Wang Nengding, Rizvydeen Saajidha, Vahedi Mithaq, Vargas Gonzalez Daysi M, Allred Amanda L, Perry Dustin W, Mirabito Peter M, Kirk Karen E
Biology Department, Lake Forest College, Lake Forest, Illinois, United States of America.
Biology Department, University of Kentucky, Lexington, Kentucky, United States of America.
PLoS One. 2014 Jun 13;9(6):e99491. doi: 10.1371/journal.pone.0099491. eCollection 2014.
Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed "telomere-anchored PCR," measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms.
端粒长度在同一生物体的生殖细胞和体细胞之间存在差异，这引发了端粒在减数分裂过程中会延长的假说。然而，对于许多生物体中减数分裂时的端粒长度，我们所知甚少。在丝状真菌构巢曲霉中，菌丝和无性孢子中的端粒长度是不变的。由于构巢曲霉中纯减数分裂细胞的丰度相对较低且存在的DNA量较少，因此尚未有研究使用现有技术确定有性子囊孢子的端粒长度。为了解决这个问题，我们开发了一种简单且灵敏的PCR策略来测量构巢曲霉减数分裂细胞的端粒长度。这种新技术被称为“端粒锚定PCR”，它使用传统末端限制片段（TRF）Southern分析所需DNA的一小部分来测量II-L染色体上的端粒长度。使用这种方法，我们确定构巢曲霉的子囊孢子端粒长度与该生物体其他细胞类型的端粒长度几乎相同，约为110碱基对，这表明构巢曲霉的主要细胞类型中存在令人惊讶的严格端粒长度调控。当在端粒酶逆转录酶（TERT）基因敲除菌株中测量菌丝端粒时，很容易检测到长度的小幅下降。因此，该技术可以检测相对罕见细胞类型中的端粒，并且在测量异常短的端粒时特别灵敏。这种快速且廉价的端粒锚定PCR方法有可能应用于其他丝状真菌和生物体类型。
