Chang H L, Zaroukian M H, Esselman W J
Department of Microbiology and Public Health, Michigan State University, East Lansing 48824-1101.
J Immunol. 1989 Jul 1;143(1):315-21.
T200 glycoproteins of lymphoid and myeloid cells exhibit cell lineage-specific structural heterogeneity. Peptide heterogeneity appears to arise from alternate 5'-exon use (Ex-4, 5, and 6), potentially giving rise to eight distinct forms of T200 mRNA containing 0 to 3 of these alternate exons. A method is described for determining the number and identity of the three alternate T200 exons expressed in cells by using the polymerase chain reaction (PCR) and the reverse transcription-polymerase chain reaction (RT-PCR) without prior purification of RNA. Synthetic primers flanking the alternate exon region of T200 were designed to yield products for each possible exon combination having unique size and restriction enzyme sites. PCR amplification of plasmids containing T200 cDNA with none (pLy-5-68) or all three (p70Z/3-3) known alternate exons resulted in the amplification of 186 and 603 bp products, respectively. That amplified products were derived from T200 cDNA was verified by restriction enzyme mapping of each PCR product. T200 cDNA prepared from cell lines utilizing no alternate exons (BW5147) or all three exons (70Z/3.12) were analyzed by RT-PCR and contained amplified products of 186 bp (zero alternate exons) and 603 bp (containing Ex-4+5+6), respectively. RT-PCR of EL4 cells revealed approximately 186 and 330 bp products suggestive of zero and one alternate exon forms. Restriction mapping confirmed that EL4 cells contained a zero-exon form and a one-exon form containing Ex-5. Analysis of the 3B3 pre-B cell line yielded 186, 330, 460, and 603 bp products; restriction mapping revealed T200 mRNA for a zero alternate exon form, two distinct one- and two-exon forms (Ex-4; Ex-5; Ex-4+5; Ex-5+6), and a three-exon form (Ex-4+5+6). Other lymphoid cell lines were heterogeneous in T200 alternate exon use, with distinct patterns distinguishing B and T cells. RT-PCR can facilitate the analysis of variations in T200 alternate exon use among developmentally and functionally distinct lymphoid and myeloid cells.
淋巴细胞和髓细胞的T200糖蛋白表现出细胞谱系特异性的结构异质性。肽的异质性似乎源于5'-外显子的交替使用（外显子4、5和6），这可能产生8种不同形式的T200 mRNA，其中包含0至3个这些交替外显子。本文描述了一种方法，可通过使用聚合酶链反应（PCR）和逆转录-聚合酶链反应（RT-PCR）来确定细胞中表达的三种交替T200外显子的数量和身份，而无需事先纯化RNA。设计了位于T200交替外显子区域两侧的合成引物，以产生具有独特大小和限制性酶切位点的每种可能外显子组合的产物。用不含任何已知交替外显子（pLy-5-68）或全部三个已知交替外显子（p70Z/3-3）的含T200 cDNA的质粒进行PCR扩增，分别得到186和603 bp的产物。通过对每个PCR产物进行限制性酶切图谱分析，验证了扩增产物源自T200 cDNA。利用不含交替外显子（BW5147）或全部三个外显子（70Z/3.12）的细胞系制备的T200 cDNA，通过RT-PCR进行分析，分别包含186 bp（零个交替外显子）和603 bp（包含外显子4+5+6）的扩增产物。EL4细胞的RT-PCR显示出约186和330 bp的产物，提示零个和一个交替外显子形式。限制性图谱分析证实EL4细胞包含零外显子形式和包含外显子5的一个外显子形式。对3B3前B细胞系的分析产生了186、330、460和603 bp的产物；限制性图谱分析揭示了零个交替外显子形式、两种不同的一个和两个外显子形式（外显子4；外显子5；外显子4+5；外显子5+6）以及一个三个外显子形式（外显子4+5+6）的T200 mRNA。其他淋巴细胞系在T200交替外显子的使用上是异质的，具有区分B细胞和T细胞的独特模式。RT-PCR有助于分析发育和功能不同的淋巴细胞和髓细胞中T200交替外显子使用的变化。
