RESUMO
AIMS: Recently, an association between two polymorphisms (1181G>C and 245T>G) of the osteoprotegerin (OPG) gene and diabetic Charcot neuroarthropathy was suggested on the basis of studies of a limited number of samples derived from subjects from one geographical region (Italy). The aim of this study was to assess the presence of various osteoprotegerin gene polymorphisms in patients with diabetes and Charcot neuroarthropathy compared with subjects with diabetic neuropathy but no Charcot foot and healthy controls from another geographical region (Poland). METHODS: DNA was isolated from 54 patients with Charcot neuroarthropathy, 35 subjects with diabetic neuropathy but no Charcot foot, and 95 healthy controls to evaluate OPG gene polymorphisms and their possible contribution to the development of Charcot neuroarthropathy. RESULTS: Statistically significant differences between the group of subjects with neuropathy but no Charcot neuroarthropathy and the control group were found for 1217C>T, 950T>C and 245T>G polymorphisms, between the group of patients with Charcot neuroarthropathy and the control group for 1181G>C and 950T>C polymorphisms, and between the group of subjects with neuropathy but no Charcot neuroarthropathy and the group of patients with Charcot neuroarthropathy for 1217C>T and 245T>G polymorphisms. CONCLUSION: We suggest that genetic factors, particularly OPG gene polymorphisms, may play a role in the development of diabetic Charcot neuroarthropathy.
Assuntos
Artropatia Neurogênica/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Neuropatias Diabéticas/genética , Osteoprotegerina/genética , Polimorfismo de Nucleotídeo Único , Adulto , Artropatia Neurogênica/sangue , Artropatia Neurogênica/epidemiologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Neuropatias Diabéticas/sangue , Neuropatias Diabéticas/epidemiologia , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Osteoprotegerina/sangue , Polônia/epidemiologiaRESUMO
Familial X-chromosome inactivation (XCI) skewing was investigated in a family in which a female mucopolysaccharidosis type II (MPS II) (Hunter syndrome, an X-linked genetic disease) occurred. Among eight related females aged under 60 years from three generations who were tested, four revealed a non-random pattern of XCI. Detailed genetic analysis failed to find mutations in genes that were previously reported as important for the XCI process. Haplotype analysis excluded linkage of non-random XCI with genes localized on the X-chromosome. We propose that analysis of the XCI pattern should be taken into consideration when assessing risk factors for X-linked recessive genetic disorders.
Assuntos
Cromossomos Humanos X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Iduronato Sulfatase/genética , Mucopolissacaridose II/genética , Inativação do Cromossomo X/genética , Adulto , Idoso , Criança , Feminino , Glicosaminoglicanos/urina , Heterozigoto , Humanos , Lactente , Masculino , Mucopolissacaridose II/urina , Mutação de Sentido Incorreto , LinhagemRESUMO
The use of bacteriophages, instead of antibodies, in the ELISA-based detection of bacterial strains was tested. This procedure appeared to be efficient, and specific strains of Salmonella enterica and Escherichia coli could be detected. The sensitivity of the assay was about 10(5) bacterial cells/well (10(6)/ml), which is comparable with or outperforms other ELISA tests detecting intact bacterial cells without an enrichment step. The specificity of the assay depends on the kind of bacteriophage used. We conclude that the use of bacteriophages in the detection and identification of bacteria by an ELISA-based method can be an alternative to the use of specific antibodies. The advantages of the use of bacteriophages are their environmental abundance (and, thus, a possibility to isolate various phages with different specificities) and the availability of methods for obtaining large amounts of phage lysates, which are simple, rapid, cheap, and easy.
Assuntos
Técnicas Bacteriológicas/métodos , Infecções por Escherichia coli/diagnóstico , Escherichia coli/isolamento & purificação , Infecções por Salmonella/diagnóstico , Fagos de Salmonella/crescimento & desenvolvimento , Salmonella enterica/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/virologia , Humanos , Salmonella enterica/virologia , Sensibilidade e EspecificidadeRESUMO
AIMS: Drug safety evaluation plays an important role in the early phase of drug development, especially in the preclinical identification of compounds' biological activity. The Vibrio harveyi assay was used to assess mutagenic and antimutagenic activity of some aminoalkanolic derivatives of xanthone (1-5), which were synthesized and evaluated for their anticonvulsant and hemodynamic activities. METHODS AND RESULTS: A novel V. harveyi assay was used to assess mutagenic and antimutagenic activity of derivatives of xanthone 1-5. Two V. harveyi strains were used: BB7 (natural isolate) and BB7M (BB7 derivative containing mucA and mucB genes on a plasmid pAB91273, products of these genes enhance error-prone DNA repair). According to the results obtained, the most beneficial mutagenic and antimutagenic profiles were observed for compounds 2 and 3. A modification of the chemical structure of compound 2 by the replacement of the hydroxy group by a chloride improved considerably the antimutagenic activity of the compound. Thus, antimutagenic potency reached a maximum with the presence of tertiary amine and chloride atom in the side chain. CONCLUSIONS: Among the newly synthesized aminoalkanolic derivatives of xanthone with potential anticonvulsant properties, there are some compounds exhibiting in vitro antimutagenic activity. In addition, it appears that the V. harveyi assay can be applied for primary mutagenicity and antimutagenicity assessment of compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained preliminary mutagenicity and antimutagenicity results encourage further search in the group of amino derivatives of xanthone as the potential antiepileptic drugs also presenting some antimutagenic potential. Furthermore, V. harveyi test may be a useful tool for compounds safety evaluation.
Assuntos
Anticonvulsivantes/farmacologia , Antimutagênicos/farmacologia , Testes de Mutagenicidade , Mutagênicos/farmacologia , Vibrio/genética , Xantonas/farmacologia , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Antimutagênicos/química , Reparo do DNA , Avaliação Pré-Clínica de Medicamentos , Mutagênicos/química , Vibrio/efeitos dos fármacos , Xantonas/químicaRESUMO
Various processes of bacteriophage lambda development in Escherichia coli cells bearing either the whole lambda exo-xis region (with truncated, thus nonfunctional, exo and xis genes) or particular genes from this region were investigated. The presence of either the exo-xis region or the ea8.5 gene on a plasmid resulted in formation of fuzzy plaques by infecting phage. Both efficiency of plating and efficiency of lysogenization were decreased in such hosts. On the other hand, neither the efficiency of adsorption nor intracellular lytic development of the infecting phage (measured in one-step-growth experiments) was affected while significantly more host cells survived the infection, when containing the exo-xis region. Although no effects of the exo-xis region on the activity of the p (L) promoter was detected, this region contributed to a decreased transcription from the cII-stimulated promoters p (I), p (aQ) and p (E). These results, together with the results of measurement of efficiency of plating of phages bearing mutations in cI, cII and cIII genes on hosts containing the exo-xis region, strongly suggest that genes from this region (especially ea8.5) are involved in the regulation of bacteriophage lambda development at the stage of the lysis-vs.-lysogenization decision.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , DNA Nucleotidiltransferases , Exodesoxirribonucleases , Regulação Viral da Expressão Gênica , Proteínas Virais/metabolismo , Bacteriólise , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , Escherichia coli/virologia , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Lisogenia , Mutação , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
A general scheme of lambda phage and plasmid DNA replication in Escherichia coli is presented, and results of in vivo experiments from the authors' laboratory are superimposed. The initiator lambda O functions in the assembly of the replication complex (RC) at ori lambda, making it a stable component of this structure. ClpP/ClpX protease-specific action on lambda O does not affect the regulation of replication; it only degrades the surplus of synthesized lambda O. The initiator lambda O becomes protected from proteolysis at a distinct step of the pathway of RC assembly. The host DnaA initiator-regulated transcriptional activation of ori lambda seems to be coupled with RC assembly at the step of chaperone-mediated rearrangement of the pre-primosome. The once-assembled RC is inherited by one of two lambda plasmid daughter copies at each round of circle-to-circle (theta) replication. The inherited, old RC-driven replication is also dependent on RNA polymerase and DnaA functions. It seems that DnaA licenses lambda plasmid DNA for only one replication round, resembling the putative eukaryotic licensing factor in this respect. The lambda O binding to ori lambda does not seem to play any role in regulation of lambda plasmid replication, and the Cro-autoregulatory loop may be deleted. The emerging picture shows lambda plasmid circles with RCs bound to their ori, awaiting a signal triggering initiation of replication. The host DnaA initiator-regulated transcriptional activation of ori lambda may be involved in signal transmission. Inactivation of DnaA function blocks initiation of lambda phage DNA replication, but the lambdoid prophage Rac compensates this defect and all parental phage DNA molecules, after one round of theta replication switch to the sigma mode and produce progeny in high yield. We suspect that DnaA-regulated transcriptional activation is involved in installation and adequate positioning of two RCs, required for bidirectional replication, but in the Rac-promoted process only one RC may be installed, leading to unidirectional replication continued in the sigma mode. In wild-type cells consumption of DnaA function by the rapidly replicating lambda phage DNA may switch replication from bidirectional theta to unidirectional theta, and later to the sigma mode; the lambda circles produced earlier may play the role of Rac, which is required only when DnaA function has been inactivated prior to phage infection.
Assuntos
Bacteriófago lambda/genética , Replicação do DNA , Replicação Viral , Sequência de Bases , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation. Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of lambda plasmid replication.
Assuntos
Bacteriófago lambda/fisiologia , Replicação do DNA , DNA Viral/biossíntese , Escherichia coli/fisiologia , Genes Bacterianos , Plasmídeos , Replicação Viral , Bacteriófago lambda/genética , Radioisótopos de Carbono , DNA Viral/isolamento & purificação , Escherichia coli/genética , Isoleucina/metabolismo , Cinética , Mutação , Timidina/metabolismo , Fatores de Tempo , Trítio , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.
Assuntos
Bacteriófago lambda/metabolismo , Replicação do DNA , DNA Viral/biossíntese , Escherichia coli/metabolismo , Proteínas Virais/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Genes Bacterianos , Cinética , Metionina/metabolismo , Mutação , Plasmídeos , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Radioisótopos de Enxofre , Fatores de Tempo , Proteínas Virais/biossínteseRESUMO
The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Origem de Replicação/genética , Streptomyces/genética , Sítio Alostérico , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/ultraestrutura , Simulação por Computador , DNA Ligases/metabolismo , Replicação do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Circular/química , DNA Circular/genética , DNA Circular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Cinética , Microscopia Eletrônica , Modelos Biológicos , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Streptomyces/química , Transformação Bacteriana/genéticaRESUMO
We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.
Assuntos
Proteínas de Bactérias/fisiologia , Bacteriófago lambda/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/fisiologia , DNA Viral/biossíntese , Proteínas de Ligação ao GTP/genética , Recombinação Genética , Origem de Replicação , Proteínas rac de Ligação ao GTPRESUMO
Lysosomal storage diseases (LSDs) is a group consisting of over 50 disorders caused mostly by dysfunctions of lysosomal proteins and resultant accumulation of particular compounds inside cells and extracellular volumes in affected organisms. Genetic diseases are among the most difficult targets for medical treatment. Nevertheless, understanding of molecular bases of LSDs made it possible to develop novel procedures of treatment, employing molecular medicine. Although various therapeutic approaches have been proposed, and some of them were introduced into clinical practice, none of them was found to be effective in correcting all symptoms in treated patients. Central nervous system and skeleton appear to be the most difficult targets to be improved. Therefore, a proposal appeared that perhaps no single therapeutic procedure may be fully effective in treatment of LSD patients, and only combination of two or more approaches could be a successful therapy. In this review, we present and discuss current stage of various combination therapies for LSDs, based on already available published data.
Assuntos
Terapia Combinada , Doenças por Armazenamento dos Lisossomos/terapia , Animais , Terapia Combinada/métodos , Humanos , Doenças por Armazenamento dos Lisossomos/diagnóstico , Doenças por Armazenamento dos Lisossomos/etiologia , Doenças por Armazenamento dos Lisossomos/metabolismoRESUMO
Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.
Assuntos
Bacteriófago lambda/genética , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Virais/genética , Escherichia coli/genética , Lisogenia , Mutação , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
The initiator of phage lambda DNA replication, the lambda O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, ori lambda and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that lambda plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at ori lambda. DnaA may act by binding to DnaA-boxes, situated around the lambda pR promoter; there are no such sequences in ori lambda. Our results being to explain in molecular terms why lambda plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203-210] and why the initiation of phage lambda DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wegrzyn et al., Genetics (1995) in press].
Assuntos
Proteínas de Bactérias/fisiologia , Bacteriófago lambda/genética , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/virologia , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Bacteriófago lambda/fisiologia , Replicação do DNA , Escherichia coli/metabolismo , Replicon , Temperatura , Replicação ViralRESUMO
We have shown that Escherichia coli pcnB mutants are lysogenized by bacteriophage lambda with lower efficiency as compared to the pcnB+ strains. Our genetic analysis revealed that expression of the lambda cII gene is decreased in the pcnB mutants. However, using various lacZ fusions we demonstrated that neither activities of pL and pR promoters nor transcription termination at tR1 were significantly impaired in the pcnB- host. On the other hand, we found that oop RNA, an antisense RNA for cII expression, is involved in this regulation. Primer protection experiments revealed that oop RNA was polyadenylated and that this polyadenylation was impaired in the pcnB mutant. We found that the oop RNA was more abundant in the pcnB mutant than in the pcnB+ strain. Furthermore, we showed that activity of the pO promoter was not stimulated in the pcnB mutant. Such findings indicated that degradation of oop RNA in the pcnB strain was slower because of inefficient polyadenylation, which could lead to more effective inhibition of cII expression by the antisense oop RNA, resulting in less efficient lysogenization of the host. The oop RNA was found previously to play a role in phage lambda development only under conditions of overproduction of this transcript. Here we demonstrate for the first time, the physiological function of oop RNA in lambda development, confirming that this short transcript plays an important role in the negative regulation of cII gene expression during lambda infection. Moreover, polyadenylation of oop RNA is one of very few known examples of specific RNA polyadenylation by PAP I in prokaryotic cells and its role in gene expression regulation.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/genética , Proteínas de Escherichia coli , Polinucleotídeo Adenililtransferase , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Primers do DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Viral da Expressão Gênica , Genes Bacterianos , Lisogenia/genética , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Antissenso/química , RNA Mensageiro/química , RNA Viral/químicaRESUMO
The oop RNA is a short (77 nucleotides (nt)) transcript encoded by bacteriophage lambda which acts as an antisense RNA for lambda cII gene expression. Recently we demonstrated that oop RNA is specifically polyadenylated at its 3' end by poly(A) polymerase I (PAP I), the pcnB gene product. Here we demonstrate that the half life of oop RNA is 3 times longer in the pcnB mutant relative to the pcnB+ host, indicating that polyadenylation of this transcript causes its accelerated degradation. Although it was proposed that polyadenylation of RNAs in bacteria leads to their enhanced degradation, in most cases stabilization of these molecules was observed only when other mutations (pnp, rnb and rne) were present in the pcnB- strain. Therefore it seems that oop RNA may serve as a very useful model in further studies on molecular mechanisms of RNA polyadenylation and degradation in bacteria. Analysis of oop RNA and its degradation product isolated from Escherichia coli cells suggests that both polyadenylated and non-modified oop transcripts can act as antisense RNA.
Assuntos
Bacteriófago lambda/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/virologia , Meia-Vida , Mutação , Polinucleotídeo Adenililtransferase/genética , Células Procarióticas/metabolismoRESUMO
There are two modes of bacteriophage lambda DNA replication during its lytic development in Escherichia coli cells. The circle-to-circle (theta) replication predominates at early stages of the phage growth, whereas rolling-circle (sigma) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of lambda DNA into phage proheads. The mechanism regulating the switch from theta to sigma replication remains unknown. Our previous genetic studies indicated that the bacteriophage lambda Pts1piA66 mutant cannot replicate at 43 degrees C in the wild-type E. coli host, but it can replicate in the dnaA46(ts) mutant. Density shift experiments suggested that the parental DNA molecules of the infecting phage enter sigma replication. Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the lambdaPts1piA66 phage at 43 degrees C, the sigma replication intermediates are highly predominant over theta replication intermediates, contrary to the wild-type conditions (wild-type bacteria infected with the lambdaP(+) phage). The initiation of replication of the lambdaPts1piA66 mutant at 43 degrees C was strongly inhibited in the dnaA(+) host, as demonstrated by electron microscopy and by pulse-labeling of the phage-derived plasmid replicon. Implications for the mechanism of the regulation of the switch from theta to sigma replication mode are discussed.
Assuntos
Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/virologia , MutagêneseRESUMO
Rifampicin is an antibiotic which binds to the beta subunit of prokaryotic RNA polymerases and prevents initiation of transcription. It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30 degrees C to 43 degrees C is not completely inhibited by this antibiotic. Here we demonstrate that while activity of a pL-lacZ fusion (pL is a sigma70-dependent promoter) in E. coli cells is strongly inhibited by rifampicin, a p(groE)-lacZ fusion, whose activity is dependent on the sigam32 factor, retains significant residual activity even at relatively high rifampicin concentrations. Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the sigma70 or the sigma32 subunit was confirmed in vitro. Since the effects of an antibiotic that binds to the beta subunit can be modulated by the presence of either the sigma70 or the sigma32 subunit in the holoenzyme, it is tempting to speculate that binding of various sigma factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme.
Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Escherichia coli/genética , Holoenzimas/metabolismo , Regiões Promotoras Genéticas/genética , Rifampina/farmacologia , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Chaperoninas , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli , Genes Reporter , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Bacteriano/biossíntese , Temperatura , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage. RESULTS: Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the lambda(imm434) prophage. CONCLUSIONS: Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains.
Assuntos
Bacteriófago lambda/fisiologia , Proteínas de Ligação a DNA , Escherichia coli/virologia , Lisogenia , Prófagos/fisiologia , Ativação Viral , Bacteriófago lambda/efeitos dos fármacos , Bacteriófago lambda/genética , Biotecnologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Regulação Viral da Expressão Gênica , Glicerol/farmacologia , Lisogenia/efeitos dos fármacos , Plasmídeos/genética , Prófagos/efeitos dos fármacos , Prófagos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , Proteínas Virais , Proteínas Virais Reguladoras e Acessórias , Ativação Viral/efeitos dos fármacosRESUMO
We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Origem de Replicação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , DnaB Helicases , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Streptomyces/genética , Streptomyces/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMO
Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.