Fitch J M, Gordon M K, Gibney E P, Linsenmayer T F
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Dev Dyn. 1995 Jan;202(1):42-53. doi: 10.1002/aja.1002020105.
The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative splicing [for alpha 1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of alpha 1(IX) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of alpha 1(IX) was from the downstream promoter (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, III, and V. For type I, the "bone/tendon" mRNA isoform of the alpha 2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of alpha 1(II). The downstream transcript of the alpha 2(I) gene (Icart) was found at high levels only in cartilage.
α1（IX）、α1（II）和α2（I）胶原蛋白链的基因可产生不同的mRNA异构体，这些异构体是通过选择性启动子的使用[对于α1（IX）和α2（I）]或选择性剪接[对于α1（II）]产生的。在本研究中，我们采用竞争性逆转录酶PCR来定量这些基因在胚胎鸡角膜从起始（约发育3.5天）到11天期间转录异构体的量。为了比较不同时间点的值，结果以“管家”酶甘油-3-磷酸脱氢酶（G3PDH）获得的值进行归一化。将这些值与从合成不同胶原蛋白异构体组合的其他组织（前视杯和软骨）获得的值进行比较。我们发现，在角膜中，α1（IX）胶原蛋白上游启动子的转录本（称为“长IX”）在第18 - 20阶段（约3.5天）占主导地位，但随后迅速下降，并维持在低水平。到5天时（基质肿胀前），α1（IX）的主要mRNA异构体来自下游启动子（称为“短IX”）。IX型胶原蛋白短形式的转录本相对量在发育约6天时升至峰值，然后下降。在此期间，II型胶原蛋白基因的主要转录异构体是IIA（即包含选择性剪接的外显子2）。这表明与I型胶原蛋白组装成异型纤维的II型胶原蛋白分子至少暂时具有与I、III和V型胶原蛋白中发现的类似的氨基末端球状结构域。对于I型，α2（I）胶原蛋白基因的“骨/肌腱”mRNA异构体占主导地位；下游启动子的转录本处于基础水平。在表达IX型和II型胶原蛋白的其他组织中，长IX在第22/23阶段在前视杯中主要与IIA形式一起表达；在14.5天的软骨中，长IX主要与α1（II）的IIB形式一起表达。α2（I）基因的下游转录本（Icart）仅在软骨中高水平存在。
