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A Novel Human Lipid Binding Protein Coding Gene: PERF15, Sequence and Cloning
Pouresmaeili Farkhondeh1, Khalili Tahereh, Jeddi Tehrani Mahmood
1Department of Genetics, Faculty of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran ; Fertility-Infertility Health Research Center (IRHRC), Taleghani Hospital, Shaheed Beheshti University of Medical Sciences, Tehran, Iran.
Introduction:
PERF15 is a testicular germ-cell specific fatty-acid binding protein (FABP) isolated from mammals, originally from rats. It encodes one of the most abundant proteins of rat spermatozoa localized in the perinuclear theca. Northern blot analysis has demonstrated that rat PERF15 mRNA is exclusively transcribed during meiosis and post-meiosis. In this study, we cloned and sequenced human PERF15 gene.
Materials And Methods:
According to the open reading frame of automated computational analysis of Homo sapiens similar to testis fatty acid binding protein nine, two specific Primers were designed to amplify human PERF15 gene. To confirm the identity of the amplified gene, PCR products of PERF15 were cloned into appropriate plasmid vectors followed by sequencing of the inserts.
Results:
A unique band of ∼3kb was obtained after PCR amplification. Restriction enzyme digestion using PvuII confirmed that the fragment was related to PERF15. Gene alignment, direct sequencing and application of specific primers to the gene showed 100% similarity between this gene and the computational data by gel extraction of the ∼3 kb band. The human PERF15 gene contained four exons and three introns. Exons one, two, three and four, respectively, coded for 24, 57, 34 and 17 amino acids. The existing three introns were composed of 2113, 461, and 168 nucleotides.
Conclusion:
In spite of the homology between exonic regions and exon-intron boundaries of human PERF15 gene and that of animals, human PERF15 gene is different in size and sequence from corresponding introns in rat and murine PERF15.
Insights
Researchers cloned and sequenced the human PERF15 gene, a testicular germ-cell specific fatty-acid binding protein (FABP). While exonic regions show homology, human PERF15 introns differ significantly from rodent counterparts.
Area of Science:
- Molecular Biology
- Genetics
- Reproductive Biology
Background:
- PERF15 is a testicular germ-cell specific fatty-acid binding protein (FABP) found in mammals.
- It is abundant in rat spermatozoa and localized in the perinuclear theca.
- Rat PERF15 mRNA is transcribed during meiosis and post-meiosis.
Purpose of the Study:
- To clone and sequence the human PERF15 gene.
- To analyze the structure of the human PERF15 gene, including exons and introns.
Main Methods:
- Computational analysis of Homo sapiens sequences to design primers.
- Polymerase Chain Reaction (PCR) amplification of the human PERF15 gene.
- Cloning of PCR products into plasmid vectors, followed by sequencing and restriction enzyme digestion for confirmation.
Main Results:
- A unique band of approximately 3kb was amplified via PCR.
- Sequencing and restriction enzyme analysis confirmed the identity of the human PERF15 gene.
- The human PERF15 gene comprises four exons and three introns, with specific nucleotide and amino acid coding.
Conclusions:
- The human PERF15 gene shares homology in exonic regions and exon-intron boundaries with animal orthologs.
- Significant differences in size and sequence were observed in the introns of the human PERF15 gene compared to rat and murine PERF15.
