Abstract
BackgroundMarek's disease virus (MDV) has a bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts. By sequencing for the promoters from 8 different strains (CVI988, 814, GA, JM, Md5, G2, RB1B and 648A), it is found, comparing with the other 7 MDV strains, CVI988 has a 5-bp (from -628 to -632) deletion in this region, which caused a Sp1 site destroyed. In order to analysis the activity of the promoter, the complete bi-directional promoters from GA and CVI988 were, respectively, cloned into pCAT-Basic vector in both directions for the recombinants pPGA(pp38)-CAT, pPGA(1.8 kb)-CAT, pPCVI(pp38)-CAT and pPCVI(1.8 kb)-CAT. The complete promoter of GA was divided into two single-direction promoters from the replication of MDV genomic DNA, and cloned into pCAT-Basic for pdPGA(pp38)-CAT and pdPGA(1.8 kb)-CAT as well. The above 6 recombinants were then transfected into chicken embryo fibroblasts (CEFs) infected with MDV, and the activity of chloramphenicol acetyltransferase (CAT) was measured from the lysed CEFs 48 h post transfection.ResultsThe results showed the activity of the divided promoters was decreased on both directions. In 1.8-kb mRNA direction, it is nearly down to 2.4% (19/781) of the whole promoter, while it keeps 65% (34/52) activity in pp38 direction. The deletion of Sp1 site in CVI988 causes the 20% activity decreased, and has little influence in pp38 direction.ConclusionThe present study confirmed their result, and the promoter for the 1.8-kb mRNA transcripts is a much stronger promoter than that in the orientation for pp38.
Highlights
Marek's disease virus (MDV) has a bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts
Complete 1.8-kb mRNA transcripts are present in oncogenic viruses but are truncated in attenuated variants [9,10], and multiple copies of the 132-bp repeats are found in vaccine strain CVI988 or attenuated viruses compared to the virulent oncogenic strains [11,12]
The complete bi-directional promoter activity in 1.8-kb mRNA direction is 15 times as that in pp38 direction To analyze the regulation activity of the bi-directional promoter for chloramphenicol acetyltransferase (CAT) reporter gene expression, plasmids pPGA(pp38)-CAT and pPGA(1.8 kb)-CAT with the promoter in opposite directions were used to transfect chicken embryo fibroblasts (CEFs) monolayers infected with rMd5, or uninfected CEF
Summary
Marek's disease virus (MDV) has a bi-directional promoter between pp gene and 1.8-kb mRNA transcripts. The complete promoter of GA was divided into two single-direction promoters from the replication of MDV genomic DNA, and cloned into pCAT-Basic for pdPGA(pp38)-CAT and pdPGA(1.8 kb)-CAT as well. Marek's disease virus (MDV) is an oncogenic herpesvirus, which causes a highly contagious neoplastic disease in chickens[1], and could be divided into 3 serogroups. Complete 1.8-kb mRNA transcripts are present in oncogenic viruses but are truncated in attenuated variants [9,10], and multiple copies of the 132-bp repeats are found in vaccine strain CVI988 or attenuated viruses compared to the virulent oncogenic strains [11,12]
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