小球藻病毒基因调控序列在大肠杆菌和真核藻中的调控活性研究

以小球藻病毒腺嘌呤甲基转移酶基因 (amt)和主要外壳蛋白VP5 4基因的 5′上游调控序列构建大肠杆菌和真核藻转化载体。以PRPL 及CaMV35S启动子为阳性对照 ,研究了小球藻病毒来源的两种调控序列在E .coli和真核藻细胞中的启动活性。发现PAMT在 4种E .coli菌株中都具有极强的调控活性 ,启动Luc基因表达而产生的酶活性高于PRPL5 0～ 40 0倍 ;PVP54 在DH5α中也具有较强的启动活性。同时PAMT在两种小球藻中启动GUS基因瞬时表达的能力也明显高于CaMV35S启动子 ,表明它们有可能在真核藻类遗传转化中具有很好的应用前景。

Expression Activity of CLCuV Bidirectional Promoter in Agrobacterium tumefaciens

Cotton leaf curl virus (CLCuV) belongs to the subgroup III of geminiviruses with single strand DNA genome. Study demonstrated that the bidirectional promoter of CLCuV had activity in Agrobacterium tumefaciens (Smith et Townsend) Conn. This is the first report for the activity of the bidirectional promoter of geminivirus in A. tumefaciens. Results showed that the activity of the complementary sense promoter was stronger than that of virion sense promoter, and was detected 2-fold higher than that of CaMV 35S promoter in A. tumefaciens. Moreover, the promoter 5' deletion analysis indicated that the mean GUS activity driven by a 287 nucleotides complementary sense promoter fragment (from-287 to the translation initiation site) is 4 times higher than that driven by the whole complementary sense promoter in A. tumefaciens. This result suggested that there might exist negative regulatory elements in this deleted fragment. The function of other cis-elements included in CLCuV complementary sense promoter was also discussed in this paper.

Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein:A potential for developing hepatitis C virus targeting gene therapy

AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter

The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expression vector which can be used in Ti plasmid system of Agro-bacterium tumefaciens. The original plasmid, which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facilitate the selection of recombinant with Ti plasmid. Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter. This chimaeric gene was introduced into integrative Ti plasmid vector pGV3850, and then transformed into Nicotiana tobaccum cells. A binary plasmid vector was also used to introduce the chimaeric gene into tobacco cells. In both cases, the expression of ocs gene was demonstrated. The amount of oc-topine was much more than the nopaline synthesized by no-paline synthase (nos) gene transferred at the same time with Ti plasmid vector. This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the promoter of nos in tobacco cells.

Plutella xylostella granulovirus late gene promoter activity in the context of the Autographa californica multiple nucleopolyhedrovirus genome

Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus(Plxy GV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.