Cattini P A, Jin Y, Sheikh F
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Mol Cell Biochem. 1998 Dec;189(1-2):33-9. doi: 10.1023/a:1006852312188.
Fibroblast growth factor-2 (FGF-2) or basic FGF is a multifunctional protein that, through interaction with specific cell surface receptors, plays important roles in the growth and development of tissues and organs. Thus, considerable attention has focused on the control of FGF-2 gene expression, including assessments of RNA levels through blotting and the use of radiolabeled FGF-2 cDNA probes. Multiple transcripts of different sizes have been reported for FGF-2 by this approach, however, more recent evidence indicates that at least one of these RNAs of about 1.5 kb, is not an authentic FGF-2 transcript. A major band of 4.7 kb and a minor band of 6.1 kb were detected in total rat glial tumor cell RNA, using the 'intact' rat ovarian FGF-2 cDNA as a probe at high stringency. This cDNA contains both coding and 5'-untranslated sequences. Although the 6.1 kb transcript levels were increased in RNA enriched for polyadenylated species, the levels of the 4.7 kb band were decreased and also shared a mobility with 28S RNA. A truncated FGF-2 cDNA probe, containing coding but not 5'-untranslated sequences, detected the 6.1 kb transcript but failed to see the 4.7 kb band. The domain responsible for detecting the 4.7 kb band was localized to a G/C-rich region containing 5'-untranslated sequences, by using different fragments of the rat FGF-2 gene, including coding and upstream flanking DNA, as probes. The degree of similarity between sequences of this G/C-rich region of the FGF-2 gene and 28S RNA from rat, human and mouse was sufficient to predict strong cross hybridization. This was confirmed by the detection of a 4.7 kb band in mouse heart RNA with the 'intact' but not truncated rat FGF-2 cDNA probes; a 6.1 kb mouse FGF-2 transcript was detected with both probes. These data indicate that the 4.7 kb RNA detected is not a bona fide FGF-2 transcript, and most likely represents cross hybridization with abundant 28S RNA through G/C-rich non-coding sequences present in the 'intact' rat FGF-2 cDNA. However, sequence comparisons suggest that this result may be the case for other species and might not be restricted to the rat FGF-2 cDNA.
成纤维细胞生长因子-2（FGF-2），即碱性成纤维细胞生长因子，是一种多功能蛋白，它通过与特定细胞表面受体相互作用，在组织和器官的生长发育中发挥重要作用。因此，对FGF-2基因表达的调控已引起了广泛关注，包括通过印迹法评估RNA水平以及使用放射性标记的FGF-2 cDNA探针。通过这种方法已报道了FGF-2的多种不同大小的转录本，然而，最近的证据表明，这些约1.5 kb的RNA中至少有一个不是真正的FGF-2转录本。使用“完整”的大鼠卵巢FGF-2 cDNA作为高严谨度探针，在大鼠神经胶质瘤细胞总RNA中检测到一条4.7 kb的主要条带和一条6.1 kb的次要条带。该cDNA包含编码序列和5'-非翻译序列。尽管在富含聚腺苷酸化物种的RNA中6.1 kb转录本水平升高，但4.7 kb条带的水平降低，并且与28S RNA具有相同的迁移率。一个截短的FGF-2 cDNA探针，包含编码序列但不包含5'-非翻译序列，检测到了6.1 kb转录本，但未检测到4.7 kb条带。通过使用大鼠FGF-2基因的不同片段，包括编码序列和上游侧翼DNA作为探针，负责检测4.7 kb条带的结构域定位于一个富含G/C的区域，该区域包含5'-非翻译序列。FGF-2基因的这个富含G/C区域的序列与大鼠、人类和小鼠的28S RNA之间的相似程度足以预测强烈的交叉杂交。用“完整”但未截短的大鼠FGF-2 cDNA探针在小鼠心脏RNA中检测到一条4.7 kb条带，证实了这一点；用两种探针均检测到一条6.1 kb的小鼠FGF-2转录本。这些数据表明，检测到的4.7 kb RNA不是真正的FGF-2转录本，很可能是通过“完整”的大鼠FGF-2 cDNA中存在的富含G/C的非编码序列与丰富的28S RNA发生了交叉杂交。然而，序列比较表明，其他物种可能也会出现这种结果，并且可能不限于大鼠FGF-2 cDNA。
