Genomic organization of ␣1 and ␤1 subunits of the mammalian soluble guanylyl cyclase genes I. G. Sharina*, J. S. Krumenacker*, E. Martin, and F. Murad† Department of Integrative Biology and Pharmacology and the Institute of Molecular Medicine, University of Texas Medical School, 6431 Fannin, Houston, TX 77030 Contributed by F. Murad, July 17, 2000 The structures of the genes encoding the ␣1 and ␤1 subunits of 1-methyl xanthine (26), endotoxin and͞or IL-1␤ (27), NO- murine soluble guanylyl cyclase (sGC) were determined. Full-length donating compounds (28), and nerve growth factor (29) affect cDNAs isolated from mouse lungs encoding the ␣1 (2.5 kb) and ␤1 the sGC mRNA levels in various cell types. There is also (3.3 kb) subunits are presented in this report. The ␣1 sGC gene is evidence that levels of sGC mRNA expression are subject to approximately 26.4 kb and contains nine exons, whereas the ␤1 developmental regulation (30). Ongoing work in our laboratory, sGC gene spans 22 kb and consists of 14 exons. The positions of to be reported elsewhere, clearly demonstrates hormonal regu- ͞ ␣ ␤ exon intron boundaries and the sizes of introns for both genes are lation of the mRNA levels for 1 and 1 sGC subunits. described. Comparison of mouse genomic organization with the Despite an increasing interest in genetic aspects of sGC Human Genome Database predicted the exon͞intron boundaries regulation, relatively little is known about the genes or the of the human genes and revealed that human and mouse ␣1 and promoter regions of mammalian sGCs. Only the genomic orga- ␤1 sGC genes have similar structures. Both mouse genes are nization of sGC for such distant species as the Anopheles localized on the third chromosome, band 3E3-F1, and are separated mosquito (31) and Medaka fish (32) has recently been described. ,by a fragment that is 2% of the chromosomal length. The 5؅ Here we report the cDNA cloning, chromosomal localization ␣ ␤ ␣ ␤ untranscribed regions of 1 and 1 subunit genes were subcloned and structure of the mouse genes for the 1 and 1 subunits of into luciferase reporter constructs, and the functional analysis of sGC and a comparative analyses with the human sGC genes, promoter activity was performed in murine neuroblastoma N1E- using the Human Genome Database (National Center for cells. Our results indicate that the 5؅ untranscribed regions for Biotechnology Information, NCBI). This information furthers 115 both genes possess independent promoter activities and, together our understanding of the mechanisms of sGC regulation through with the data on chromosomal localization, suggest independent alternative splicing and͞or modulation of mRNA levels. regulation of both genes. Materials and Methods ͉ nitric oxide chromosomal localization Isolation of a cDNA Clone for Mouse sGC␣1 Subunit. A mouse lung ␭ Triplex cDNA library (CLONTECH) was screened by hybridiza- ␣ itric oxide-dependent signal transduction is associated with tion using a 1.3-kb rat sGC 1 cDNA fragment obtained by PCR Na number of important physiological processes, including using Taq polymerase (GIBCO) and the oligonucleotide primers Ј 91 Ј Ј 520 smooth muscle relaxation (1), platelet aggregation (2), neuro- 5 - TGCACTTCAGAGAACCTTG-3 and 5 - CTCCACCTT- Ј transmission (3), cellular differentiation (4), and apoptotic cell GTAGACATCCA-3 (superscript indicates position of codon at death (5–7). Soluble guanylyl cyclase (sGC), a NO-stimulated which the primers start). Six positive clones were identified from hemoprotein, which converts GTP to cyclic guanosine mono- approximately 1 ϫ 106 independent phage plaques. Positive clones phosphate, is a key element in these processes. were subsequently purified, sequenced bidirectionally for positive sGC is a heterodimer consisting of ␣ and ␤ subunits (8), clone identification, and analyzed by using DNASTAR software which are encoded by separate genes (9). A heme prosthetic (DNAstar, Madison, WI). After analysis, the clone was defined as ␣ group is crucial for the stimulation of the enzyme by NO (10, mouse 1 sGC and submitted to the NCBI database (accession no. 11). The enzyme has been purified from various animal tissues AF297082). This clone was used in all subsequent experiments and (10, 12, 13), and corresponding cDNAs were cloned from alignments in this article. various vertebrate species, including rat (14, 15), human (16), ␣ ␤ bovine (17, 18), and fish (19). At least two isoforms for each Isolation of Genomic Clones for Mouse sGC 1 and 1 Subunits. A subunit of the enzyme have been identified in various species, bacterial artificial chromosome (BAC) high-density membrane prompting a recent revision of the nomenclature of sGC mouse library was purchased from Genome Systems (St. Louis). subunits (20). Although isoforms for both subunits were The hybridization was performed overnight at 46°C in standard detected at the mRNA level in various tissues and were found hybridization solution (33). A random primer-labeled ␣32P- ␤ to have an overlapping tissue distribution, until recently only dCTP-labeled cDNA fragment (0.9 kb) for the 1 sGC probe was ␣ ͞␤ the 1 1 heterodimeric enzyme has been isolated from native generated by reverse transcription–PCR from a total RNA sources. However, Russwurm and coworkers (21) described an preparation from murine neuroblastoma N1E-115 cells, using ␣ ͞␤ additional 2 1 heterodimer, which was shown previously to be catalytically active in vitro. Alternatively spliced transcripts ␣ ␤ Abbreviations: sGC, soluble guanylyl cyclase; NCBI, National Center for Biotechnology for both human (22) and (23) subunits also have been ␤ ␤ ␣ Information; BAC, bacterial artificial chromosome; -gal, -galactosidase. reported. mRNA for the human 1 subunit undergoes alter- Data deposition: The sequences reported in this paper have been deposited in the NCBI native splicing, resulting in several mRNA species that are database (accession nos. AF297082 and AF297083). ␣ N-terminally truncated. The 2I subunit, an alternatively ␣ *I.G.S. and J.S.K. contributed equally to this paper. spliced variant of 2, has been detected in several mammalian ␤ †To whom reprint requests should be addressed. E-mail: [email protected]. cell lines and tissues at the mRNA level (24), and a 1 cDNA The publication costs of this article were defrayed in part by page charge payment. This splice variant has been detected in humans (25). article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Evidence that sGC activity is regulated both at the protein and §1734 solely to indicate this fact. mRNA levels has begun to emerge. Several groups have reported Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.190331697. that such treatments as forskolin, dibutyryl-cAMP or 3-isobutyl- Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.190331697 10878–10883 ͉ PNAS ͉ September 26, 2000 ͉ vol. 97 ͉ no. 20 Downloaded by guest on September 24, 2021 the oligonucleotides 5Ј--3GACACCATGTACGGTTTCGTG-3Ј extended to the first identified exon for each gene, DNA and 5Ј-243CCCTTCCTTGCTTCTCAGTAC-3Ј (superscript in- fragments were obtained by PCR using the specific genomic dicates the base pairs upstream of the start codon or the position clones as templates and Pfu Turbo DNA Polymerase (Strat- of the codon at which the primers start). agene). Positive-strand oligonucleotide primers for each ␣ ␣ Ј -1901 The membranes then were rehybridized with an 1 sGC cDNA construct were: 1.6 kb, 1 5 - GTCAGTGTCAGACCT- Ј ␤ Ј -1528 probe (1.3 kb containing coding sequence) under the same GAAGATGCTG-3 and 1.4 kb, 1 5 - CTCTCTGTGTGT- conditions. Positive BAC clones were identified by using the GAGAGAGAG-3Ј (superscript indicates the base pairs manufacturer’s procedure and purchased from Genome Sys- upstream of the start codon). Each of these positive-strand tems. A BAC plasmid purification kit (CLONTECH) was used oligonucleotide primers contained a KpnI restriction site linker for BAC DNA isolation from bacterial culture. BAC DNA was sequence at the 5Ј end. The negative-strand primers were: Ј -104 Ј ␣ subjected to restriction and Southern blot hybridization analysis 5 - CATGATGCGATCACAGGAGGC-3 for the 1 con- (33) using the same hybridization probes to confirm isolation of struct and 5Ј--105CGCCCGGAGCCTAGGAAGCAG-3Ј for the ␤ positive clones (data not shown). 1 construct (superscript indicates the base pairs upstream of the start codon). Each of the negative-strand primers contained a ␣ Ј Determination of Boundaries and Sizes of Introns. Based on the 1 BglII restriction site linker sequence at the 5 end. After restric- ␤ and 1 sGC cDNA sequences, sequencing oligonucleotide prim- tion digestion of the ends, the PCR fragments were directionally ers were designed to determine the genomic structure of each cloned into the luciferase reporter vector pGL3-Basic (Promega) subunit. All sequencing analyses were performed at the Molec- between the KpnI and BglII restriction sites upstream of the ular Core Sequencing Facility at the University of Texas- luciferase gene. Houston Medical School on an ABI Prism 377 DNA sequencer with the DigDye Terminator cycle sequencing kit (Applied Transfection and Detection of Luciferase Activity. N1E-115 mouse Biosystems). Primers positioned in the exons of both subunits neuroblastoma cells were maintained in DMEM with 4 mM were used to determine the intron sizes by PCR with Pfu-Turbo L-glutamine, 4.5 g͞liter glucose, 1% penicylin-streptomicyn DNA polymerase (Stratagene) from BAC DNA templates. PCR mixture, and 10% FBS (HyClone). Cells were transiently trans- ␣ ␤ conditions were: (i) melting step at 95°C for 1 min, (ii) primer fected with each ( 1 and 1 sGC) luciferase plasmid construct by annealing at 55°C for 1 min, and (iii) extension step at 72°C for using Fugene-6 transfection reagent (Roche Molecular Bio- 3 min, repeated for 35 cycles. PCR products were separated by chemicals). Cultures were incubated in the presence of Fugene GENETICS electrophoresis on 1% agarose gels. and DNA (1 ␮g) for 48 h and assayed for luciferase activity by using a luciferase reporter assay (Promega).