) Bands of 85-88 bases long were seen. (iii) Transcription exon 3. The region that constitutes the core of the glucose- initiation was localized over a range of 4 bases. (iv) The binding domain [amino acids 144-171 (7)] is encoded by parts strongest band, 88 nucleotides long, corresponds to initiation of exons 4 and 5. at an adenine on the glucokinase gene. Since this base is near Potential Regulatory Motifs. It is likely that multiple tran- the center of the size range seen for the S1 nuclease protec- scription factors, some of which may be liver-specific, inter- tion experiment (compare Fig. 4 A and B), it was designated act with DNA elements in the glucokinase promoter. The as position + 1 for numbering the nucleotides in the glucoki- sequence TATTT, located at positions -29 to -25, may be nase gene. a "TATA box," although it is not a good consensus sequence Glucokinase Promoter Sequence. The sequence ofthe DNA (17). The TATA box is generally considered to be important on both sides of the transcription initiation site was deter- for the precise positioning oftranscription initiation (17). The mined. A Bgl II site located 1448 bp upstream of adenine + 1 rather poor TATA homology may explain the observation was the limit for sequence determination in the 5' direction. that the glucokinase gene initiates over several bases. The This sequence is shown in Fig. 5 and the strategy used to sequence CCCCCGCCCC (at positions -442 to -433) is a Table 1. Identification of intron-exon boundaries Intron number 5' intron junction 3' intron junction 1 TTGTTGACTCTG/gtaagggccatt cccccgcacag/GTCGAGCAGATC 2 CAGAAGGCTCAG/gtaccgcaggtt ctotgcctgcag/AAGTCGGAGACT 3 ACTGCCGAGATG/gtgagcagcctg tgcaactcctag/CTCTTTGACTAC 4 GACCTAGACAAG/gtgagccgggtg cctaccttacag/GGCATCCTCCTC 5 AAGAGGAGAGGG/gtgagcacagcg acttcttggcag/GACTTTGAGATG 6 GCATGATTGTGG/gtaagggcttot ccctccctctag/GCACTGGCTGCA 7 CGGTCAGCAGCT/gtaaggatgctc ttctgtatccag/GTACGAGAAGAT 8 ACAAGTGGAGAG/gtgcctgcaggg tcctgcccgcag/CGACTCCGGGGA 9 GCTGCACCCGAG/gtcagcttccac cctcctgttcag/CTTCAAGGAGCG The consensus sequences determined for the splice donor and acceptor sites of exons from class II genes are MAG/gtragt and yyyyyynyag/GK, respectively (17), where M is A or C, R is G or A, Y is T or C, K is G or T, and N is any base. Exon sequences are designated by uppercase letters, intron sequences are designated by lowercase letters, and splice sites are designated by the slashes (/). Downloaded by guest on October 1, 2021 Biochemistry: Magnuson et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4841

A A B C D E F B A BC D E F _ _ _~' 100 I~~~~~~~ 100 - _ o _m s _ T T 90 : _ A.4-A G _-T t , 80- _ cJ _f-i GJ

70- 70- _- Si Nuclease Protection Primer Extension

FIG. 4. Identification ofthe transcription initiation site ofthe hepatic glucokinase gene. (A) An S1 nuclease protection assay was performed. Lanes A-D show A, C, G, and T dideoxynucleotide sequencing reactions used to define nucleotide positions. The S1 nuclease protection experiment was performed using 20 ,ug of poly(A)+ RNA isolated from livers of diabetic rats treated with insulin for 4 hr (lane E) or using 20 Ag of poly(A)+ RNA isolated from livers ofuntreated diabetic rats Oane F). The length ofthe DNA fragments protected is indicated by the size scale to the left. The nucleotides surrounding the transcription initiation site are shown to the right. (B) A primer-extension assay was performed. Lanes A-D show A, C, G, and T dideoxynucleotide sequencing reactions used to define the nucleotide positions. The primer-extension experiment was performed using 20 j&g of poly(A)+ RNA isolated from livers of diabetic rats treated with insulin for 4 hr (lane E) or using 20 ,ug ofpoly(A)' RNA isolated from livers of untreated diabetic rats (lane F). The length of the DNA fragments generated is indicated by the size scale to the left. The nucleotides surrounding the transcription initiation site are shown to the right. 9/10 match for the reverse complement of the consensus primarily at a transcriptional level. The magnitude and ki- binding site (G/T)GGGCGG(G/A)(G/A)(C/T) of the Spl netics of the increase, at least a 20-fold change within 2 hr, transcription factor (18, 19). correspond well with data reported (22). The increase in The 5' flanking DNA sequence ofthe glucokinase gene was transcription is rapid; it occurs within 1 hr of injection of the searched for the presence of elements found in several animal with insulin. The rapidity of insulin's action on the liver-specific genes (20, 21). Hepatocyte nuclear factor I transcription of the glucokinase gene suggests that a direct binds to the DNA sequence ATTAAC in a region of the effect is involved, as is the case in the suppression of a1-antitrypsin gene demonstrated to be essential for tissue- transcription of the PEPCK gene by insulin (23). Transcrip- specific expression (20). Elements containing the same AT- tion of the PEPCK gene was inhibited while the glucokinase TAAC core sequence are located in the promoters of the a- gene was stimulated by insulin treatment, but the kinetics of and P-fibrinogen genes and appear to bind the same protein the changes in the transcription rates for the two genes were (20). The sequence ATTAAC is located in the promoter remarkably similar. In the diabetic animal the PEPCK gene region of the glucokinase gene (positions -171 to -166). is maximally stimulated (almost 7000 ppm) while the glucoki- Liver factor Al, another protein that may be important for nase gene is virtually inactive. Thus, these promoters have liver-specific gene expression, binds to the DNA sequence dramatically different basal activities in absence of insulin, TG(G/A)(A/C)CC (21). This motif is present in the regula- and in the presence of insulin the transcription of one gene is tory region of the human a1-antitrypsin, apolipoprotein Al, stimulated while that of the other is inhibited. and haptoglobin-related genes (21). Several copies of this Insulin response elements (IREs) probably reside up- DNA sequence are located in the 5' flanking region of the stream of genes regulated by insulin. In addition to PEPCK glucokinase gene. Single copies of the sequence lie at posi- (23) and glucokinase, transcription of the growth hormone tions -795 to -790 and -684 to -679, while three tandem (24), glyceraldehyde-3-phosphate dehydrogenase (25), gene repeats of the sequence, TGGCCC, occur between positions 33 (26, 27), c-fos (28), and amylase (29) genes is regulated by -70 and -53. insulin. A consensus IRE has not been identified; however, Insulin Regulates Glucokinase mRNA Synthesis in the Liver. when 5' flanking DNA from some of these genes is linked to Our results indicate that the increase of glucokinase mRNA reporter genes and expressed either in responsive cell lines or in response to insulin treatment of diabetic rats occurs in transgenic mice, the expected effect of insulin is conferred

-1448 hG&TtrC&TcTAhfGCAL...ii,.L..1IALb,,L'.AA.J.LLi.,L'.Af'". -1400 cAGTACT -1300_ -1200 GGAGGACACTT ATACA -1100 Ac_ -1000

900 GGcC&GCTrrGCTCTATr!TGGGMAGcGATCMT.TGTCAAGGGGTGAcC&TACTGCcCAATCrrGGGAGMcCTGGC&TrccX&CAGGC&ccCYA.G - 800A FIG. 5. Sequence of the glucokinase - 700 AGGAAGGTcTGCTCTAMGCTCCTGGTrGCGCMT-R-S XAAT promoter region. This figure shows the sequence of the 1448 bases of DNA that - 500 flank the 5' side of the transcription ini- - 500 tiation site and the 130 bases of sequence (b) initia- - 400 COGGCAGGTGECC>(ATCCCACCGGGTCCCCA on the 3' side of the transcription tion site. Putative regulatory motifs, de- - 300 TATTCACAATTAAGCCT CAC~:IG&CC~:GCATGAAGGTG scribed in the Discussion, are underlined and designated as: (a), TATA box; (b), (C ) ----_ -- Spl binding site; (c), hepatocyte nuclear 100 (d) (d) (d) (a) factor I binding site; and (d), liver factor- + 1 AGrGlCCTGAGTTCrrCrTTCFGGcATcCTcGGcCGAcAGTCCrGcAC&I ccCATc cTCTACc Al binding site. The translation initiation +128 + 101 MC~~alT codon, ATG, is located at position and is designated as (e). Downloaded by guest on October 1, 2021 4842 Biochemistry: Magnuson et al. Proc. Natl. Acad Sci. USA 86 (1989) 3. lynedjian, P. B., Mobius, G., Seitz, H. J., Wollheim, C. B. & Renold, A. E. (1986) Proc. Nati. Acad. Sci. USA 83, 1998- 2001. G) c 4. Sibrowski, W. & Seitz, H. J. Chem. - 0 (1984) J. Biol. 259, .2 343-346. 5. Bedoya, F. J., Matschinsky, F. M., Shimizu, T., O'Neill, J. J. cs & Appel, M. C. (1986) J. Biol. Chem. 261, 10760-10764. -- 6. E V 0 Lawrence, G. M. & Trayer, I. A. (1984) Comp. Biochem. a- _~~~~~~~~~~~~~~~~ Physiol. B 79, 233-238. 0%a - _. 7. Andreone, T. L., Printz, R. L., Pilkis, S. J., Magnuson, M. A. o~~~~~~~~~~~~~0 & Granner, D. K. (1989) J. Biol. Chem. 264, 363-369. 8. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular CD Cloning: A Laboratory Manual (Cold Spring Harbor Labs., Cold Spring Harbor, NY). 9. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, 0 30 60 90 120 W. J. (1979) Biochemistry 18, 5294-5299. Insulin Treatment Time 10. Dente, L., Cesareni, G. & Cortese, R. (1983) Nucleic Acids (min) Res. 11, 1645-1655. 11. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. FIG. 6. Effect of insulin on transcription of the glucokinase and Acad. Sci. USA 74, 5463-5467. PEPCK genes. Rats made diabetic with streptozotocin were treated 12. Beale, E., Andreone, T., Koch, S., Granner, M. & Granner, with insulin for various times, then the hepatic nuclei were isolated D. K. (1984) Diabetes 33, 328-332. and used to quantitate transcription of the PEPCK and glucokinase 13. Schibler, U., Hagenbuchle, O., Wellaur, P. K. & Pittet, A. C. genes. Each data point represents the average ofduplicate transcrip- (1983) Cell 33, 501-508. tion assays using nuclei isolated from two rats for each treatment 14. Sasaki, K., Cripe, T. P., Koch, S. R., Andreone, T. L., Pe- condition. The scale, in parts per million (ppm) ofRNA transcription, tersen, D. D., Beale, E. G. & Granner, D. K. (1984) J. Biol. is expanded 10 times for the glucokinase gene (right axis) compared Chem. 259, 15242-15251. to that for the PEPCK gene (left axis). 15. McKnight, G. S. & Palmiter, R. D. (1979) J. Biol. Chem. 254, 9050-9058. 16. Beale, E. G., Chrapkiewicz, N. B., Scoble, H. A., Metz, R. J., upon the reporter genes (30). It will be interesting to compare Quick, D. P., Noble, R. L., Donelson, J. E., Biemann, K. & the IREs from a gene stimulated by insulin (e.g., glucokinase) Granner, D. K. (1985) J. Biol. Chem. 260, 10748-10760. with one inhibited by insulin (e.g., PEPCK) to determine 17. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. 50, whether similar or different IREs are involved and whether 349-383. similar or different transcription factors are involved. 18. Kadonaga, J. T., Jones, K. A. & Tjian, R. (1986) Trends Biochem. 11, 20-23. Concluding Statement. The availability of the glucokinase 19. Dynan, W. S. & Tjian, R. (1985) Nature (London) 316, 774- gene, including significant 5' flanking DNA, makes possible 778.- the construction of fusion genes that can be used to identify 20. Courtois, G., Morgan, J. G., Campbell, L. A., Fourel, G. & the elements that confer tissue-specific and hormone- Crabtree, G. R. (1987) Science 238, 688-692. regulated expression. The characterization of this gene also 21. Hardon, E. M., Frian, M., Paonessa, G. & Cortese, R. (1988) EMBO J. represents an 7, 1711-1719. important step toward elucidating the evolution 22. lynedjian, P. B., Ginovci, A. & Renold, A. E. (1988) J. Biol. of the hexokinase enzyme family. Chem. 263, 740-744. 23. Magnuson, M. A. & Granner, D. K. (1988) Recept. Biochem. Note Added in Proof. Two recent papers present evidence in favor of Methodol. 12B, 189-209. the hypothesis that the mammalian hexokinases of -100 kDa evolved 24. Yamashita, S. & Melmed, S. (1986) J. Clin. Invest. 78, 1008- from an -50-kDa ancestral molecule through a process of gene 1014. duplication and tandem ligation (31, 32). 25. Alexander, M. C., Lomanto, M., Nasrin, N. & Ramaika, C. (1988) Proc. Natl. Acad. Sci. USA 85, 5092-5096. We thank Emmanuel Eusebio, Charles Davis, III, Deborah Ca- 26. Messina, J. L., Hamlin, J., Azizkahn, J. & Larner, J. (1985) plenor, and Elizabeth Zimmerman for their assistance. This work Biochem. Biophys. Res. Commun. 133, 1168-1174. was supported by Grant DK 35107 (D.K.G.) from the National 27. Chu, D. T., Davis, C. M., Chrapkiewicz, N. B. & Granner, Institutes of Health, by the Vanderbilt Diabetes Research and D. K. (1988) J. Biol. Chem. 263, 13007-13011. Training Center (DK 07061), and grants from the Juvenile Diabetes 28. Stumpo, D. J., Stewart, T. N., Gilman, M. Z. & Blackshear, Foundation P. J. (1988) J. Biol. Chem. 263, 1611-1614. (M.A.M. and D.K.G.). M.A.M. is the recipient of a 29. Osborne, L., Rosenberg, M. J., Keller, S. A. & Meisler, M. H. Research Career Development Award from the American Diabetes (1987) Mol. Cell. Biol. 7, 326-334. Association. 30. Magnuson, M. A., Quinn, P. G. & Granner, D. K. (1987) J. Biol. Chem. 262, 14917-14920. 1. Weinhouse, S. (1976) Curr. Top. Cell. Regul. 11, 1-50. 31. Nishi, S., Seino, S. & Bell, G. I. (1988) Biochem. Biophys. Res. 2. Meglasson, M. D., Burch, P. T., Berner, D. K., Najafi, J., Commun. 157, 937-943. Vogin, A. P. & Matschinsky, F. M. (1983) Proc. Natl. Acad. 32. Schwab, D. A. & Wilson, J. E. (1989) Proc. Natl. Acad. Sci. Sci. USA 80, 85-89. USA 86, 2563-2567. Downloaded by guest on October 1, 2021