RESUMEN
Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.
Asunto(s)
Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/genética , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura , Clonación Molecular/métodos , Cartilla de ADN , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Cinética , Células L , Luciferasas/biosíntesis , Mamíferos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Transfección/métodosAsunto(s)
Clonación Molecular/métodos , Endonucleasas , Saccharomyces cerevisiae/enzimología , Animales , Chlamydomonas/enzimología , Chlamydomonas/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Physarum/enzimología , Physarum/genética , Plásmidos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
CHO SSF3 cells grow as a suspension culture in unmodified commercial medium with only low-molecular weight ingredients. Continuous serum-free culture unexpectedly induced expression of a low dihydrofolate reductase activity in the originally dhfr- CHO cells. Nevertheless, it was possible with methotrexate to induce amplification of a gene coding for the hybrid plasminogen activator K2tu-PA cotransfected with a dhfr gene. Expression of K2tu-PA expression was proportionally increased to that of dhfr, which was measured with fluorescent methotrexate. Because no serum proteases were present, secreted K2tu-PA was not converted to the enzymatically active form, but was exclusively recovered in proenzyme form.
Asunto(s)
Células CHO/fisiología , Medio de Cultivo Libre de Suero , Amplificación de Genes , Regulación de la Expresión Génica , Activadores Plasminogénicos/biosíntesis , Tetrahidrofolato Deshidrogenasa/genética , Adaptación Biológica , Animales , Cricetinae , ADN Recombinante , Resistencia a Medicamentos , Metotrexato/farmacología , Activadores Plasminogénicos/genética , Selección Genética , Tetrahidrofolato Deshidrogenasa/biosíntesis , TransfecciónRESUMEN
The folA gene, the chromosomal dhfr gene of Escherichia coli, was engineered for expression in mammalian cells. In contrast to plasmid-derived bacterial dhfr genes previously used as selection markers in mammalian cells, the folA gene product is inhibitable by methotrexate (MTX) and trimethoprim (TMP). Therefore, this dhfr may present an alternative to mammalian dhfr species currently used as amplifiable selection markers. Transfected E. coli folA dhfr could complement the lack of endogenous DHFR in Chinese hamster ovary (CHO) cells lacking a functional dhfr gene. Both MTX and TMP inhibited growth of E. coli folA dhfr-transfected CHO cells. Expression of E. coli folA DHFR could be visualized by incubating the transfected cells with a fluorescent methotrexate derivative (F-MTX). Binding of F-MTX to E. coli folA DHFR was inhibitable as by both MTX and TMP, whereas MTX but not TMP blocked binding of F-MTX to recombinant mouse DHFR.
Asunto(s)
Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Marcadores Genéticos , Tetrahidrofolato Deshidrogenasa/genética , Animales , Células CHO , Cricetinae , Citomegalovirus/genética , Escherichia coli/enzimología , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Metotrexato/farmacología , Ratones , Regiones Promotoras Genéticas , Conejos , Mapeo Restrictivo , Transfección , Trimetoprim/farmacologíaRESUMEN
Development of a CHO cell-based production system for the hybrid plasminogen activator K2tu-PA is described. Using the major immediate-early promoter of mouse cytomegalovirus (MCMV) transient and stable expression levels were 3-10-fold higher than those obtained with several other strong promoters. Splicing and polyadenylation signals from the rabbit beta-globin gene were used downstream of the DNA segment coding for K2tu-PA. The strong enhancer moiety of the MCMV promoter also stimulated strongly the promoter of the dihydrofolate reductase (DHFR) gene, placed adjacently for selection/gene amplification purposes. One construct with opposing K2tu-PA and DHFR RNA transcripts yielded the highest expression level with a single copy of the plasmid, but K2tu-PA expression was consistently lost after amplification of such genes, possibly as a result of the formation of antisense RNA. With other constructs, K2tu-PA production leveled off at 6.5 micrograms per million cells per day despite a high gene copy number. This was due to a combination of inefficient mRNA translation and mRNA instability, caused by elements from the untranslated portions of tissue-type and urokinase-type plasminogen activator cDNA which were included in the expression vector. After elimination of these inhibitory DNA segments, 4-5-times higher expression levels were reached.
Asunto(s)
Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Secuencia de Bases , Células CHO , Cricetinae , Citomegalovirus/genética , ADN Complementario , Elementos de Facilitación Genéticos , Amplificación de Genes , Genes Inmediatos-Precoces , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Conejos , Proteínas Recombinantes de Fusión/genética , Tetrahidrofolato Deshidrogenasa/genética , TransfecciónRESUMEN
Rabbits were repeatedly immunized with a chimeric plasminogen activator composed of the kringle-2 domain of human tissue-type plasminogen activator (t-PA) attached to the serine protease domain of the human urokinase-type plasminogen activator (u-PA). IgG from these rabbits was purified, biotinylated and subjected to affinity chromatography on K2tu-PA covalently attached to Sepharose. Roughly half the antibodies recovered from the K2tu-PA column could subsequently be bound to a column with similarly immobilized t-PA, whereas the other half bound to a u-PA column. Less than 0.01% of the biotinylated anti-K2tu-PA antibodies did not bind to either t-PA or u-PA and perhaps are directed against neoantigenic determinants on K2tu-PA, not present in the natural plasminogen activators.
Asunto(s)
Antígenos/inmunología , Kringles , Proteínas Recombinantes de Fusión/inmunología , Activador de Tejido Plasminógeno/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Animales , Biotina , Cromatografía de Afinidad , Inmunoglobulina G/aislamiento & purificación , ConejosRESUMEN
A process was developed to produce recombinant human renin for X-ray analysis and enzyme inhibition studies. An expression vector containing a human prorenin cDNA and expressing a mouse dihydrofolate reductase selection marker was transfected into dhfr-minus Chinese hamster ovary cells. After selection of cell strains with an increased gene copy number with methotrexate, cultures of the recombinant cells were scaled-up in serum-free media. Major improvements in cellular productivity were achieved by using continuous suspension cultures with cell recycling instead of an adherent culture system or batch-mode suspension cultures. The recombinant zymogen prorenin was purified and preparatively activated with trypsin. Enzymatic properties of the recombinant active renin are described.
Asunto(s)
Proteínas Recombinantes/biosíntesis , Renina/biosíntesis , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , Cristalización , Precursores Enzimáticos/biosíntesis , Humanos , Datos de Secuencia Molecular , Renina/química , Renina/aislamiento & purificaciónRESUMEN
This two-plasmid system for transient gene expression can be used in a wide variety of primate cells. It consists of a cDNA expression vector and a helper plasmid. The cDNA cloned in the expression vector is transcribed by the powerful major immediate-early promoter of murine cytomegalovirus. A segment of the rabbit beta-globin gene placed downstream of the cDNA provides signals for splicing and polyadenylation. The helper plasmid provides SV40 T antigen and adenovirus VA RNA. The T antigen induces replication of the expression vector, which contains an SV40 origin of replication, and VA RNA enhances translation of the transcribed mRNA. In monkey kidney cells, with human tissue-type plasminogen activator (t-PA) cDNA as reporter gene, the helper plasmid boosted t-PA production 30-fold and up to 500 ng/ml t-PA accumulated in the medium in the 5 days following transfection of the two plasmids. In nonprimate cells the helper plasmid stimulated expression 3- to 5-fold.
Asunto(s)
ADN/metabolismo , Expresión Génica , Plásmidos , Animales , Antígenos Transformadores de Poliomavirus/genética , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Citomegalovirus/genética , Vectores Genéticos , Riñón/citología , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Virus 40 de los Simios/genética , TransfecciónRESUMEN
A recombinant human plasminogen activator hybrid variant K2tu-PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asn12 (A chain, kringle-2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N-linked carbohydrate chains by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb-NH2, and analysed by 500-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (8%), disialylated diantennary (45%), disialylated tri- and tri'-antennary (1%), trisialylated tri- and tri'-antennary (28%), and tetrasialylated tetra-antennary (18%) structures, all having fucose in alpha(1-6)-linkage at the Asn-bound N-acetylglucosamine. Sialic acid occurred exclusively in alpha(2-3)-linkage to galactose, and consisted of N-acetylneuraminic acid (94%), N-glycolylneuraminic acid (3%), and N-acetyl-9-O-acetylneuraminic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K2tu-PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K2tu-PA with that of tissue-type plasminogen activator from different biological sources showed significant differences. Profiling studies on different K2tu-PA production batches demonstrated that the structures of N-linked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.
Asunto(s)
Oligosacáridos/química , Activador de Tejido Plasminógeno/química , Activador de Plasminógeno de Tipo Uroquinasa/química , Secuencia de Aminoácidos , Animales , Asparagina , Células CHO , Conformación de Carbohidratos , Secuencia de Carbohidratos , Quimera , Cricetinae , Variación Genética , Glicopéptidos/aislamiento & purificación , Glicosilación , Humanos , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Mapeo Peptídico , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/genética , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
uK2t-PA is a hybrid plasminogen activator in which the epidermal growth factor-like domain of the urokinase-type plasminogen activator precedes the kringle 2 and catalytic domains of tissue-type plasminogen activator. The molecules are expressed in Chinese hamster ovary cells in two variant forms, a type II form in which only the protease domain is glycosylated, and a type I form in which both the kringle 2 and the protease domains carry N-acetyllactosamine type glycans. The two forms differed slightly in their affinity for fibrin and fibrinogen, which allowed their separation, but the stimulation of plasminogen activation of the type II form by fibrin was up to eight-fold lower than that of the type II form. The sensitivity to fibrin could be restored by treatment of the type I form with N-glycanase or sialidase. Enzymatic activity vs low molecular weight substrates was not influenced by the glycosylation of kringle 2.
Asunto(s)
Fragmentos de Péptidos/química , Activadores Plasminogénicos/química , Animales , Sitios de Unión , Células CHO , Cricetinae , Fibrina/metabolismo , Fibrinógeno/metabolismo , Glicósido Hidrolasas , Glicosilación , Isoenzimas , Neuraminidasa , Proteínas Recombinantes/químicaRESUMEN
As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.
Asunto(s)
Farmacorresistencia Microbiana/genética , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Bleomicina/farmacología , Línea Celular , ADN/genética , Elementos Transponibles de ADN , Elementos de Facilitación Genéticos , Expresión Génica , Vectores Genéticos , Humanos , Higromicina B/farmacología , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Transformación GenéticaRESUMEN
Fibrinolytic properties of four hybrids of u-PA and t-PA, all containing the u-PA growth factor domain and binding to recombinant human u-PA receptor expressed in CHO cells, were compared. Highest fibrin stimulation was observed with uK2tPA which when compared to t-PA in the rabbit system, had a considerably prolonged circulatory half-life in vivo. Compared to an equimolar dose of t-PA, 0.4 mg/kg uK2tPA caused a similar consumption of alpha 2-antiplasmin and fibrinogen and a considerably greater prolongation of the ex-vivo blood clotting time. Nevertheless, this dose of uK2tPA was inactive in the jugular vein thrombosis assay. This lack of thrombolytic activity is presumably due to the presence of a functional u-PA growth factor domain, which in binding uK2tPA to cellular blood elements possibly retards its penetration into the blood clot and in this manner could neutralize the potential thrombolytic activity of the t-PA kringle 2 and protease domains in uK2tPA.
Asunto(s)
Activadores Plasminogénicos/metabolismo , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Secuencia de Bases , Células CHO , Cricetinae , Femenino , Venas Yugulares , Masculino , Tasa de Depuración Metabólica/fisiología , Datos de Secuencia Molecular , Conejos , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Trombosis/sangre , Activador de Plasminógeno de Tipo Uroquinasa/farmacocinéticaRESUMEN
Shuttle vectors expressing resistance to hygromycin B in both E. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of the neo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive in E. coli.
Asunto(s)
Farmacorresistencia Microbiana/genética , Higromicina B/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cricetinae , Elementos Transponibles de ADN , ADN Recombinante/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos , Marcadores Genéticos , Vectores Genéticos , Datos de Secuencia Molecular , Fosfotransferasas/genética , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
A serum-dependent and two serum-independent variants of the Bowes melanoma cell line, RPMI7272, were transfected with plasmids containing a geneticin-resistance (neo) gene transcribed by the HSV thymidine kinase promoter and an SV40 T antigen gene under control of the mouse metallothionein I promoter. T-antigen increased the cloning efficiency of the serum-dependent cell line in soft-agar more than 50-fold, but cloning efficiency of serum-independent lines was not increased. Trypsinization of serum-independent lines required 100 times lower concentrations of trypsin than serum-dependent cells. Human metal-inducible T-antigen-producing (HMT) melanoma cells supported replication of transfected plasmids containing an SV40 origin of replication. Transient expression of interferon or plasminogen activator from such plasmids was 40-fold higher than in untransformed melanoma cells and could be enhanced 30-fold more by stimulation of transcription of the T antigen gene with cadmium chloride. HMT cells can be grown in suspension and thus may represent an attractive alternative to monkey kidney COS cells.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Neoplásica , Transformación Celular Viral , Melanoma Experimental/patología , Virus 40 de los Simios/inmunología , Animales , Antígenos Transformadores de Poliomavirus/biosíntesis , Antígenos Transformadores de Poliomavirus/genética , División Celular , Melanoma Experimental/genética , Ratones , Virus 40 de los Simios/genética , Transfección , Células Tumorales CultivadasRESUMEN
At 39 to 40 degrees C, selection of antibiotic-resistant transfected mammalian cell lines or Escherichia coli required lower aminoglycoside antibiotic concentrations than at 37 degrees C. The thermosensitivity of antibiotic susceptibility was much more manifest during genetic selection experiments than in conventional growth inhibition assays.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Células Eucariotas/efectos de los fármacos , Células Procariotas/efectos de los fármacos , Animales , Células CHO , Cricetinae , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/genética , Temperatura , TransfecciónRESUMEN
We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.
Asunto(s)
Enfermedad de Hodgkin/genética , Proteínas de Filamentos Intermediarios/genética , Proteínas Asociadas a Microtúbulos , Proteínas de Neoplasias/genética , Células de Reed-Sternberg/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Línea Celular , Células Cultivadas , Clonación Molecular , Cricetinae , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Biblioteca de Genes , Enfermedad de Hodgkin/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/aislamiento & purificación , Proteínas de Filamentos Intermediarios/metabolismo , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/metabolismo , Monocitos/patología , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Transfección , Vimentina/análisisRESUMEN
Bowes melanoma cells, which naturally produce tissue-type plasminogen activator (t-PA), were transfected with a plasmid containing a human t-PA cDNA under transcriptional control of the promoter/enhancer of the major immediate early gene of human cytomegalovirus (CMV) plus genes expressing geneticin (G418) resistance and dihydrofolate reductase (DHFR). In one of the initial geneticin-resistant transformants, t-PA mRNA transcribed from the chromosomally integrated plasmid had the same short half-life, 20-30 min, as did mRNA transcribed from the endogenous t-PA gene compared to 7-8 h for total poly(A)+ mRNA. After subsequent selection of such cells with methotrexate, a cell line was obtained in which the t-PA cDNA construct was co-amplified with the DHFR gene and which produced 10 times more t-PA protein than the original Bowes melanoma cells.
Asunto(s)
Amplificación de Genes , ARN Mensajero/metabolismo , Activador de Tejido Plasminógeno/genética , Clonación Molecular , Resistencia a Medicamentos/genética , Semivida , Humanos , Melanoma , Metotrexato/farmacología , Plásmidos , Biosíntesis de Proteínas , Transfección , Células Tumorales CultivadasRESUMEN
A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.
Asunto(s)
ADN Recombinante , Amplificación de Genes , Genes Virales , Marcadores Genéticos , Virus de la Hepatitis B/genética , Plásmidos , Animales , Línea Celular , Cricetinae , Cricetulus , Femenino , Antígenos de la Hepatitis B/biosíntesis , Kanamicina Quinasa , Ovario/citología , Fosfotransferasas/genética , Tetrahidrofolato Deshidrogenasa/genética , Proteínas Virales/biosíntesisRESUMEN
Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.