鯉魚cdc2 cDNA 之選殖及其cdk 基因結構之研究

Abstract

CDC2蛋白激脢的活化與高等動物細胞週期的分裂期進行有關。而在動物組織中，已知至少存在六種結構和CDC2相似的CDK分子，配合著不同的cyclin擔任調節細胞週期的不同角色。 利用RT-PCR在鯉魚組織找到數種類似cdk的DNA片段，並在卵巢組織cDNA基因庫篩選出一個全長1.3Kb的cDNA clone，可轉譯一個含302個氨基酸的CDC2蛋白激脢，此蛋白激脢和各種真核生物的CDC2比較，相同的氨基酸約佔80％。為了研究CDC2的基因調控，我們進一步從鯉魚染色體工基因庫中篩選出含CDC2基因的phage clone，並定出長約5Kb的部份DNA序列，此序列涵蓋CDC2基因的啟動子區域和CDC2基因的蛋白質轉譯區，分析CDC2基因的啟動子區域DNA序列，發現存在多種和基因轉錄有關的核酸序列，如：和細胞週期有關的E2F, p53結合序列，和左右生長分裂或細胞分化的c-Myb結合序列，以及與急性狀態反應有關的NF-IL6, H-APF-1, HNF-5結合序列。 另外利用RT-PCR在鯉魚不同組織也找到類似cdk6的DNA片段，經核酸定序發現來自脾臟的Sp82植株其序列與來自卵巢組織的0v153植株完全相同，但多出87bp。利用Sp82所標定之核酸探針，也從鯉魚染色體基因庫中篩選出含鯉魚cdk6基因的phage clone。完全定出長5kb的部份DNA序列後，發現Sp82植株所多出的87bp屬於intron的序列,因此這87bp可能由於alternative splicing所造成，或由mRNA imcomplete processing所造成。 比較鯉魚CDC2, cdk6和人類的cdc2基因結構，發現它們的intron splice sites很類似，但intron大小不同，如此cdc2和cdk6基因可能是由同一古老的基因演化而來。

Cyclin-dependent kinases (CDKs) control cell cycle progression in all eukaryotes. For full enzymatic activity, CDKs require physical association with regulatory subunits called cyclins. Moreover, CDK activities are regulated by both phosphorylation and dephosphorylation events. p34cdc2 is the prototypic member of the CDK family and controls entry into mitosis in all eukaryotes. Other distinct CDKs, such as cdk2, cdk4 and cdk5, seem to control G1 and S phase functions. In order to study the transcriptional regulation of these cdk genes, in this study we reported the genomic structure of carp cdc2 and cdk6 genes. By using RT-PCR, several amplified DNA fragments containing cdk-related sequences were obtained from different carp tissues. Using these DNA fragments as probe, a carp ovary lamda-gt10 cDNA library was screened and one cDNA clone was obtained. This cDNA clone is 1.3 Kb in length and encodes the carp CDC2 protein of 302 amino acid residues. By comparing with known CDC2 sequences from other species, carp CDC2 is 82%, 81%, 83.5%, 69% and 62.6% identical to human CDC2, chicken CDC2, frog CDC2, fruit fly CDC2 and fission yeast CDC2, respectively. The structural organization of carp cdc2 gene was also determined. Two phage clones containing carp cdc2 gene were picked up from a carp FIXII genomic library. The nucleotide sequences of the genomic DNA spanning 5 Kb were then determined. This DNA fragment was found to cover about 2 Kb promoter region and most of the coding region of the carp cdc2 gene. By the aid of computer search, many kinds of mammalian transcription factor binding sequences were found in the promoter region. Among these sequences, there are (1) E2F, p53-binding sequences which are relevant to cell cycle control; (2) c-Myb binding sequences which play roles in cell proliferation and differentiation and (3) NF-IL6, H-APF-1, HNF-5 binding sequences which response to acute phase stimulation. In some RT-PCR products using carp ovary and spleen cDNA as template, we also found cdk6-related sequences. The nucleotide sequences of spleen clone, Sp82, are the same as those of the ovary clone, 0v153 except for one insertion of 87 bp. By using Sp82 clone as a probe, we rescreened the same carp FIXII genomic library and fortunately, we isolated another clone containing the carp cdk6 gene. After RE mapping, Southern blotting and sequencing, the genomic DNA spanning 5 Kb was found to contain four exons and three introns. By comparing the nucleotide sequences of this genomic DNA fragment and those of Sp82, the 87 bp insertion present in Sp82 turned out to be the intron sequences. It is interesting to notice that one possible specific alternative splicing may be present in the processing of carp cdk6 mRNA transcript. The comparison of genomic organization between human cdc2 gene, carp cdc2 and cdk6 gene was discussed in the text.