[[alternative]]Molecular Cloning of Very Early Gene of Hz-1 Virus

[[abstract]]Hz-1 病毒是Baculovirus 的一種。由於它是所有動植物裡面極少數能在不 同細胞株中進行明顯的急性及持續感染 ( productive and persistent infection ) 的病毒。因此，成為研究病毒急性及持續感染機制的良好模 式。為瞭解病毒急性及持續感染的分子轉換機制，有必要比較急性特異性 及持續感染相關性之起動子，以便知道其控制機制。由於持續感染相關性 起動子已被發現，若能找到急性感染特異性起動子，則將極有助於此轉換 機制的研究。Hz-1 病毒之絕大部分極早期基因皆位於 HindIII-A、D、I 三片段上，但由於其可能對細菌有毒，以前曾有學者用質體，cosmid，及 phage 皆無法選殖成功。於是本實驗改用 low copy number 的 pWSK-29 質體，才選殖到 HindIII-A、D兩片段。Southern analysis 的結果確定質 體所插入之 DNA 確實分別為 16.1 kb 的 HindIII-A ( HA ) 及 13.4 kb 的HindIII-D ( HD ) 片段。此二質體的限制酵素圖譜已被完成，在 HD 片 段上的所有轉錄子 ( transcripts ) 及其方向也完整的經過解析。HD 片 段區域內含有 1.35 kb、1.52 kb、1.78 kb、2.33 kb 及 2.91 kb 的早期 轉錄子及 4.60 kb、4.81 kb、6.05 kb 及 7.33 kb 的晚期轉錄子。HD 片 段並且含有一個極早期基因，此基因轉錄了一個 5.54 kb 的轉錄子。這個 極早期基因經進一步分析發現位於 XhoI-H 片段到 XhoI-W 片段的區域， 其轉錄方向已確定。而 5.54 kb 的轉錄子上游含有 TATA 及 CAGT 之區域 則可能是起動子。此為 Hz-1病毒之第一個被發現的極早期基因及起動子 。 Hz-1 virus is a non-occluded baculovirus. It is one of the few animal and plant viruses which can induce both productive and persistent viral infections in the culture cells. In order to understand the molecular switches of productive and persistent viral infections, promoters specifically associated to these two phases should be compared. Since the persistent-associated gene is driven by an very early type promoter, the cloning of productive-specific very early promoters is needed. According to previous results, most of the very early type genes of Hz-1 virus are located on HindIII-A, D, and I fragments. For some unknown reasons, these fragments are not possible to be cloned by conventional plasmids and cosmids.However in my study, HindIII-A and D fragments are successfully cloned by using a low copy number plasmid, pWSK-29. Southern analysis confirms that the two viral DNA fragments, HindIII -A of 16.1kb and HindIII-D of 13.4 kb, have been inserted into pWSK-29 plasmids. Restriction maps of these two contructed plasmids are produced and all the transcripts and their directions on the HD fragment have been identified. Hind III-D fragment contains early and late transcripts. A very early gene, which transcripts a 5.54kb RNA, was also found to be contained in this fragment. This very ealy gene is located between XhoI-H and XhoI-W fragments of viral genome. Furthermore, the promotor may locate at the TATA and CAGT areas upstream of 5.54 kb transcript. These are the first productive-infection associated very early gene and promotor being reported in Hz-1 virus. Hz-1 virus is a non-occluded baculovirus. It is one of the