Cloning, Chromosome Mapping And Biological Characterization Of Porcine SCAP And INSIG Family

Objective:SCAP, INSIG1 and INSIG2 are involved in the regulation of lipid and cholesterol metabolism. To investigate the molecular basis of fat deposition in pig, we herein report the cloning, chromosome mapping and biological characterization of these three genes. Our data also lay foundation for the functional study of these genes.Methods:1. Based on the sequence of conserved region in genes from different species, primers were designed to amplify the corresponding genes in pig;2. cDNA of sepcific genes were amplified by RT-PCR;3. 5'and 3'ends of the cDNA were obtained by performing 5'and 3' RACE, respectively;4. Amplified products were purified, cloned and subsequently sequenced;5. Tissue distributions of specific genes were determined by RT-PCR;6. Genomic fragments of genes were obtained by PCR of genomic DNA;7. Chromosome location of genes were determined by RH mappingResults:1. A 505-bp partial INSIG1 open reading frame and a partial INSJG1 variant (INSIG1 variant 3) were obtained by RT-PCR. The splice vairant of INSIG1 is produced by a 292-bp deletion from the widetype JNSIG1 (INSIG1 variant 1) . Sequence comparisonshowed that pig INSIG1 shares 97.6% with human INSIG1. RT-PCR detection showed that JNSIG1 is expressed in liver, adipose tissue, heart, kidney, testis, gut, brain, muscle lung and spleen. INSIG1 variant 3 was not detected only in heart and muscle. A search of human, mouse and rat EST databases and RT-PCR detection of total RNA from mouse indicated that the splice event of INSIG1 occures in pig and human, and not in rodents.2. A 1295-bp INSIG2 cDNA containing a 675-bp full coding region was obtained by the combination of RT-PCR and 3' RACE. The encoded pig INSIG2 shares 99.1% homology with human INSIG2. RT-PCR detection showed that INSIG2 is expressed in heart, brain, spleen, lung, liver, muscle, kidney, testis, gut, and adipose tissue. Moreover, an expressed INSIG2 pseudogene, which shares 96.0% identity with the functional INSIG2 was identified. The pseudogene is intronless and contains several substitutions, deletions and a poly (A) tail at the 3' end. The BLAST searches in the human, mouse and rat genomic databases and PCR of monkey genomic DNA indicated that the JNSIG2 pseudogene resides uniquely in the pig genome.3. A 4045-bp full-length SCAP cDNA containing a 3870-bp full coding region was obtained by the combination of RT-PCR, 5' RACE and 3'RACE. The encoded pig SCAP shares 92.2% homology with human SCAP. RT-PCR detection showed that SCAP is expressed in liver, lung, kidney, gut, testis, brain, adipose tissue and muscle. The analysis of SCAP genomic structre showed that pig SCAP covers over 10 Kb in the genome, containing 18 exons and 17 introns. Comparison of the genomic structure of pig SCAP and its counterparts in human and mouse revealed intron losses in pig SCAP gene. Moreover, two splice variants of SCAP were identified. These variants are produced by deletion of 193 bp (SCAP variant 2) and 291 bp (SCAP variant 3) from the widetype SCAP (SCAP variant 1), respectively. SCAP variant 2 is expressed specificly in liver and muscle, and SCAP variant 3 is expressed exclusively in testis. A search of human, mouse and rat EST databases and RT-PCR detection of total RNA from mouse indicated that the splice events of SCAP occur exclusively in pig.4. By radio hybridization mapping, pig JNSIG1, INSIG2, INSIG2 pseudogene and SCAP were located to pig chromosome 18q21-23, 15ql4-15, 7q21-q23 and 13q21-22, respectively.Conclusion:1. Pig INSIG1, INSIG2 and SCAP show high homology with their counterparts in other species.2. A splice event of INSIG1, which is not conserved across species, ocurres in pig.3. An INSIG2 pseudogene, which exists uniquely in pig genome, resides on pig chromosme 7q21-q23.4. There two splice variants of SCAP, which exist exculsively in pig.5. Intron losses happened in pig SCAP gene during evolution6. Mapping data of INSIG1, INSIG2 and SCAP further support comparative map of human and pig, in which the synteny between HSA7 and SSC18, HSA2 and SSC15, HSA3 and SSC13 was established.