Ybot-Gonzalez P, Runswick S, Smyth N, Edgar D
Department of Human Anatomy and Cell Biology, University of Liverpool, UK.
Differentiation. 1995 Nov;59(4):215-23. doi: 10.1046/j.1432-0436.1995.5940215.x.
In order to define specific laminin variants implicated in organogenesis, we have undertaken a systematic search to detect and characterize novel laminin subunits, the expression of which is both developmentally regulated and tissue-specific. cDNA prepared from embryonic chick tissues was amplified by the polymerase chain reaction (PCR) with degenerate primers based on conserved sequences in domains V and VI of members of the laminin beta subunit family. Restriction mapping, cloning and sequencing of PCR products demonstrated a novel cDNA, the derived protein sequence of which displayed greatest homology with laminin beta subunits. However, the degree of amino acid divergence, comparison of sequence motifs and pattern of expression indicates that the cloned cDNA does not code for the avian orthologue of a previously characterized laminin beta subunit. Northern blot analysis showed that the expression of the 6-kb mRNA coding for the novel subunit was restricted to the skin. The mRNA was not detectable before day 5 of chick embryonic development, after which in situ hybridization showed expression only in surface ectoderm cells and subsequently in the epidermis. The developmentally regulated ectodermal expression of the novel beta subunit, prior to condensation of mesenchymal cells to form the dermis, is consistent with a specific role for this laminin isoform in the development and maintenance of the skin.
为了确定参与器官形成的特定层粘连蛋白变体，我们进行了系统的搜索，以检测和表征新型层粘连蛋白亚基，其表达受发育调控且具有组织特异性。基于层粘连蛋白β亚基家族成员结构域V和VI中的保守序列，用简并引物通过聚合酶链反应（PCR）扩增从胚胎鸡组织制备的cDNA。PCR产物的限制性图谱分析、克隆和测序显示了一个新的cDNA，其推导的蛋白质序列与层粘连蛋白β亚基具有最大的同源性。然而，氨基酸差异程度、序列基序比较和表达模式表明，克隆的cDNA并不编码先前鉴定的层粘连蛋白β亚基的禽类直系同源物。Northern印迹分析表明，编码该新型亚基的6kb mRNA的表达仅限于皮肤。在鸡胚胎发育第5天之前检测不到该mRNA，之后原位杂交显示仅在表面外胚层细胞中表达，随后在表皮中表达。在间充质细胞凝聚形成真皮之前，新型β亚基在发育过程中受调控的外胚层表达，与这种层粘连蛋白异构体在皮肤发育和维持中的特定作用一致。
