Isolation and Sequencing of a Putative Promoter Region of the Murine G Protein β1 Subunit ( GNB1 ) Gene

Abstract

The expression of the heterotrimeric GTP-binding protein beta 1 subunit gene (GNB1) is regulated by psychostimulants such as cocaine and amphetamines. Since the up-regulation appears to be one of the candidate processes of sensitization, it is necessary to elucidate the cellular and molecular mechanism of the GNB1 gene regulation for a better understanding the establishment of sensitization. In the present study, we describe the isolation and nucleotide sequence analysis of the GNB1 gene promoter region. We have isolated approximately 10 kb of the 5'-flanking region of the mouse of GNB1 gene and found potential elements involved in putative transcriptional control of the GNB1, such as AP1, AP2, Sp1, cyclic AMP response element, and nuclear factor kappa B recognition sites, within the sequences 0.3 kb upstream from the putative transcription start site. This region was highly rich in G + C content, but lacked TATA or CATT boxes. Comparing the nucleotide sequence of the cDNA clone with the human genome databases using the BLAST program a region containing putative exon 1 and promoter of the human GNB1 gene in chromosome 1 was found. The cloning and sequence analysis of an extensive portion of the 5'-flanking regulatory region of the GNB1 gene provides new insights into the factors involved in the regulation by psychostimulants of GNB1 expression.

Comparative Inter-strain Sequence Analysis of the Putative Regulatory Region of Murine Psychostimulant-regulated GeneGNB1(G Protein β1 Subunit Gene)

“…We previously isolated and characterized a cDNA for a putative promoter region of the murine GNB1 gene (designated G#2; Kitanaka et al, 2002a). Using the cDNA G#2 as a PCR template, we cloned the 341 bp DNA fragment of the GNB1 gene by PCR [designated L2; corresponding to the region between nucleotide 739 and 1079 of the deposited sequence data (GenBank accession number, AB066210)].…”

“…The hybridization membranes were exposed to an X-ray film with an intensifying screen at 2 808C for 5-7 h for GNB1 or for 3 -5 h for G3PDH, respectively. The bands of mouse GNB1 mRNA and G3PDH mRNA were detected with a size of 3.5 kb and 1.6 kb, respectively, estimated by comparison with an RNA size marker (Kitanaka et al, 2002a). The developed autoradiograms were analyzed using an Apple Macintosh computer-based image analysis system with the ATTO Densitograph software program (version 4.0, ATTO Co., Tokyo, Japan).…”

“…The DNA fragment from C11 was subcloned into pBluescript II-KS(þ ). The plasmid clone derived from C11 was sequenced in both directions using the dideoxy chain termination method (Sanger et al, 1977) in an automated DNA sequencer (Perkin Elmer-Applied Biosystems, Inc., model 377XL) as described previously (Kitanaka et al, 2001;2002a). Primers included the 5 0 -TGT AAA ACG ACG GCC AGT-3 0 (-21M13), 5 0 -CAG GAA ACA GCT ATG ACC-3 0 (M13RP1), and a series of custom designed primers.…”

“…The PCR reaction was initiated at 948C for 1 min, then carried out with 30 cycles at 948C for 15 s and 608C for 10 min, followed by the final polymerization period at 728C for 10 min using a primer set as follows: G2C57F1, 5 0 -AAT CAC GGG TCC TTT AAG CC-3 0 and E1R1, 5 0 -CTC GCC CTC GCC CGG TCT-3 0 . Then, clones from a C57BL/6 mouse genomic library, partially Sau3AI-digested and constructed in lambda DASH II vector at a Bam HI site (Stratagene, La Jolla, CA) were screened using the cDNA clone L2 as described previously (Kitanaka et al, 2002a). A positive candidate phage DNA clone (designated C11) after autoradiography was amplified and the DNA was isolated and purified.…”

“…In line with these observations, the GNB1 gene product appears to respond to a stimulation of drugs of abuse, and modulates the signal transduction pathways towards some phenomena associated with drug addiction. To investigate the different degrees of contribution of GNB1 gene to behavioral sensitization between murine strains, firstly, we attempted to analyze the nucleotide sequence of GNB1 gene locus from a genomic DNA library of C57BL/6 strain which is available commercially, because we previously isolated the gene locus from 129Sv strain (Kitanaka et al, 2001;2002a). We carefully compared the sequences to identify some polymorphisms between those two strains.…”