Abstract

1. Lesson from the SFTPB gene: evolutionary mechanism of exonization, Alu recombination-mediated deletion (ARMD), and alternative splicing could be occurred more frequently in diverse primate lineage Ja-Rang Lee1, Jae-Won Huh1,2, Dae-Soo Kim3, Hong-Seok Ha1, Kung Ahn1, Yun-Ji Kim1, Kyu-Tae Chang2, and Heui-Soo Kim1,* Cerebellum Cerebellum Salivary gland Salivary gland Skeletal muscle Skeletal muscle Bone marrow Bone marrow Adrenal gland Adrenal gland Whole brain Whole brain Spinal cord Spinal cord Marker Marker Fetal brain Fetal brain Fetal liver Fetal liver Prostate Prostate Thymus Thyroid Thymus Trachea Thyroid Trachea Placenta Placenta Heart Heart Lung Marker Kidney Liver Testis Uterus Liver Kidney Lung Marker Testis Uterus 1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea 2 National Primate Research Center (NPRC), KRIBB, Ochang, Chungbuk 363-883, Republic of Korea 3 Korea Bioinformation Center, KRIBB, Daejeon 305-806, Korea Results & Discussion Abstract The SFTPB (surfactant, pulmonary-associated protein B) gene on human chromosome 2p11.2 encodes an amphipathic surfactant protein essential for lung function and homeostasis after birth. The SFTPB gene has been investigated to have transcript variants related with transposable elements (LTR7B and AluSx). We investigated the evolutionary aspect and molecular characterization of SFTPB gene using genomic DNAs and RNAs from various primate samples. LTR7B and AluSx elements within the SFTPB gene have been integrated into ancestor genome before the divergence of simian and prosimian. Alu recombination-mediated deletion (ARMD) event has been occurred after the divergence of New World monkeys and Old World monkeys in SFTPB gene 3’ UTR region. Transcript variants (306 bp and 463 bp) were newly identified by comparative RT-PCR analysis. Original transcript (419 bp) shows ubiquitous expression in various tissues and TE fusion transcript (841 bp) shows the lung specific expression in human. Taken together, integration event of LTR7B and AluSx on SFTPB gene seem to cause a lineage specific exonization event via general exonization event (New World monkeys and prosimian), ARMD event (Old World monkeys and hominoids), and alternative splicing (human) during the primate evolution. ……………………. Chr. 2 SFTPB Gene Structure 2p11.2 638 bp NM_198843.1 AS2 S1 11_A 10 1 2 AS2 NM_000542.2 S1 638 bp 11_B 1 2 10 S AS 841 bp NM_198843.1 New Transcript 841 bp 463 bp 419 bp 306 bp G3PDH 120 bp G3PDH 120 bp Introduction Other region SINE 13% 16% LINE 20% SFTPB Gene Expression in Various Tissues of Human ARMD event and evolutionary aspect of the SFTPB gene S 419 bp NM_000542.2 New Transcript Gene-related Sequence HERV element 8% 36% DNA element Pseudogene Coding sequence 3% 1% 3% 463 bp 419 bp 306 bp G3PDH 120 bp G3PDH 120 bp Marker Marker Marker Marker Cerebellum Brain Cerebellum Cerebellum Kidney Heart Kidney Kidney Kidney Heart Heart Heart Lung Uterus Liver Liver Lung Lung Lung Uterus 463 bp 419 bp 306 bp 306 bp G3PDH 120 bp G3PDH 120 bp G3PDH 120 bp G3PDH 120 bp Integration lineage of HERV-H LTR and AluSx in the SFTPB gene Materials & Methods AS Marker HU CH RH OR GO JM TA RL NM SFTPB Gene Expression in Various Tissues of Rhesus monkey GI Cerebrum Stomach Pancreas Cerebellum Heart Adrenal gland Salivary gland Urine bladder Duodenum Marker Kidney Trachea Liver Gullet Jejunum Cecum Lung Marker Rectum Tongue Ileum Testis Spleen Colon 638 bp Marker Cerebrum Stomach Pancreas Cerebellum Adrenal gland Heart Salivary gland Urine bladder Duodenum HU CH Marker Kidney Trachea Liver Gullet Jejunum Cecum RH OR GO TA JM NM RL Lung Marker Rectum Tongue Testis Spleen Colon Ileum GI 763 bp 392 bp AluSg AluJ AluSx LTR7B SFTPB Gene Expression in Various Tissues of New World monkeys ARMD event ? http://www.primate.or.kr/HESAS/ AluSg vs AluSx AluJ AluSx LTR7B Bioinformatics Genomic DNA of various primate and human cDNA of various tissues of human, rhesus monkey & marmoset Genomic DNA & cDNA Human, Rhesus monkey & Marmoset(30 cycle) ; Various tissues RT-PCR Alternative splicing 30 cycle; 94 ℃- 40 sec, 56 ℃- 30 sec, 72 ℃- 40 sec Qiaquick Gel-extraction, Promega T-easy Vector, EcoR-I(DH5α) PCR & Cloning of PCR product ARMD event References Insertion of TEs Genome Information Lab. 1. Han, K., Lee J., Meyer, T. J., Wang, J., Sen, S. K., Srikanta, D., Liang, P. & Batzer, M. A. (2007). Alu recombination-mediated structural deletions in the chimpanzee genome. PLoS Genet.3, 1939-1949. 2. Clark JC, Wert SE, Bachurski CJ, Stahlman MT, Stripp BR, Weaver TE, Whitsett JA. 1995. Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc. Natl. Acad. Sci. USA. 92: 7794-7798.