Abstract
The frequency in human genomic DNA of three human interferon (IFN) alpha genes which differ from each other by a single base substitution was examined in 11 normal individuals. The polymerase chain reaction (PCR) was used to amplify the coding sequence for the Hu-IFN-alpha 2, Hu-IFN-alpha A, and Hu-IFN-alpha 2(Arg) sequences from genomic DNA. The PCR products were then cloned and individual clones were sequenced. PCR products were also analyzed by restriction endonuclease analysis for the IFN-alpha 2 and IFN-alpha A genes by use of a HinfI site which is eliminated by the substitution of an A for a G in IFN-alpha A. The IFN-alpha A gene which was cloned from the myeloblastoid cell line KG-1 was not observed in any of the 201 clones sequenced from normal individuals or in the Namalwa cell line. It was detected in KG-1 cell genomic DNA where it represented 49% of the clones sequenced. Similarly the IFN-alpha 2(Arg) gene was not detected in normal individuals or in the KG-1 cell line but only in the lymphoblastoid cell line from which it was cloned. In Namalwa cells the IFN-alpha 2(Arg) sequence represented 35% of the clones sequenced while the IFN-alpha 2 sequence comprised 59%. Therefore, both the IFN-alpha A and IFN-alpha 2(Arg) sequences represent alleles of the IFN-alpha 2 gene.
Highlights
The frequency in human genomic DNA of three human interferon (IFN) alpha genes which differ from each other by a single base substitution was examined in 11 normal individuals
It can be seen that the sequence for the a A gene defined by the aA-specific substitution of an A for G at nucleotide number 67 corresponding to amino acid 23of the protein coding sequence was not detected in any of the normal individuals examined in this study
These sequences were not examined in thisstudy, these changes may be unique to the KG-1 cell line from which it was isolated
Summary
After incubation for 1h at 37 "C, the reaction was extracted once with phenol/chloroform/ isoamyl alcohol (25:24:1) and precipitated with ethanol as above. Orientation of the insert was ascertained by utilizing an asymmetric EglII site at position 175 of the insert To this end, miniprep DNAs were digested with restriction endonucleases EcoRI and BglII and electrophoresed in 4% NuSieve 3:l gels (FMC Bioproducts, Rockland, ME). The DNA pellet was dried and sequenced by the dideoxy chain termination procedure [25] with the Sequenase enzyme (version 2.0, United States Biochemicals, Cleveland, OH) as described with C X - ~ ~ S - ~ A(26T)P. First strand cDNA synthesis was accomplished as described [29] from 2 pg of RNA template, 0.75 p M Primer I, and 200 units of (Moloney murine leukemia virus) reverse transcriptase in the following reaction: 4 pl of 5 X buffer
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