Isolation of a cDNA and characterization of the 5′ flanking region of the gene encoding the type I regulatory subunit of the cAMP‐dependent protein kinase

Ines NOWAK, Katja SEIPEL, Manuela SCHWARZ, David A. JANS, Brian A. HEMMINGS

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Abstract

A bovine cDNA probe for the type I regulatory subunit of the cAMP‐dependent protein kinase [Lee et al. (1983) Proc. Natl Acad. Sci. USA 80, 3608–3612] was used to screen two λgt11 libraries constructed from poly(A)‐rich RNA from the porcine kidney cell line, LLC‐PK1. A series of overlapping clones were isolated and characterized. The largest clone, λR115, of 1426 bp was found to code for the entire R1 protein but was apparently missing the 3′ end of the mRNA. The porcine cDNA codes for a protein of 389 amino acids that shows 99% homology to bovine R1 and hybridizes to two major mRNA transcripts of approximately 2.0 kb and 4.5 kb from LLC‐PK1 cells. The porcine cDNA for R1 was used to screen a genomic library of LLC‐PK1 DNA constructed in the EMBL‐3 vector and several clones were isolated and characterized. By using a probe from the 5′ end of the R1 cDNA we isolated the 5′ end of the gene and 700 bp of the promoter region of the gene were sequenced. The promoter region lacks a characteristic TATA box but contains two inverted CAAT boxes and is rich in G + C residues. Several sequence motifs were identified in the 5′ promoter region which could be responsible for the regulation of synthesis of this gene. Multiple transcription initiation sites were identified by S1 nuclease mapping.

Original languageEnglish
Pages (from-to)27-33
Number of pages7
JournalEuropean Journal of Biochemistry
Volume167
Issue number1
DOIs
Publication statusPublished - 1 Jan 1987
Externally publishedYes

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