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首页> 外文期刊>Nucleic Acids Research >EXPRESSION OF HERPES VIRUS THYMIDINE KINASE IN NEUROSPORA CRASSA
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EXPRESSION OF HERPES VIRUS THYMIDINE KINASE IN NEUROSPORA CRASSA

机译：疱疹病毒胸腺激酶在神经管ASS中的表达

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The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene (tk) was expressed in Neurospora crassa.tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2-tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [H-3]thymidine or [C-14]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N.crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.
机译：通常缺乏这种酶的真菌中胸苷激酶的表达将大大有助于DNA代谢的研究，并提供有用的药物敏感性表型。单纯疱疹病毒1型胸苷激酶基因（tk）在Neurospora crassa中表达.tk表达为与N.crassa arg-2调控序列的融合体，以及潮霉素磷酸转移酶-胸苷激酶融合基因在巨细胞病毒和SV40序列。仅含有tk的菌株显示胸苷激酶活性。在含有arg-2-tk基因的菌株中，在精氨酸培养基中的生长会同时降低酶的活性和mRNA的水平，这与通过arg-2调控序列进行的控制一致。在生长培养基中添加[H-3]胸苷或[C-14]胸苷后，在N.crassa中胸苷激酶的表达促进了复制DNA的放射性标记。 DNA的胸腺嘧啶核苷标记可证明羟基脲可用于阻止复制并同步N.crassa有丝分裂周期。表达胸苷激酶的菌株比缺乏胸苷激酶的菌株对被胸苷激酶激活的抗癌和抗病毒核苷药物敏感，包括5-氟-2'-脱氧尿苷，1-（2-脱氧-2-氟-β-D -阿拉伯呋喃糖基）-5-碘尿苷和三氟胸苷。最后，胸腺嘧啶核苷激酶在N.crassa中的表达使溴脱氧尿苷掺入DNA的水平足以通过平衡离心将新复制的DNA与旧的DNA分离。

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