DIRECTED MUTAGENESIS OF A CLONED EYE-SPECIFIC GENE

提出了开发聚合酶的新应用

It is proposed to develop a novel application of the polymerase chain reaction (PCR) technique to isolate insertion mutations in a gene suspected of playing a role in Drosophila eye development. PCR is a sensitive method used to amplify particular DNA segments flanked by oligonucleotides. A model system has been used to demonstrate the efficacy of using PCR to detect known P-element transposon insertions. It is proposed to adapt this technique to isolate new insertion mutations. Briefly, multiple oligonucleotides complementary to a single strand of the gene of interest would be included with an oligonucleotide that primes synthesis away from the terminal repeats of the P-element transposable element. A substrate suitable for amplification would arise only if a transposition event led to the insertion of a P- element within about 100-1,000 nt of one of the gene oligonucleotides, yielding an appropriate segment of DNA flanked by primers. The subject of this study will be the gene encoding a differentiation antigen of photoreceptor cells, expressed very early in their development. This gene is expressed about thirty hours earlier than previously studied photoreceptor differentiation antigens. It is expressed at about the same time as other early neuronal differentiation antigens, but it is exquisitely specific for photoreceptor cells. Its time of appearance and tissue specificity suggest that the antigen may play an important role in some aspect of photoreceptor cell differentiation, perhaps in distinguishing photoreceptors from other neurons. This gene has been cloned, and partial sequence information is available. Use of the PCR technique to isolate insertion mutations in this gene will aid in the identification of its function during photoreceptor cell development. If successful, this technique should find broad application to the isolation of insertion mutations in Drosophila genes of importance in the development and function of the visual system.