[Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli].
The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.
Chistiakov
D A
DA
Arsenian
S G
SG
Braga
E A
EA
Lapidus
A D
AD
Kozlov
Iu I
IuI
Mashko
S V
SV
Nosikov
V V
VV
Sverdlov
E D
ED
Debabov
V G
VG
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D005798:Genes, Bacterial; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D007375:Interleukin-1; D004247:DNA
Udalenie intronov metodom amplifikatsii DNK iz genomnogo gena interleĭkina 1 beta i ékspressiia gena interleĭkina 1 beta v Escherichia coli]