Cloning and characterization of the CDKN2A and p19ARF genes from Monodelphis domestica.
Cloning and characterization of the CDKN2A and p19ARF genes from Monodelphis domestica.
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DOI：
10.1089/dna.1998.17.975
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发表时间：
1998-11
影响因子：
3.1
通讯作者：
T. E. Sherburn;J. M. Gale;R. Ley
中科院分区：
文献类型：
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作者：
T. E. Sherburn;J. M. Gale;R. Ley
肿瘤抑制基因CDKN2A(P16)编码一种细胞周期蛋白依赖的激酶抑制物，在视网膜母细胞瘤途径中起负调控作用，阻止细胞周期从G1期进展。该基因已被发现在广泛的肿瘤类型中被缺失、截断、突变或被启动子甲基化沉默。在黑色素瘤CDKN2A突变的特征中，发现了C-->；T和CC-->；TT转变，这表明紫外线辐射(UVR)诱导的嘧啶二聚体在一些肿瘤的形成中起直接作用。南美负鼠Monodelphis home tica已经被我们的团队和其他人证明，仅在长期暴露于UVR的情况下，它就容易诱发黑色素瘤。为了研究CDKN2A基因及其外显子1β交替转录本p19ARF在紫外线诱导的黑色素瘤和非黑色素瘤发生中的作用，对家蝇CDKN2A基因及其外显子1β交替转录本p19ARF进行了克隆和测序。这两个基因首先通过聚合酶链式反应(PCR)从负鼠角膜肿瘤细胞系文库中获得，并根据人、小鼠和大鼠的CDKN2A基因序列设计简并引物进行扩增。为了验证这些基因是否为正常序列，随后从培养的正常负鼠黑素细胞mRNA中RT-PCR扩增了这两个基因。当比较肿瘤和黑素细胞的序列时，我们在角膜肿瘤细胞系的第二外显子内发现了一个UVR标志点突变，即C->；T转变。在其他肿瘤中，该位点的相同突变已被证明改变了CDKN2A蛋白与CDK4激酶结合的能力，这可能导致细胞周期失控。比较负鼠CDKN2A的氨基酸序列，与人、小鼠和大鼠的同源性在57%到63%之间，当考虑保守的氨基酸替换时，同源性在63%到67%之间。P19ARF的氨基酸同源性和相似性在39%~49%之间。
The tumor suppressor gene, CDKN2A (p16), encodes a cyclin-dependent kinase inhibitor and functions as a negative regulator in the retinoblastoma pathway that blocks cell cycle progression from the G1 phase. The gene has been found to be deleted, truncated, mutated, or silenced by promoter methylation in a wide range of tumor types. Where melanoma CDKN2A mutations have been characterized, C --> T and CC --> TT transitions were found, indicating a direct role for ultraviolet radiation (UVR)-induced pyrimidine dimers in the formation of some tumors. The South American opossum, Monodelphis domestica, has been shown by our group and others to be susceptible to the induction of melanoma on chronic exposure to UVR alone. The CDKN2A gene and its exon 1beta alternate transcript p19ARF were cloned and sequenced from M. domestica to investigate the role of these genes in the development of UVR-induced melanoma and non-melanoma tumors. Both genes were first amplified by polymerase chain reaction (PCR) using cDNA from an opossum corneal-tumor cell-line library and degenerate primers based on human, mouse, and rat CDKN2A gene sequences. To verify these as normal sequences, both genes were then RT-PCR amplified from cultured normal opossum melanocyte mRNA. When comparing the tumor and melanocyte sequences, we found a UVR signature point mutation, a C --> T transition, within exon 2 in the corneal tumor cell line. The same mutation at this site in other tumors has been shown to alter the CDKN2A protein's ability to bind CDK4 kinase, which may lead to uncontrolled cell cycling. A comparison of the amino acid sequence of opossum CDKN2A showed identities relative to human, mouse, and rat between 57% and 63%, and when conserved amino acid substitutions are considered (similarity), the range is 63% to 67%. The amino acid identity and similarity for p19ARF ranged from 39% to 49%.