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• W3048383877 abstract "•In the 1970s we developed technologies to elucidate the regulation of translation of α- and β-globin mRNAs.•In parallel we determined the primary defect in beta thalassemia.•In the 1980s we developed technologies to clone and characterize the Epo receptor and the red cell membrane proteins GLUT1 and Band 3.•In the 2000s we developed other technologies to identify and elucidate the function of many microRNAs and long noncoding RNAs in red cell development.•We currently use single-cell transcriptomics to elucidate how BFU-E progenitors balance the need for more progenitors with the need for terminally differentiated erythroid cells.•We have identified drugs potentially useful for treating Epo-resistant anemias including Diamond Blackfan anemia.•Throughout I describe some of the lessons I learned in managing a large number of diverse fellows and projects. I am deeply honored to receive the International Society for Experimental Hematology (ISEH) 2020 Donald Metcalf Lecture Award. Although I am not a physician and have had no formal training in hematology, I have had the privilege of working with some of the top hematologists in the world, beginning in 1970 when Dr. David Nathan was a sabbatical visitor in my laboratory and introduced me to hematological diseases. And I take this award to be given not just to me but to an exceptional group of MD and PhD trainees and visitors in my laboratory who have cloned and characterized many proteins and RNAs important for red cell development and function. Many of these projects involved taking exceptionally large risks in developing and employing novel experimental technologies. Unsurprisingly, all of these trainees have gone on to become leaders in hematology and, more broadly, in molecular cell biology and molecular medicine. To illustrate some of the challenges we have faced and the technologies we had to develop, I have chosen several of our multiyear projects to describe in some detail: elucidating the regulation of translation of α- and β-globin mRNAs and the defect in beta thalassemia in the 1970s; cloning the Epo receptor and several red cell membrane proteins in the 1980s and 1990s; and more recently, determining the function of many microRNAs and long noncoding RNAs in red cell development. I summarize how we are currently utilizing single-cell transcriptomics (scRNAseq) to understand how dividing transit-amplifying burst-forming unit erythroid progenitors balance the need for more progenitor cells with the need for terminally differentiated erythroid cells, and to identify drugs potentially useful in treating Epo-resistant anemias such as Diamond Blackfan anemia. I hope that the lessons I learned in managing these diverse fellows and projects, initially without having grants to support them, will be helpful to others who would like to undertake ambitious and important lines of research in hematology. I am deeply honored to receive the International Society for Experimental Hematology (ISEH) 2020 Donald Metcalf Lecture Award. Although I am not a physician and have had no formal training in hematology, I have had the privilege of working with some of the top hematologists in the world, beginning in 1970 when Dr. David Nathan was a sabbatical visitor in my laboratory and introduced me to hematological diseases. And I take this award to be given not just to me but to an exceptional group of MD and PhD trainees and visitors in my laboratory who have cloned and characterized many proteins and RNAs important for red cell development and function. Many of these projects involved taking exceptionally large risks in developing and employing novel experimental technologies. Unsurprisingly, all of these trainees have gone on to become leaders in hematology and, more broadly, in molecular cell biology and molecular medicine. To illustrate some of the challenges we have faced and the technologies we had to develop, I have chosen several of our multiyear projects to describe in some detail: elucidating the regulation of translation of α- and β-globin mRNAs and the defect in beta thalassemia in the 1970s; cloning the Epo receptor and several red cell membrane proteins in the 1980s and 1990s; and more recently, determining the function of many microRNAs and long noncoding RNAs in red cell development. I summarize how we are currently utilizing single-cell transcriptomics (scRNAseq) to understand how dividing transit-amplifying burst-forming unit erythroid progenitors balance the need for more progenitor cells with the need for terminally differentiated erythroid cells, and to identify drugs potentially useful in treating Epo-resistant anemias such as Diamond Blackfan anemia. I hope that the lessons I learned in managing these diverse fellows and projects, initially without having grants to support them, will be helpful to others who would like to undertake ambitious and important lines of research in hematology. Throughout my entire adult life, I have been fascinated by red blood cells, their membrane proteins, and their biogenesis. It all began in the summer of 1958, after 11th grade, when I spent the first of three summers at Western Reserve (now Case Western Reserve) Medical School with Dr. Robert Eckel studying potassium transport in red blood cells. We were trying to determine the glycolytic intermediates that powered K+ uptake, and among other techniques I used flame photometry to measure the K+ concentration in red cells and the Warburg apparatus to measure oxygen uptake and CO2 emission. This led to my first scientific publications [1Eckel RE Lodish H Metabolism during potassium transport in human red cells.J Clin Invest. 1961; 40: 1035-1036Google Scholar,2Eckel RE Rizzo SC Lodish H Berggren AB Potassium transport and control of glycolysis in human erythrocytes.Am J Physiol. 1966; 210: 737-743Crossref PubMed Scopus (22) Google Scholar], and I have had membranes and red blood cells constantly in my mind ever since! This Metcalf award essay is a tribute to an exceptional group of trainees in my laboratory who have cloned and characterized many proteins and RNAs important for red cell development and function, and who took exceptionally large risks in developing and employing novel experimental technologies. To begin, I describe the primitive technologies we used in the 1970s to elucidate the regulation of translation of α- and β-globin mRNAs in reticulocytes and the primary defect in beta thalassemia. Then I review the technologies we had to develop in the 1980s to clone and characterize the Epo receptor and the red cell membrane proteins GLUT1 and Band 3. In the 2000s, we had to develop yet other technologies to identify and elucidate the function of many microRNAs and long noncoding RNAs in red cell development. Finally, I summarize how we are currently utilizing single-cell transcriptomics (scRNAseq) to understand how transit-amplifying burst-forming unit erythroid progenitors balance the need for more progenitors with the need for terminally differentiated erythroid cells and to identify drugs potentially useful for treating Epo-resistant anemias such as Diamond–Blackfan anemia. Initially we had no funding for most of these projects. But once the key goals were achieved, grant funds flowed in. The first of these projects I did mostly myself, but most were undertaken by an exceptional group of graduate students and postdoctoral fellows, many of whom were MDs. These trainees took exceptionally large risks with their career paths, but all have gone on to become leaders in molecular or cellular biology or molecular medicine. Throughout this article, I describe some of the lessons I learned in managing these and other diverse fellows and projects, hoping that this will be helpful to others who want to undertake ambitious and important lines of research in molecular hematology. But first I took a detour, as I majored in mathematics and chemistry at Kenyon College. My PhD thesis under Norton Zinder at the Rockefeller University focused on a genetic analysis of the RNA bacteriophage f2, generating and analyzing nonsense and temperature-sensitive mutants; I identified mutations in three phage genes—two coat proteins and a subunit of the RNA polymerase [3Lodish HF Zinder ND Semi-conservative replication of bacteriophage f2 RNA.J Mol Biol. 1966; 21: 207-209Crossref PubMed Scopus (1) Google Scholar, 4Lodish HF Zinder ND Mutants of the bacteriophage f2: 8. Control mechanisms for phage-specific syntheses.J Mol Biol. 1966; 19: 333-348Crossref PubMed Scopus (49) Google Scholar, 5Lodish HF Zinder ND Replication of the RNA of bacteriophage f2.Science. 1966; 152: 372-377Crossref PubMed Scopus (14) Google Scholar]. My work as a postdoctoral fellow under Sydney Brenner and Francis Crick focused on understanding the regulation of translation of these three f2 genes [6Lodish HF Bacteriophage f2 RNA: control of translation and gene order.Nature. 1968; 220: 345-350Crossref PubMed Scopus (77) Google Scholar,7Lodish HF Independent translation of the genes of bacteriophage f2 RNA.J Mol Biol. 1968; 32: 681-685Crossref PubMed Scopus (27) Google Scholar], and I continued this line of research during the first 2 years after I joined the MIT faculty in 1968. An important issue during the late 1960s was the mechanism of initiation of translation of mammalian mRNAs. Bacterial cells were known to initiate protein synthesis with N-formyl methionyl tRNA, but eukaryotic cells did not have this initiator aminoacylated tRNA. It was known that at least some eukaryotic cells contained two species of methionyl tRNA and that one of them could be formylated by the Escherichia coli transformylase. David Housman, my second graduate student, together with Tom RajBhandary, a new MIT faculty colleague, showed that enzymically formylated yeast N-formyl methionyl tRNA initiated synthesis of hemoglobin in a rabbit reticulocyte cell-free system and that the normal initiator methionine, but not the initiator N-formyl methionine, was subsequently removed by a cellular enzyme [8Housman D Jacobs-Lorena M Rajbhandary UL Lodish HF Initiation of haemoglobin synthesis by methionyl-tRNA.Nature. 1970; 227: 913-918Crossref PubMed Scopus (163) Google Scholar]. Thus, both α- and β-globin polypeptides initiate with an initiator methionyl tRNA that is not formylated, establishing well before there was any nucleotide sequence that an AUG codon initiated eukaryotic mRNA translation, the final letter in the genetic code. I had no grant to support this research; we just did it using money that was supposed to be spent on other projects. Once the article was published, I had no difficulty in obtaining National Institutes of Health (NIH) support for a long series of studies on the regulation of translation of α- and β-globin mRNAs. Working at the bench myself, I could show (well before globin mRNAs were cloned or could be quantified directly) that reticulocytes had more mRNA encoding the α-globin chain than the β-globin chain but that translation of α-globin mRNA was initiated less efficiently than that of β-globin mRNA. These studies all made use of rabbit reticulocytes; in culture, reticulocytes synthesize large amounts of hemoglobin at a linear rate for many minutes, and, more importantly, they are regulated so as to make essentially equal amounts of α- and β-globin polypeptides. Work by Tim Hunt et al. [9Hunt T Hunter T Munro A Control of haemoglobin synthesis: distribution of ribosomes on the messenger RNA for alpha and beta chains.J Mol Biol. 1968; 36: 31-45Crossref PubMed Scopus (48) Google Scholar] had shown that, in intact cells, β chains are made on polyribosomes that contain 30% to 40% more ribosomes than those synthesizing α chains [9Hunt T Hunter T Munro A Control of haemoglobin synthesis: distribution of ribosomes on the messenger RNA for alpha and beta chains.J Mol Biol. 1968; 36: 31-45Crossref PubMed Scopus (48) Google Scholar]. We soon confirmed this result for intact human and rabbit reticulocytes and also for crude lysates from rabbit reticulocytes, which synthesize hemoglobin at a linear rate for long periods [10Lodish HF Alpha and beta globin messenger ribonucleic acid: different amounts and rates of initiation of translation.J Biol Chem. 1971; 246: 7131-7138PubMed Google Scholar]. These experiments, in hindsight, were ridiculously complex because we had to use very indirect ways of measuring the two mRNAs by monitoring the localization on polyribosomes of nascent α- and β-globin polypeptides. In brief, we labeled either intact reticulocytes or a reticulocyte cell-free protein-synthesizing lysate with huge amounts of [35S]methionine. (At the time, [35S]methionine was not commercially available, and I made it myself starting with a culture of bakers’ yeast and 50 mCi of 35SO4; Fred Sanger himself taught me the protocol when I was at the MRC Laboratory of Molecular Biology [LMB].) After protein synthesis had proceeded for a few minutes, we added cycloheximide to freeze polypeptide chain elongation and then lysed the intact cells. The cell lysates were then layered atop a sucrose gradient and centrifuged in an ultracentrifuge such that the larger polyribosomes (containing up to 10 ribosomes) were at the bottom of the tube and the single 80s ribosomes and 60s and 40s subunits were at the top. Using a long needle and a pump, we collected fractions from the gradient and measured the amount of nascent α- and β-globin chains on each size of polysome. To do this we added large amounts of [3H]methionine- labeled hemoglobin to each fraction as an internal control and then precipitated all of the protein in each fraction. We dissolved the protein pellets in buffer and digested them with trypsin, then resolved the tryptic peptides by paper electrophoresis in giant gasoline-filled tanks. We focused on one internal methionine-containing peptide from the α polypeptide and one from the β polypeptide; from the ratio of [35S]- to [3H]methionine in these two peptides we could then calculate the relative abundance of nascent α and β polypeptides on each size of polysome. In both intact human and mouse reticulocytes, as well in lysates of rabbit reticulocytes synthesizing hemoglobin at a constant linear rate, we found that β chains are made on larger polyribosomes than those synthesizing α chains. Each experiment took about 2 weeks to complete; now of course, measurements of α and β mRNAs can be accomplished in an afternoon by reverse transcription polymerase chain reaction (RT-PC). But how could one explain the fact that reticulocytes synthesized equal amounts of α- and β-globin chains yet that β chains are made on larger polyribosomes than those synthesizing α chains? Hunt et al. had measured the rate of elongation of α- and β-globin chains in rabbit reticulocytes and concluded that α chains are translated 30% to 70% faster than β. They noted that this difference in translation rate would be sufficient to account for the difference in polysome size and suggested that the rate and mechanism of polypeptide initiation are the same for the two chains. In contrast, I used [14C]tyrosine to label α- and β-globin chains synthesized in rabbit reticulocyte lysates and measured the time it took for [14C]tyrosine incorporated into different positions in nascent α- and β-globin chains to be incorporated into released, full-length polypeptides. I found no difference in the rates of translation of α- and β-globin chains in these cell-free extracts of rabbit reticulocytes (about 200 s per chain at 25°C) nor in the rate of release of completed globin chains from polyribosomes (about 15 s per chain) [11Lodish HF Jacobsen M Regulation of hemoglobin synthesis: equal rates of translation and termination of α- and β-globin chains.J Biol Chem. 1972; 247: 3622-3629PubMed Google Scholar]. These results implied that each β-globin mRNA initiates synthesis of about 40% more β-globin than each α-globin mRNA initiates synthesis of α chains. Hence, I concluded that reticulocytes must contain 30% to 40% more α-globin mRNA than β to synthesize equal numbers of the two hemoglobin chains. I thus expected that nonspecific inhibitors of polypeptide chain initiation would inhibit translation of the more poorly initiating mRNA, in this case α-globin mRNA. Indeed, a large number of synthetic polynucleotides inhibited globin synthesis in cell-free extracts of rabbit reticulocytes; without exception, all of these polymers preferentially inhibited initiation of α-globin chains [12Lodish HF Nathan DG Regulation of hemoglobin synthesis: preferential inhibition of alpha and beta globin synthesis.J Biol Chem. 1972; 247: 7822-7829PubMed Google Scholar]. My training in mathematics led to a widely cited article I published in Nature in 1974 [13Lodish HF Model for the regulation of mRNA translation applied to haemoglobin synthesis.Nature. 1974; 251: 385-388Crossref PubMed Scopus (274) Google Scholar]. Using one of the original programmable calculators (desktop computers were still 10 or more years away), I wrote a set of kinetic differential equations for initiation and elongation of protein synthesis; the essence of this model was that once a ribosome initiated polypeptide synthesis, another ribosome could not initiate on the same mRNA until the first had moved an arbitrary distance along the mRNA, which I set at 25 codons, so that the initiation codon was no longer buried by the ribosome. The model predicted that treatments that reduce the overall rate of polypeptide chain initiation would inhibit translation of mRNAs with lower rate constants for polypeptide chain initiation. Conversely, treatments that reduce the rate of polypeptide elongation would increase the ratio of α- to β-globin chains, and several experiments I conducted confirmed this prediction. An important contribution to my education as a scientist was made by my first sabbatical visitor, during the 1970–1971 academic year. David Nathan, MD, was then a young associate professor in the Hematology Division at Children's Hospital Boston. He was a student (the only one wearing a jacket and tie) in the graduate molecular biology course (the first ever taught at MIT) I was teaching, and we soon started talking about the potential implications of the emerging discipline of molecular biology for understanding several human diseases. David was studying beta thalassemia, a genetic syndrome in which the rate of production of β-globin chains is depressed. The cause of the reduction was unknown, but two hypotheses were commonly discussed. The first was decreased levels of β mRNA because of its instability or diminished transcription of the β-globin gene. The second was production of an abnormal β mRNA with one or more codon substitutions that would cause a delay in initiation, elongation, or termination of the nascent β-globin chain. We realized that we could resolve this issue by measuring the sizes of polysomes synthesizing β-globin; the first hypothesis predicted fewer but normal sized polysomes synthesizing β globin. In contrast the second hypothesis predicted abnormally sized polysomes synthesizing β chains— smaller β polysomes if initiation of polypeptide synthesis was reduced. Together with my PhD student David Housman and David Nathan's fellow Y. W. Kan we performed the experiment [14Nathan DG Lodish HF Kan YW Housman D Beta thalassemia and translation of globin messenger RNA.Proc Natl Acad Sci USA. 1971; 68: 2514-2518Crossref PubMed Scopus (34) Google Scholar]. David Nathan would show up in the lab around 10:00 am with a vial of blood that he collected from a local patient; because of the anemia, these individuals had a lot of reticulocytes in their blood that synthesized α- and β-globins. Controls were blood samples from patients with other genetic anemias that also contained many reticulocytes. (One day David arrived disheveled about 2 hours late. The owner of a gas station became concerned when David emerged from the rest room with another man and with a vial of blood in his hands, and called the police.) These intact cells were incubated with [35S]methionine; as I described above, the relative amounts of β and α nascent chains on polysomes of different sizes were measured by tryptic digestion of pooled polysomes and by determination of the specific activities of β and α peptides that contain methionine. Strikingly, β-globin chain synthesis predominated on normal-size heavy polysomes in thalassemic as well as non-thalassemic cells. Thus, we concluded that the defect in thalassemia does not involve reduction in the rate of initiation of translation caused by the production of an abnormal β message and suggested that the decreased production of β-globin chains results from a decreased amount of functional β-globin mRNA. Gratifyingly, we were proven correct when, a decade later, cloning of the globin mRNAs allowed direct measurements of their levels in normal and diseased reticulocytes. Increased levels of α-globin mRNA relative to β mRNA were also confirmed by direct analysis; in part this is due to the fact that the diploid human genome contains four copies of the α-globin gene but two copies of the β-globin gene. This study had two important long-term consequences. David Nathan realized that molecular biology would revolutionize medicine. Starting with his hematology fellows at Boston Children's and then extending to physician scientists training in other subspecialties, David sent them to MIT or Harvard laboratories to master emerging molecular, biochemical, and cell biological techniques. To import these new technologies, he then hired the best of these fellows as assistant professors in their own labs at Boston Children's Hospital. For this and other innovations David introduced into the training of physician scientists, he was awarded the National Medal of Science in 1990. In turn, I realized the impact that studies of the molecular basis of human disease were going to have both on the development of novel therapies and on an understanding of basic molecular mechanisms. I have trained in my laboratory more than 30 MD and MD/PhD scientists, most of whom have gone on to become eminent leaders in fields as diverse as hematology, cardiology, nephrology, and diabetes and metabolism. I have been part of a National Heart, Lung, and Blood Institute (NHLBI) Program Project with hematologists at Children's Hospital for almost 45 years, and in 2016, the grant was renewed for 5 more years. In 2006, I joined the Board of Trustees at Boston Children's Hospital, where I headed the Board Research Committee and do my best to help the hospital raise philanthropic gifts to support basic, translational, and clinical research. Whether by accident or design I still do not know, but upon arrival at MIT, I was given an office next door to David Baltimore, my old friend from Rockefeller days, and we shared three large research laboratories. David and his postdoc (later wife) Alice Huang introduced me to vesicular stomatitis virus (VSV). One VSV gene, the G or glycoprotein, became invaluable in studies David Knipe carried out in the early 1970s defining the endoplasmic reticulum (ER) to Golgi to plasma membrane pathway for biosynthesis of the G-protein as a model for all cell surface glycoproteins [15Knipe DM Baltimore D Lodish HF Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.J Virol. 1977; 21: 1149-1158Crossref PubMed Google Scholar,16Knipe DM Lodish HF Baltimore D Localization of two cellular forms of the vesicular stomatitis viral glycoprotein.J Virol. 1977; 21: 1121-1127Crossref PubMed Google Scholar]. Later, in collaboration with Günter Blobel's group, Flora Katz and Jim Rothman developed in vitro cell-free protein synthesizing systems where they could translate the VSV G mRNA and insert it into ER membranes [17Katz FN Rothman JE Lingappa VR Blobel G Lodish HF Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein.Proc Natl Acad Sci USA. 1977; 74: 3278-3282Crossref PubMed Scopus (190) Google Scholar]. Jim then used this system to demonstrate obligatory co-translational insertion of this transmembrane glycoprotein into the ER membrane and co-translational attachment of the two asparagine-linked oligosaccharides [18Rothman JE Katz FN Lodish HF Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein.Cell. 1978; 15: 1447-1454Abstract Full Text PDF PubMed Scopus (45) Google Scholar,19Rothman JE Lodish HF Synchronised transmembrane insertion and glycosylation of a nascent membrane protein.Nature. 1977; 269: 775-780Crossref PubMed Scopus (270) Google Scholar]. Contemporaneously we worked on the biogenesis of several erythrocyte “membrane” proteins, that is the major proteins in a purified red cell membrane pellet, or “ghost.” We showed that several, now known to be cytoskeletal proteins, are made on membrane-free polysomes [20Lodish HF Small B Membrane proteins synthesized by rabbit reticulocytes.J Cell Biol. 1975; 65: 51-64Crossref PubMed Scopus (64) Google Scholar]. One of my all-time favorite experiments revealed that the major red cell membrane and cytoskeleton proteins are made at different times during development. This involved injecting a mouse with several millicuries of [35S]methionine (the pulse), then (chase) bleeding it every ∼12 hours for a few days and preparing membrane “ghosts,” followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography. The logic was that the last proteins to be made during the multiday developmental period would be the first to be found in mature red cells released into the blood [21Chang H Langer PJ Lodish HF Asynchronous synthesis of erythrocyte membrane proteins.Proc Natl Acad Sci USA. 1976; 73: 3206-3210Crossref PubMed Scopus (58) Google Scholar]. Oldtimers will recognize this as a whole-organism version of the “Dintzis experiment.” But to further understand how membrane proteins are made, we needed to know the amino acid sequences of normal cell membrane proteins and to have molecular techniques to study individual membrane proteins in detail. In the early 1980s, the only membrane protein whose sequence was known was glycophorin, and no membrane protein had been cloned (remember, even globin mRNAs were cloned only in 1977!). So I picked three membrane proteins that were expressed in reasonable abundance: the liver asialoglycoprotein receptor studied by Gil Ashwell [22Ashwell G Harford J Carbohydrate-specific receptors of the liver.Annu Rev Biochem. 1982; 51: 531-554Crossref PubMed Scopus (1499) Google Scholar] and two major red cell membrane proteins—Band 3, the anion exchanger that could be more or less purified by SDS-PAGE of red cell ghosts, and the glucose transporter that had been purified by Gus Lienhard. But aside from doing a lot of peptide purification and amino acid sequencing and making degenerate pools of DNA to screen a cDNA library by hybridization, which was the only existing cDNA cloning technology and a forbidding undertaking with little guarantee of success, there was no obvious way to clone these proteins. The solution came from Rick Young, a postdoc at Stanford whom we were recruiting to the MIT and Whitehead faculty; he developed a general technique for using antibodies as probes for proteins encoded by cloned DNA (Figure 1). His vector, lambda gt11, allowed insertion of libraries of foreign DNA in-frame into the β-galactosidase structural gene lacZ and promoted synthesis of chimeric proteins. Induction of lysogens of these libraries produced large quantities of protein, and clones could be detected by blotting filters of colonies with specific antibodies. However, no one had actually used these technologies to clone mammalian proteins, and we were unaware of the technical hurdles that awaited us. But three postdocs—Mike Mueckler, Ron Kopito, and Martin Spiess, all of whom joined my laboratory in the early 1980s—were up to the challenge, especially given all of the help Rick provided us. Using an antibody provided by Gus Lienhard, Mike Mueckler showed that the “erythrocyte glucose transporter” was expressed in the HepG2 hepatoma cell line. Then, from a HepG2 cDNA library he made in lambda gt11, Mike was quickly able to clone the protein now known as GLUT1. But DNA sequencing was still in its infancy; we did this in our own laboratory using the Sanger technique and with huge gels that were dried and subjected to autoradiography, and errors in reading the gel autoradiograms were many. We were afraid that our initial sequence contained errors and, thus, that our derived amino acid sequence would be wrong. So we enlisted the help of Howard Morris and his group in London, one of the pioneers of protein mass spectrometry (another technique in its infancy). They obtained sufficient amino acid sequences from peptides generated from Gus’ purified protein to show that our derived amino acid sequence was correct [23Mueckler M Caruso C Baldwin SA et al.Sequence and structure of a human glucose transporter.Science. 1985; 229: 941-945Crossref PubMed Scopus (1126) Google Scholar]. Indeed, the amino acid sequence and the 12 membrane-spanning α-helixes we predicted were confirmed by the much later three-dimensional molecular structure [24Deng D Xu C Sun P et al.Crystal structure of the human glucose transporter GLUT1.Nature. 2014; 510: 121-125Crossref PubMed Scopus (462) Google Scholar]. Researchers in the diabetes and metabolism fields immediately realized the importance of having a glucose transporter clone in hand to study glucose uptake and metabolism. Several of my postdocs took up these projects. Bernard Thorens used low-stringency hybridization to isolate from a rat liver cDNA library a cDNA encoding a protein—now known as GLUT2—that led to a long series of stu" @default.
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• W3048383877 title "Over 60 Years of Experimental Hematology (without a License)" @default.
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