Prokopenko S N, He Y, Lu Y, Bellen H J
Program in Developmental Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Genetics. 2000 Dec;156(4):1691-715. doi: 10.1093/genetics/156.4.1691.
In our quest for novel genes required for the development of the embryonic peripheral nervous system (PNS), we have performed three genetic screens using MAb 22C10 as a marker of terminally differentiated neurons. A total of 66 essential genes required for normal PNS development were identified, including 49 novel genes. To obtain information about the molecular nature of these genes, we decided to complement our genetic screens with a molecular screen. From transposon-tagged mutations identified on the basis of their phenotype in the PNS we selected 31 P-element strains representing 26 complementation groups on the second and third chromosomes to clone and sequence the corresponding genes. We used plasmid rescue to isolate and sequence 51 genomic fragments flanking the sites of these P-element insertions. Database searches using sequences derived from the ends of plasmid rescues allowed us to assign genes to one of four classes: (1) previously characterized genes (11), (2) first mutations in cloned genes (1), (3) P-element insertions in genes that were identified, but not characterized molecularly (1), and (4) novel genes (13). Here, we report the cloning, sequence, Northern analysis, and the embryonic expression pattern of candidate cDNAs for 10 genes: astray, chrowded, dalmatian, gluon, hoi-polloi, melted, pebble, skittles, sticky ch1, and vegetable. This study allows us to draw conclusions about the identity of proteins required for the development of the nervous system in Drosophila and provides an example of a molecular approach to characterize en masse transposon-tagged mutations identified in genetic screens.
在探寻胚胎外周神经系统(PNS)发育所需新基因的过程中,我们以终末分化神经元的标志物单克隆抗体22C10开展了三项遗传筛选。共鉴定出66个正常PNS发育所需的必需基因,其中包括49个新基因。为获取这些基因分子特性的信息,我们决定用分子筛选来补充我们的遗传筛选。从基于其在PNS中的表型鉴定出的转座子标签突变中,我们选择了代表第二和第三条染色体上26个互补群的31个P因子菌株,以克隆和测序相应基因。我们使用质粒拯救来分离和测序这些P因子插入位点两侧的51个基因组片段。利用从质粒拯救末端获得的序列进行数据库搜索,使我们能够将基因归为四类之一:(1)先前已表征的基因(11个),(2)克隆基因中的首次突变(1个),(3)已鉴定但未进行分子表征的基因中的P因子插入(1个),以及(4)新基因(13个)。在此,我们报告了10个基因(迷途、拥挤、达尔马提亚、胶子、乌合之众、熔化、卵石、彩虹糖、粘性ch1和蔬菜)候选cDNA的克隆、序列、Northern分析及胚胎表达模式。这项研究使我们能够得出关于果蝇神经系统发育所需蛋白质身份的结论,并提供了一个分子方法的实例,用于大规模表征在遗传筛选中鉴定出的转座子标签突变。