Wu Shaobo, De Croos J N Amritha, Storey Kenneth B
Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6.
Gene. 2008 Nov 15;424(1-2):48-55. doi: 10.1016/j.gene.2008.07.023. Epub 2008 Jul 29.
Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific changes in gene expression. The present study used differential display PCR to find cold-responsive genes in wood frog skin. A cDNA was retrieved from skin that was in higher amounts in cold- versus warm-acclimated frogs. The cDNA was used to probe a wood frog liver cDNA library and retrieve a long sequence that, after the further application of 5'RACE, was shown to encode the full sequence of the ribosomal large subunit protein 7 (RPL7) (GenBank accession number AF175983). Wood frog RPL7 contained 246 amino acids and shared 90% identity with Xenopus laevis RPL7, 82-83% with chicken and zebrafish homologues, and 79% with mammalian RPL7. Multiple binding domains found in human RPL7 showed differing degrees of conservation in the frog protein. Transcript levels of rpl7 were elevated up to 4-fold in skin of cold-acclimated frogs as compared with warm-acclimated animals. Organ-specific responses by rpl7 transcripts also occurred when frogs were given survivable freezing exposures. Transcripts rose by 1.8-3.3 fold in brain and skeletal muscle during freezing but were unaffected in central organs such as liver and heart. Up-regulation of rpl7 also occurred in brain of anoxia-exposed frogs and RPL7 protein levels increased strongly in heart under both freezing and dehydration stresses. Cold- and freezing-responsive up-regulation of the rpl7 gene and RPL7 protein in selected organs suggests that targeted changes in selected ribosomal proteins may be an integral part of natural freeze tolerance.
林蛙（Rana sylvatica）的自然冷冻存活涉及基因表达中多个器官特异性的变化。本研究利用差异显示PCR技术寻找林蛙皮肤中的冷响应基因。从冷驯化青蛙与暖驯化青蛙相比含量更高的皮肤中获取了一个cDNA。该cDNA用于探测林蛙肝脏cDNA文库，并获得一个长序列，在进一步应用5'RACE后，显示其编码核糖体大亚基蛋白7（RPL7）的完整序列（GenBank登录号AF175983）。林蛙RPL7包含246个氨基酸，与非洲爪蟾RPL7的同源性为90%，与鸡和斑马鱼同源物的同源性为82 - 83%，与哺乳动物RPL7的同源性为79%。在人类RPL7中发现的多个结合结构域在蛙类蛋白中显示出不同程度的保守性。与暖驯化动物相比，冷驯化青蛙皮肤中rpl7的转录水平升高了4倍。当青蛙接受可存活的冷冻暴露时，rpl7转录本也出现了器官特异性反应。在冷冻过程中，大脑和骨骼肌中的转录本增加了1.8 - 3.3倍，但在肝脏和心脏等中央器官中未受影响。在缺氧暴露的青蛙大脑中也出现了rpl7的上调，并且在冷冻和脱水应激下，心脏中的RPL7蛋白水平都大幅增加。rpl7基因和RPL7蛋白在选定器官中的冷响应和冷冻响应上调表明，选定核糖体蛋白的靶向变化可能是自然耐冻性的一个重要组成部分。
