Genomic organization and promoter analysis of the gene encoding the mouse chemoattractant-like receptor, CMKLR1.
Chemoattractant-like receptor 1 (CMKLR1) is a functionally unknown ("orphan") G-protein coupled receptor. It has been implicated in osseous and cartilage development, and it also has a pathophysiological role as one of the minor coreceptors involved in human immunodeficiency virus type I (HIV-1)/simian immunodeficiency virus (SIV) infection of CD4(+) immune cells. Here we report the cloning of the mouse cmklr1 gene, the characterization of its genomic structure for comparison with the human gene, and the mapping and functional analysis of its 5' flanking sequence. The gene was found to contain three exons intercepted by one larger and one smaller intron. The overall structure resembles the human orthologue. The promoter lacks classical TATA and CCAAT boxes but contains several GC-rich regions as well as AP-4 elements, C/EBP motifs, and GATA-1 and GATA-2 binding sites. Promoter function was analyzed in mouse neuroblastoma (NB4 1A3) cells, endogenously expressing CMKLR1, as well as in mouse embryonic fibroblastic (3T3 clone A31) cells not expressing CMKLR1. 5' Deletion analysis and luciferase reporter gene assays of the promoter indicated that a 280-bp sequence adjacent to the transcription start site (established through 5'-RACE) is essential for initiating transcription. Within this region it was possible to identify four potential Sp1-binding sites that may be active in the transcription of the gene. Thus, we show that the mcmklr1 gene has several conserved features in common with its human counterpart, which suggests that they are regulated in a similar manner. The promoter does not seem to be tissue specific but other elements or enhancers may be missing. The results provide a necessary basis for further studies of the gene regulation and function of this chemoattractant-like receptor and will be useful when manipulating the gene in the development of transgenic animal models.
Mårtensson
Ulrika E A
UE
Division of Molecular Neurobiology, Wallenberg Neuroscience Center, BMC A12, SE-221 84, Lund, Sweden. Ulrika.Martensson@mphy.lu.se
Owman
Christer
C
Olde
Björn
B
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D019707:Receptors, Chemokine; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D024363:Transcription Initiation Site
C486456:CMKLR1 protein, mouse; D009687:Nuclear Proteins; D019707:Receptors, Chemokine; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases