RESUMO
Early postnatal life is a critical period for development of the endocrine pancreas, involving remodelling of islet cells and maturation of secretory responses. Factors that regulate these processes are undefined. Somatostatin-secreting delta-cells are abundant in the developing pancreas and, because somatostatin inhibits growth, the hormone may regulate islet expansion in early life. The aim of this study was to investigate effects of somatostatin-deficiency on proliferation, apoptosis and pancreas expansion in the first 3 weeks of life in mice. Pancreases from control or somatostatin-knockout mice were analysed for beta cell, alpha cell and pancreatic volumes by morphometry, proliferation by BrdU incorporation and apoptosis by TUNEL labelling. Signalling pathways associated with proliferation and apoptosis were studied by immunohistochemistry and Western blotting. Knockout mice grew normally in the first 3 weeks of life, but had high circulating insulin that normalised by day 21. Beta cell, alpha cell and pancreatic volumes were decreased in knockout mice, accompanied by reduced proliferation and increased apoptosis in the pancreas. Decreased growth was not due to impaired Akt signalling, as Akt phosphorylation and nuclear cyclin-D2 increased in the knockout pancreas. Levels of TGF-ß1, a factor implicated in tissue remodelling, together with SMAD phosphorylation through which TGF-ß mediates its effects, were increased in the knockout pancreas. Beta cell expansion was impaired in knockout mice, potentially compensating for increased insulin secretion from islets lacking inhibitory effects of somatostatin, and was associated with increased TGF-ß1 levels. TGF-ß1 may represent an important regulator of beta cell mass in early life.
Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Transdução de Sinais , Somatostatina/deficiência , Fator de Crescimento Transformador beta/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Peso Corporal , Bromodesoxiuridina/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Fosforilação , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Smad/metabolismo , Somatostatina/metabolismoRESUMO
Offspring of rats fed high-fat diets during pregnancy and lactation develop glucose intolerance and islet dysfunction in adulthood. Because other models of developmental programming of glucose intolerance are associated with defective islet development, we investigated whether high-fat exposure during fetal or neonatal life impairs islet development and function, thereby contributing to islet dysfunction in later life. Female rats were fed control or high-fat diets and their pups cross-fostered after birth to represent 4 groups with each combination of control and high-fat diet for the natural and foster mother. In a time course study, pups were kept with the natural mother until weaning. Pancreases were analysed for insulin content, beta cell mass, and islet number. Isolated islets were studied for insulin secretory responses and susceptibility to palmitate-induced apoptosis assessed by caspases 3/9 activity. Pancreatic insulin content and beta cell mass were increased in pups exposed to maternal high-fat diets after birth, whereas glucose-stimulated insulin secretion from islets of high-fat offspring at 5 and 11 days of age was lower than controls. Islets from control rats of 2-14 days of age were resistant to the pro-apoptotic effects of palmitate seen in older animals. The immature beta cell is therefore insensitive to toxic effects of palmitate and may compensate for the inhibitory effects on insulin secretion by increasing beta cell mass. The data suggest that susceptibility to glucose intolerance in offspring of dams fed high-fat diets may not be a consequence of deleterious effects on beta cell mass in early life.
Assuntos
Dieta , Gorduras na Dieta/toxicidade , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Animais , Animais Recém-Nascidos , Animais Lactentes , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocinas/farmacologia , Ácidos Graxos/toxicidade , Feminino , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/anatomia & histologia , Ilhotas Pancreáticas/enzimologia , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The replication system of bacteriophage T7 is remarkable in that the 40,000 nucleotide genome is replicated over 100-fold in a matter of minutes. In order to accomplish this feat T7 has evolved an efficient and economical process for the replication of its DNA. The T7 replisome provides a model system to study DNA replication. Four proteins are sufficient for reconstitution of the functional replication complex, yet the assembled replisome recapitulates all the key features of more complex prokaryotic and eukaryotic systems. In this review, we describe chemical mechanisms employed by individual proteins at the replication fork. Integration of structural, biochemical, and single-molecule data reveals a compelling view on how a nearly 1-MDa molecular machine acts as a unit to synthetize the two antiparallel DNA strands in a coordinated fashion.
Assuntos
Bacteriófago T7/genética , Replicação do DNA , DNA Viral/biossíntese , Bacteriófago T7/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Virais/metabolismoRESUMO
Polynucleotide kinase catalyzes the transfer of a phosphate group from ATP to the 5'-hydroxyl termini of polynucleotides. Selective labeling of the 5'-hydroxyl termini of DNA with polynucleotide kinase has been used to study the number and the identity of the 5'-terminal residues of bacteriophage DNA's, and to examine the nature of the phosphodiester bond cleavages produced by endonucleases and by sonic irradiation. The intact strands of T7 DNA bear 5'-phosphoryl end-groups; only deoxyadenylate and deoxythymidylate are present as 5'-terminal residues. The intact strands of native lambda-DNA bear 5'-hydroxyl end-groups. M13 DNA, a circular molecule, cannot be phosphorylated. End-group labeling of DNA provides a method for determination of molecular weight; calibration against other DNA preparations is not required. The molecular weight of a single strand of T7 DNA, determined by end-group labeling, is 13.1 x 10(6); the molecular weight of a single strand of lambda-DNA is 16.0 x 10(6). These values are in agreement with molecular weight estimates by sedimentation analysis and electron microscopy. Sonic irradiation of DNA has been shown to favor the production of polynucleotides terminated by 5'-phosphomonoester groups. All four deoxyribonucleotides are present as 5'-terminal residues of sonicated DNA.
Assuntos
DNA Viral , Polinucleotídeos , Trifosfato de Adenosina , Fosfatase Alcalina , Bacteriófagos , Fenômenos Químicos , Química , Colífagos , Desoxirribonucleases , Escherichia coli/enzimologia , Peso Molecular , Fosfotransferases , UltrassomRESUMO
Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.
Assuntos
Replicação do DNA , Fagos T/genética , Sequência de Bases , Clonagem Molecular , DNA Viral/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli , Genes Virais , Dados de Sequência Molecular , PlasmídeosRESUMO
We have determined the genome sequences of two closely related lytic bacteriophages, SP6 and K1-5, which infect Salmonella typhimurium LT2 and Escherichia coli serotypes K1 and K5, respectively. The genome organization of these phages is almost identical with the notable exception of the tail fiber genes that confer the different host specificities. The two phages have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized. The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats. About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage. The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved. Low levels of nucleotide similarity between genomes in the T7 and SP6 groups suggest that they diverged a long time ago but, on the basis of this conservation of genome organization, they are expected to have retained similar developmental strategies.
Assuntos
Bacteriófago T7/genética , Escherichia coli/virologia , Éxons/genética , Íntrons/genética , Fagos de Salmonella/genética , Proteínas da Cauda Viral/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Genes Reguladores/genética , Genoma , Dados de Sequência Molecular , Podoviridae/genética , Regiões Promotoras Genéticas/genética , Salmonella typhimurium , Homologia de Sequência do Ácido Nucleico , Proteínas da Cauda Viral/química , Vírion/genéticaRESUMO
A new mouse model of stage-specific bone growth failure and fracture has been recovered as an autosomal recessive mutation, designated spontaneous fracture (sfx). The sfx/sfx mice are phenotypically normal until shortly after weaning, when reduced mobility and impaired somatic growth are first noted. By 6 weeks of age, body, spleen, and thymus weights, as well as hematocrits and serum calcium, inorganic phosphate, total alkaline phosphatase, insulin-like growth factor-I, and osteocalcin levels are decreased. The sfx/sfx mice also show reduced femoral cortical density and diaphyseal circumference, as well as a paucity of mature osteoblasts on bone surfaces. Histological analyses of the femur and tibia in the mutants show subtle reduction of chondrocyte numbers in epiphyseal-plate columns, reduction of matrix, and near absence of osteoid below the differentiated chondrocytes. Trabeculae in proximal tibiae, iliacs, and vertebral bodies are sparse and thin. Cortical bone thickness of mutants is markedly thinned in all sites examined. By 7-8 weeks, radiographic films routinely show spontaneous impact fractures of the distal femur accompanied by callus formation, whereas complete fractures are less commonly observed. Volumetric bone mineral density (BMD) of mutant femurs is similar to +/? littermates in the center of the femoral diaphysis, but BMD declines as either end of the femoral diaphysis is approached. We have mapped the gene responsible for this phenotype to central Chromosome 14. Reduced bone mass, impaired bone formation, abnormalities of bone architecture, and a disposition to spontaneous fracture identify sfx/sfx mice as a useful model for understanding the mechanisms responsible for peripubertal bone formation.
Assuntos
Desenvolvimento Ósseo/genética , Fraturas Espontâneas/genética , Modelos Genéticos , Animais , Mapeamento Cromossômico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , FenótipoAssuntos
Colífagos , DNA Viral , Nucleotídeos , Fosfatase Alcalina , Fenômenos Químicos , Química , FosfotransferasesAssuntos
Colífagos/análise , DNA Viral/análise , Nucleotídeos/análise , Fosfatase Alcalina/farmacologia , Isótopos de Carbono , Centrifugação com Gradiente de Concentração , Colífagos/efeitos dos fármacos , Desoxirribonucleases/farmacologia , N-Glicosil Hidrolases/farmacologia , Monoéster Fosfórico Hidrolases/farmacologia , Isótopos de Fósforo , Fosfotransferases/farmacologia , UltracentrifugaçãoRESUMO
AIMS/HYPOTHESIS: Although cells expressing insulin are detected early in human fetal development, islets isolated from fetal pancreases show poor insulin secretory responses to glucose, which may be the result of deficient glucose sensing. We have used dual and triple immunolabelling of human fetal and adult pancreas sections to investigate the presence of proteins that participate in glucose sensing in the pancreatic beta cell, namely glucose transporter 1 (GLUT 1, also known as SLC2A1), glucose transporter 2 (GLUT2, also known as SLC2A2), glucokinase (GCK) and inwardly rectifying K+ channel (KIR6.2, also known as KCNJ11) and sulphonylurea receptor 1 (SUR1, also known as ABCC8) subunits of ATP-sensitive K+ channels (K+(ATP) channels). MATERIALS AND METHODS: Pancreases obtained with ethical approval from human fetuses from 11 to 36 weeks of gestation, from infants and from adults were formalin-fixed and embedded in paraffin. Sections were labelled with antibodies to proteins of interest. Co-production of antigens was examined by dual and triple immunolabelling. RESULTS: GLUT2 and K+(ATP) channel labelling was detected in the 11-week pancreas, but largely within the pancreatic epithelium, whereas no labelling for GLUT1 was observed. From 15 weeks, GLUT1, GCK and K+(ATP) channel labelling was detected in an increasing proportion of insulin-positive cells and epithelial labelling with K+(ATP) channel antibodies diminished. GLUT2 was seen in the majority of beta cells only after 7 months of age. CONCLUSIONS/INTERPRETATION: The results demonstrate that only a subpopulation of beta cells in the human fetal pancreas produce all key elements of the glucose-sensing apparatus, which may contribute to poor secretory responses in early life.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Adulto , Desenvolvimento Fetal , Idade Gestacional , Glucoquinase/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Humanos , LactenteRESUMO
AIMS/HYPOTHESIS: We investigated the production of kisspeptin (KISS1) and the KISS1 receptor, GPR54, in pancreatic islets and determined the effects of exogenous kisspeptin on insulin secretion. METHODS: RT-PCR and immunohistochemistry were used to detect expression of KISS1 and GPR54 mRNAs and the production of KISS1 and GPR54 in human and mouse islets and in beta (MIN6) and alpha- (alphaTC1) cell lines. The effects of KISS1 on basal and glucose-induced insulin secretion from mouse and human islets were measured in a perifusion system. RESULTS: KISS1 and GPR54 mRNAs were both detected in human and mouse islets, and GPR54 mRNA expression was also found in the MIN6 and alphaTC1 endocrine cell lines. In sections of mouse pancreas, KISS1 and GPR54 immunoreactivities were co-localised in both beta and alpha cells within islets, but were not detected in the exocrine pancreas. Exposure of mouse and human islets to KISS1 caused a stimulation of glucose-induced (20 mmol/l) insulin secretion, but had no effect on the basal rate of secretion at a sub-stimulatory concentration of glucose (2 mmol/l). In contrast, KISS1 inhibited insulin secretion from MIN6 cells at both 2 and 20 mmol/l glucose. KISS1 had no significant effect on glucagon secretion from mouse islets. CONCLUSIONS/INTERPRETATION: This is the first report to show that the GPR54/KISS1 system is expressed in the endocrine pancreas, where it influences beta cell secretory function. These observations suggest an important role for this system in the normal regulation of islet function.
Assuntos
Ilhotas Pancreáticas/metabolismo , Proteínas/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Linhagem Celular Tumoral , Expressão Gênica , Glucagon/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Kisspeptinas , Camundongos , Proteínas/genética , Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Kisspeptina-1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/farmacologiaRESUMO
In vivo, replication of T7 DNA does not occur after infection of Escherchia coli tsnC mutants (CHAMBERLIN, M. (1974) J. Virol. 14, 509-516). In vitro, extracts of tsnC mutant E. coli infected with T7 hage are incapable of replicating duplex T7 DNA, although extracts of wild type E. coli infected with T7 phage support replication of T7 DNA. In addition, extracts of the infected tsnC mutant are deficient in T7 DNA polymerase activity. Extracts prepared from uninfected E.coli tsnC-+ cells restore the ability of the infected tsnC extracts to replicate duplex T7 DNA, and also restore normal levels of the phage DNA polymerase activity. A 12,000-dalton heat-stable protein responsible for this complementation has been purified to near homogeneity from uninfected tsnC+ extracts and it is designated "TsnC protein."
Assuntos
Proteínas de Bactérias/farmacologia , Colífagos/metabolismo , DNA Nucleotidiltransferases/metabolismo , Replicação do DNA , Escherichia coli/metabolismo , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Etilmaleimida/farmacologia , Exonucleases/farmacologia , Peso Molecular , Mutação , Nucleotídeos/metabolismo , Papaína/farmacologia , Radioisótopos de Fósforo , Ribonucleases/farmacologia , Espectrofotometria Ultravioleta , Tosilina Clorometil Cetona/farmacologia , Trítio , Tripsina/farmacologiaRESUMO
The DNA polymerase induced after infection of Escherichia coli by phage T7 has been purified 500-fold to near homogeneity as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme complements extracts of cells infected with a T7 gene 5 mutant to permit cell-free replication of duplex T7 DNA. In contrast, purified T4 DNA polymerase or E. coli DNA polymerase I is unable to do so, thus suggesting a specific requirement for the T7 enzyme in the replication of the viral DNA. E. coli TsnC protein is present in purified T7 DNA polymerase in one-to-one stoichiometry with T7 gene 5 protein, and can be isolated in homogeneous form from heat-denatured enzyme by chromatography on DEAE-cellulose. The inactive form of T7 gene 5 protein that accumulates in tsnC hosts has been partially purified. When partially purified gene 5 protein is mixed with purified TsnC protein, DNA polymerase activity is restored, and formation of a one-to-one complex between the two proteins occurs. These results indicate that the functional form ofT7 DNA polymerase is a complex composed of phage- and host-specified subunits.
Assuntos
Colífagos/metabolismo , DNA Nucleotidiltransferases/metabolismo , Replicação do DNA , Escherichia coli/metabolismo , Proteínas de Bactérias/farmacologia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , DNA Nucleotidiltransferases/isolamento & purificação , Desoxirribonucleotídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Mutação , Dodecilsulfato de Sódio , Tiorredoxinas/farmacologiaRESUMO
The T7 gene 4 protein, a protein known from genetic analysis to participate in phage DNA replication in vivo, has been purified approximately 500-fold with an in vitro complementation assay. The protein, purified from cells infected with a T7 gene 4 temperature-sensitive mutant, is thermolabile, establishing that the complementation activity is in the protein product of the phage gene 4. The purified protein has no detectable nuclease, DNA polymerase, or RNA polymerase activity. However, in addition to stimulating the rate of DNA replication in crude extracts of T7 gene 4 mutant-infected cells, the gene 4 protein effects a marked stimulation of DNA synthesis by the purified T7 DNA polymerase when duplex T7 DNA is used as template. This effect is not observed when denatured T7 DNA is used as template, or when phage T4 DNA polymerase or Escherichia coli DNA polymerase I, II, OR III is substituted for the T4 enzyme. Analysis of the DNA synthesized by the T7 DNA polymerase in the presence of the gene 4 protein indicates that much of the product is in short DNA chains which are not covalently attached to the template. This result suggests a novel mechanism for the initiation of DNA chains in this reaction.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Colífagos/metabolismo , DNA Nucleotidiltransferases/metabolismo , Replicação do DNA , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Genes , Peso Molecular , Mutação , Radioisótopos de Fósforo , Dodecilsulfato de Sódio , TrítioRESUMO
Escherichia coli encodes a dGTP triphosphohydrolase (dGTPase) that cleaves dGTP to deoxyguanosine and tripolyphosphate. dGTP is hydrolyzed with a Michaelis constant (Km) of 5 microM and a maximal velocity (Vmax) of 1.8 mumols/min/mg. The ribonucleotide GTP is a poor substrate with a much lower affinity. It is hydrolyzed with a Km of 150 microM and Vmax of 0.07 mumols/min/mg. Bacteriophage T7 encodes a specific inhibitor of dGTPase, the gene 1.2 protein, that forms a tight complex with the enzyme. The enzyme-inhibitor complex binds dGTP with a dissociation constant (KD) of 1.5 microM, but the bound dGTP is not hydrolyzed. It remains stably bound to the complex with a half-life of approximately 5 min. In contrast, dGTP is unable to bind to gene 1.2 protein alone, and dGTP bound to dGTPase alone is quickly hydrolyzed and released. Surprisingly, the dGTPase-gene 1.2 protein complex has a higher affinity for GTP than for dGTP. GTP is stably bound to the dGTPase-gene 1.2 protein complex with a half-life greater than 30 min and KD of 0.8 microM; GTP is not stably bound to either dGTPase or gene 1.2 protein alone. Both GTP and dGTP bind to and stabilize the dGTPase-gene 1.2 protein complex, inhibiting its dissociation. Although the presence of dGTP induces conformation changes in dGTPase so that it is unable to associate with the gene 1.2 protein, saturating concentrations of GTP have no such effect. The enzyme efficiently associates with its inhibitor in the presence of GTP. These results indicate that E. coli dGTPase and gene 1.2 protein interact to form a high affinity GTP-binding site. dGTP is most effective in preventing the association of the enzyme with the inhibitor whereas GTP is most effective in preventing the dissociation of the enzyme-inhibitor complex.
Assuntos
Escherichia coli/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fagos T/análise , Proteínas Virais/metabolismo , Sítios de Ligação , Cátions Bivalentes , Nucleotídeos de Desoxiguanina/metabolismo , Nucleotídeos de Desoxiguanina/farmacologia , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Meia-Vida , Hidrólise , Cinética , Manganês/farmacologia , Nucleotídeos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Conformação ProteicaRESUMO
Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.