Subject(s)
Chromosomes, Human, Pair 13 , Cosmids , Genetic Markers , Genomic Library , Oligonucleotides , Base Sequence , Blotting, Southern , DNA , Humans , In Situ Hybridization, Fluorescence , Metaphase , Microsatellite Repeats , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Genetic , Sequence Tagged SitesABSTRACT
Construction of human GM-CSF gene was conducted by the PCR technique. Four exons of GM-CSF gene were synthesized on the basis of human blood DNA using thermostable Tth DNA polymerase. Synthetic oligonucleotides were used as primers. The oligonucleotides contained sequences complementary to the ends of exons. Joining of exons was conducted by reciprocal complementation of the terminal sequences, followed by filling and amplification of the joined products. In most cases the effective synthesis of exons and joined products was possible only after optimizing the polymerase reaction conditions for each primer pair. Determination of the nucleotide sequence of the synthetic gene showed complete identity with the natural one. The gene was introduced into an expression vector under control of the promoter tandem (tac+lac). Expression of the GM-CSF gene was obtained in Pseudomonas putida cells. The recombinant protein had biological activity.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Pseudomonas putida/genetics , Base Sequence , Cloning, Molecular , Exons , Genetic Complementation Test , Humans , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Promoter Regions, GeneticABSTRACT
From a blood serum of patients with chronic posttransfusional non-A, non-B hepatitis the genomic RNA of hepatitis C virus (HCV) was isolated. Using RT-PCR (reverse transcription-polymerase chain reaction) there were synthesized and cloned cDNA fragments, representing 3 regions of the genome of a new virus isolate (HCV-R): 5'-nontranslating region, a core gene and a part of the nonstructural region NS3/NS4. Analysis of the nucleotide and of the amino acid sequences of a core and NS3/NS4 regions revealed significant difference between isolates from Russia (HCV-R) and from Japan (HCV-J). Nucleotide sequence homology between them was 90.0-90.87%, while homology between Russian and American isolates (USA-PT) complised 95.27-97.32%. No essential variations were found in the nucleotide sequences of 5'-nontranslating region of all three HCV isolates.
