Insights into the human cDNA: A descriptive study using library screening in yeast

IF 3.5 Q3 Biochemistry, Genetics and Molecular Biology
Zina Alaswad , Nayera E. Attallah , Basma Aboalazm , Eman S. Elmeslhy , Asmaa S. Mekawy , Fatma A. Afify , Hesham K. Mahrous , Ashrakat Abdalla , Mai A. Rahmoon , Ahmed A. Mohamed , Ahmed H. Shata , Rana H. Mansour , Fareed Aboul-ela , Mohamed Elhadidy , Biola M. Javierre , Sherif F. El-Khamisy , Menattallah Elserafy
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引用次数: 0

Abstract

The utilization of human cDNA libraries in yeast genetic screens is an approach that has been used to identify novel gene functions and/or genetic and physical interaction partners through forward genetics using yeast two-hybrid (Y2H) and classical cDNA library screens. Here, we summarize several challenges that have been observed during the implementation of human cDNA library screens in Saccharomyces cerevisiae (budding yeast). Upon the utilization of DNA repair deficient-yeast strains to identify novel genes that rescue the toxic effect of DNA-damage inducing drugs, we have observed a wide range of transcripts that could rescue the strains. However, after several rounds of screening, most of these hits turned out to be false positives, most likely due to spontaneous mutations in the yeast strains that arise as a rescue mechanism due to exposure to toxic DNA damage inducing-drugs.
The observed transcripts included mitochondrial hits, non-coding RNAs, truncated cDNAs, and transcription products that resulted from the internal priming of genomic regions. We have also noticed that most cDNA transcripts are not fused with the GAL4 activation domain (GAL4AD), rendering them unsuitable for Y2H screening. Consequently, we utilized Sanger sequencing to screen 282 transcripts obtained from either four different yeast screens or through direct fishing from a human kidney cDNA library. The aim was to gain insights into the different transcription products and to highlight the challenges of cDNA screening approaches in the presence of a significant number of undesired transcription products. In summary, this study describes the challenges encountering human cDNA library screening in yeast as a valuable technique that led to the identification of important molecular mechanisms. The results open research venues to further optimize the process and increase its efficiency.
洞察人类 cDNA:利用酵母中的文库筛选进行描述性研究
在酵母遗传筛选中利用人类 cDNA 文库是一种方法,已被用于通过使用酵母双杂交(Y2H)和经典 cDNA 文库筛选的正向遗传学方法来鉴定新的基因功能和/或遗传和物理相互作用伙伴。在此,我们总结了在酿酒酵母(芽生酵母)中实施人类 cDNA 文库筛选过程中观察到的几个挑战。在利用 DNA 修复缺陷酵母菌株鉴定能挽救 DNA 损伤诱导药物毒性作用的新基因时,我们观察到了一系列能挽救菌株的转录本。然而,经过几轮筛选后发现,这些基因大部分都是假阳性的,很可能是由于酵母菌株在暴露于有毒的 DNA 损伤诱导药物后作为一种拯救机制而产生的自发突变。我们还注意到,大多数 cDNA 转录本没有与 GAL4 激活域(GAL4AD)融合,因此不适合进行 Y2H 筛选。因此,我们利用 Sanger 测序技术筛选了 282 个转录本,这些转录本来自四个不同的酵母筛选或直接从人类肾脏 cDNA 文库中获取。我们的目的是深入了解不同的转录产物,并强调在存在大量非预期转录产物的情况下,cDNA 筛选方法所面临的挑战。总之,本研究描述了在酵母中筛选人类 cDNA 文库所遇到的挑战,这是一项宝贵的技术,它导致了重要分子机制的鉴定。研究结果为进一步优化该过程并提高其效率开辟了研究领域。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Genetic Engineering and Biotechnology
Journal of Genetic Engineering and Biotechnology Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
5.70
自引率
5.70%
发文量
159
审稿时长
16 weeks
期刊介绍: Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts
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