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A point mutation in the splice donor site of intron 7 in the αs2‐casein encoding gene of the Mediterranean River buffalo results in an allele‐specific exon skipping
Author(s) -
Cosenza G.,
Pauciullo A.,
Feligini M.,
Coletta A.,
Colimoro L.,
Di Berardino D.,
Ramunno L.
Publication year - 2009
Publication title -
animal genetics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.756
H-Index - 81
eISSN - 1365-2052
pISSN - 0268-9146
DOI - 10.1111/j.1365-2052.2009.01897.x
Subject(s) - biology , exon , intron , genetics , splice , primer (cosmetics) , microbiology and biotechnology , genotype , gene , point mutation , allele , restriction site , restriction enzyme , mutation , chemistry , organic chemistry
The CSN1S2 cDNA of 10 unrelated MediterraneanRiver Buffaloes reared in Southern Italy was amplifiedby RT-PCR, while the region from the 6th to the 8th exonof the CSN1S2 gene was amplified from genomic template.cDNA sequence comparisons showedthat five individuals had a normal transcript only (named CSN1S2A), one had adeleted transcript only (named CSN1S2B), because of the splicing out of the 27-bp ofexon 7, and the remaining four had a heterozygous pattern.Analysis of the genomic sequences revealed a FM865620:g.773G>C transversion that caused inactivation of the intron 7splice donor site and, consequently, the allele-specific exon skippingcharacteristic of the CSN1S2B allele. The g.773G>Cmutation creates a new AluI restriction site enabling a PCR–RFLP rapid genotyping assay. The cDNA sequences showed three additionalexonic mutations forming an extended haplotype withthe g.773G>C polymorphism: FM865618: c.459C>T,c.484A>T and c.568A>G homozygous and heterozygousrespectively in the CSN1S2BB and CSN1S2AB buffaloes. Thefirst is silent, while the remaining two are non-conservative(p.Ile162Phe and p.Thp200Ala respectively). The genotype frequencies (37 CSN1S2A/A,15 CSN1S2A/B and one CSN1S2B/B) are in agreement withHardy–Weinberg equilibrium, with thefrequency of the deleted B allele being 0.16.The predicted bubaline as2B proteinis 198 aa long instead of 207 aa and would also be characterizedby the presence of Phe at position 147 and Ala at 185