RESUMO
2'-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the action of DNA glycosylases. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of an 11-mer DNA containing a single dAr.T base pair at its center. Analysis of the spectra revealed the existence of a right-handed duplex in solution, stabilized by Watson-Crick hydrogen bonding and base-stacking interactions. The carbocyclic sugar adopted a C1'-exo conformation and sugars of the 3'-flanking base pair had puckers in the O4'-endo range. The dAr.T base pair was mildly propeller twisted, and the dAr analogue showed a positive roll with the 3'-flanking base. Our findings indicate that the observed resistance of dAr-containing oligodeoxynucleotides to the catalytic action of DNA glycosylases relates to its electronic properties rather than structure, and validate the use of dAr and related carbocyclic nucleoside analogues for biological and structure/function relationship studies.
Assuntos
DNA/química , Desoxiadenosinas/química , Simulação por Computador , Desoxiadenosinas/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fósforo/química , Prótons , EspectrofotometriaRESUMO
Carbocyclic nucleosides have been of great interest as antiviral agents and in studies in the area of antisense technology. The recent finding that the replacement of a single 2'-deoxynucleoside in DNA by a carba analogue does not alter the Watson-Crick base pairing, yet at the same time provides a chemically and enzymatically stable "glycosidic" linkage, led us to examine this class of compound as enzyme inhibitors of the DNA-repair enzymes involved in oxidative damage. We now report the synthesis and incorporation into oligomeric DNA via suitable derivatives, the carbanucleosides 8-oxo-7,8-dihydro-2'-deoxycarbainosine, 8-oxo-7,8-dihydro-2'-deoxycarbaguanosine, and 2'-deoxyaristeromycin. Aristeromycin (1) was deoxygenated at the 2'-position as follows. Treatment of 1 with TPDSCl2 gave the 3',5'-protected derivative 3 (76%) which on phenylthiocarbonylation at the 2'-position gave 4 in 51% yield. The latter compound on reduction with Bu3SnH led to the 2'-deoxy derivative 5 (90%). Benzoylation followed by deprotection with TBAF in THF then gave the desired intermediate (6) in 65% yield. N2-Isobutyryl-8-oxo-7,8-dihydro-2'-deoxycarbaguanosine (16) was synthesized from 3-chloro-2'-deoxycarbainosine (9). Treatment of 9, either with hydrazine followed by catalytic reduction of the 2-hydrazino derivative or with 1-(2-nitrophenyl)ethylamine followed by photolysis of the resulting 2-substituted derivative, in both instances gave the desired 2'-deoxycarbaguanosine (12) in approximately 50% overall yield in each case. Bromination of 12 gave 13 (90%) which, when treated with BnONa in DMSO at 65 degrees C, led to the 8-benzyloxy derivative 14 (46%). Isobutyrylation of 14 followed by catalytic reduction then afforded 16. 8-Oxo-7,8-dihydro-2'-deoxycarbainosine (23) was prepared in four steps. Bromination of 2'-deoxyaristeromycin (19) at the 8-position gave 20 (> 95%) which was converted to the 8-benzyloxy derivative 21 (61%) using BnONa/DMSO at 80 degrees C. Reductive debenzylation of 21 then led to 8-oxo-7,8-dihydro-2'-deoxyaristeromycin (approximately 100%) which, when treated with adenosine deaminase, provided the desired carbainosine derivative 23 in quantitative yield. Compounds 6, 16, and 23 were converted to their respective 5'-O-DMT, 3'-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine] derivatives (8, 18, and 25) in excellent overall yields. The latter were then used to synthesize a series of DNA oligomers by automated procedures.
Assuntos
Inibidores Enzimáticos/síntese química , Nucleosídeos/síntese química , Adenosina/análogos & derivados , Reparo do DNA , GlicosilaçãoRESUMO
Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E. coli was examined. The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase. Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor. These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane. In the latter case, a large amount of the unprocessed "precursor" was accumulated. The combination of these modifications, however, did not work additively. An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion. These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region. Moreover, the overall balance of the physicochemical properties of respective regions is important.
Assuntos
Toxinas Bacterianas/genética , Enterotoxinas/genética , Fator de Crescimento Epidérmico/metabolismo , Proteínas de Escherichia coli , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Expression plasmids were constructed containing chemically synthesized human epidermal growth factor (EGF) gene fused in a frame to a leader sequence of human interleukin-2 (IL-2) gene under the control of a viral promoter. COS7 cells transfected with the plasmids synthesized and secreted EGF. Transfection of mouse A9 cells or BALB/3T3 clone A31 cells with the plasmids permitted the isolation of cell lines secreting the product which showed EGF activity. In particular, A31 transformed cells secreting human EGF grew well even in a medium containing a minimal level of serum. Using similar vectors having IgE cDNA (C2-C4) in place of EGF gene, a human IgE Fc fragment was also produced and secreted in mouse cells. These results show that heterologous leader sequences are useful for the expression and secretion of proteins whose genes lack leader sequences.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Imunoglobulina E/metabolismo , Interleucina-2/genética , Sinais Direcionadores de Proteínas/genética , Animais , Sequência de Bases , Linhagem Celular , DNA Recombinante , Fator de Crescimento Epidérmico/análise , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Humanos , Imunoglobulina E/análise , Imunoglobulina E/biossíntese , Imunoglobulina E/genética , Camundongos , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
An approach using a new combination of protecting groups in RNA oligomer synthesis is proposed, in which 5'-hydroxyl group of ribose moiety is temporarily protected with the alkaline labile 9-fluorenylmethoxycarbonyl (Fmoc) group and the 2'-hydroxyl group is protected with the acid labile 1-ethoxyethyl (EE) group. The adoption of this method presented great selectivity in removing the 5'-hydroxyl protecting group and facilitated the RNA oligomer synthesis. A RNA pentamer was synthesized by the phosphotriester method in solution.
Assuntos
Oligorribonucleotídeos/síntese química , RNA/síntese química , Cromatografia Líquida de Alta Pressão , Fluorenos , Hidroxilação , Indicadores e Reagentes , Oligorribonucleotídeos/isolamento & purificaçãoRESUMO
A high level of expression in yeast of a chemically synthesized human lysozyme (hL) gene was achieved by introducing an A-rich DNA fragment just upstream from the ATG start codon. The synthesized recombinant human lysozyme (r-hL) was insoluble and biologically inactive. It was solubilized with 7 M urea (pH 9) from yeast cells and its lytic activity was efficiently regenerated by oxidative renaturation. This renaturation experiment and Western blotting analysis under reducing and non-reducing conditions indicate that the insoluble form might be caused by the formation of incorrect intra- or intermolecular disulfide bonds. The N-terminal amino acid sequence of the purified r-hL was identical with that of authentic hL.
Assuntos
Muramidase/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Regulação da Expressão Gênica , Humanos , Muramidase/isolamento & purificação , Plasmídeos , Desnaturação Proteica , SolubilidadeRESUMO
Treating carbocyclic N1-methoxymethy-inosine and 2'-deoxy-inosine with 1N-NaOH/aq.EtOH gave the carbocyclic 5-amino-4-imidazolecarboxamide-riboside and -2'-deoxyriboside respectively. Reaction of both useful key intermediates with PhCONCS afforded the corresponding 5-(N-isothiocarbamoyl) derivatives in high yield. Methylation of the isothiocarbamoyl groups, followed by treatment with NaOH led to the purine ring-closure (guanine, isoguanine, and xanthosine) reaction.
Assuntos
Nucleosídeos de Purina/síntese química , Aminoimidazol Carboxamida/análogos & derivados , Indicadores e Reagentes , Metilação , RibonucleosídeosRESUMO
The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease.
Assuntos
Clonagem Molecular , Endopeptidases/genética , Genes Bacterianos , Genes , Serratia/genética , Sequência de Aminoácidos , Sequência de Bases , Códon , Enzimas de Restrição do DNA , Metaloendopeptidases , Plasmídeos , Serratia/enzimologiaRESUMO
Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.
Assuntos
Clonagem Molecular , Proteína Quinase C/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/enzimologia , DNA/análise , Proteína Quinase C/análise , RatosRESUMO
The chemical structure of aristeromycin M, a new carbocyclic nucleoside, was elucidated by spectroscopic analysis and chemical transformation from aristeromycin.
Assuntos
Adenosina/análogos & derivados , Adenosina/síntese química , Adenosina/isolamento & purificação , Adenosina/farmacologia , Animais , Fenômenos Químicos , Química , Espectroscopia de Ressonância Magnética , CamundongosRESUMO
A modified nucleoside antibiotic on polymer support was employed in the solid-phase synthesis of two oligodeoxynucleotides to substitute acid labile 2'-deoxyadenosine(A) at the 3'-terminus of oligomers with acid resistant 2'-deoxyaristeromycin(Ar). The duplex of the decamer having sticky ends was inserted successfully into the Eco R1 site of pBR322. E. coli DH 1 was transformed with the Ar-containing plasmid. Inspection of the cloned plasmid pBR2552 by restriction endonucleases revealed that no deletion and insertion occurred near the inserted sequence. This result indicates that Ar can behave as A in DNA replication of the host cells. Therefore, Ar can be used as a substitute for 3'-terminal A in the solid-phase oligonucleotide synthesis.
Assuntos
Desoxiadenosinas/análogos & derivados , Oligodesoxirribonucleotídeos/síntese química , Clonagem Molecular , Enzimas de Restrição do DNA , Escherichia coli/genética , Indicadores e Reagentes , PlasmídeosRESUMO
A promoter with the consensus sequence(TTGACA and TATAAT) at -35 and -10 regions was constructed, and the distance between the two consensus sequences was independently altered at two restriction sites. In an in vitro transcription system, a maximum activity was observed at the spacer length of 17 base-pairs regardless of the sites of space adjustment. Evidence was also provided that the start point of transcription was fixed by the consensus sequence at the -10 region.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Genes , Óperon , Transcrição Gênica , Composição de Bases , Sequência de Bases , Enzimas de Restrição do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Relação Estrutura-Atividade , Fagos T/enzimologiaRESUMO
A 809 bp Sau 3A - Hpa I fragment containing a complete HBsAg gene and fragments 744 bp Hinc II - Hpa I and 712 bp Xba I - Hpa I containing a truncated HBsAg gene lacking the sequence encoding the NH2-terminal hydrophobic domain were prepared from a composite plasmid pHBV933 containing the 3.2 kb Eco RI DNA fragment of the entire HBV/adw genome and inserted into an expression vector pTRP801 to give plasmids pTRP SS-6, pTRP SS-39, and pTRP SS-50, respectively. The growth of a recombinant having pTRP SS-6 was greatly inhibited and the transformant expressed a low level of HBsAg, which is reactive to human anti-HBsAg antibody. Interestingly, the growth of transformants harbouring pTRP SS-39 and pTRP SS-50 was not inhibited and these transformants expressed a considerable level of the HBsAg. Minicells harbouring pTRP SS-6, pTRP SS-39, and pTRP SS-50 formed specific polypeptides of about 24 K, 23 K, and 22 K daltons, respectively.
Assuntos
Escherichia coli/genética , Genes , Antígenos de Superfície da Hepatite B/genética , Complexo Antígeno-Anticorpo , Composição de Bases , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Antígenos de Superfície da Hepatite B/análise , Humanos , Cinética , PlasmídeosRESUMO
A phosphorylating reagent o-chlorophenyl phosphoro-p-anisi-dochloridate was synthesized to phosphorylate the 3'-hydroxyl group of N, 5'-protected deoxynucleosides. These nucleotides served as 3'-terminal units for the synthesis of oligonucleotide blocks. By condensation of these oligonucleotide blocks the partially complementary deoxypentadecanucleotides dAGCTTATAATGC-TCG and dAGCTCGAGCATTATA, which contained the ideal Pribnow sequence TATAATG, were synthesized.