Bultman S J, Klebig M L, Michaud E J, Sweet H O, Davisson M T, Woychik R P
Biology Division, Oak Ridge National Laboratory, Tennessee 37831-8077.
Genes Dev. 1994 Feb 15;8(4):481-90. doi: 10.1101/gad.8.4.481.
The agouti gene regulates the differential production of eumelanin (black or brown) and phaeomelanin (yellow) pigment granules by melanocytes in the hair follicles of mice. The original nonagouti (a) allele, which confers a predominantly black coat color, has been shown to revert to two other more dominant agouti alleles, black-and-tan (a(t)) and white-bellied agouti (Aw), with an exceptionally high frequency. The a(t) and Aw alleles confer phenotypes in which the pigmentation is not uniformly distributed over the dorsal and ventral surfaces of the animal; in both cases the ventral surface of the animal is markedly lighter than the dorsal surface due to an increase in phaeomelanin production. To understand the unusually high reversion rate of a to a(t) or Aw, and to decipher the molecular events associated with the different pigmentation patterns associated with these three agouti alleles, we have characterized a, a(t) and Aw at the molecular level. Here, we report that insertions of 11, 6, and 0.6 kb are present at precisely the same position in the first intron of the agouti gene in a, a(t), and Aw, respectively. The a insertion consists of a 5.5-kb VL30 element that has incorporated 5.5 kb of additional sequence internally; this internal sequence is flanked by 526-bp direct repeats. The a(t) allele contains only the VL30 element and a single, internal 526-bp repeat. The Aw allele has only a solo VL30 LTR. Based on the comparison of the structure of the a(t) and Aw insertions, we propose that reverse mutations occur by excision of inserted sequences in a through homologous recombination, utilizing either the 526-bp direct repeats to generate a(t) or the VL30 LTRs to generate Aw. Moreover, the analysis of these three alleles has allowed us to identify additional exons of the agouti gene that give rise to alternatively processed forms of agouti mRNA. We demonstrate that the distinct insertions in a, a(t) and Aw cause pigmentation differences by selectively inactivating the expression of different forms of agouti transcripts.
刺豚鼠基因调控小鼠毛囊中黑素细胞产生真黑素（黑色或棕色）和褐黑素（黄色）色素颗粒的差异。最初的非刺豚鼠（a）等位基因赋予小鼠主要为黑色的毛色，现已证明它以极高的频率回复突变为另外两个更具显性的刺豚鼠等位基因，即黑褐相间（a(t)）和白腹刺豚鼠（Aw）。a(t)和Aw等位基因赋予的表型中，色素沉着在动物的背腹表面分布不均；在这两种情况下，由于褐黑素产生增加，动物的腹面明显比背面颜色浅。为了解a突变为a(t)或Aw的异常高回复率，并破译与这三个刺豚鼠等位基因相关的不同色素沉着模式的分子事件，我们在分子水平上对a、a(t)和Aw进行了表征。在此，我们报告分别在a、a(t)和Aw的刺豚鼠基因第一内含子的精确相同位置存在11 kb、6 kb和0.6 kb的插入片段。a插入片段由一个5.5 kb的VL30元件组成，该元件内部整合了5.5 kb的额外序列；此内部序列两侧是526 bp的同向重复序列。a(t)等位基因仅包含VL30元件和一个单一的内部526 bp重复序列。Aw等位基因只有一个单独的VL30长末端重复序列。基于对a(t)和Aw插入片段结构的比较，我们提出反向突变是通过同源重组切除a中插入的序列而发生的，利用526 bp的同向重复序列产生a(t)，或利用VL30长末端重复序列产生Aw。此外，对这三个等位基因的分析使我们能够鉴定出刺豚鼠基因的其他外显子，这些外显子产生了刺豚鼠mRNA的可变加工形式。我们证明a、a(t)和Aw中不同的插入片段通过选择性地使不同形式的刺豚鼠转录本的表达失活而导致色素沉着差异。
