Flisikowski Krzysztof, Szymanowska Małgorzata, Zwierzchowski Lech
Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wólka Kosowska, Poland.
Cell Mol Biol Lett. 2003;8(3):831-40.
The STATs are a family of transcription factors. STAT5A, previously known as MGF, transduces prolactin signals to the milk protein genes. Here, we describe the detection of nucleotide sequence polymorphism in exon 16 of the bovine STAT5A gene, coding for the SH2 domain. SSCP was found in a 281-bp PCR amplified gene fragment, lying between positions 12,525 and 12,806, and encompassing parts of intron 15 and exon 16 of the bovine STAT5A gene (GenBank AJ 237937). Three SSCP patterns (genotypes) were identified in a group of 108 animals of different cattle breeds. The DNA sequencing showed that they differed by a CCT deletion at position from 12,549 in intron 15, and a T-->C substitution at position 12,743 in exon 16. The latter mutation changes an amino acid sequence in the STAT5A protein - a Val/Ala substitution at position 686. Since T-->C substitution creates a new MslI site, genetic variants in the bovine STAT5A gene can be distinguished with RFLP analysis. The frequency of alleles T and C varied between the different cattle breeds studied; the CC genotype was the least frequent and the frequency of alleles T and C was 0.842 and 0.158, respectively. Proteins were extracted from the cell nuclei of liver tissues derived from bulls of different STAT5A genotypes and subjected to EMSA in order to study if the amino acid substitution might change the DNA-binding capacity of STAT5A transcription factor. Statistically significant (p<0.05) differences in nuclear protein binding to DNA were observed between genotypes TT and CC; nuclear proteins derived from CC animals always showed less DNA protein complexing than those of TT animals. EMSA competition experiments confirmed that STAT5 transcription factors take part in the formation of the DNA-protein complexes.
信号转导及转录激活因子（STATs）是一类转录因子。STAT5A，以前称为MGF，可将催乳素信号转导至乳蛋白基因。在此，我们描述了牛STAT5A基因第16外显子中核苷酸序列多态性的检测，该外显子编码SH2结构域。在一个281 bp的PCR扩增基因片段中发现了单链构象多态性（SSCP），该片段位于12525至12806位之间，涵盖牛STAT5A基因（GenBank AJ 237937）的第15内含子和第16外显子的部分区域。在一组108头不同牛品种的动物中鉴定出三种SSCP模式（基因型）。DNA测序表明，它们的差异在于第15内含子12549位的CCT缺失以及第16外显子12743位的T→C替换。后一种突变改变了STAT5A蛋白中的氨基酸序列——686位的缬氨酸/丙氨酸替换。由于T→C替换产生了一个新的MslI位点，因此可以通过限制性片段长度多态性（RFLP）分析来区分牛STAT5A基因中的遗传变异。在所研究的不同牛品种中，等位基因T和C的频率有所不同；CC基因型最不常见，等位基因T和C的频率分别为0.842和0.158。从不同STAT5A基因型公牛的肝脏组织细胞核中提取蛋白质，并进行电泳迁移率变动分析（EMSA），以研究氨基酸替换是否可能改变STAT5A转录因子的DNA结合能力。在TT和CC基因型之间观察到核蛋白与DNA结合的统计学显著差异（p<0.05）；来自CC动物的核蛋白与DNA的复合物形成总是比TT动物的少。EMSA竞争实验证实STAT5转录因子参与了DNA-蛋白质复合物的形成。
