ULTRASTRUCTURAL-LOCALIZATION OF NUCLEIC-ACID SEQUENCES IN SACCHAROMYCES-CEREVISIAE NUCLEOLI
ULTRASTRUCTURAL-LOCALIZATION OF NUCLEIC-ACID SEQUENCES IN SACCHAROMYCES-CEREVISIAE NUCLEOLI
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DOI：
10.1007/bf00352202
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发表时间：
1991-09-01
期刊：
影响因子：
1.6
通讯作者：
HAMKALO, BA
中科院分区：
文献类型：
--
作者：
DVORKIN, N;CLARK, MW;HAMKALO, BA
在电子显微镜下，可以看到酿酒酵母中假定的核仁是一个与核膜相关的深染的新月形结构。单倍体酵母基因组包含一个9.1kb的核糖体DNA(RDNA)重复序列的100-200个串联拷贝，预计位于该结构中。我们将非同位素标记的探针与分离的酿酒酵母细胞核进行原位杂交和免疫胶体金检测相结合，在电子显微镜(EM)水平上定位细胞核中的rDNA和rRNA前体序列。金粒子被限制在原子核的特定区域，这些区域看起来比核的主体更致密，并且通常呈现出被认为是核仁的典型结构的新月形。此外，哺乳动物U3的酵母同源物SnR17是一种核仁限制性小核RNA(SnRNA)，定位于细胞核的同一电子致密区。这些数据结合发表的核仁相关抗原的免疫荧光定位，提供了致密新月是核仁的确凿证据。最后，所描述的技术适用于探测遗传可操纵系统中的核组织。
The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope. The haploid yeast genome contains 100-200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure. We combined in situ hybridization of non-isotopically labeled probes to isolated S. cerevisiae nuclei with immunogold detection to localize rDNA and rRNA precursor sequences in nuclei at the electron microscope (EM) level. Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus. In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus. These data, in conjunction with published immunofluorescent localizations of nucleolar-associated antigens, provide definitive proof that the dense crescent is the nucleolus. Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.