Tanaka S, Halter D, Livingstone-Zatchej M, Reszel B, Thoma F
Institut für Zellbiologie, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Nucleic Acids Res. 1994 Sep 25;22(19):3904-10. doi: 10.1093/nar/22.19.3904.
When the function of origins of replication in yeast was compromised by placing ARS sequences downstream of strong promoters, ARS activity might have been affected either by transcription or by an altered chromatin configuration induced by the construct. To distinguish between these possibilities, derivatives of the yeast TRP1ARS1 minichromosome were constructed that contained either the DED1 or the PET56 promoter firing against ARS1 (DEDARS and PETARS constructs). PETARS constructs transformed yeast at high frequencies and were maintained as minichromosomes consistent with efficient ARS1 function, but DEDARS constructs transformed at low frequencies and had to be rescued as minichromosomes by insertion of a second ARS (H4-ARS). Chromatin analysis revealed that the ARS1 regions in PETARS and H4-DEDARS constructs were indistinguishable from the ARS1 region of the host TRP1ARS1 circle showing a nuclease sensitive region flanked by a nucleosome. However, RNA-analysis in the ARS region showed high and low levels of transcripts in H4-DEDARS and PETARS, respectively. Transcription elongated through the A, B1, and B2 elements and ended in B3, the binding site for ABFI. We conclude that transcription through ARS1 and not an altered chromatin structure affected ARS activity in these constructs.
当通过将自主复制序列（ARS）置于强启动子下游来破坏酵母中复制起点的功能时，ARS活性可能受到转录的影响，也可能受到该构建体诱导的染色质构型改变的影响。为了区分这些可能性，构建了酵母TRP1ARS1微型染色体的衍生物，其包含针对ARS1的DED1或PET56启动子（DEDARS和PETARS构建体）。PETARS构建体以高频转化酵母，并作为微型染色体维持，这与高效的ARS1功能一致，但DEDARS构建体以低频转化，并且必须通过插入第二个ARS（H4-ARS）作为微型染色体进行拯救。染色质分析表明，PETARS和H4-DEDARS构建体中的ARS1区域与宿主TRP1ARS1环的ARS1区域没有区别，显示出一个被核小体侧翼包围的核酸酶敏感区域。然而，ARS区域的RNA分析分别显示H4-DEDARS和PETARS中存在高水平和低水平的转录本。转录延伸穿过A、B1和B2元件，并在ABFI的结合位点B3处终止。我们得出结论，在这些构建体中，通过ARS1的转录而非染色质结构改变影响了ARS活性。
