RESUMEN
The developmental brain gene NPAS3 stands out as a hot spot in human evolution because it contains the largest number of human-specific, fast-evolving, conserved, non-coding elements. In this paper we studied 2xHAR142, one of these elements that is located in the fifth intron of NPAS3. Using transgenic mice, we show that the mouse and chimp 2xHAR142 orthologues behave as transcriptional enhancers driving expression of the reporter gene lacZ to a similar NPAS3 expression subdomain in the mouse central nervous system. Interestingly, the human 2xHAR142 orthologue drives lacZ expression to an extended expression pattern in the nervous system. Thus, molecular evolution of 2xHAR142 provides the first documented example of human-specific heterotopy in the forebrain promoted by a transcriptional enhancer and suggests that it may have contributed to assemble the unique properties of the human brain.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Evolución Molecular , Regulación de la Expresión Génica/genética , Proteínas del Tejido Nervioso/genética , Prosencéfalo/metabolismo , Factores de Transcripción/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biología Computacional , Secuencia Conservada/genética , Cartilla de ADN/genética , Galactósidos , Humanos , Inmunohistoquímica , Hibridación in Situ , Indoles , Operón Lac/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Pan troglodytes/genética , Elementos de Nucleótido Esparcido Corto/genética , Especificidad de la Especie , Factores de Transcripción/metabolismoRESUMEN
Malpighian tubules constitute the main excretion organ of insects. Infection by egt(-) recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt(-), and used another two recombinants (one egt(-)) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt(-) viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.
Asunto(s)
Lepidópteros/anatomía & histología , Lepidópteros/virología , Túbulos de Malpighi/patología , Túbulos de Malpighi/virología , Nucleopoliedrovirus/patogenicidad , Animales , Muerte Celular , Línea Celular , Regulación Viral de la Expresión Génica , Genes de Insecto/genética , Genes Virales/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Operón Lac/genética , Larva/anatomía & histología , Larva/virología , Túbulos de Malpighi/ultraestructura , Microscopía Electrónica de Transmisión , Plásmidos/genéticaRESUMEN
The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.
Asunto(s)
Codón/genética , Biosíntesis de Proteínas/genética , ARN de Transferencia/metabolismo , Secuencia de Bases , Codón de Terminación/genética , Escherichia coli/genética , Operón Lac/genética , Terminación de la Cadena Péptídica Traduccional/genética , Plásmidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
We have shown that apotransferrin (aTf) promotes the differentiation of two oligodendroglial cell (OLGc) lines, N19 and N20.1, representing different stages of OLGc maturation. Although in both cell lines aTf promoted myelin basic protein (MBP) expression, an increase in cAMP levels and CREB phosphorylation was observed only in the less mature cells (N19), suggesting that the maturation induced by aTf is achieved probably through different signaling pathways. We transfected both cell lines with the proximal region of the human MBP promoter fused to the lacZ reporter gene. In both transfected cell lines, addition of aTf produced an activation of the promoter. To elucidate the mechanisms involved in this action, Western blot analysis, EMSAs, and RT-PCR were performed for different transcription factors involved in mbp regulation. In the N20.1 line, treatment with aTf increased the expression and the DNA-binding capacity of thyroid hormone (TH) receptors, Sp1, and nuclear factor-kappaB (NFkappaB). For these cells we found that an inductor of NFkappaB (tumor necrosis factor-alpha) promoted MBP messenger synthesis, whereas mithramycin, a specific inibitor of Sp1, and a cAMP analog (db-cAMP) inhibited its transcription. In the N19 cell line, aTf stimulated NF-I and NFkappaB activation, but, aside from aTf, only db-cAMP induced mbp transcription. These data suggest that, depending on the OLGc maturational stage, aTf modulates MBP expression and OLGc differentiation through different signaling pathways and different transcription factors.
Asunto(s)
Apoproteínas/fisiología , Proteína Básica de Mielina/biosíntesis , Oligodendroglía/metabolismo , Factores de Transcripción/fisiología , Transferrina/fisiología , Western Blotting , Línea Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN Complementario/biosíntesis , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Operón Lac/genética , FN-kappa B/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/aislamiento & purificación , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/genética , Sales de Tetrazolio , Tiazoles , Hormonas Tiroideas/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A genetic transformation system for penaeid shrimp could provide a powerful technique for the improvement of different production traits of importance for a sustainable aquaculture. The development of a successful transformation system depends on the ability to efficiently introduce exogenous DNA into the target species. The ability of the nuclear localization signal (NLS) peptide of the SV40 T antigen to facilitate nuclear import and transient gene expression is known from vertebrate systems and for the first time, is shown here to be efficient in a crustacean species, i.e. the shrimp Litopenaeus schmitti. Electroporation was used to introduce the pCMV-lacZ plasmid that contains the human cytomegalovirus promoter/enhancer (CMV) fused to the beta-galactosidase (lacZ) coding region, into L. schmitti zygotes. Supercoiled DNA was used at 50 or 500 ng/microl naked or bound to NLS peptide. The hatching rate of electroporated zygotes was around 60% for all groups, except from the pCMV-lacZ:NLS group at 500 ng/microl (43%). Based on Southern blot analyses of polymerase chain reaction (PCR) products the gene transfer frequency was 2-fold higher using DNA:NLS complexes than with naked DNA (23.8% vs. 11.5%, with 50 ng/microl of plasmid DNA, 44.3% vs. 28.8% with 500 ng/microl). The beta-galactosidase activity assay indicated that nuclear uptake is faster for the DNA:NLS complexes than for naked DNA. The beta-galactosidase activity was always higher in the DNA:NLS groups than in the naked DNA groups. To our knowledge, this is the first report on the use of an NLS peptide to improve gene transfer and nuclear uptake in crustaceans.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Núcleo Celular/metabolismo , Embrión no Mamífero/metabolismo , Señales de Localización Nuclear/genética , Penaeidae/genética , Transporte Activo de Núcleo Celular , Animales , Southern Blotting , ADN/administración & dosificación , ADN/genética , ADN/metabolismo , ADN Recombinante/genética , ADN Recombinante/metabolismo , Electroporación , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario , Femenino , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Operón Lac/genética , Óvulo/efectos de los fármacos , Óvulo/crecimiento & desarrollo , Óvulo/metabolismo , Penaeidae/embriología , Reacción en Cadena de la Polimerasa , Factores de Tiempo , Transfección/métodos , beta-Galactosidasa/metabolismoRESUMEN
An important characteristic of promoters used in recombinant protein production in Escherichi coli is their inducibility in a simple and cost-effective manner. The IPTG inducible promoters lac, tac, and trc are powerful and widely used for basic research. However, the use of IPTG in large-scale production is undesirable due to its high cost and toxicity. The promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source. Nevertheless, the use of this sugar in industrial processes has several drawbacks, which result in low volumetric yields and difficulties in process control. We have genetically engineered a BL21 strain to allow the efficient use of lactose as inducer in fed-batch cultures. Two modifications were introduced, the exchange of the wild-type lac operator by a constitutive one (lacO(c)) and the replacement of the gal alleles to recover the Gal(+) phenotype. The constitutive expression of the lac operon overcame the negative effects that the Lac nongenetic heterogeneity of wild-type E. coli introduces when lactose is used as inducer. The gal(+) genotype allowed the complete use of the lactose as carbon and energy source. The relevance of these two modifications in the efficient utilization of lactose as inducer was demonstrated in fed-batch cultures of the novel recombinant strain MP101 harboring expression vectors containing the calf prochymosin gene or the pccB gene, which encodes for the carboxyltransferase component of a propionyl-CoA carboxylase complex from Streptomyces coelicolor. Similar levels of recombinant protein production (up to 16 g/L) were obtained by using either lactose or IPTG as inducers, which confirmed the success of the genetics modifications introduced.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Lactosa/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/clasificación , Escherichia coli/crecimiento & desarrollo , Operón Lac/genética , Lactosa/genética , Regiones Promotoras Genéticas/genética , Especificidad de la EspecieRESUMEN
The phosphoglucomutase (pgm) gene codes for a key enzyme required for the formation of UDP-glucose and ADP-glucose, the sugar donors for the biosynthesis of glucose containing polysaccharides. A Mesorhizobium loti pgm null mutant obtained in this study contains an altered form of lipopolysaccharide (LPS), lacks exopolysaccharide (EPS), beta cyclic glucan, and glycogen and is unable to nodulate Lotus tenuis. The nonnodulating phenotype of the pgm mutant was not due to the absence of glycogen, since a glycogen synthase (glgA) null mutant effectively nodulates this legume. In M. loti, pgm is part of the glycogen metabolism gene cluster formed by GlgP (glycogen phosphorylase), glgB (glycogen branching), glgC (ADP-glucose pyrophosphorylase), glgA, pgm, and glgX (glycogen debranching). The genes are transcribed as a single transcript from glgP to at least pgm under the control of a strong promoter (promoter I) upstream of glgP. An alternative promoter (promoter II), mapping in a 154-bp DNA fragment spanning 85 bp upstream of the glgA start codon and the first 69 bp of the glgA coding region, controls the expression of glgA and pgm, independently of the rest of the upstream genes. Primer extension experiments showed that transcription starts 19 bp upstream of the glgA start codon.
Asunto(s)
Glucógeno Sintasa/genética , Glucógeno/metabolismo , Lotus/microbiología , Fosfoglucomutasa/genética , Rhizobiaceae/genética , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Agrobacterium tumefaciens/enzimología , Agrobacterium tumefaciens/genética , Secuencia de Bases , Genes Bacterianos/genética , Prueba de Complementación Genética , Glucosa-1-Fosfato Adenililtransferasa , Sistema de la Enzima Desramificadora del Glucógeno/genética , Glucógeno Fosforilasa/genética , Operón Lac/genética , Datos de Secuencia Molecular , Mutación , Nucleotidiltransferasas/genética , Operón/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhizobiaceae/enzimología , Simbiosis/genética , Transcripción GenéticaRESUMEN
Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.
Asunto(s)
Bacteriocinas/biosíntesis , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Anaerobiosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Secuencia de Bases , Medios de Cultivo Condicionados/química , Escherichia coli/metabolismo , Guanosina Pentafosfato/genética , Guanosina Pentafosfato/metabolismo , Concentración de Iones de Hidrógeno , Factores de Integración del Huésped , Operón Lac/genética , Operón Lac/fisiología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Datos de Secuencia Molecular , Oxígeno/farmacología , Pirofosfatasas/genética , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADNRESUMEN
In different bacterial species, ccmIEFH genes have been suggested to code for subunits of a bacterial haem-lyase catalyzing the covalent attachment of haem to c-type apoproteins. In Rhizobium etli CE3 there are two copies of ccmIEFH: one in the chromosome and the other located in plasmid pf. However, the null phenotype of chromosomal ccmF mutant indicates that the gene locus of plasmid pf is not functional. Two ccmI chromosomal mutants, previously isolated, produced detectable levels of c-type cytochromes under certain culture conditions in contrast with the ccmF mutant, suggesting that ccmF could be transcribed independently. The transcriptional organization of ccmIEFH operon was established. Two promoters from the chromosomal locus were mapped by primer extension, one located upstream of ccmI and the second located upstream of ccmF. The regulation of the expression of both promoters was studied using appropriate lacZ gene fusions (ccmI-lacZ and ccmEF-lacZ). The ccmI-lacZ gene fusion was expressed in complex medium, during exponential growth, under microaerobic conditions and in a R. etli mutant that accumulates reducing power, conditions where a higher respiration rate could be limited by c-type cytochrome content. The ccmEF-lacZ fusion was also primarily expressed in complex medium and under microaerophilic conditions. The finding of two independent promoters in this gene locus could suggest that the step catalyzed by CcmFH could be a rate-limiting step for c-type cytochrome assembly under certain culture conditions.
Asunto(s)
Grupo Citocromo c/metabolismo , Liasas/genética , Operón , Rhizobium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Bacterianos/genética , ADN Recombinante/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Operón Lac/genética , Liasas/metabolismo , Datos de Secuencia Molecular , Mutación , Fenotipo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Rhizobium/enzimologíaRESUMEN
Resistance of haploid yeast to hydrogen peroxide and to tert-butylhydroperoxide strongly increases when 4% glucose is replaced by glycerol or ethanol as the carbon source of the complex medium. Using a GSH1-promoter-lacZ-fusion reporter construct we could demonstrate that GSH1 is one of the genes that are up-regulated during the shift from fermentative to oxidative metabolism. A gsh1 mutant did not exhibit respiratory growth resistance to H2O2, whereas it was only slightly impaired in acquiring resistance against t-BOOH in the same experimental conditions. An isogenic deltayap1 mutant, although more sensitive to oxidative stress than the wild-type (WT), could increase resistance to both peroxides by a similar factor as observed for the WT when shifted from 4% glucose to a non-fermentable carbon source. This indicates that in this case induction of resistance to oxidative stress is independent from Yap1 and from the Yap1-mediated stress response via the STRE motif.
Asunto(s)
Alcoholes/farmacología , Proteínas de Unión al ADN/fisiología , Glucosa/farmacología , Glutatión/fisiología , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efectos de los fármacos , Factores de Transcripción/fisiología , Adaptación Fisiológica , División Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Proteínas de Unión al ADN/genética , Etanol/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , Glicerol/farmacología , Peróxido de Hidrógeno/farmacología , Operón Lac/genética , Mutación , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/genética , Transformación Genética , terc-Butilhidroperóxido/farmacologíaRESUMEN
Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.
Asunto(s)
Bacterias/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Genoma Bacteriano , Proteínas Virales , Acetiltransferasas/genética , Bacterias/efectos de los fármacos , Cloranfenicol/farmacología , Cloranfenicol O-Acetiltransferasa/genética , ADN Recombinante , Resistencia a Múltiples Medicamentos/genética , Escherichia coli/efectos de los fármacos , Eliminación de Gen , Marcadores Genéticos , Vectores Genéticos , Gentamicinas/farmacología , Integrasas/genética , Kanamicina/farmacología , Kanamicina Quinasa/genética , Operón Lac/genética , Mutagénesis Insercional , Plásmidos/genética , UDPglucosa 4-Epimerasa/genéticaRESUMEN
The Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins. The size of the groESL-specific transcript (2.3 kb) suggested that groES and groEL of C. crescentus are organized in a bicistronic operon. Heat-shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40 degrees C. Primer extension experiments showed that transcription initiated at two sites. Only the start site closer to the groES coding region was highly induced during heat shock. The promoter corresponding to the heat-shock-inducible transcript has -10 and -35 regions very similar to Escherichia coli sigma 32 promoters. At normal temperatures, transcription of the groESL operon is cell-cycle controlled and both transcripts increase co-ordinately in pre-divisional cells. Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat-shock promoter are necessary for temporal control. An 11 bp inverted repeat, located between the heat-shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat-shock genes of other bacteria, may play a role in cell-cycle control of C. crescentus groESL expression.
Asunto(s)
Caulobacter crescentus/metabolismo , Ciclo Celular/genética , Regulación Bacteriana de la Expresión Génica/genética , Secuencia de Bases , Northern Blotting , Caulobacter crescentus/genética , Chaperonina 10/química , Chaperonina 60/química , Clonación Molecular , Immunoblotting , Operón Lac/genética , Datos de Secuencia Molecular , Operón/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genética , Mapeo Restrictivo , Análisis de Secuencia , Eliminación de Secuencia/genética , Temperatura , Transcripción Genética/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
El operon lac de escherichia coli, es un sistema genético que ha sido útil para elucidar principios básicos de variación y expresión genética y para la construcción de cepas microbianas de proyección industrial. En este contexto, hemos derivado por clonación in vivo, un plasmidio de amplio rango de hospedero (pUCV3), que contiene los genes lac de e. coli, los cuales pueden ser ahora transmitidos con facilidad a todos los microorganismos capaces de incorporar replicones IncPa, grupo de compatibilidad al que pertenece pUCV3. Como ejemplo este plasmidio fue transmitido a bacterias marinas y a cytophaga johsonae, microorganismos en los cuales se apreció la expresión regulada del sistema lac. En consecuencia, pUCV3, puede ser usado como sonda para evaluar en comportamiento del operón lac en variados transfondos genéticos microbianos
Asunto(s)
Sondas de ADN/genética , Escherichia coli/genética , Operón Lac/genética , Clonación Molecular , Plásmidos/genéticaRESUMEN
Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination