Ward J G, Blomberg P, Hoffman N, Yao M C
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, 1124 Columbia Street, Seattle, WA 98104, USA.
Chromosoma. 1997 Sep;106(4):233-42. doi: 10.1007/s004120050244.
In the ciliated protozoan, Tetrahymena thermophila, the diploid germinal micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA). During genesis of new somatic macronuclei the germline rDNA gene is excised by developmentally programmed chromosome breakage and preferentially amplified to approximately 9, 000 copies. We have studied this process by fluorescence in situ hybridization. We find that initially rDNA amplification is restricted to two separate and highly confined regions of the nucleus. Analysis of nuclei that are hemizygous for the rDNA locus reveals that each focus of hybridization is derived from a single allele of the rDNA. As rDNA amplification progresses these two foci of hybridization disperse and spread throughout the macronucleus, eventually forming approximately 100-500 new nucleoli. These events are correlated with morphologically distinct developmental stages. We investigated the amplification of the C3 allele of the rDNA that confers a replication advantage over the B allele during vegetative propagation, and find no evidence for preferential amplification of the C3 early in rDNA maturation. We also show that the rmm 11 rDNA mutant allele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and heterozygotes, but fails to amplify further and disperse into multiple nucleoli. These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occur. We propose that rDNA amplification is a two-step process. First, the two rDNA alleles are independently amplified, while allelic copies remain closely associated. Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, generating fully mature rDNA minichromosomes that are able to replicate to high copy number.
在纤毛原生动物嗜热四膜虫中，二倍体生殖微核包含核糖体RNA（rDNA）基因的两个等位基因拷贝。在新的体大核形成过程中，生殖系rDNA基因通过发育程序控制的染色体断裂被切除，并优先扩增至约9000个拷贝。我们通过荧光原位杂交研究了这一过程。我们发现，最初rDNA扩增局限于细胞核的两个独立且高度受限的区域。对rDNA基因座半合子的细胞核分析表明，每个杂交位点都来自rDNA的单个等位基因。随着rDNA扩增的进行，这两个杂交位点分散并扩散到整个大核中，最终形成约100 - 500个新的核仁。这些事件与形态上不同的发育阶段相关。我们研究了在营养繁殖期间赋予比B等位基因复制优势的rDNA的C3等位基因的扩增，发现在rDNA成熟早期没有优先扩增C3的证据。我们还表明，在发育程序控制的rDNA切除方面有缺陷的rmm 11 rDNA突变等位基因，在突变纯合子和杂合子的双位点阶段可以扩增，但无法进一步扩增并分散到多个核仁中。这些数据表明，rmm 11等位基因的扩增在最初几轮扩增中没有延迟，这表明这种扩增发生并不需要有效的切除。我们提出rDNA扩增是一个两步过程。首先，两个rDNA等位基因独立扩增，而等位基因拷贝保持紧密关联。后来，rDNA拷贝分散并进一步扩增，推测是因为rDNA切除已经发生，产生了能够复制到高拷贝数的完全成熟的rDNA小染色体。
