ABSTRACT
A streptavidin-coated TSK-gel support, with the loading capacity of 50-70 nmol of biotinylated substance per gram of dry support, and biotinylated oligonucleotides, containing the 4,9-dithiadodecane-6,7-dihydroxy-1,12-diphosphate insert, were prepared for the reversible immobilization of DNA. A non-nucleotide link can be located either at 5'- or 3'-end of the DNA fragment between the biotin moiety and the nucleotide sequence and is subjected to the selective periodate cleavage at the glycol group, which takes 45 min in solution and 3 h in heterophase. For the incorporation of the cleavable and biotin moieties into synthetic oligonucleotides, the corresponding phosphoramidite reagents and biotinylated CPG support were synthesized.
Subject(s)
DNA/chemistry , Oligodeoxyribonucleotides/metabolism , Base Sequence , Indicators and Reagents , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistryABSTRACT
Prolonged therapy of AIDS patients with azidothymidine results in the development of resistance to the drug. This phenomenon is accompanied by the appearance of point mutations in the pol gene coding for reverse transcriptase (RT). Kinetic studies of interactions of wild type RT and its forms containing the above-mentioned mutations with substrates and azidothymidine 5'-triphosphate have been carried out. Considerable differences in the affinities of RT and the mutants for RNA and DNA heteropolymer templates were established. The mutations did not affect the RT affinity for dNTP; however, its location influenced considerably the inhibition of the reaction with azidothymidine 5'-triphosphate.
Subject(s)
HIV-1/drug effects , RNA-Directed DNA Polymerase/metabolism , Thymine Nucleotides/metabolism , Zidovudine/analogs & derivatives , Zidovudine/pharmacology , Amino Acid Sequence , Base Sequence , DNA, Viral/metabolism , Dideoxynucleotides , Drug Resistance, Microbial/genetics , Genes, pol , HIV Reverse Transcriptase , HIV-1/enzymology , Humans , Kinetics , Molecular Sequence Data , Point Mutation , RNA, Viral/metabolism , RNA-Directed DNA Polymerase/genetics , Substrate Specificity , Zidovudine/metabolismABSTRACT
To investigate the biochemical basis of the HIV-1 resistance to AZT we obtained the RT mutant containing four amino acid substitutions by an oligonucleotide-directed mutagenesis technique. Enzymatic properties of the wild type and mutant RTs were compared. 'AZT-resistant' mutations in RT were shown to be associated with the reduced capability of AZT-TP to block the DNA- but not RNA-directed DNA synthesis.
Subject(s)
Antiviral Agents/pharmacology , HIV-1/enzymology , RNA-Directed DNA Polymerase/genetics , Thymine Nucleotides/pharmacology , Zidovudine/analogs & derivatives , DNA, Viral/biosynthesis , Dideoxynucleotides , Drug Resistance, Microbial/genetics , HIV Reverse Transcriptase , HIV-1/drug effects , Mutagenesis, Site-Directed , RNA-Directed DNA Polymerase/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Inhibitors , Substrate Specificity , Zidovudine/pharmacologyABSTRACT
Using the oligonucleotide directed mutagenesis, a human lymphotoxin (TNF beta) mutant gene lacking 21 N-terminal codons has been obtained. Recombinant plasmid pLT21 for expression of the mutant gene has been constructed. The mutant gene in the plasmid was placed under control of a tandem of constitutive promoters from coliphage T7. A simple procedure for isolation of recombinant protein was developed. The procedure allows to obtain the highly purified biologically active mutant protein with a good yield. During biosynthesis the recombinant protein undergoes a posttranslational processing resulted in the cleavage of N-terminal methionine and leucine residues.
Subject(s)
Escherichia coli , Gene Expression , Lymphotoxin-alpha/genetics , Mutagenesis, Site-Directed , Amino Acid Sequence , Base Sequence , Codon , DNA, Viral , Humans , Lymphotoxin-alpha/metabolism , Molecular Sequence Data , Plasmids , Promoter Regions, Genetic , Protein Processing, Post-TranslationalABSTRACT
Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.
Subject(s)
Escherichia coli/genetics , Genes, Synthetic , Recombinant Fusion Proteins/genetics , Thymosin/analogs & derivatives , Tumor Necrosis Factor-alpha/genetics , Amino Acid Sequence , Animals , Chromatography, DEAE-Cellulose , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Gene Expression , Mice , Molecular Sequence Data , Plasmids , Thymalfasin , Thymosin/geneticsABSTRACT
We have found, with the aid of 2-D gel electrophoresis, that double-stranded human telomeric repeat, (T2AG3)12.(C3TA2)12, being cloned within a plasmid, forms a protonated superhelically-induced structure. Experiments on chemical and enzymatic probing also indicate that the human telomeric repeats adopt an unusual structure. We have proposed an eclectic model for this structure in which four different elements coexist: a non-orthodox intramolecular triplex stabilized by the canonical protonated C.G*C+ base-triads and highly enriched by noncanonical base-triads; the intramolecular quadruplex formed by a portion of the G-rich strand; the single-stranded region encompassing a portion of the G-rich strand and, probably, the (C,A)-hairpin formed by a portion of the C-rich strand.
Subject(s)
DNA, Superhelical/chemistry , Repetitive Sequences, Nucleic Acid , Base Sequence , Electrophoresis, Gel, Two-Dimensional , Hydrogen-Ion Concentration , Molecular Probes , Molecular Sequence Data , Molecular Structure , Oligodeoxyribonucleotides/metabolism , TelomereABSTRACT
Gene estA coding for thermostable enterotoxin of Escherichia coli has been cloned. It is shown that in the E. coli strain SA162 this gene is located on the chromosome. Using polymerase chain reaction a site-directed mutagenesis of the cloned gene has been carried out, resulted in a recombinant strain--producer of the thermostable enterotoxin.
Subject(s)
Bacterial Toxins/genetics , Enterotoxins/genetics , Escherichia coli Proteins , Escherichia coli/metabolism , Amino Acid Sequence , Bacterial Toxins/isolation & purification , Base Sequence , Blotting, Southern , Chromosomes, Bacterial , Cloning, Molecular , DNA, Bacterial/genetics , Enterotoxins/isolation & purification , Gene Expression , Immunoenzyme Techniques , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids , Polymerase Chain ReactionABSTRACT
The cDNA sequence for human tumor necrosis factor (hTNF) was reconstructed in vitro from genomic sequence. Using the oligonucleotide directed mutagenesis, a site for restriction endonuclease ClaI was introduced into the end of the first exon. The nucleotide sequence representing the second and third exons flanked with restriction sites ClaI and XhoI was obtained by means of chemical enzymatic synthesis. Assembly of the total gene coding for precursor of hTNF was accomplished in pTNF33 plasmid containing semisynthetic gene for mature hTNF with appropriate restriction sites.
Subject(s)
Tumor Necrosis Factor-alpha/genetics , Amino Acid Sequence , Base Sequence , DNA/genetics , Genetic Engineering , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids , Restriction MappingABSTRACT
Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.
Subject(s)
Aphthovirus/genetics , Epitopes/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Viral , Genes, Viral , Amino Acid Sequence , Antigens, Viral , Aphthovirus/immunology , Base Sequence , Blotting, Western , Cloning, Molecular , DNA, Viral/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genes, Bacterial , Molecular Sequence Data , Plasmids , Restriction MappingABSTRACT
Recombinant plasmids coding for hybrid proteins between human interferon gamma and human tumour necrosis factor alpha or beta have been constructed using site-directed mutagenesis. The genes were fused via a synthetic oligonucleotide linker coding for tetrapeptide Pro-Val-Gly-Pro. The fused genes were expressed in Escherichia coli under control of early promoters of bacteriophage T7. E. coli cells harbouring the plasmids with the hybrid genes gave rather high level of the fused proteins biosynthesis. The hybrid recombinant proteins proved to be unstable in E. coli cells.
Subject(s)
Interferon-gamma/genetics , Plasmids , Tumor Necrosis Factor-alpha/genetics , Base Sequence , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Humans , Molecular Sequence Data , Recombination, GeneticABSTRACT
A number of recombinant plasmids coding for fusion proteins between human interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha) or beta (TNF beta) were constructed by using site-directed mutagenesis and ligation of the respective genes. In these proteins the whole IFN-gamma sequence of the molecule is linked at the N terminus via a short polypeptide linker to the TNF alpha sequence lacking two N-terminal amino acid residues or to the whole TNF beta sequence. A series of mutants with deletions in the interferon part of the fusion proteins were also produced. All the fusion genes obtained were efficiently expressed in Escherichia coli under the control of early promoters of bacteriophage T7. The recombinant fusion proteins were found to be unstable inside bacterial cells. Bacterial cell lysates expressing these fusion genes or their deletion mutants showed both biological activities in vitro: the antiviral activity of IFN-gamma and the cytotoxic activity of TNF.
Subject(s)
Interferon-gamma/genetics , Lymphotoxin-alpha/genetics , Recombinant Fusion Proteins/genetics , Tumor Necrosis Factor-alpha/genetics , Base Sequence , Cell Line , Cell Survival/drug effects , Cloning, Molecular , DNA , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Immunoblotting , Interferon-gamma/biosynthesis , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Lymphotoxin-alpha/biosynthesis , Lymphotoxin-alpha/metabolism , Lymphotoxin-alpha/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Vesicular stomatitis Indiana virus/drug effectsABSTRACT
DNA fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. The isomers were separated by the reversed-phase HPLC (RPC), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. The RPC-retention time values for Rp-isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P- and OH-components in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the amidophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp-isomer, whereas Rp-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphate by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereomers do take part in the ligation. As a result of the ligation of the modified fragments on unmodified templates a set of 31-mers was obtained. They contain FokI and EcoRI recognition sites with the cleavage points of both endonucleases coinciding and being amidated. Upon treatment of duplex DNA consisted of unmodified and amidated strands with these endonucleases Sp-configuration did not hinder the cleavage of the unmodified strand, whereas Rp-configuration inhibited the EcoRI and did not affect the FokI cleavage.
Subject(s)
DNA/chemistry , Base Sequence , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid HeteroduplexesABSTRACT
The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron. It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci. The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.
Subject(s)
Escherichia coli/genetics , Operon , Protein Biosynthesis , Ribosomes/physiology , Base Sequence , Genes, Bacterial , Molecular Sequence Data , Plasmids , RNA, Messenger/genetics , beta-Galactosidase/biosynthesisABSTRACT
We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.
Subject(s)
DNA/chemistry , Mutation , Purine Nucleotides/genetics , Pyrimidine Nucleotides/genetics , Repetitive Sequences, Nucleic Acid , Base Composition , Base Sequence , Cloning, Molecular , Electrophoresis, Gel, Two-Dimensional , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , Plasmids , Single-Strand Specific DNA and RNA Endonucleases , ThermodynamicsABSTRACT
Alterations of the C-terminal amino acid sequence of human tumour necrosis factor-alpha (hTNF-alpha) caused significant changes in its biological activity. Thus shortening of the C-terminus by removal of two or three amino acids led to a very marked loss of cytotoxic activity. Other, more subtle changes introduced by site-directed mutagenesis resulted in a less drastic reduction in cytotoxicity. The mitogenic activity towards human fibroblasts of the hTNF-alpha was reduced in parallel with the loss of cytotoxicity. These results suggest that the C-terminal amino acids of hTNF-alpha are critical for its biological actions and that they may be part of the receptor-binding site.
Subject(s)
Tumor Necrosis Factor-alpha/chemistry , Amino Acid Sequence , Cytotoxicity, Immunologic , Humans , Mitosis/immunology , Molecular Sequence Data , Mutagenesis, Site-Directed/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Tumor Necrosis Factor-alpha/immunologyABSTRACT
A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.
Subject(s)
DNA/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Base Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA/genetics , DNA Ligases , Escherichia coli/genetics , Molecular Sequence Data , Plasmids , Promoter Regions, Genetic , Spectrophotometry, UltravioletABSTRACT
Chemical-enzymatic synthesis of an artificial gene encoding leader peptide and 22 N-terminal amino acids of mature carboxypeptidase G2 from Pseudomonas sp. followed by enterokinase signal sequence (Asp4Lys) has been accomplished. The resulted DNA was fused with semi-synthetic gene coding for polypeptide 4-157 of mature human tumour necrosis factor (TNF) and then placed under control of early promoters of T7 bacteriophage. The expression products of the construct obtained was analysed using anti-TNF anti-serum. In E.coli leader peptide was cleaved off during translocation through inner membrane and the resultant product was found in membrane fraction.
Subject(s)
Bacterial Proteins/metabolism , DNA/genetics , Genes, Synthetic , Gram-Negative Bacteria/metabolism , Protein Sorting Signals/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA/chemical synthesis , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genes, Bacterial , Molecular Sequence Data , Plasmids , Protein Processing, Post-Translational , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.