Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.
Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.
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非洲爪蟾纤维蛋白原 Bβ 亚基 cDNA 的分子克隆，该纤维蛋白原是协调调控基因家族的产物。
DOI：
10.1016/0303-7207(91)90225-h
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发表时间：
1991
影响因子：
4.1
通讯作者：
Holland,LJ
中科院分区：
文献类型：
--
作者：
Bhattacharya,A;Shepard,AR;Moser,DR;Roberts,LR;Holland,LJ
纤维蛋白原是主要的凝血蛋白，由肝脏中合成的三种不同亚基组成。在体外给予糖皮质激素的肝细胞从蛙爪蟾laeviscauses一个显着增加纤维蛋白原的合成。在mRNA水平上研究这种激素刺激的分子机制需要与编码三种纤维蛋白原亚基（称为Aα、Bβ和γ）的mRNA互补的cDNA克隆。本文描述了Xenopus纤维蛋白原Bβ亚基cDNA克隆的分离和鉴定。以蛙肝mRNA为材料，构建了质粒（pBR 322）和噬菌体（λ gt 10）克隆载体中的cDNA文库，并以大鼠Bβ cDNA为模板进行筛选。如此分离的克隆与两种2500和1800碱基长的异种肝mRNA杂交，这是先前确定的Bβ mRNA的大小。通过杂交选择体外翻译成Bβ多肽的互补mRNA（根据大小和对凝血酶切割的敏感性确定）来确认质粒克隆Bβ-27的身份。λ/Bβ10是从噬菌体cDNA文库中克隆到的一个克隆，它代表了几乎所有的Bβ mRNA。该克隆的3 '-末端包括一个多聚腺苷酸化信号，位于一段34个腺苷残基上游约20个残基处，可能代表信使RNA的3'-poly（A）尾。λ/Bβ10在5 '端仅缺少全长Bβ mRNA的20个核苷酸，并且有一个主要的转录起始位点。2500个碱基的Bβ mRNA在3 '端具有700个碱基的延伸，这在1800个碱基的mRNA中不存在。非洲爪蟾的基因组含有两个或三个Bβ纤维蛋白原亚基的基因。以该cDNA克隆为探针，用糖皮质激素处理原代培养的纯化的异种肝实质细胞，可使Bβ mRNA的表达增加至少20倍。
Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frogXenopus laeviscauses a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called Aα, Bβ, andγ. We describe here the isolation and characterization of cDNA clones for the Bβ subunit ofXenopusfibrinogen. cDNA libraries in both plasmid (pBR322) and phage (λgt10) cloning vectors were constructed from frog liver mRNA and screened with a rat Bβ cDNA. Clones thus isolated hybridized to twoXenopusliver mRNAs 2500 and 1800 bases long, the previously-determined sizes for Bβ mRNAs. The identity of the plasmid clone Bβ-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the Bβ polypeptide, as determined by size and susceptibility to thrombin cleavage. λ/Bβ10, a clone representing nearly all of the 2500-base Bβ mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. λ/Bβ10 lacks only 20 nucleotides of full-length Bβ mRNA at the 5'-end and there is one major start site of transcription. The 2500-base Bβ mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. TheXenopus laevisgenome contains two or three genes for the Bβ fibrinogen subunit. Using the cDNA clone as a probe, Bβ mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells ofXenopusliver maintained in primary culture.