RESUMO
The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F(1) hybrids to ENU. The results show that most F(1) hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.
Assuntos
Etilnitrosoureia/farmacologia , Camundongos/genética , Animais , Relação Dose-Resposta a Droga , Etilnitrosoureia/toxicidade , Hibridização Genética , Infertilidade Masculina/induzido quimicamente , Injeções Intraperitoneais , Masculino , Camundongos Endogâmicos , Camundongos MutantesRESUMO
New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.
Assuntos
Alelos , Embrião de Mamíferos/anormalidades , Etilnitrosoureia/toxicidade , Genes Letais , Mutagênicos , Mutação , Animais , Distribuição de Qui-Quadrado , Mapeamento Cromossômico , Cruzamentos Genéticos , Feminino , Genes Recessivos , Teste de Complementação Genética , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Recombinação GenéticaRESUMO
Mutant mice exhibiting heritable hyperphenylalaninemia have been isolated after ethylnitrosourea mutagenesis of the germ line. We describe one mutant pedigree in which phenylalanine hydroxylase activity is severely deficient in homozygotes and reduced in heterozygotes while other biochemical components of phenylalanine catabolism are normal. In homozygotes, injection of phenylalanine causes severe hyperphenylalaninemia and urinary excretion of phenylketones but not hypertyrosinemia. Severe chronic hyperphenylalaninemia can be produced when mutant homozygotes are given phenylalanine in their drinking water. Genetic mapping has localized the mutation to murine chromosome 10 at or near the Pah locus, the structural gene for phenylalanine hydroxylase. This mutant provides a useful genetic animal model affected in the same enzyme as in human phenylketonuria.
Assuntos
Fígado/enzimologia , Mutação , Fenilalanina Hidroxilase/deficiência , Animais , Southern Blotting , Cruzamentos Genéticos , DNA/genética , DNA/isolamento & purificação , Feminino , Genótipo , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Mutantes , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Recombinação GenéticaAssuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Etilnitrosoureia/toxicidade , Fenilalanina/sangue , Fenilcetonúrias/genética , Erros Inatos do Metabolismo dos Aminoácidos/induzido quimicamente , Animais , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Mutantes , Mutação , Fenótipo , Fenilcetonúrias/induzido quimicamenteRESUMO
The t region of mouse chromosome 17 exhibits recombination suppression with wild-type chromatin. However, the region has resisted classical genetic dissection because of a lack of defined variants. Mutations induced by N-ethyl-N-nitrosourea (ENU) at the Brachyury (T), quaking (qk), and tufted (tf) loci of the mouse tw5 haplotype have now allowed the analysis of crossovers between two complete t haplotypes. A classical breeding analysis of the complete t haplotypes, tw5 and t12, utilizing the newly induced markers, reveals two inversions in t chromatin: one involving T and qk, and one involving tf and the H-2 complex. Moreover, the recombination frequency between the loci of T and qk is reduced compared to the frequency reported in normal chromatin. These two inversions are a sufficient explanation for the recombination inhibition with normal chromatin exhibited by t haplotypes isolated from the wild. Furthermore, the reduced recombination frequency between T and qk may indicate that the proximal gene rearrangement is not a simple inversion.
Assuntos
Inversão Cromossômica , Etilnitrosoureia/toxicidade , Haplótipos , Mutagênicos , Mutação , Animais , Mapeamento Cromossômico , Genes Letais , Camundongos , Camundongos Endogâmicos , Camundongos Quaking , Recombinação Genética , Supressão GenéticaRESUMO
Ethylnitrosourea mutagenesis of spermatogonial stem cells and a three-generation breeding scheme were used to screen for recessive mutations that cause defects in phenylalanine metabolism leading to elevated serum levels of this amino acid. This paper describes the isolation of such a mutation, hph-1, causing a heritable hyperphenylalaninemia in the neonate and weanling and an inability to effectively clear a phenylalanine challenge in the adult. Micro-pedigree analysis of the original mutant mouse and data obtained from crosses of affected and unaffected animals indicate that the mutation segregates in an autosomal recessive manner. An interspecies mouse backcross mapping experiment places the mutant gene locus on mouse chromosome 14 very near Np-1 and a backcross experiment with a conventional inbred mouse strain involving a nearby locus confirms the chromosome 14 assignment. The initial symptomatology of the mutant phenotype suggests this mutant may represent a useful animal model for the study of hyperphenylalaninemia in man.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Mutação , Fenilalanina/metabolismo , Animais , Mapeamento Cromossômico , Cruzamentos Genéticos , Etilnitrosoureia , Feminino , Ligação Genética , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Linhagem , FenótipoRESUMO
A hyperphenylalaninemic mouse mutant, hph-1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid-dihydropteridine reductase activity is also normal. GTP-cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP-cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4-dihydroxyphenylalanine (DOPA) and serotonin.
Assuntos
Aminoidrolases/deficiência , GTP Cicloidrolase/deficiência , Fenilalanina/sangue , Animais , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Genótipo , Fígado/enzimologia , Camundongos , Camundongos Mutantes , Mutação , Oxirredutases/metabolismo , Fenilalanina Hidroxilase/metabolismoRESUMO
A mutation, resulting in a deficiency of liver GTP-cyclohydrolase activity, has been induced in the laboratory mouse. Mice homozygous for this mutation exhibit hyperphenylalaninemia under the following conditions: 1) early in life and 2) throughout life when exposed to phenylalanine. A phenylalanine loading regimen was used to discriminate between mutant and wild type mice on the basis of the resultant phenylalanine and tyrosine serum levels. Subjecting mice to this regimen reveals several distinguishing characteristics. Mutant mice exhibit approximately 2-fold higher peak phenylalanine levels than wild-type mice. In wild-type mice the hyperphenylalaninemic state is transient and rapidly abates while in mutant mice it is persistent and remains for a prolonged period. Mutant mice exhibit normal serum tyrosine levels after a loading challenge, while wild-type mice experience an increase in tyrosine levels. The loading regimen was also used to gauge the response of mutant hyperphenylalaninemic mice to exposure to chemical compounds required for normal phenylalanine catabolism (i.e. pteridine cofactors of the phenylalanine hydroxylase reaction). Mutant mice exposed to native enzyme cofactor or cofactor precursors exhibit a sharp decline in serum phenylalanine levels relative to their uninjected counterparts coupled with a tyrosine increase. By contrast, mutant mice exposed to nonprecursor compounds that are structurally related to the native cofactor, experience no diminution of serum phenylalanine levels.
Assuntos
Aminoidrolases/deficiência , GTP Cicloidrolase/deficiência , Fenilalanina/sangue , Animais , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Modelos Animais de Doenças , GTP Cicloidrolase/genética , Meia-Vida , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Mutantes , Fenilalanina/farmacocinética , Tirosina/sangueAssuntos
Alelos , Etilnitrosoureia/toxicidade , Ligação Genética , Mutagênicos , Mutação , Animais , Lactoilglutationa Liase/genética , CamundongosRESUMO
Ethylnitrosourea mutagenesis of spermatogonia in male mice is very efficient and makes it practical to isolate new desired mutant alleles by subsequent progeny screening. This is demonstrated for three genes in the t region of chromosome 17. The first, a mutation designated t-int, interacts with the dominant mutation, T (Brachyury), to produce a tailless mouse. Previously, mutant alleles of the t-int gene were available only in t haplotypes, where they are part of a t chromatin block within which recombination with wild-type chromosomes is inhibited. In addition to t-int, new mutations at the quaking and tufted loci were obtained, as well as at several loci not on chromosome 17, e.g., an X-linked lethal that causes a mottled phenotype in the heterozygote and four new mutant W alleles on chromosome 5. In the experiment, an average of one fertilizing spermatozoan in 1500 was mutant at a given locus and an average of one male in five was able to sire mutants at that locus.
Assuntos
Etilnitrosoureia/farmacologia , Camundongos/genética , Mutagênicos/farmacologia , Compostos de Nitrosoureia/farmacologia , Alelos , Animais , Fertilidade , Genes/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos CBA/genética , Camundongos Mutantes/genética , Camundongos Mutantes Neurológicos/genética , Pigmentação , Espermatogônias/efeitos dos fármacosRESUMO
The biological containment of the lambda gt family of cloning vectors has been enhanced by conditionally blocking DNA replication as well as head and tail morphogenesis. The vector, lambda gtALO.lambda B, was constructed by crossing the Oam29, Aama1 and Lam439 mutations into lambda gt.lambda B. The mutation blocking phage DNA replication, Oam29, is suppressed by suII+ or suIII+. The head gene mutation, Aama1, is suppressed by suIII+ but not by suII+ and the tail gene mutation, Lam439, is suppressed by suII+ but not by suIII+. This allows the option of increasing the biological containment by producing heads when a large amount of cloned DNA is being prepared from an individual isolate. A model recombinant, lambda gt Aama1 Lam439 Oam29.KmR' (lambda gtALO.KmR') was constructed and the containment of the vector was evaluated by the series of standardized experiments required for EK2 certification.
Assuntos
Colífagos/genética , DNA Recombinante/metabolismo , Cruzamentos Genéticos , Replicação do DNA , Enzimas de Restrição do DNA , DNA Viral/biossíntese , Escherichia coli/genética , Genes Virais , Genótipo , Mutação , Especificidade da EspécieAssuntos
Colífagos/crescimento & desenvolvimento , Adsorção , Membrana Celular/microbiologia , Clorofórmio/farmacologia , Colífagos/metabolismo , Colífagos/ultraestrutura , DNA Viral/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Magnésio/farmacologia , Putrescina/farmacologia , Espermina/farmacologia , TemperaturaAssuntos
Colífagos/metabolismo , DNA Viral/metabolismo , Adsorção , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Membrana Celular/microbiologia , Sistema Livre de Células , Clorofórmio/farmacologia , Colífagos/crescimento & desenvolvimento , Colífagos/ultraestrutura , Escherichia coli/metabolismo , Putrescina/farmacologia , TemperaturaAssuntos
Colífagos/metabolismo , DNA Viral/metabolismo , Escherichia coli/metabolismo , Poliaminas/farmacologia , Putrescina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colífagos/efeitos dos fármacos , Ácido Edético/farmacologia , Escherichia coli/efeitos dos fármacos , Magnésio/farmacologia , Espermina/farmacologia , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Ethidium-binding isotherms for purified circular lambda DNA, isolated from a superinfected lysogen, and for linear lambda DNA, isolated from the purified phage, were constructed from fluorescence measurements of ethidium-DNA MIXTURES. The measurements were made in 0.01 M Tris-HC1-0.001 M EDTA,pH 7.1, buffer at 20 degrees and in the same buffer containing 0.1, 0.4, or 1.0 M NaC1. When NaC1 was present, differences in the binding affinity for supercoiled and linear DNA could be quantitated. As the ethidium concentration was increased, supercoiled lambda DNA molecules bound the intercalating dye first more and then less avidly than nonsupercoiled ones. The number of potential supercoils in a circular lambda DNA molecular in the absence of dye was calculated from the amount of dye bound when it exhibited the same affinity for dye as its linear counterpart. The point of equivalent affinity shifted from 0.053 mol of dye bound per mol of nucleotide in 0.1 M NaC1 to 0.067 mol in 1.0 M NaC1. This corresponds to the removal of 164 and 206 supercoiling turns per molecule and superhelix densities in the absence of dye equal to 0.036 and 0.045 superhelical turns per 10 base pairs. If this difference in the number of supercoils reflects a salt-dependent change in the average rotation angle between base pairs of the Watson-Crick helix the angle differs by 0.32% in the two ionic environments.
Assuntos
Colífagos/análise , DNA Circular , DNA Viral , Etídio , Cloreto de Sódio , Sítios de Ligação , Vírus de DNA/análise , DNA Bacteriano , Escherichia coli/análise , Cinética , Matemática , Conformação de Ácido Nucleico , Concentração Osmolar , Espectrometria de Fluorescência , TemperaturaRESUMO
The sedimentation of circular lambda DNA suggests that the molecular undergoes significant changes in shape and super-coiling as the NaC1 concentration increases. Closed circular lambda DNA, species I, isolated and purified from superinfected immune bacteria, sediments in sucrose gradients of low ionic strength at a rate 2.0 times faster than linear lambda DNA, species III. The addition of ethidium causes the sedimentation rate of species I DNA to decrease until enough dye is bound to remove 121 supercoils per molecule. At this point, species I co-sediments with nicked and nonsupercoiled species II. Futher additions of ethidium cause the sedimentation rate to increase until the relative rate of species I is again at least twice that of species III. This classical behavior is altered when NaC1 is present in the buffer. In 1.0 M NaC1 the changes in S are complex. Initially, species I sediments 1.55 times faster than species III. Titration with ethidium caused a decrease in S to an early minimum value, than an increase to a first maximum, followed by a decrease to the S of species II. At this point enough dye has intercalated to remove 208 superhelical turns. Further additions of dye introduce supercoils and cause S to increase again. In 0.1 to 0.4 M NaC1 the relative S of species I is 1.69 and 1.59, respectively. If titrated with ethidium, S first increases to a maximum value then decreases to the minimum rate when enough dye is bound to remove 158 and 183 supercoils, respectively. The results indicate an increase in the superhelix density from 0.026 turns per 10 base pairs in buffer alone to 0.045 in the same buffer with 1.0 M NaC1. If this change in superhelix density results from a concomitant change in the average rotation angle between base pairs in the Watson-Crick helix, the addition of 1.0 M NaC1 alters the rotation angle by 0.68 degrees per base pair.
Assuntos
Colífagos/análise , DNA Circular , DNA Viral , Etídio , Cloreto de Sódio , Sítios de Ligação , Centrifugação com Gradiente de Concentração , Vírus de DNA/análise , DNA Circular/isolamento & purificação , DNA Viral/isolamento & purificação , Escherichia coli/análise , Cinética , Lisogenia , Conformação de Ácido Nucleico , Concentração Osmolar , Espectrometria de Fluorescência , SacaroseAssuntos
Colífagos/crescimento & desenvolvimento , Mutação , Proteínas Virais/biossíntese , Adsorção , Centrifugação com Gradiente de Concentração , Colífagos/metabolismo , Vírus de DNA , DNA Viral/metabolismo , Teste de Complementação Genética , Radioisótopos de Fósforo , Timidina/metabolismo , Trítio , Ensaio de Placa Viral , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Amber mutants previously mapped at seven sites in head gene F exhibit two contrasting phenotypes. Mutant F423, located in the right half of gene F, makes defective head particles with normal size lambdaDNA. These particles are complemented by crude lysates of mutants in other head genes. Mutants F785, F471 and F730 map in three adjacent sites in the left half of gene F. They are not complemented by lysates of other head mutants and do not produce headlike particles. Their lysates complement the incomplete heads present in F423. In vivo all three mutants are complemented by F423 but not by each other.