Vocke C D, Cox E C
Department of Molecular Biology, Princeton University, NJ 18544.
Dev Genet. 1990;11(5-6):427-38. doi: 10.1002/dvg.1020110517.
A series of monoclonal antibodies were previously raised against developing Polysphondylium pallidum cells. In this work, six of these antibodies have been used as probes to identify and characterize antigens regulated during development. Soluble and membrane fractions of P. pallidum cells at six stages of development or three stages of cyclic adenosine monophosphate (cAMP)-induced development were run in sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analysis. Three of the monoclonals, anti-Tp200, anti-Tp423, and anti-Pg101, stain sorogen tips. Tp423 and Tp200 are membrane-associated antigens; both are stable to urea extraction, and Tp200 remains in the membrane after NaOH extraction. Tp423 is present in starved cells but is more prominent in sorogens and particularly in cAMP-developed cells. In contrast, Tp200 is first detected in early to mid-aggregation and is more abundant late in development. Pg101, which is expressed as a gradient with its highest concentration in tips, first appears in tight aggregates but is much more abundant in sorogens; unlike the Tp antigens, Pg101 is not greatly induced in cAMP-developed cells. All three of these antigens undergo changes in apparent molecular weight at the tight aggregate or sorogen stage: The gel mobilities of Tp200 and Pg101 increase, whereas that of Tp423 decreases. In addition to the tip-specific monoclonals, two monoclonals that stain all but the tips of sorogens have been used for analysis. One of these, anti-3D10Pnk stains most cells within secondary tips, whereas anti-3D10Dif does not. 3D10Dif is membrane associated; it is present very early in development, increasing two- to threefold through the sorogen stage and diminishing in late cAMP-developed cells. 3D10Pnk is a mostly soluble species first detected in late streaming. Anti-1c3, a sixth monoclonal, which stains nuclei uniformly throughout sorogens, is also developmentally expressed. 1c3 is mainly membrane associated and is expressed from late streaming through the sorogen stage.
此前制备了一系列针对发育中的苍白聚黏菌细胞的单克隆抗体。在这项研究中，其中六种抗体被用作探针，以鉴定和表征发育过程中受调控的抗原。处于六个发育阶段或三个环磷酸腺苷（cAMP）诱导发育阶段的苍白聚黏菌细胞的可溶性和膜部分，在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶中进行电泳，并进行蛋白质免疫印迹分析。其中三种单克隆抗体，抗-Tp200、抗-Tp423和抗-Pg101，可对孢子梗顶端进行染色。Tp423和Tp200是与膜相关的抗原；两者对尿素提取均稳定，且在氢氧化钠提取后，Tp200仍保留在膜中。Tp423存在于饥饿细胞中，但在孢子梗中更为显著，尤其在cAMP诱导发育的细胞中。相比之下，Tp200最早在聚集早期至中期被检测到，在发育后期更为丰富。Pg101以梯度形式表达，在顶端浓度最高，最早出现在紧密聚集体中，但在孢子梗中更为丰富；与Tp抗原不同，Pg101在cAMP诱导发育的细胞中不会大量诱导产生。在紧密聚集体或孢子梗阶段，所有这三种抗原的表观分子量都会发生变化：Tp200和Pg101的凝胶迁移率增加，而Tp423的凝胶迁移率降低。除了顶端特异性单克隆抗体外，另外两种可对除孢子梗顶端外的所有部分进行染色的单克隆抗体也被用于分析。其中一种，抗-3D10Pnk可对次生顶端内的大多数细胞进行染色，而抗-3D10Dif则不能。3D10Dif与膜相关；它在发育早期就存在，在整个孢子梗阶段增加两到三倍，并在cAMP诱导发育后期的细胞中减少。3D10Pnk主要是可溶性物质，最早在后期的原生质流动中被检测到。第六种单克隆抗体抗-1c3，可对整个孢子梗中的细胞核进行均匀染色，也在发育过程中表达。1c3主要与膜相关，从后期的原生质流动阶段到孢子梗阶段均有表达。
