The human erythrocyte glucose-transporterprotein is a heterogeneously
glycosylated protein of Mr 55,000 (3). The proteins present in rat adipose tissue
and ...
Proc. Natl. Acad. Sci. USA
Vol. 83, pp. 5784-5788, August 1986 Biochemistry
Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein MoRRis J. BIRNBAUM*, HOWARD C. HASPEL, AND ORA M. ROSEN Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division of the Cornell University, Graduate School of Medical Sciences, 1275 York Avenue, New York, NY 10021
Communicated by Jerard Hurwitz, April 15, 1986
Antibody raised against the human erythroABSTRACT cyte glucose transporter identified a recombinant Xgtll bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain. The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier. The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein. As assayed by blot-hybridization analysis, mRNA from insulin-responsive and nonresponsive tissues are indistinguishable. These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver.
MATERIALS AND METHODS Materials. Avian myeloblastosis virus reverse transcriptase was from Life Sciences (St. Petersburg, FL). Mung bean nuclease, nucleoside triphosphates, and EcoRI linkers were from P-L Biochemicals. DNA polymerase I, EcoRI methylase, T4 DNA ligase, and restriction enzymes were from New England Biolabs. Xgtll bacteriophage and Escherichia coli host strain Y1090 were obtained from Promega Biotec (Madison, WI). The X phage packaging extract and radionucleotides were from Amersham. Calf intestinal phosphatase was from Boehringer Mannheim. Sprague-Dawley rats were obtained from the Charles River Laboratories. Measurements of Glucose-Transporter Protein. The specificity of rabbit polyclonal antisera against the purified human erythrocyte transporter, as well as its use for the immunoprecipitation of the products of in vitro translation and the immunoblotting of total cellular membranes, has been described (5). Cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Human liver tissue was obtained from pathological specimens of therapeutic partial hepatectomy and were grossly free of tumor. When used for immunoblotting, the human liver was depleted of erythrocytes by finely mincing the tissue and washing four times (30 min) with sonication buffer at 40C (5). Construction and Screening of a cDNA Library. RNA was
Virtually all mammalian cell plasma membranes contain a system for the stereospecific transport of glucose (1). However, different cell types have at least several functionally distinguishable transport systems with properties consistent with their tissue distribution. In the brush border membranes of kidney and small intestines, which transfer glucose from luminal compartments into the circulation, the transporter is a sodium-dependent active carrier, in contrast to the facilitated transport system present in nonepithelial cells (1). These two transport proteins are also immunologically distinguishable (2). The passive carrier, of which the human erythrocyte protein has been best studied, is stimulated by insulin in specialized tissues (e.g., fat and skeletal muscle). However, there is no evidence for intrinsic differences in the transporter proteins of insulin-responsive and nonresponsive cells (1). The structural basis of these differences in transporter function is a problem of considerable interest. The human erythrocyte glucose-transporter protein is a heterogeneously glycosylated protein of Mr 55,000 (3). The proteins present in rat adipose tissue and cultured mouse fat cells are about the same size and are related immunologically (4, 5). Both murine and human mRNA code for a glucosetransporter protein whose primary translation product is Mr 38,000 as assayed by in vitro translation or metabolic labeling in the presence of an inhibitor of N-linked glycosylation (5). Since polyclonal antisera prepared against the human erythrocyte glucose transporter recognized the nonglycosylated rat protein (unpublished observations) as well as the mature protein on immunoblots (4, 5), it seemed that the antibody might be a useful tool for obtaining the cDNA for the rat glucose transporter. This report describes the primary structure of such a cDNA clone, its remarkable sequence homology to the human glucose transporter (6), and the indication of a genetically unrelated transport system active in human and rat liver.
prepared by guanidinium isothiocyanate (Fluka) disruption of cells and centrifugation through 5.7 M cesium chloride (7), with further purification by oligo(dT)-cellulose chromatography (8). mRNA was translated by using a wheat germ system
(9).
Total adult brain polysomes were prepared as described except that the initial homogenization buffer contained 1% Triton X-100 and 1% sodium deoxycholate and the polysomes were stored at -70°C in buffer containing 500 units of RNasin (Promega Biotec) per ml (10). Immunoadsorption and elution of RNA was by the method of Shapiro and Young (11). cDNA was constructed by oligo(dT) priming of the first strand and self priming of the second strand with cleavage of the hairpin loop by mung bean nuclease (12). The doublestranded cDNA was fractionated by 4% polyacrylamide gel electrophoresis, and all material with mobility slower than a 1.3-kilobase (kb) standard was electroeluted. This material was concentrated by precipitation with ethanol and inserted in Xgtll, which was then packaged and used to infect E. coli Y1090 (12). The library was screened with rabbit antisera, and the second antibody-biotin system (ABC) of Vector Laboratories (Burlingame, CA). Positive clones were plaquepurified twice, from which DNA was prepared (ref. 13, pp. 371-372) and subcloned into pUC19 (14). Restriction frag-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: kb, kilobase(s). *To whom correspondence should be addressed.
5784
Biochemistry: Birnbaum et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
ments were cloned into M13 mpl8 or M13 mpl9 and sequenced by the dideoxy chain termination technique (15). Analysis of RNA and DNA. For blot-hybridization analysis, RNA was denatured, size-fractionated on 1% formaldehyde/ agarose gels and transferred to Schleicher & Schuell BA85 nitrocellulose membranes according to the recommendations of the manufacturer. Probes were prepared by nick-translation (16) or from single-stranded M13 templates by using a 17-nucleotide sequencing primer (New England Biolabs). Hybridizations were performed in solutions containing 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, and 1 M NaCl at 420C, and the blots were washed in 0.1% NaDodS04/0.1x NaCl/Cit (lx NaCl/Cit is 0.15 M NaCl/0.015 M sodium citrate) at 51'C. Blots were exposed to Kodak XAR5 film at -70'C with a Cronex intensifying screen (DuPont). High molecular weight DNA (ref. 13, pp. 280-281) treated with the appropriate restriction endonuclease was submitted to 1% agarose gel electrophoresis, transferred to nitrocellulose, and hybridized to a nick-translated probe (17). Highstringency hybridization was as described above. Lowstringency conditions were the same except that the hybridization solution contained 25% formamide and the blots were washed at room temperature.
RESULTS Preparation and Screening of an Enriched cDNA Library. Screening normal rat tissues for glucose transporter mRNA by in vitro translation and immunoprecipitation (5) revealed that rat brain was a relatively abundant source of transporter mRNA. In vitro translation of poly(A)+ RNA from rat brain showed a product of Mr 38,000 and an apparently lessabundant species of Mr =58,000 (Fig. 1, lane 1). The presence of nonradioactive human erythrocyte transporter during treatment with antibody completely interfered with precipitation of the Mr 38,000 protein but only partially competed with the larger polypeptide for antibody (Fig. 1, lane 2). As described previously, the Mr 38,000 species represents the primary translation product of the glucose-transporter protein (5). Total polysomes were prepared from adult rat brain and enriched for glucose-transporter mRNA by using rabbit 1 2 3 4 5
6
7
FIG. 1. In vitro translation and immunoprecipitation of polysomal RNA. Poly(A)' RNA was translated, precipitated with antisera against the glucose transporter, and submitted to 11% polyacrylamide gel electrophoresis (5). Lanes: 1, 2, and 3, rat brain RNA (1 Mg); 4, polysomal RNA (0.5 Mg); 5, immunoselected polysomal RNA (0.2 Mg); 6, nonselected polysomal RNA (0.6 Mg); 7,
molecular weight standards 200,000, 116,000, 97,400, 68,000, and 43,000. Nonradioactive erythrocyte glucose transporter (1 Mig) was present during the immunoprecipitation of the protein in lane 2. Lane 3 represents protein precipitated with nonimmune serum. Amounts of polysomal RNA translated were calculated from the incorporation of [35S]methionine into protein relative to 1 ug of brain RNA.
5785
polyclonal antisera against the human erythrocyte transporter. While the isolation of polysomes resulted in some selective loss of transporter mRNA (Fig. 1, lane 4), the subsequent immunoadsorption provided an overall enrichment of -25fold as estimated by quantitative laser densitometry (Fig. 1, lane 5). In other experiments, enrichments as high as 50-fold were obtained. This RNA was used to construct a cDNA library in the bacterial expression vector Xgtll. Unamplified plaques (-2 x 105) were screened with antisera, yielding 15 positive signals, all of which were found to cross-hybridize. DNA was prepared from these, and the largest insert of about 1.9 kb was subcloned into pUC19 to give prGTH-14. The insert was sequenced and the 5' end was used to make a probe with which to rescreen the amplified Xgtll library by nucleic acid hybridization. Thus, a bacteriophage containing a 2.6-kb insert was identified, and the cDNA was cloned into the EcoRI site of pUC19 to give prGT4-12. Deduced Primary Structure of the Rat Brain Glucose Transporter. The nucleotide sequence and deduced amino acid sequence of the rat brain glucose transporter is shown in Fig. 2. It represents the complete sequence of the insert in prGTH-14 as well as the 5' 800 base pairs of the insert in prGT4-12. The remainder of the two cDNAs were identical, based on analysis with multiple restriction enzymes. There is a single long open reading frame extending from nucleotide 208 to 1683 that predicts a 492-amino acid protein of Mr 56,133. The discrepancy between the deduced molecular mass and apparent Mr of the transporter and its anomalous behavior on polyacrylamide gel electrophoresis have been noted (5, 6). Comparison of the rat sequence to that predicted from a cDNA obtained from a library prepared from a human hepatoma cell line (HepG2) (6) indicated identity at 97.6% of amino acid residues. Of the few amino acid differences, most were conservative substitutions (Fig. 2). A comparison of the rat brain and human hepatoma noncoding sequence is shown in Fig. 3. The homology between the nucleotide sequence, which is about 89% in the coding region, extends only partly into the 5' untranslated region. The nucleotide homology is 78% over the 36 nucleotides immediately adjacent to the putative ATG initiation codon, and there is essentially no homology in the 5'-most sequence available for comparison. On the other hand, the 3' untranslated regions show strong homology from the putative termination codon to the polyadenylylation site in the rat cDNA. At this point the sequences diverge, the human cDNA extending for at least 300 additional nucleotide pairs. The sequence ATATAAA, which closely resembles the proposed consensus sequence for termination (19), is located about 20 nucleotides upstream from the putative poly(A) addition site in the rat cDNA and is present in the human sequence. Three additional rat brain cDNAs were sequenced at their 3' ends and found to have the same terminal sequence as the insert from prGTH-14 (data not shown). Structure of the Glucose-Transporter Gene. Southern blot analysis of high molecular weight DNA isolated from rat brain after cleavage with three different restriction enzymes revealed a simple pattern consistent with the presence of a single gene (Fig. 4). A similarly prepared blot showed an identical hybridization pattern when probed at low stringency; this is strong evidence against the presence of other unique sequences related to the transporter cDNA in the rat genome. Tissue Distribution of Glucose-Transporter mRNA. Blothybridization analysis of total RNA from rat brain revealed a single hybridizing species of 2.9 kb (Fig. 5A, lane 1). Prolonged exposure of similar blots of brain poly(A)+ RNA showed another less-abundant RNA of 5.8 kb, which hybridized to the transporter cDNA (data not shown). Surprisingly, an equivalent amount of RNA from rat liver showed only a faint band hybridizing to the same probe. RNA from Fao
Proc. Natl. Acad Sci. USA 83
Biochemistry: Birnbaum et al.
5786
(1986)
1
GGGCGGCCAAT;GCGGCGGTCCTATMAAAGGCAGCTCCGCGCGCTCTCTTCCTAAGAACACAAGATCCCTTGTGGAGTGTCGGTTAGGTTGCAGGGTCTTAAGTGAGTCAGGGCGC
121
GGAGGTCCGGCGGvGAGACGCATAGTCACAGAACGTCCATTCTCCGTTTCACAGCCCG;CACAGCTTGAGCCTCGAGCGCAGCGCGGCCAT(GGAGCCCAGCAGCAAGAAGGTGACGGGCCGC
Leu
MetGluProSerSerLysLysValThrGlyArg Val
241
Leuk~etLeuAlaValGlyGlyAlaValLeuGlySerLeuGlnPheGlyTyrAsnThrGlyValIleAsnAlaProGlnLysValIleGluGluPheTyrAsnGlnThtTrpAsnHisArg
CTTATGTTGGCCGTGGGAGGGGCAGTGCTCGGATCCCTGCAGTTCGGCTATAACACCGGTGTCATCAACGCCCCCCAGAAGGTAATTGAGGAGTTCTACAATCAAACATGGAACCACCGC LeuPro
TyrGlyGluSerIleProSerThrThrLeuThrThrLeuTrpSerLeuSerValAlaIlePheSerValGlyGlyMetIleGlySerPheSerValGlyLeuPheValAsnArgPheGly
361
TATGGAGAGTCCATECCATCCACCACACTCACCACACTCTGGTCTCTCTCCGTGGCCATCTTCTCTGTCGGGGGCATGATTGGTTCCTTCTCTGTGGGCCTCTTTGTTAATCGCTTTGGC
481
ArgArgAsnSerMetLeutinetMetAsnLeuLeuAlaPheValSerAlaValLeuMetGlyPheSerLysLeuGlyLysSerPheGluMetLeuIleLeuiGlyArgPheIleIleGlyVal AGGCGGACTCCATGCTATGAirAAccTGTTGGCCTTTGTGTCTG;CCGTG.CTTAT.GGGTTCTCCAAACTCflGGCAAGTCCTTTGAGATGCTGATCCTGGGCCGCTTCATC-ATTGGAGTG Phe
TyrCysGlyLeuThrThrGlyPheVilP~roMetTyrValGlyGluValSerProThrAlaLeuArgGlyAlaLeu~lyThrLeuHisGlnLeuGlyIleValValGlyIleLeuIleAla
TACT~GoGCC>GACCACCGGCTTTGMCCCATGTATGTGGGGGAGGTGTCACCCACAGCTCTTCGTGGAGCCCTGGGCACCCTGCACCAGCTGGGCATCGTCGTTGGGATCCTTATTGCC $: Lys Ile Val GClsValPIt'kGlyLeuAXspLer lelZuLGlyAenAliAepLeu'rpProLueueubuSerV4l l lePhe l lePruAa lhLeuLeuG I riCysIeLeuLeuPruPlieCysPruGluSerPru 721 CAGGqwGTTAGACTCCATCA'f:GCATGCAGACTltGTGCCTCTACTGCTCAGTCTCATsTCAGCCCTCCTACAGTGTATCCIlurrsCCTICTGCCCTGAGAGCCC
601
His
Ser
ArgPheLeuLeuIleAsnArgAsnGluGluAsnArgAlaLysSerValLeuLysLysLeuArgGlyThrAlaAspValThrArgAspLeuGlnGluMetLysGluGluGlyArgGlnMet b4l
CGiCTTCCTGCTCACATCGTAACGAGGAGAACCGGGCCAAGAGGTGWClm
961
AIrGGGAAGAAGG
AAAGC' rTCGAGGGACAGCCGATGTGACCCGAG;ACCTGCAGGAGATGAMGAAGAGGGTCGGCAGATG
MetArgGluLbysLysValThrIleLeuGluLeuPheA~rgSerProAlaTyrArgGlnProIleLeuIleAlaValValLeuGlnLeuSerGlnGlnLeuSerGlyIleAsnAlaValPhe
TCACCATT~ rT:GAGCTGTTCCGCTCACCCGCCTACCGCCAGCCCATCCTCATCGCCGTGGTGCTGCAGCTGTCCCAGCAGCTGTCGGGCATCAAT:CoTGTGTTC
TyrTyrSerThrSerIlePheGluLysAlaGlyValGlnGlnProValTyrAlaTh~rIleGlySerGlyIleValAsnThrAlaPheThrValValSerLeuPheValValGluArgAla 1081
TACTACTCAACGAGCATCTTCGAGAAGGCAGGTGTGCAGCAGCCTGTGTATGCCACCATCGGCTCGGGTATCGTCAACACGGCCTTCACTGTGGTGTCGCTGTTCGTCGTGGAGCGAGCT Ile
1201
GlyArgArgThrLeuHisLeuIleGlyLeuAlaGlyMetAlaGlyCysAlaValLeuMetThrIleAlaLeuAlaLeuLeuGluGlnLeuProTrpMetSerTyrLeuSerIleValAla
GGCCGTCGGACCCTGCATCTCATTGGTCTGGCTGGCATGGCGGGCTGTGCTGTGCTCATiGACCATCGCCCTGGCCCTGCTGGAGCAGCTGCCCTGGATGTCCTATCTGAGTATCGTGGCC lle
IlePheGlyPheValAlaPhePheLGluValGlyProGlyProIleProTrpPheIleValAlaGluLeuPheSerGlnGlyProArgProAlaAlaValAlayaLAlaGlyPheSerAsn
1321
ATCTTTGCTTriTGoGCCTTCTTTGAAGTAGGCCCTGGTCCTA1TCATGGTTCATTGTGGCCGAGCTGTTCAGCCAGGGGCCCCGACCTGCTGCTGTTGCTGTGGC1'GGCTTCTCTAAC
1441
TGGACCTCAAACTTCATCGTGGGCATGTGCTTCCAATATGTGGAGCAACTGTGTGGCCCCTACGTCTTCATCATCTTCACGGTGCTGCTGGTACTCTTCTTCATCTTCACCTACTTCMAA
1561
GTTCCTGGCCAAAGGCCGCACCql'CGATGAGATCGCIIITCCGGCTTCCGGCAGGGCGGGTGCCAGCCAGAGCGACAAGACACCIIGAGGAGCTC'ln'ICCACCC''CluGCGGCl'GACTCCCAA
TrpThrSerAsnPhelleValGlyt~etCysPheGlnTyrValGluGlnLeuCysGlyProTyrValPheIleIlePheThrValL~euLeuValLeuPhePheIlePheThr'IyrPheLys
Voal~rcAluTtbrl~ysGlyAryThrPtleAspGlulleAlciSerGlyPheAryGlnGlyGlyAldSearGlnSerAspLy..;,rhrProGltiGluLeuPhetlisProLeuGlyAla~spSerGln
ValOP 1681 1 801
1921 2041 2161
2281 2401 2521
GTGTCAGGAGCCCACAGCCAGTCCCGCCTGCTCCCAGCAGCCCCGAGGATCTCTCTGGAGCACAGGCAGCTAGATGAGACCTCTTCCMAACTGACAGATCTCGGGCGAGCCGGGCCTGGG CACCi-ltTATCAGCAAT GAGTCCAGAAGAATATTCAGGACT TTATGGCTCCAGAATTTTTM~AATGAsCAAGACTGTTGCTCAGATCTATTCAGATAAGCAGCAGATTTTATAA TTT ATA TGTTTGTTATTA'11rTTTTTl~iTTTTATCAGCCACTCTCCTATCTCCACACTGTAGTCTTCACCTTGATTGGCCTAGTGCCTGAGGGTGGAGACCACGCCCTGTCCAGA CACA~r.C TCqGCAAGCTAATCTGCTAGGGCTGGACCTTTGGCCAAGGACACACGAATACTGAACAATGGCTAGGAGGCT'rrACCGCAGG;AGGCGGTAGCTGCCACCCACTTCTGCA
GGCCTrpATCTCGACACCATAGGGGTCCAGGCTCCATTTAGGATTCGCCCATTCCTGTCTCTTCCAACTCAACCAACCACTCGATTMATCTTTCCTTGCCTGAGACCAGTTGAAAGCACT GGAGTCCAGGGAGGAGAGGGAAGGGCCAGGCToGGGCTGCCAGGTTCAGGTCTCCTGTGCACTGAGGGCCAGACAAACACCA'I'AGAAGGACCTCGGAGGCTGAGAAC1'TACTiGCTGAAGA cAcGGAcAcTdCTG;cccTGcTGTGTATAGATGG.,AAGATATTTATATATTTTTTGGTTGTCTATTAAATACAGACACTAAGTTATAGTATATATCTGGACAAACCCACTTGTAAATACACC M
C.AAAcTCCmTAMcTTTAccTMAGCAGATATAAATGGCTGGTTTTTAGAAAAAAnAAAAAAAAAAA
FIG. 2. Translated nucleotide sequence of the rat brain glucose-transporter' cDNA. Where the human hepatoma transporter amino acid sequence (6) is different from the rat sequence, the residue present in the former is indicated over the correspondent rodent amino acid.
cells, a Well-differentiated rat hepatoma cell line (20), had levels of-transporter that were less than brain levels by only a factor of 2-3 (Fig. 5A). The disparity between transporter mRNA levels in adult rat brain (and liver was also true for 10-day-old rats (Fig. SB). In both cases, the presence of intact liver RNA was verified by reprobing the blots with a rat albumin cDNA probe (data not shown). Enrichment of mRNA by oligo(dT)-celiulose selection confirmed the presence of relatively small amounts of transporter mRNA in rat liver (Fig.. 5C). Two micrograms of poly(A)+ RNA from brain (lane 1) or Fao cells (lane 5) produced a much stronger hybridization signal than did 10 ,ug of poly(4) liver RNA. By using scanning densitometry, it was estimated that in poly(A)+ RNA, glucose transporter mRNA was less abundant in liver than in Fao cells and brain by factors of 14 and 40, respectively. Fig. 5D shows a blot-hybridization of total RNA from several additional tissues; rat brain, adipose tissue, and kidney all contained a 2.9-kb transcript, the latter two in sligltly lower abundance, which hybridized to the transporter cDNA. It was important to correlate the tissue differences in transporter mRNA levels with immunologically identifiable protein. As assayed by immunoblotting, no glucose-transporter protein was detected in rat liver membranes under conditions that readily demonstrated transporter of the appropriate electrophoretic mobility in membranes from rat brain and Fao cells (Fig. 6). There was no detectable transporter protein in normal human liver in spite of substantial amounts in IlepG2 membranes (Fig. 6). HepG2 cells contain glucose transporter mRNA levels similar to that in rat brain when
blot-hybridizations were probed at moderately high stringen-
cy with the rat cDNA probe; no transporter mRNA has been detected in RNA prepared from normal human liver, though it has been difficult to obtain human hepatic RNA completely
free of degradation (data not shown). The two species present in rat brain (Fig. 6, lane 6) probably represent alternative glycosylation, since treatment with endoglycosidase F reduced them to a single polypeptide that comigrated with the deglycosylated human erythrocyte protein (data not shown).
DISCUSSION Since facilitated glucose transport represents a process fundamental to energy metabolism in all vertebrate cells, one might expect significant conservation in the primary structure of the transporter protein among distantly related species. However, the >97% sequence identity over 492 amino
acids between the human and rat hexose carrier is extraordinary. Based on current ideas about evolution and sequence divergence, this would suggest that essentially all regions of the protein, including the putative hydrophobic transmembrane domains (6), are functionally important. In addition to transporting glucose, the carrier protein is regulated at the level of its subcellular distribution or concentration by such perturbations as insulin treatment (21, 22), cellular starvation (23), and possibly oncogenic transformation (24); thus, it is likely that certain domains- of the protein are conserved because of their importance to these regulatory functions. Also of interest is the striking nucleotide homology between the rat and human cDNAs in the 3' untranslated sequence, though the human mRNA apparently extends for at least 300 nucteotides further. Whether the alternative sites of
Biochemistry: Birnbaum et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
5787
rgt
WGGGCGGGCCAAI'GGCGGCGGTCCTATAAAAAGGCAGCTCCGCGCGCTCTCTTCCTAAGAACACAAGAATCCCl'TGTGGAGTGTCGGTTAGGTTGCAGGGTCTTA-AGTGAGT-CAGGG
hgt
TAGTCGCGGGTCCCCGAGTGAGCACGCCAGGGAGCAGGAGACCAAACGACGGGGGI'CGGAGTCAGAGTCGCAGTGGGAGTCCCCGG 10
rgt
30
40
50
60
70
80
CGCCGAGGTCCGGCGGGAGACGCATA--GTCAC-AGAACGTCCATTCTCCGTTTCACAGCCCGCACAGCTTGAGCCTCGAGCGCAGCGCGGCC ::: ;::::: ::: :: X ,::: :: :X ACCGGAGCACGAGCCTGAGC;G&GAGAGCGCCGCTCGCACGCCCGTCGCCACCCGCGTACCCGGCGCAGCCAGAGCCACCAGCGCAGCGCTGCC 90 ::
:
hgt
20
:
:
:
:
11Q
100
:
120
::
130
140
::
:::::
150
160
:
:
:
:
170
180
rgt
GGAGCCCACAGCCAGTCCCGCCTGCTCCCAGCAGCCCCGAGGATCTCTCTGGAGCACAGGCAGCTAGATGAGACCTCTT
hgt
AGCTGTTCCATCCCCTGzGGGGCTG;ATTCCCAAGTGTGAGTCGCCCCAGATCACCAGCCCGGCCTGCTCCCAGCAGCCCTAAGGATCTCTCAGGAGCACAGGCAGCTGGAI'GAGACTTC-1630
1640
1650
1660
1670
1680
1690
1700
1710
1720
1730
rgt
CCAACTGACAGAlTCTCGGGCGAGCCGGGCCT-GGGCACCT8TTCTTCAGTCAGCAATGAAGTCCAGAAGAATATTCAGGACaY TGATGGCTCCAGAATTTTTAATGAAAGCAAGACTGTT
h.gt
CAACCTCAGATGTCAGCCGAGCCGGGCCTGG ,GGCTCCTTTCTCCAGCCAGCAATGATGTCCAGAAGAATAT'rCAGGAC-TTAACGGCTCCAGGA-TrrTACAAAAGCAAGACl'TT 1740 1750 1760 1770 1780 1790 1800 1810 1820 1830 1840 1850
-GATCTATTCAGATAGCAGCAGATTATAAMMATTACTGATTGTTATTA TCAC-ACTCTCCTATCTCCACACTGTAGTCTTCACCIT2GATTG
rgt
GCT
tigt
GCTCAATCTATTCAGACAAGCAACAGGTTTATAATTTTTTTATACTGATGTTATTT1860
rgt h gt
1870
hg t
1900
1910
X
.....
ATAICAGCCTGAGTCTCCTGGCCCACATCCCAGGCTTCACCCTCAATG 1920
1930
1940
1950
1960
GCCTALGII;CCTGAGGGfGAGACCACGCCCTGTCCAGACACATGCCTTCTTTGCCAAGCTAATCTGTAGGGCTGGACCq'MTGGCC-AAGGACACACGAATACTGAACAATG-GCTAGGAG GTTCCATG;CC~xGGGTS;GAGACTAAGCCCTGTCGAGACACTTGCCTTCTTCACCCAGCTAATCTGTAGGGCl (;GAC CTA1S TCCTAAGGACACACTAAT- -CGAACTATGAACTACAAA 1970
rgt
1890
1880
1980
1990
2000
2010
2020
2030
2040
2050
2070
2060
2080
GC7'T-TACCGCAGGAGGCGGTAGCTGCCACCCACTTCTGCAGGCCTGGATCTCGACACCATAGGGGTCCAGGCTCCATTTAGGATTCGCCCATTCCTGTCTCTTCCAACTCAACCAACCA GCTTTTCCCAGGAGGaT;GCTATCGGCCACCC-GTTCTGCTUGGCCTGGATCTCCCCACTCTAGGGGT-CAGGCTCCA -TTAGGATTTGCCCCTTCCCATCTCTTCCTACCCAACCACTCA 2120
2110
2100
2090
2140
2130
2150
2170
2160
2180
2190
2200
hg t
CTCGATTAATCTTTCCTTGCCCTGAGACCAGTT'GAAAGCACTGGAGTGC-AGGGAGGAGA-GGGAAGGGCCAGGCTrGGGCTGCCAGG'rfCAGGTCTCCfrGTGCACTGAGGGCCA.GAr-AtAAC - - -ATTAATCT7'TCTTTACCTGAGACCAGTTGGGAGCACTGGAGTGCAGGGAGGAGAGGGGAAGGGCCAGTCTGGGCTGCCGGGTTCTAGTCTCCTTTrGCACTGAGGGCCACACTATTrA
rgt
CCAAGAA- -GGACCT- -CGGAGGC7WZMACTTACTGCT- --GAAGACACGGACACTCCTiGcCCTGCTGTGTATrAGATGGAAGATAT'1'1'ATATAT-'I'rTTTGGTTGTC--TATTAAAT
hgt
CCAlG8GCCAAGAGGCAG~vACCTGC AAACTCACTGCTCAAGAAGACATGGAGACTCC'ICCC'YTGTTGTG'l'A'l'AGA'1YS(-AAGATAlM"I'Al'ATATA'rlrGTTIGTCAATAiTTAAAT 2340 2350 2330 2360 2370 2380 2390 2400 :2410 2420 2430 2440
r gt
ACAGCACTAAGTTsATAGTATAVTATCTGACAAAC CCAC TTG TAATA CAC CAACAAAC TCCTG TAACTTTAC C '1'AAG CAGA TAT'AAA TGG C 1x;GTlwl'TAGAAAMAAMAAMAA_
hgt
ACAGlACACTAACTTATAG--TATATC7W;ACAAGCCAACTlGTAAATACACCACCTCACTCCTiGTTAC-TTACCTAAACAGATATAAATGGCTGG~rMAGAAACATzGGTT7GAATG
r gt
2210
2220
2450
2460
2240
2230
2470
2250
2480
2490
2280
2270
2260
2510
2500
2290
2300
2530
2520
2310
2540
2320
2550
FIG. 3. Comparison of the 5' nontranslated (Upper) and 3' nontranslated (Lower) sequence of the rat brain glucose-transporter cDNA (lines rgt) and the human hepatoma glucose-transporter cDNA (6) (lines hgt) by the algorithm of Lipman and Pearson (18). The numbering is according to the human sequence, starting with the first base in the cDNA.
polyadenylylation represent differences between the species or true alternative termination within a single organism remains to be determined; however, blot-hybridization analyses indicate a single mRNA of similar size in rat brain and
-
aE
M
M
23.1
(D
i
9.46.64.4-
-0
I- I~-A
E -0 al C I
a
m
_ _
6,
_
:t N
>
._
0 0 LL.
.
-i
m
:Lt
0
s,
,P..1
*:I
I
ZL
I.. m
C
2 U
.'
@
°
..J0E
0
m
mm
B .'
'-4
D
C A
B
A
HepG2 cells (unpublished observation). The 5' flanking sequence of the human and rat cDNAs do not show homology
o @1 a -2 V
0-
C Y
4
LI-
*v
Ir
,"s *'_
2.3 -
2.0
0.6
41
-
0 FIG. 4. Southern blot analysis of rat genomic DNA. High molecular weight DNA (10 ug) from rat brain treated with the indicated restriction endonuclease was fractionated by 1% agarose electrophoresis, transferred to nitrocellulose, and hybridized to nick-translated prGT4-12 under either high stringency (A) or low stringency (B). The molecular sizes in kilobase pairs of the standards (M) are indicated.
1 2 3 1 2 1 2 3 4 5 1 2 3 FIG. 5. Blot-hybridization analysis of the glucose-transporter mRNA. RNA was size-fractionated on formaldahyde/agarose gels, transferred to nitrocellulose, and hybridized to nick-translated prGT4-12. RNA was visualized by ultraviolet shadowing to ensure that the total RNA was intact and to allow calculation of molecular weight based on migration of the 28S and 18S ribosomal subunits. (A) Total RNA (20 jig). Lanes: 1, rat brain; 2, rat liver; 3, Fao cells. (B) Total RNA from 10-day-old rats (20 ,g). Lanes: 1, brain; 2, liver. (C) Lanes: 1, poly(A)+ rat brain (2 ug); 2, poly(A)+ rat liver (5 Mig); 3, poly(A)+ rat liver (10 ,ug); 4, total Fao (20 ,ug); 5, poly(A)+ Fao (2 M1g). (D) Total RNA (20 Mg). Lanes: 1, rat brain; 2, rat kidney; 3, rat adipose tissue.
5788
Biochemistry: Birnbaum et al. a)
) 0
cL
I
g _
.>
4).
.L o
