Orr S L, Hughes T P, Sawyers C L, Kato R M, Quan S G, Williams S P, Witte O N, Hood L
Division of Biology, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11869-73. doi: 10.1073/pnas.91.25.11869.
A cDNA sequencing project was initiated to characterize gene expression in human bone marrow and develop strategies to isolate novel genes. Forty-eight random DNAs from total human bone marrow were subjected to single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed no similarity to known sequences. Information from DNA sequence analysis was used to develop a differential prescreen to subtract unwanted cDNAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negative with a complex probe were subject to single-pass DNA sequence determination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA expression analysis using 25 different human leukemic cell lines. Of 13 unknown cDNAs tested, 10 were expressed in all cell types tested and 3 revealed a hematopoietic lineage-restricted expression pattern. Interestingly, while a total of only 96 bone marrow cDNAs were sequenced, 31 of these cDNAs represent sequences from unknown genes and 12 showed significant similarities to sequences in the data bases. One cDNA revealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may represent a previously unknown kinase. Differential screening techniques coupled with single-pass cDNA sequence analysis may prove to be a powerful and simple technique to examine developmental gene expression.
启动了一个cDNA测序项目，以表征人类骨髓中的基因表达并制定分离新基因的策略。从人类骨髓总DNA中选取48个随机DNA进行单通道DNA序列分析，以确定骨髓中表达的mRNA的有限复杂性。总体而言，8个cDNA（17%）与已知序列无相似性。DNA序列分析所得信息用于开发差异预筛选，以去除不需要的cDNA并富集未知cDNA。48个与复杂探针呈阴性的cDNA进行单通道DNA序列测定。在这些预筛选的cDNA中，未知序列的数量增加到23个（48%）。未知cDNA还通过使用25种不同人类白血病细胞系的RNA表达分析进行表征。在测试的13个未知cDNA中，10个在所有测试细胞类型中均有表达，3个显示出造血谱系限制性表达模式。有趣的是，虽然总共只对96个骨髓cDNA进行了测序，但其中31个cDNA代表未知基因的序列，12个与数据库中的序列有显著相似性。一个cDNA在氨基酸水平上与丝氨酸/苏氨酸蛋白激酶有显著相似性（123个氨基酸中有56%的同一性），可能代表一种以前未知的激酶。差异筛选技术与单通道cDNA序列分析相结合可能被证明是一种强大而简单的技术，用于研究发育基因表达。
