Cloning And Expression Of LASS1 Gene And Primary Study On The Association Of Its Expression With Neuron Aging In Rat Cerebral Cortex

Background and Objective Longevity-assurance gene 1 (LAG1) is the first of several longevity genes identified and cloned from the baker's yeast Saccharomyces cerevisia, which was differentially expressed during the yeast replicative life span and was shown to play a role in determining yeast longevity[1,2,3]. LASS1 is its mammalian homolog and is expressed only in brain, skeletal muscle, and testis[4,5,6]. Studies have revealed that lag1p and its homologs possess a characteristic substrate preference for a particular fatty acyl-CoA in the process of ceramide synthesis[5,6]. The LASS1 protein (Lass1p) selectively regulates the synthesis of C18:0-containing sphingolipids, and C18:0-ceramide is the major ceramide in brain[7,8]. Ceramide functions as a second messenger in a variety of cellular events including apoptosis and differentiation[9,10], LASS1 regulates C18:0-ceramide synthesis, and is expressed in brain abundantly and specifically. We wonder whether LASS1 may participate in the process of mammalian neuron aging. In this study, we intended to:(1) Clone and characterize the rat LASS1 mRNA sequence;(2) Analyze the LASS1 transcription levels in rat cerebral cortex tissue at varying ages;(3) Express the fusion protein with full or part of LASS1 cDNA in the prokaryotic cells, and to prepare the antibody against the fusion protein. It may lay strong foundations for exploring the role of LASS1 in mammalian neuron aging.Materials and methods (1) Total RNA was isolated from rat cerebral cortex tissue, First-strand cDNA was synthesized by RT-PCR method, LASS1 gene specific primers were designed and PCR amplifications were performed for acquiring the full length sequence of LASS1 mRNA. The sequences of LASS1 cDNA and its deduced amino acid were further analyzed. (2) Semi-quantitative RT-PCR analysis, real-time RT-PCR assays and Northern blot were performed to analyze the LASS1 transcription levels in rat cerebral cortex tissue at varying ages: fetus (embryonic day 18), newborn rats (postnatal age, 1 day), 1 month, 3 months, 6...