Gene Cloning and Gene Expression of Pyruvate Kinase from Drosophila melanogaster

• Main Authors: Yu-Jing Zhu, 朱玉菁
• Other Authors: Yi-Chih Chien
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/04316674516527820524

碩士 === 國立彰化師範大學 === 科學教育研究所 === 87 === Abstract 5’ Rapid Amplification of cDNA End (5’RACE) analysis was applied to identify the transcription start point (tsp) of pyruvate kinase gene (PyK) from Drosophila melanogaster. Screening a EMBL-3 genomic library of D.melanogaster using PyK genomic DNA as a probe, a genomic DNA clone (pk61), which contains the 5’ untranscribed region of D. melanogaster PyK gene was isolated. The insert was reconfirmed to contain Pyk gene by Southern blot analyses. A sequence of 734 bp upstream from tsp of Pyk was determined, from which some core sequences that might regulate transcription or might be recognized by proteins were proposed. The PyK expression of D. melanogaster from different developmental stages and sexes was analyzed by Northern blot analyses. The results that the amounts of PyK transcripts produced in larvae and adults was higher than those in pupae, indicates that Pyk expression is low in pupal stages. The fact that similar level of PyK expression in both male and female adults, suggests that there is a dosage compensation phenomenon in Pyk gene of D. melanogaster male adults. In addition, the results that there is a single band present in all samples of the Northern blot analyses reveals that there is only a single PyK mRNA in different developmental stages and sexes of D. melanogaster. RT-PCR analysis was performed to determine the number of species of PyK transcript in adults of D. melanogaster. The result that only a single amplicon appeared in each amplication, suggests that there might be a single PyK transcript expressed in adults of D. melanogaster. Furthermore, this might suggest that there is only a single PyK gene in D. melanogaster.