Background and purpose: Polymerase chain reaction (PCR) is a rather quick and accurate method employed for gene detection and isolation. Primer designing is an important issue in this technique and plays a critical role in considering both the genome properties and cloning of the isolated genes. Streptomycin antibiotic is produced by Streptomyces griseus using str gene cluster with more than 25 genes. This gene cluster contains StrR gene encoding a specific protein regulator of this cluster. The pathway specific transcriptional activator then induces transcription of other genes in the str gene cluster. In this study, the researchers aimed at isolating promoterless StrR2 gene and then cloning it.Materials and methods: To isolate promoterless StrR gene, a set of primers (StrR2) was designed. One pair of these primers (St Nes) detected the StrR from the genome. Moreover, Nested-PCR was then used to detect amplified StrR2 gene. Sites for BamHI and XbaI were designed in other primers (Str nP1and Str nP2). These primers not only amplified the StrR2 gene but also created restriction enzyme sites in the amplified fragments.Results: Using PCR, the promoterless StrR2 gene was amplified and its structure was confirmed. This gene was successfully cloned, too. The correct structure of the recombinant plasmids was confirmed using different techniques such as gel electrophoresis, PCR and restriction digestion analysis.Conclusion: Using this vector, one can subclone the promoterless StrR2 gene in the Streptomyces expression vectors containing inducible promoters.

Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene (EGFP) could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, GFP gene was expressed, compared to pLOX/CWgfp-transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing “ from the start of the GFP gene to 5’ LTR R” . The GFP gene was again expressed. Therefore, our findings suggest the EGFP does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment.