Mirzayan C, Copeland C S, Snyder M
Department of Biology, Yale University, New Haven, Connecticut 06511.
J Cell Biol. 1992 Mar;116(6):1319-32. doi: 10.1083/jcb.116.6.1319.
In an attempt to identify structural components of the yeast nucleus, subcellular fractions of yeast nuclei were prepared and used as immunogens to generate complex polyclonal antibodies. One such serum was used to screen a yeast genomic lambda gt11 expression library. A clone encoding a gene called NUF1 (for nuclear filament-related) was identified and extensively characterized. Antibodies to NUF1 fusion proteins were generated, and affinity-purified antibodies were used for immunoblot analysis and indirect immunofluorescence localization. The NUF1 protein is 110 kD in molecular mass and localizes to the yeast nucleus in small granular patches. Intranuclear staining is present in cells at all stages of the cell cycle. The NUF1 protein of yeast is tightly associated with the nucleus; it was not removed by extraction of nuclei with nonionic detergent or salt, or treatment with RNAse and DNAse. Sequence analysis of the NUF1 gene predicts a protein 945 amino acids in length that contains three domains: a large 627 residue central domain predicted to form a coiled-coil structure flanked by nonhelical amino-terminal and carboxy-terminal regions. Disruption of the NUF1 gene indicates that it is necessary for yeast cell growth. These results indicate that NUF1 encodes an essential coiled-coil protein within the yeast nucleus; we speculate that NUF1 is a component of the yeast nucleoskeleton. In addition, immunofluorescence results indicate that mammalian cells contain a NUF1-related nuclear protein. These data in conjunction with those in the accompanying manuscript (Yang et al., 1992) lead to the hypothesis that an internal coiled-coil filamentous system may be a general structural component of the eukaryotic nucleus.
为了鉴定酵母细胞核的结构成分，制备了酵母细胞核的亚细胞组分，并将其用作免疫原以产生复杂的多克隆抗体。其中一种血清用于筛选酵母基因组λgt11表达文库。鉴定出一个编码名为NUF1（与核丝相关）基因的克隆，并对其进行了广泛表征。制备了针对NUF1融合蛋白的抗体，并使用亲和纯化的抗体进行免疫印迹分析和间接免疫荧光定位。NUF1蛋白的分子量为110kD，定位于酵母细胞核中的小颗粒斑。在细胞周期的所有阶段，细胞核内均有染色。酵母的NUF1蛋白与细胞核紧密相关；用非离子去污剂或盐提取细胞核，或用RNA酶和DNA酶处理均不能将其去除。对NUF1基因的序列分析预测其编码的蛋白质长度为945个氨基酸，包含三个结构域：一个由627个残基组成的大的中央结构域，预计形成卷曲螺旋结构，两侧为非螺旋的氨基末端和羧基末端区域。破坏NUF1基因表明它对酵母细胞生长是必需的。这些结果表明，NUF1在酵母细胞核内编码一种必需的卷曲螺旋蛋白；我们推测NUF1是酵母核骨架的一个组成部分。此外，免疫荧光结果表明哺乳动物细胞含有一种与NUF1相关的核蛋白。这些数据与随附论文（Yang等人，1992年）中的数据共同提出了一个假设，即内部卷曲螺旋丝状系统可能是真核细胞核的一个普遍结构成分。
