RESUMO
BACKGROUND: High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing. RESULTS: Using gRNA/Cas9-mediated editing, we introduced a three bp deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as causative variant for the semi-dominant color dilution phenotype observed in Galloway and Highland cattle. Calves generated from cells with homozygous edits revealed a strong color dilution effect. Instead of the characteristic black and white markings of control calves generated from unedited cells, the edited calves displayed a novel grey and white coat pattern. CONCLUSION: This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. Although only one of the calves was healthy at birth and later succumbed to a naval infection, the study showed the feasibility of generating such edited animals with the possibility to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change.
Assuntos
Mudança Climática , Transtornos de Estresse por Calor , Animais , Bovinos , Edição de Genes , Resposta ao Choque Térmico , FenótipoRESUMO
Transgenic technology allows for the stable introduction of exogenous genetic information into livestock genomes. With its ability to enhance existing or introduce entirely novel characteristics at unprecedented magnitude and speed this emerging technology is expected to have a profound impact on the genetic improvement of livestock in the future. The continual advances in animal genomics towards the identification of genes that influence livestock production traits and impact on human health will increase its ability and versatility for the purposeful modification of livestock animals to enhance their welfare, produce superior quality food and biomedical products and reduce the environmental impact of farming. In contrast to biomedicine, which has so far been the main driver for this technology platform, the potential opportunities for animal agriculture are more challenging because of the greater demands on cost, efficiency, consumer acceptance and relative value of the product. While various transgenic concepts for the genetic improvement of livestock animals for agriculture are being evaluated the integration of this technology into practical farming systems remains some distance in the future.
Assuntos
Animais Domésticos/genética , Animais Geneticamente Modificados/genética , Engenharia Genética/tendências , Imunidade Inata/genética , Criação de Animais Domésticos/tendências , Animais , Tecnologia de Alimentos/tendências , HumanosAssuntos
Bovinos/embriologia , Bovinos/genética , Quimerismo/veterinária , Transferência Embrionária/veterinária , Feto , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA/genética , Feminino , Globinas , Masculino , Troca Materno-Fetal/genética , Gravidez , Quimeras de Transplante , Cromossomo YRESUMO
As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.
Assuntos
Clonagem de Organismos/métodos , Transferência Embrionária , Zona Pelúcida/metabolismo , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Bovinos , Linhagem Celular , Núcleo Celular/metabolismo , Embrião de Mamíferos/fisiologia , Feminino , Fertilização in vitro , Fibroblastos/metabolismo , Oócitos/metabolismoRESUMO
Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G(0), G(1), and different phases within G(1). We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G(0) results in a significantly higher percentage of viable calves at term than synchronization in early G(1) or late G(1). For transgenic fibroblasts, however, cells selected in G(1) show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G(0). This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.
Assuntos
Bovinos/genética , Ciclo Celular , Clonagem de Organismos , Técnicas de Transferência Nuclear , Animais , Bovinos/embriologia , Transferência Embrionária/veterinária , Desenvolvimento Embrionário e Fetal , Feminino , Fibroblastos/ultraestrutura , Fase G1 , Fase G2 , Mitose , Gravidez , Fase de Repouso do Ciclo CelularRESUMO
The development of multi-cellular organisms is regulated by the ordered definition of gene expression programmes that govern cell proliferation and differentiation. Although differential gene activity is mainly controlled by transcription factors, it is also dependent upon the underlying chromatin structure, which can stabilize transcriptional "on" or "off" states. We have recently isolated human (SUV39H1) and mouse (Suv39h1) histone methyltransferases (HMTases) and shown that they are important regulators for the organization of repressive chromatin domains. To investigate whether a SUV39H1-induced modulation of heterochromatin would affect mammalian development, we generated transgenic mice that over-express the SUV39H1 HMTase early during embryogenesis. SUV39H1 transgenic mice are growth retarded, display a weak penetrance of skeletal transformations and are largely characterized by impaired erythroid differentiation, consistent with highest transgene expression in foetal liver. Ex vivo transgenic foetal liver cultures initially contain reduced numbers of cells in G1 but progress to immortalized erythroblasts that are compromised in executing an erythroid differentiation programme. The outgrowing SUV39H1-immortalized erythroblasts can maintain a diploid karyotype despite deregulation of several tumour suppressor proteins and dispersed distribution of the heterochromatin component HP1. Together, these data provide evidence for a role of the SUV39H1 HMTase during the mammalian development and indicate a possible function for higher-order chromatin in contributing to the balance between proliferation and differentiation potentials of progenitor cells.
Assuntos
Diferenciação Celular , Divisão Celular , Eritroblastos/fisiologia , Eritropoese , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Peso Corporal , Osso e Ossos/anormalidades , Ciclo Celular , Células Cultivadas , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Desenvolvimento Embrionário e Fetal , Expressão Gênica , Heterocromatina/metabolismo , Histonas/metabolismo , Cariotipagem , Fígado/citologia , Fígado/embriologia , Fígado/enzimologia , Metiltransferases/genética , Camundongos , Camundongos Transgênicos , Penetrância , Proteínas Repressoras/genética , Proteína do Retinoblastoma/metabolismo , Baço/citologia , Baço/enzimologia , Transgenes , Proteína Supressora de Tumor p53/metabolismoRESUMO
Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, the Suv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although both Suv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.
Assuntos
Cromatina/genética , Histona-Lisina N-Metiltransferase , Metiltransferases/genética , Metiltransferases/metabolismo , Fosfoproteínas/genética , Testículo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Cromatina/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Embrião de Mamíferos/metabolismo , Fibroblastos , Expressão Gênica , Células Germinativas/metabolismo , Células HeLa , Histona Metiltransferases , Humanos , Immunoblotting , Hibridização in Situ Fluorescente , Masculino , Metiltransferases/química , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Filogenia , Proteínas Metiltransferases , RNA/metabolismo , Cromossomos Sexuais/metabolismo , Testículo/anatomia & histologia , Testículo/químicaRESUMO
SUV39H1, a human homologue of the Drosophila position effect variegation modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of SUV39H1 mutant proteins in transfected cells, we now show that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization. First, forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and metaphase chromatin. These properties depend on the C-terminal SET domain, although the major portion of the SUV39H1 protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the M31 interaction surface, which was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. Second, cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-specific distribution of phosphorylated histone H3 at serine 10 (phosH3) is dispersed in these cells. This phosH3 shift is not observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated M31. Together, our data reveal a dominant role(s) for the SET domain of SUV39H1 in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal SU(VAR) protein and histone H3.
Assuntos
Segregação de Cromossomos , Proteínas de Drosophila , Heterocromatina , Metiltransferases , Mitose , Proteínas Repressoras/metabolismo , Proteínas de Schizosaccharomyces pombe , Proteínas de Ciclo Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Aberrações Cromossômicas , Cromossomos Humanos Par 1/ultraestrutura , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genéticaRESUMO
The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila , Proteínas Repressoras/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Primers do DNA/genética , Drosophila/genética , Proteínas Fúngicas/genética , Genes de Insetos , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Proteínas de Insetos/genética , Camundongos , Dados de Sequência Molecular , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieAssuntos
Mapeamento Cromossômico , Proteínas de Drosophila , Genes Homeobox , Família Multigênica , Proteínas Nucleares/genética , Proteínas/genética , Proteínas Repressoras/genética , Animais , Sequência de Bases , Cruzamentos Genéticos , Primers do DNA , Proteínas de Ligação a DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Histona-Lisina N-Metiltransferase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Complexo Repressor Polycomb 2 , Fatores de TranscriçãoRESUMO
The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals. Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture. These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin--a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain. Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV. Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g. HOM-C).
Assuntos
Cromatina/metabolismo , Heterocromatina/metabolismo , Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona , Sequência Conservada , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Supressão Genética , Fatores de TranscriçãoRESUMO
Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes.
Assuntos
Proteínas de Drosophila , Regulação da Expressão Gênica , Heterocromatina/genética , Proteínas de Insetos/genética , Proteínas Nucleares/genética , Proteínas Repressoras , Telômero/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Drosophila/genética , Feminino , Genes Homeobox , Teste de Complementação Genética , Impressão Genômica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Transcription of the variable region of the rearranged immunoglobulin mu gene is dependent on an enhancer sequence situated within one of the introns of the gene. Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer. As this mu gene enhancer can establish local areas of accessible chromatin, we investigated whether the MARs can extend accessibility to more distal positions. We eliminated interactions between enhancer- and promoter-bound factors by linking mu enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer. The mu enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter. This long-range accessibility correlates with extended demethylation of the gene construct but not with whether it is being actively transcribed. MARs thus collaborate with the mu enhancer to generate an extended domain of accessible chromatin.
Assuntos
Cromatina/fisiologia , Cadeias mu de Imunoglobulina/genética , Matriz Nuclear/fisiologia , Sequências Reguladoras de Ácido Nucleico , Animais , Sítios de Ligação , Linhagem Celular , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonuclease I , Elementos Facilitadores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagos T/enzimologia , Transcrição GênicaRESUMO
A study of the expression of a bean phenylalanine ammonia-lyase (PAL) promoter/beta-glucuronidase gene fusion in transgenic tobacco has shown that the PAL2 promoter has a modular organization. Expression of the PAL2 promoter in the vascular system involves positive and negative regulatory cis elements. Among these elements is an AC-rich motif implicated in xylem expression and a suppressing cis element for phloem expression. Using radiolabelled complementary oligonucleotides bearing the AC-rich motif, a cDNA clone encoding a DNA-binding protein has been isolated from a tobacco lambda gt11 expression library. This factor, named AC-rich binding factor (ACBF), showed binding specificity to the AC-rich region. The specificity of ACBF for the AC-rich region was also shown using a gel retardation assay with an ACBF recombinant protein extract. The deduced amino acid sequence from ACBF contains a long repeat of glutamine residues as found in well characterized transcription factors. Interestingly, ACBF shared sequence similarity to conserved amino acid motifs found in RNA-binding proteins. Genomic gel blot analysis indicated the presence of a small gene family of sequences related to ACBF within the tobacco nuclear genome. Analysis of tobacco mRNA using the ACBF cDNA as probe showed that while ACBF mRNA was present in all tissues examined, the highest transcript accumulation occurred in stem tissues. The functional characteristics of the AC-rich sequence were examined in transgenic tobacco. A heptamer of the AC-rich sequence, in front of a minimal 35S promoter from cauliflower mosaic virus (-46 to +4), conferred specific expression in xylem.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Plantas , Fenilalanina Amônia-Liase/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Plantas Tóxicas , Ligação Proteica/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia , NicotianaRESUMO
A genetically engineered water-soluble derivative of PBP2x of Streptococcus pneumoniae has been produced, purified and crystallized in a form suitable for X-ray diffraction analysis. The best crystals have been grown at 15 degrees C, from solutions containing 8% polyethylene glycol 10,000 at pH values ranging from 3.9 to 6.0. These crystals diffract to a resolution of 3.5 A and have a space group P6(1)22 (or enantiomorph) with unit cell dimensions of a = b = 162.2 A, c = 171.8 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one molecule of enzyme per asymmetric unit. The breakdown of a chromogenic cephalosporin derivative diffused into a crystal reveals clearly that the enzyme is active in the crystalline state.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/química , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/química , Peptidil Transferases , Streptococcus pneumoniae/enzimologia , Cristalização , Proteínas de Ligação às Penicilinas , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Solubilidade , Água , Difração de Raios XRESUMO
A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases , Streptococcus pneumoniae/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Fracionamento Celular , Clonagem Molecular , DNA Bacteriano , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/isolamento & purificação , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas , Plasmídeos , Regiões Promotoras GenéticasRESUMO
Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Mutação , Penicilinas/metabolismo , Peptidil Transferases , Streptococcus pneumoniae/genética , Sequência de Bases , Proteínas de Transporte/metabolismo , Cefotaxima/farmacologia , Desoxirribonucleotídeos , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às PenicilinasRESUMO
Penicillin resistance in pneumococci is due to the appearance of high molecular-weight penicillin-binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX 2x genes (pbpX) of one penicillin-susceptible and five penicillin-resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low-affinity form of PBP 2x. The 2 kb region of the two penicillin-susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (less than 3% diverged), alternating with regions that were very different (18-23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson et al., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to beta-lactam antibiotics, indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Peptidil Transferases , Recombinação Genética/genética , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Variação Genética/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Humanos , Dados de Sequência Molecular , Proteínas de Ligação às Penicilinas , Infecções Pneumocócicas/microbiologia , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Streptococcus pneumoniae/efeitos dos fármacos , Transformação Bacteriana/genéticaRESUMO
Isolates of serotype 23F Streptococcus pneumoniae with high levels of resistance of penicillin have been commonly recovered in Spain for more than a decade. Recently penicillin-resistant serotype 23F S. pneumoniae strains were also isolated from children attending a day-care center in Cleveland. A number of Spanish and Cleveland isolates were compared by electrophoretic analysis of penicillin-binding protein (PBP) profiles and DNA restriction endonuclease cleavage profiles of the PBP 2X and 2B genes amplified with the polymerase chain reaction and by multilocus enzyme electrophoresis. All strains were identical by these criteria. The findings demonstrate that the Spanish and Cleveland isolates are clonally related and suggest that this antibiotic resistant clone of serotype 23F S. pneumoniae has spread intercontinentally from Spain to the United States.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/análise , DNA Bacteriano/análise , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/análise , Resistência às Penicilinas , Peptidil Transferases , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/classificação , Sequência de Bases , Proteínas de Transporte/genética , Resistência ao Cloranfenicol , Impressões Digitais de DNA , DNA Bacteriano/química , Resistência Microbiana a Medicamentos , Eletroforese em Gel de Amido , Enzimas/análise , Eritromicina/farmacologia , Humanos , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Ohio , Oligonucleotídeos/química , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Reação em Cadeia da Polimerase , Sorotipagem , Espanha , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Resistência a Tetraciclina , Combinação Trimetoprima e Sulfametoxazol/farmacologiaRESUMO
Four out of the five high molecular weight penicillin-binding proteins (PBPs) of Streptococcus pneumoniae are involved in the development of intrinsic penicillin resistance. In beta-lactam resistant laboratory mutants, point mutations in the PBP 2x-genes were identified that result in low penicillin-affinity mutant proteins. In contrast, PBPs 1a, 2x, and 2b of resistant clinical isolates are highly altered as can be recognized biochemically and immunologically; DNA sequence analysis of the PBP 2x gene from resistant strains confirmed these results. The variability of the three PBPs analyzed implies a very heterogeneous gene pool accessible to the pneumococcus that is used for recruitment of resistant PBP genes in wild type strains.