Abstract
The mouse Cig30 gene codes for a 30-kDa membrane glycoprotein, which appears to have a role in the recruitment of brown adipose tissue. To elucidate the structure of the Cig30 gene, we have isolated a lambda phage genomic DNA clone containing the entire mouse gene and found that Cig30 consists of four exons that are spread over 4 kilobase pairs of genomic sequence. Using a fluorescence in situ hybridization assay and interspecific backcross panel mapping, we have localized the Cig30 locus to the distal region of mouse chromosome 19, between the Tlx1 and Ins1 loci. Sequencing of the corresponding lambda clone to completion revealed that the insert contained yet another gene in the opposite orientation. It turned out to be the newly identified homeobox gene Pitx3. Interestingly, the genes are very tightly linked, so that the 3' ends of their transcripts are complementary. Thus, our results provide evidence for bidirectional transcription of a several hundred base pair-long DNA region as a result of the extremely tight linkage between Cig30 and Pitx3.
Highlights
The first mammalian member of this gene family [1]
The present knowledge comes almost exclusively from studies of three paralogous yeast genes of this family. Two of these yeast genes, known as FEN1/GNS1/VBM2/ELO2 and SUR4/ APA1/SRE1/ELO3, have been implicated in a complex pleiotropic phenotype suggesting a defect in a plasma membrane function and/or interaction between the plasma membrane and cytoskeleton [2]
Cig30 is the first mammalian member of a gene family that has been suggested to be involved in the elongation of very long chain fatty acids
Summary
Mouse Cig Genomic Cloning—Genomic clones of Cig were isolated by plaque hybridization of a commercial mouse 129 strain liver genomic DNA library in the Lambda FIX II vector (Stratagene catalog no. 946308) with a 32P-labeled probe corresponding to 1.2 kb from the 5Ј end of the Cig cDNA Mouse Cig Genomic Cloning—Genomic clones of Cig were isolated by plaque hybridization of a commercial mouse 129 strain liver genomic DNA library in the Lambda FIX II vector Phage DNA was prepared on a large scale by the polyethylene glycol precipitation method as described previously [5]. Restriction fragments generated by various combinations of restriction enzymes were analyzed by Southern blotting, and the presence of the 3Ј end of the Cig gene was checked by hybridization with a 3Ј untranslated region probe (0.8 kb from the 3Ј end of the Cig cDNA). Sequencing was performed with an ABI 373A automatic DNA sequencer (Applied Biosystems) on reactions prepared by the dye-termination method, using the ABI Prisms dye terminator cycle sequencing ready reaction kit (Perkin-Elmer). The nucleotide consensus sequence of the introns’ splice junctions are shown in boldface. The amino acid positions encoded by spliced codons are indicated in parentheses
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