Couwenhoven R I, Schwartz S A, Snead M L
Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, Los Angeles 90033.
J Craniofac Genet Dev Biol. 1993 Oct-Dec;13(4):259-69.
An important issue in craniofacial biology is understanding the molecular mechanisms that regulate the transcription of genes during development. Low concentrations of the thymidine analogue, 5-bromodeoxyuridine (BrdU), have been used to perturb transcription of tissue-specific genes in a variety of tissue types, although the molecular mechanism for this inhibition has not been elucidated. The purpose of the present study was to examine the following: (1) if amelogenin transcription is inhibited in mouse molars cultured in the presence of BrdU, (2) if changes in methylation patterns of the amelogenin gene can be detected with terminal differentiation of ameloblasts in vivo and in vitro; and (3) if changes in methylation patterns of the amelogenin gene can be detected in mouse molars cultured in the presence of BrdU. Northern blot hybridization and RNA phenotyping analysis revealed that bromodeoxyuridine (BrdU) incorporation into the DNA of developing mouse mandibular first molars (M1) in vitro inhibited amelogenin transcription. Restriction endonuclease digestion of M1 genomic DNA followed by Southern blot hybridization analysis revealed that amelogenin transcriptional activity in vivo and in vitro did not correlate with changes in methylation of the amelogenin gene. These results suggested that, unlike several other developmentally regulated genes, transcriptional regulation of the amelogenin gene may not be associated with changes in DNA methylation patterns.
颅面生物学中的一个重要问题是了解在发育过程中调控基因转录的分子机制。低浓度的胸苷类似物5-溴脱氧尿苷（BrdU）已被用于干扰多种组织类型中组织特异性基因的转录，尽管这种抑制作用的分子机制尚未阐明。本研究的目的是检验以下几点：（1）在BrdU存在下培养的小鼠磨牙中，釉原蛋白转录是否受到抑制；（2）在体内和体外成釉细胞终末分化过程中，是否能检测到釉原蛋白基因甲基化模式的变化；以及（3）在BrdU存在下培养的小鼠磨牙中，是否能检测到釉原蛋白基因甲基化模式的变化。Northern印迹杂交和RNA表型分析表明，体外将溴脱氧尿苷（BrdU）掺入发育中的小鼠下颌第一磨牙（M1）的DNA中会抑制釉原蛋白转录。对M1基因组DNA进行限制性内切酶消化，随后进行Southern印迹杂交分析，结果表明，体内和体外的釉原蛋白转录活性与釉原蛋白基因的甲基化变化无关。这些结果表明，与其他一些发育调控基因不同，釉原蛋白基因的转录调控可能与DNA甲基化模式的变化无关。
