IDENTIFICATION OF A NONHISTONE CHROMOSOMAL PROTEIN ASSOCIATED WITH HETEROCHROMATIN IN DROSOPHILA-MELANOGASTER AND ITS GENE
IDENTIFICATION OF A NONHISTONE CHROMOSOMAL PROTEIN ASSOCIATED WITH HETEROCHROMATIN IN DROSOPHILA-MELANOGASTER AND ITS GENE
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DOI：
10.1128/mcb.6.11.3862
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发表时间：
1986-11-01
影响因子：
5.3
通讯作者：
ELGIN, SCR
中科院分区：
文献类型：
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作者：
JAMES, TC;ELGIN, SCR
针对果蝇核蛋白中与DNA紧密结合的一部分制备了单抗。这些抗体中有四个是针对一个19千道尔顿的核蛋白；多线染色体的免疫荧光染色将抗原定位在α、β和中间异染色区。用其中一种单抗筛选λgt11表达文库，鉴定出一个重组DNA噬菌体克隆，其产生的融合蛋白在免疫学上与异染色质相关蛋白相似。针对细菌LacZ融合蛋白的多克隆血清在Western blotts上识别相同的核。从lambda.gt10文库中克隆了一个全长cDNA克隆，并获得了其DNA序列。对开放阅读框的分析表明，该cDNA编码一个18,101-道尔顿蛋白。从黑斑潜蝇的Charon4文库中分离到两个与之重叠的基因组DNA克隆，并获得了限制性内切酶图谱。与这些探针的原位杂交结果表明，该基因定位于2L染色体上29A的单一染色体位置。这一一般策略对于克隆基因和确定染色体蛋白质的遗传位点应该是有效的，而这些基因不能用其他方法轻易地检测出来。
Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the .alpha., .beta., and intercalary heterochromatic regions. Screening of a .lambda.gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear on Western blots. A full-length cDNA clone was isolated from a .lambda.gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melangaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.