RESUMO
OBJECTIVE: To determine the association between maternal oral health and Early Childhood Caries (ECC) and discover the role of maternal and child behaviours in explaining this association. BASIC RESEARCH DESIGN: A cross-sectional analytic study. CLINICAL SETTING: Public Healthcare Services in Pasto, Colombia. PARTICIPANTS: 384 mothers and their 2-5-year-old children. METHODS: Mothers completed a questionnaire to provide information on sociodemographic and behavioural factors and were examined for caries experience (DMFS index) and periodontal status (Community Periodontal Index, CPI). Children were examined for dental caries (dmfs index). The association between maternal dental indicators and child dmfs was assessed in negative binomial regression adjusting for confounders. RESULTS: About 96% and 33% of mothers had caries experience and periodontal disease, respectively. The mean dmfs was 7.4 (SD: 9.5, range: 0-71). Maternal DMFS, but not CPI, was positively associated with children's dmfs (Rate Ratio: 2.51, 95%CI: 1.59-3.97) after adjustments for sociodemographic and behavioural factors. CONCLUSIONS: Maternal caries experience, but not periodontal status, was positively associated with ECC in their children. Maternal and child behaviours explained little of this association.
Assuntos
Cárie Dentária , Saúde Bucal , Criança , Pré-Escolar , Estudos Transversais , Índice CPO , Feminino , Humanos , PobrezaRESUMO
The HaeIII methyltransferase (MTase) gene from Haemophilus aegyptius (recognition sequence: 5'-GGCC-3') was cloned into Escherichia coli in the plasmid vector pBR322. The gene was isolated on a single EcoRI fragment and on a single HindIII fragment. Clones carrying additional adjacent fragments were found to code also for the HaeII restriction endonuclease and HaeII modification MTase (recognition sequence: 5'-PuGCGCPy-3'). The sequence of the HaeIII modification gene was determined. The inferred amino acid sequence of the protein was found to share extensive similarity with other sequenced m5C-MTases. The central 'non-conserved' region of the M.HaeIII MTase, thought to form the nucleotide sequence-specificity domain, is almost identical to that of the M.BsuRI, M.BspRI and M.NgoPII MTases, which also recognize the sequence 5'-GGCC-3'.
Assuntos
Proteínas de Bactérias/genética , DNA-Citosina Metilases/genética , Genes Bacterianos , Haemophilus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , DNA-Citosina Metilases/metabolismo , Genes , Haemophilus/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
The xmnIRM genes from Xanthomonas manihotis 7AS1 have been cloned and expressed in Escherichia coli. The nucleotide (nt) sequences of both genes were determined. The XmnI methyltransferase (MTase)-encoding gene is 1861 bp in length and codes for 620 amino acids (aa) (68660 Da). The restriction endonuclease (ENase)-encoding gene is 959 bp long and therefore codes for a 319-aa protein (35275 Da). The two genes are aligned tail to tail and they overlap at their respective stop codons About 4 x 10(4) units/g wet cell paste of R.XmnI was obtained following IPTG induction in a suitable E. coli host. The xmnIR gene is expressed from the T7 promoter. M.XmnI probably modifies the first A in the sequence, GAA(N)4TTC. The xmnIR and M genes contain regions of conserved similarity and probably evolved from a common ancestor. M.XmnI is loosely related to M.EcoRI. The XmnI R-M system and the type-I R-M systems probably derived from a common ancestor.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Xanthomonas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Bacteriano , Escherichia coli , Evolução Molecular , Expressão Gênica , Genes Bacterianos , Homologia de Genes , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Xanthomonas/genéticaRESUMO
The nucleotide sequence of the lambda gt11 SacI-KpnI region, surrounding the unique EcoRI cloning site, was directly determined. This sequence previously had to be compiled from several diverse sources. The direct sequence confirms the sequence predicted from the compilation and pinpoints other unique restriction enzyme targets in the region for use in subcloning.
Assuntos
Vetores Genéticos , Bacteriófago lambda , Sequência de Bases , Clonagem Molecular/métodos , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
The organization and nucleotide (nt) sequences were determined for the genes encoding the SalI restriction and modification (R-M) system (recognition sequence 5'-GTCGAC-3') from Streptomyces albus G. The system comprises two genes, salIR, coding for the restriction endonuclease (ENase, R.SalI; probably 315 amino acids (aa), a predicted M(r) of 35,305; product, G'TCGAC) and salIM, coding for the methyltransferase (MTase, M.SalI; probably 587 aa, a predicted M(r) of 64,943; product, GTCGm6AC). The genes are adjacent, they have the same orientation, and they occur in the order salIR then salIM. R.SalI contains a putative magnesium-binding motif similar to those at the active sites of R.EcoRI and R.EcoRV, but otherwise it bears little aa sequence similarity to other ENases. M.SalI is a member of the m6A gamma class of MTases. In aa sequence it resembles M.AccI, another m6A gamma-MTase whose recognition sequence includes the SalI recognition sequence as a subset.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Genes Bacterianos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Streptomyces/enzimologia , Streptomyces/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Mapeamento Cromossômico , Códon , Desoxirribonucleases de Sítio Específico do Tipo II/biossíntese , Genes Reguladores , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/biossíntese , Transcrição GênicaRESUMO
The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R.FokI) and methyltransferase (M.FokI) are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M.FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a 'tandem' structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M. FokI were constructed by deleting the 5'- or 3'-ends of the fokIM gene. Deleting most of the C-terminus of M.FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M.FokI for methylating the two strands of the recognition sequence are largely separate.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Flavobacterium/enzimologia , Metiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Análise Mutacional de DNA , Escherichia coli/genética , Flavobacterium/genética , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Vetores Genéticos , Proteínas de Transporte de Monossacarídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Cromatografia de Afinidade , Proteínas Ligantes de Maltose , Plasmídeos , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34,950 Da); the MTase is predicted to be 309 aa (35,550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible lambda pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.
Assuntos
Desoxirribonuclease HindIII/genética , Haemophilus influenzae/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Escherichia coli , Genes Bacterianos , Haemophilus influenzae/enzimologia , Dados de Sequência MolecularRESUMO
Bg/II, a type II restriction-modification (R-M) system from Bacillus globigii, recognizes the sequence 5'-AGATCT-3'. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R-M system. Oligonucleotide probes specific for the 5' end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R-M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R-M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R-M system.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias , Clonagem Molecular , Enzimas de Restrição-Modificação do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Evolução Molecular , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Primers do DNA/química , Enzimas de Restrição-Modificação do DNA/química , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Eletroforese em Gel de Ágar , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência , DNA Metiltransferases Sítio Específica (Adenina-Específica)/químicaRESUMO
SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.
Assuntos
Proteínas de Bactérias , Metilases de Modificação do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Streptomyces/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Escherichia coli , Expressão Gênica , Dados de Sequência Molecular , Mapeamento por Restrição , Streptomyces/genéticaRESUMO
NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC. The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli [Van Cott and Wilson, Gene 74 (1988) 55-59]. However, none of these clones expressed detectable levels of the restriction endonuclease (ENase). The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans. The complete NaeI system was finally cloned using E. coli AP1-200 [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] and less stringent MTase-selection conditions. The naeIR gene was expressed first by cloning into S. lividans, and later by cloning under control of a regulated promoter in an E. coli strain preprotected by the heterologous MspI MTase (M.MspI). The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo II/biossíntese , Escherichia coli/genética , Dados de Sequência Molecular , Nocardia/genética , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Streptomyces/genéticaRESUMO
We have cloned into Escherichia coli the genes for 38 type-II bacterial modification methyltransferases. The clones were isolated by selecting in vitro for protectively modified recombinants. Most of the clones modify their DNA fully but a substantial number modify only partially. In approximately one-half of the clones, the genes for the corresponding endonucleases are also present. Some of these clones restrict infecting phages and others do not. Clones carrying endonuclease genes but lacking methyltransferase genes have been found, in several instances, to be viable.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Metilases de Modificação do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Genes Bacterianos , Proteínas Recombinantes/genética , DNA Bacteriano/metabolismo , DNA Viral/metabolismo , Escherichia coli/genéticaRESUMO
CircumVent thermal cycle and standard DNA sequencing protocols utilizing the cloned and highly thermostable VentR (exo-) DNA polymerase are described. The thermal cycle sequencing procedures are advantageous because they allow fast and simple semiautomation of the sequencing reaction; make possible the direct DNA sequencing of PCR products, bacterial colonies and phage plaques; require only femtomoles of template DNA; eliminate the requirement of an independent primer annealing step; remove the requirement of denatured plasmids for sequencing double-stranded templates; and use a highly thermostable DNA polymerase for sequencing through potential recalcitrant secondary structure domains and large linear double-stranded DNA templates such as lambda derivatives. More standard methods of DNA sequencing (i.e., a one-step protocol and a labeling-termination protocol) are also presented. For each protocol, alternatives for choice of label and method of labeling are presented, including the use of 5' biotinylated primers for chemiluminescent DNA sequencing and fluorinated primers for automated sequencing using the BaseStation Automated DNA Sequencer.
Assuntos
DNA Polimerase Dirigida por DNA , Análise de Sequência de DNA/métodos , Autorradiografia , DNA/análise , Eletroforese em Gel de PoliacrilamidaRESUMO
A chemiluminescent DNA detection method is described and its application shown for both single-vector and multiplex DNA sequencing using the standard dideoxy chain-termination process. This recently developed detection method, which utilizes the light emitted by an enzyme-catalyzed dioxetane reaction, is highly sensitive and affords significant advantages in safety and speed over the traditional radioactive labeling method. When adapted to a multiplex strategy, this chemiluminescent detection method constitutes a safe, simple and rapid method for increasing the throughput of DNA sequencing procedures.
Assuntos
Sequência de Bases , DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas Genéticas , Vetores Genéticos/genética , Medições LuminescentesRESUMO
A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.
Assuntos
DNA Polimerase I/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Polimerase I/genética , DNA Polimerase I/isolamento & purificação , Primers do DNA/genética , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/isolamento & purificação , Exodesoxirribonucleases/metabolismo , Genes Bacterianos , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.
Assuntos
Clonagem Molecular , Enzimas de Restrição do DNA/genética , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Genes Bacterianos , Sequência de Aminoácidos , Bacteriófago lambda/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular/métodos , Códon , Enzimas de Restrição do DNA/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Especificidade da Espécie , Thermus/enzimologia , Thermus/genéticaRESUMO
The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.
Assuntos
Enzimas de Restrição do DNA/genética , DNA-Citosina Metilases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Western Blotting , Clonagem Molecular , Enzimas de Restrição do DNA/biossíntese , Enzimas de Restrição do DNA/química , Endonucleases/genética , Escherichia coli/fisiologia , Expressão Gênica/fisiologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genéticaRESUMO
BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/genética , Clonagem Molecular , Desoxirribonuclease BamHI/genética , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular/métodos , Metilases de Modificação do DNA/genética , Sondas de DNA/genética , Sondas de DNA/isolamento & purificação , Elementos de DNA Transponíveis , DNA Bacteriano/isolamento & purificação , Desoxirribonuclease BamHI/isolamento & purificação , Desoxirribonuclease BamHI/fisiologia , Escherichia coli/genética , Rearranjo Gênico , Dados de Sequência Molecular , Plasmídeos , Mapeamento por Restrição/métodos , Transformação GenéticaRESUMO
The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.
Assuntos
DNA-Citosina Metilases/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Sequência de Aminoácidos , Bacillus subtilis/genética , Clonagem Molecular , Códon , DNA Bacteriano/genética , DNA-Citosina Metilases/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The DNA polymerase gene from the Archaea Thermococcus litoralis has been cloned and expressed in Escherichia coli. It is split by two intervening sequences (IVSs) that form one continuous open reading frame with the three polymerase exons. To our knowledge, neither IVS is similar to previously described introns. However, the deduced amino acid sequences of both IVSs are similar to open reading frames present in mobile group I introns. The second IVS (IVS2) encodes an endonuclease, I-Tli I, that cleaves at the exon 2-exon 3 junction after IVS2 has been deleted. IVS2 self-splices in E. coli to yield active polymerase, but processing is abolished if the IVS2 reading frame is disrupted. Silent changes in the DNA sequence at the exon 2-IVS2 junction that maintain the original protein sequence do not inhibit splicing. These data suggest that protein rather than mRNA splicing may be responsible for production of the mature polymerase.