RESUMO
Certain genes are only expressed at one allele, a phenomenon called imprinting. Although it is well established that one allele of certain imprinted genes is silenced through methylation, this does not appear to be the case for all imprinted genes. In a thoughtful Perspective, Thorvaldsen and Bartolomei discuss new findings showing that insertion of insulator elements (boundary regions) between the promoter of a gene and its enhancer (a sequence that boosts gene expression) may be another way in which genes are silenced during imprinting.
Assuntos
Metilação de DNA , Inativação Gênica , Impressão Genômica , RNA não Traduzido , Proteínas Repressoras , Alelos , Animais , Fator de Ligação a CCCTC , Proteínas de Ligação a DNA/metabolismo , Fosfatos de Dinucleosídeos , Elementos Facilitadores Genéticos , Pai , Feminino , Humanos , Fator de Crescimento Insulin-Like II/genética , Masculino , Modelos Genéticos , Mães , Proteínas Musculares/genética , Óvulo/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante , Sequências Reguladoras de Ácido Nucleico , Espermatozoides/metabolismo , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol 3-kinase. These early events lead to activation of the serine-threonine protein kinase Akt, also known as protein kinase B. We show that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on liver and skeletal muscle. These data establish Akt2 as an essential gene in the maintenance of normal glucose homeostasis.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Resistência à Insulina , Insulina/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Glicemia/metabolismo , Desoxiglucose/metabolismo , Feminino , Marcação de Genes , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Homeostase , Insulina/administração & dosagem , Insulina/sangue , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de SinaisRESUMO
The yeast Candida glabrata harbors two distinct gene families that encode metallothioneins (MTs). One of these loci, the MT-IIa locus, exhibits selective and tandem amplification in many wild type strains of C. glabrata. The present paper demonstrates that the amplified MT-IIa gene contains autonomously replicating sequences (ARS). These ARS elements have been used to construct vectors capable of replicating in C. glabrata. The ARS element(s) in the MT-IIa gene were localized to a 457-bp segment downstream from the MT-IIa coding sequence. Although plasmids containing this fragment transform C. glabrata with high frequency, the stability of the transformants and the copy number of the plasmid improve when the entire 1.25-kb MT-IIa gene is used. Transformation of C. glabrata with plasmids carrying the 2 microns circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2 microns plasmids replicate only to a limited extent in C. glabrata. Conversely, a C. glabrata plasmid carrying three copies of the MT-IIa gene was able to transform S. cerevisiae.
Assuntos
Candida/genética , Replicação do DNA/genética , Genes Fúngicos , Metalotioneína/genética , Sequência de Bases , Clonagem Molecular/métodos , Amplificação de Genes , Dados de Sequência Molecular , Família Multigênica , Oligodesoxirribonucleotídeos , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Transformação GenéticaRESUMO
Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.
Assuntos
Candida/genética , Cobre/farmacologia , Metalotioneína/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Candida/efeitos dos fármacos , Sulfato de Cobre , Análise Mutacional de DNA , Resistência Microbiana a Medicamentos/genética , Metalotioneína/química , Dados de Sequência Molecular , Mutagênese/genéticaRESUMO
The ribonuclease protection assay (RPA) is a sensitive technique for the analysis of total cellular RNA. It involves generating a specific antisense riboprobe, hybridizing the probe to total RNA, removing unprotected RNA by RNases, and finally isolating and analyzing the protected RNA on a denaturing gel. Although the RPA is somewhat more labor-intensive than Northern analysis, it has the advantage of being more sensitive (as little as 0.1 pg of target RNA can be detected with ideal hybridization conditions). RPAs are also more tolerant of partially degraded RNA (provided the area that is protected is intact). Although RPAs are not as sensitive as polymerase chain reaction (PCR)-based RNA analyses, the target RNA is analyzed directly; a reverse transcription step is not required. Finally, the RPA is quantitative as long as the probe is in excess. More important for the study of imprinted genes, the RPA can be designed to detect allele-specific expression of the target gene of interest.
Assuntos
Ensaios de Proteção de Nucleases/métodos , Ribonucleases/metabolismo , Animais , Humanos , Sondas RNA/genéticaRESUMO
The N-terminal metal-binding domains of the copper-activated yeast transcription factors, ACE1 and AMT1, bind to specific DNA sequences in a Cu-dependent fashion. Recombinant AMT1 and ACE1 metal-binding domains are isolated as Cu4Zn1-protein complexes. Site-directed mutagenesis of AMT1 was used in this study to map the ligands of the Cu(I) and Zn(II) ions. The results are consistent with the N-terminal halves of AMT1 and ACE1 consisting of two independent submodules, one binding a single Zn(II) ion and the second binding the tetracopper cluster. The basis of this conclusion is, first, that mutations of two cysteinyl codons and a histidyl codon in the first 42 residues of AMT1 do not alter DNA binding. In contrast, serine substitutions at four cysteine positions at codons 43, 61, 90, and 98 abolish DNA binding. We demonstrated previously that population of the Zn(II) site in AMT1 does not alter the ability of the protein to bind DNA but bound Cu(I) ions are essential for DNA binding [Thorvaldsen, J. L., et al. (1994) Biochemistry 33, 9566-9577]. Second, mutations in the N-terminal 42 residue segment reduce the Zn(II) content of purified mutant AMT1 molecules. Third, a synthetic peptide consisting of the N-terminal 42 residues in AMT1 forms a stable Zn(II) complex and substitution with Co(II) reveals an electronic spectrum identical to that of the Co-substituted intact Cu4AMT1 protein. 113Cd(II) NMR studies reveal that the divalent metal site consists of ligands provided by three cysteinyl thiolates and a single histydyl imidazole. The sequence homology between AMT1, ACE1, and MAC1 in the N-terminal 42 residues suggests that ACE1 and MAC1 will, likewise, contain N-terminal Zn modules. A 42-residue ACE1 synthetic peptide gives identical metal binding properties to the corresponding AMT1 synthetic peptide. Thus, AMT1 and likely ACE1 consist of two contiguous modules, residues 1-42 forming an independent Zn(II) module and residues 43-110 enfolding a tetracopper cluster.
Assuntos
Cobre/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metaloproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Candida/genética , Sequência Conservada , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas Fúngicas , Ligantes , Metaloproteínas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genéticaRESUMO
Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.
Assuntos
Metilação de DNA , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Proteínas Musculares/genética , RNA não Traduzido , Alelos , Animais , Feminino , Heterozigoto , Homozigoto , Fator de Crescimento Insulin-Like II/biossíntese , Masculino , Camundongos , Modelos Genéticos , Proteínas Musculares/biossíntese , RNA Longo não Codificante , Deleção de Sequência , Fatores SexuaisRESUMO
The AMT1 metalloregulatory trans-acting factor from Candida glabrata was found to functionally mimic the ACE1 metalloregulatory trans-acting factor from Saccharomyces cerevisiae in the copper-induced expression of the chromosomal S. cerevisiae metallothionein gene. Plasmid constructs with promoters of various metal-inducible genes fused to the bacterial beta-galactosidase (lacZ) reporter gene were used in S. cerevisiae to evaluate the roles of ACE1 and AMT1 in mediating metal-stimulated expression. Promoters from the S. cerevisiae CUP1 gene and Cu,Zn-superoxide dismutase (SOD1) and from the C. glabrata MT genes MTI, MTIIa, and MTIIb were used. The ACE1 factor was effective in the metalloregulation of the two S. cerevisiae promoters, CUP1 and SOD1, but of only one C. glabrata promoter, MTI. AMT1 was found to be effective in the metalloregulation of all three C. glabrata MT promoters and the two S. cerevisiae promoters tested. The regulation mediated by both ACE1 and AMT1 was copper-dependent and copper-specific. Episomally expressed SWI5, a distinct trans-acting factor of S. cerevisiae, enhanced only the basal expression from promoters. The SWI5 enhancement was not metal dependent. In conclusion, AMT1 and ACE1 are functionally homologous in metal-specific regulation, AMT1 appears to be more promiscuous than ACE1 in this function.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Metalotioneína/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Candida/genética , Clonagem Molecular , Cobre/farmacologia , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Cinética , Metais/farmacologia , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/efeitos dos fármacos , Superóxido Dismutase/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBbeta, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728-1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBalpha, display a conspicuous impairment in organismal growth. Akt1(-/-) mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBbeta null mice, Akt1/PKBalpha-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 and Akt2 genes with respect to organismal growth and insulin-regulated glucose metabolism.
Assuntos
Glucose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Alelos , Animais , Glicemia/metabolismo , Southern Blotting , Fibroblastos/metabolismo , Genótipo , Heterozigoto , Camundongos , Camundongos Knockout , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt , Radioimunoensaio , Fatores Sexuais , Transdução de Sinais , Fatores de TempoRESUMO
Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTIIb is more highly inducible than MTIIa and cells harboring a single MTIIb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between -03 and -292 upstream of the MT coding sequences. Expression of MTI is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.
Assuntos
Candida/genética , Metalotioneína/genética , Plasmídeos/genética , Sequência de Bases , Candida/efeitos dos fármacos , Mapeamento Cromossômico , Cobre/farmacologia , DNA Fúngico/genética , Resistência Microbiana a Medicamentos/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido NucleicoRESUMO
The molecular mechanism leading to the imprinted expression of genes is poorly understood. While no conserved cis-acting elements have been identified within the known loci, many imprinted genes are located near directly repetitive sequence elements, suggesting that such repeats might play a role in imprinted gene expression. The maternally expressed mouse H19 gene is located approximately 1.5 kb downstream from a 461-bp G-rich repetitive element. We have used a transgenic model to investigate whether this element is essential for H19 imprinting. Previous results demonstrated that a transgene, which contains 14 kb of H19 sequence, exhibits parent-of-origin specific expression and methylation analogous to the endogenous H19 imprinting pattern. Here, we have generated transgenes lacking the G-rich repeat. One transgene, containing a deletion of the G-rich repetitive element but which includes an additional 1.7 kb of 5' H19 sequence, is imprinted similarly to the endogenous H19 gene. To determine whether the G-rich repeat is conserved in other imprinted mammalian H19 homologues, additional 5' flanking sequences were cloned from the rat and human. This element is conserved in the rat but not in human DNA. These results suggest that the 461-bp G-rich repetitive element is not essential for H19 imprinting.
Assuntos
Impressão Genômica , Proteínas Musculares/genética , RNA não Traduzido , Sequências Repetitivas de Ácido Nucleico , Animais , Composição de Bases , Sequência de Bases , DNA/química , DNA/metabolismo , Metilação de DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Guanina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Longo não Codificante , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/fisiologia , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transgenes/genéticaRESUMO
We have examined heterologous protein secretion from Candida glabrata with the aid of a stable C. glabrata vector and a secretion reporter cassette comprising the Saccharomyces cerevisiae PGK-gene promoter and a Kluveromyces Iactis secretion signal to drive secretion of Escherichia coli beta-lactamase. Abundant secretion of beta-lactamase from C. glabrata indicates that the S. cerevisiae PGK promoter functions in C. glabrata. Furthermore, we show that C. glabrata processes the secreted beta-lactamase in a manner similar to, but not identical with, S. cerevisiae and K. lactis. C. glabrata may be a suitable new host for the expression of foreign genes.
Assuntos
Candida/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Sequência de Bases , Candida/metabolismo , Clonagem Molecular , DNA Recombinante , Dados de Sequência Molecular , Fosfoglicerato Quinase/genética , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , beta-Lactamases/metabolismoRESUMO
AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata. The copper-binding, DNA-binding domain of AMT1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a gel shift assay. The Cu-activated AMT1 was shown to contain a Cu(+)-thiolate tetracopper center and a single Zn2+ site. AMT1 is purified as a Cu-Zn protein from bacterial cultures grown in the presence of CuSO4. Chemical analysis suggested that 4.2 +/- 0.2 and 1.2 +/- 0.2 molar equiv copper and zinc ions bound, respectively. Electrospray mass spectrometry was used to verify that a uniform species was present with 4 Cu+ ions and 1 Zn2+ ion bound per AMT1 molecule. Cu+ binding to form a tetracopper center occurs cooperatively as shown by electrospray MS of apoAMT1 samples reconstituted with increasing equivalency of Cu+. Copper-thiolate coordination was indicated by Cu-S charge-transfer transitions in the ultraviolet, luminescence typical of Cu-thiolate clusters and EXAFS. Analysis of the EXAFS of CuZnAMT1 revealed predominantly trigonal Cu+ coordination and the presence of a polycopper cluster by virtue of a short Cu-Cu distance of 2.7 A. Zn K-edge EXAFS of Cu4Zn1AMT1 and electronic spectroscopy of AMT1 with Co2+ substituted for the single Zn2+ ion are consistent with tetrahedral Zn2+ coordination with thiolate ligands. The Cu-activated AMT1 exhibited a conformation distinct from that of metal-free AMT1 as shown by circular dichroism. DNA binding by AMT1 was dependent on the tetracopper center but was independent of occupancy of the Zn2+ site. This is the first report of a single, uniform tetracopper center in a metal-activated transcription factor.