Research Information System
Internatioanl DNA Day and Genome Congress, Kırşehir, Turkey, 24 - 28 April 2017, pp.168
Aims and Scopes:
pHIG22 is a small, novel, cryptic, multicopy and double strand plasmid which has 2222 bp in long and with a total G + C content of 62,78 from Thermus scotoductus K6. The aim of this study is to detect the transcripts in both strands of pHIG22 and to characterize these transcripts identified by using LACE and RACE techniques.
Materials and Methods:
In order to determine the transcripts encoded by the plasmid pHIG22, cDNA was synthesized by designing specific primers from different regions at specific intervals in
both strands of the plasmid using Thermus scotoductus K6 Total RNA as template and the obtained cDNAs were amplified by PCR, then the orientation and possible sizes of the transcripts were determined. The 5'/3' RACE 2nd Generation (Ver. 13, Roche) kit was
used to detect the 5 'ends and LACE technique was used to detect the 3' ends of the identified transcripts of pHIG22. In the RACE technique, the first strand DNAs were generated using specific primers designed, and then the generated cDNAs were purified, the homopolymeric tail was added, and the target cDNAs were amplified by PCR. The amplified 5 'ends of the transcripts were cloned into the pGEM-T Easy vector. In the LACE technique, a 5 'phosphorylated primer (P1) was first ligated to the 3' end of the respective transcript with RNA ligase. Subsequently, another primer (P2) was used as a template (P2 is complementer to P1) and the cDNA was obtained by reverse transcription, which was complementary to the P1 primer. The generated cDNAs were amplified by PCR using a third primer (P3), and the resulting fragments were cloned into the pGEM-T Easy vector. All clones were sent to Macrogen (South Korea) for DNA sequence analysis.
Results and Discussion:
The use of Thermus scotoductus K6 Total RNA as a template revealed that it generated a total of 2 transcripts, one encoded in the first strand and one encoded in the opposite strand of the pHIG22 plasmid. As a result of studies with RACE and LACE techniques, it has been determined that Transcript 1 encoded by pHIG22 begins at nucleotide 329 and terminates at nucleotide 1950; Transcript 2 encoded in the opposite strand was also found to start at nucleotide 2103 and terminate at nucleotide 329. It has been found that the transcripts whose start and end points are determined are complementary to each another. Transcript 1 of 1622 bp in length appears to overlap with a region of Transcript 2 and 1559 bp of 1712 bp in length. It is known from the literature that interactions between RNAs can be regulated by the pRNAs required for replication. Antisense RNAs that control the number of plasmid copies are complementary to a 5 'end region of the target transcript (preprimer RNA or Rep mRNA required for replication).
These are referred to as "contrary transcript RNAs" (ctRNA). In some cases, inhibition by ctRNAs is carried out in the matching region of the target RNAs [1] . The complementation of the 3' end of Transcript 2 to the 5' end of Transcript 1 enhances the belief that these two transcripts are effective at controlling the number of plasmid copies and at the initiation of replication, as opposed to that of the transcripts identified from the pHIG22 plasmid. The complementation of the 3 'end of Transcript 2 to the 5' end of Transcript 1 enhances the idea that these two transcripts are effective at controlling the number of plasmid copies and at the initiation of replication, as the transcripts identified from the pHIG22 plasmid are in different chains and opposite to each other.
Keywords:pHIG22 transcripts, LACE and RACE techniques
Acknowledgements:This study was supported by TUBITAK (Project No: 112T277).
References:[1] del Solar, G., Giraldo, R., Ruiz-Echevarria, M., J., Espinosa, M. ve Diaz-Orejas, R. Replication and control of circular bacterial plasmids,Microbiol. Mol. Biol.Rev., 1998, 62, 434–464.