期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1978
卷号：75
期号：8
页码：3688-3692
DOI：10.1073/pnas.75.8.3688
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The structural ovalbumin DNA sequences are not contiguous and are separated by multiple "intervening regions" in native chicken DNA. EcoRI, a restriction endonuclease that does not cleave the structural ovalbumin DNA sequences, digests the natural ovalbumin gene into three distinct fragments of 2.4, 1.8, and 9.5 kilobase pairs in length by cleaving within these "intervening regions." The 2.4-kilobase pair fragment contains only about 450 nucleotide pairs of coding sequence, with the rest being intervening sequences. This DNA fragment was cloned in bacteria by using the certified EK2 vector {lambda}gtWES{middle dot}{lambda}B after enrichment from total EcoRI-digested chicken DNA by a combination of RPC-5 column chromatography and preparative agarose gel electrophoresis. Five out of approximately 20,000 recombinant phage plaques were capable of hybridizing with a 32P-labeled Hha I fragment of a recombinant plasmid pOV230 containing the entire structural ovalbumin gene. DNA amplified in these recombinant phages, {lambda}gtWES{middle dot}OV2.4, was shown to contain the same restriction endonuclease cleavage sites as in the 2.4-kilobase pair EcoRI fragment previously determined by restriction mapping of total genomic chicken DNA. The intervening sequences were allowed to hybridize with excess total chicken DNA and oviduct nuclear RNA after nick-translation. They were found to be unique chicken DNA sequences, and appeared to be transcribed in their entireties during gene expression. Like the structural gene sequences, the expression of the intervening sequences is also inducible by steroid hormones.
