RETROTRANSPOSON AND ENDOGENOUS RETROVIRUS ANALYSES IN HUMAN GENOME NERMIN GOZUKIRMIZI
Contents Purpose Introduction MaterialsandMethodology Results Discussion
Purpose Detection of Sukkula, Nikita, BAGY2 andSIRE1 in thehumangenomewith IRAP - PCR analysis. Detection of movements of HERV-K6 and HERV-K11 endogenousretroviruswith IRAP- PCR analysis. This is thefirststudyto investigate plant-specific retrotransposons in the human genome.
PlantRetrotransposons Sukkula Detected in thebarleygenome. Contain 4.4 kbLTRs. Nikita Detected in thebarleygenome. Found in thegenome as solo LTR. Genome size is approximately 6.5 kb.
BAGY2 Detected in thebarleygenome. BAGY2 containsenv gene in additiontogagandpol. Genome size is approximately 10 kbexceptenv. SIRE1 Detected in thesoybeangenome. Genome size is approximately 7036-9805 kb. SIRE1containsenv gene in additiontogagandpol.
Human EndogenousRetroviruses (HERVs) Theyinserted in thehumangenome 2-70 My ago, andpassdown as Mendel’slaws. Therearemanyhumanendogenousretrovirusfamily in thehumangenome. AlthoughHERVsactivity is knownverylimited in humans, therearemanyactive HERV elements in thegenome. HERV-K is known as themostactive HERV elements in genome.
16 Determination of RetrotransposonMovements Using IRAP Marker Technique sample 1 IRAP-PCR sample 2
Integration polymorphism of human endogenous retrovirus H (HERV-H) Purpose: In the normal human population (HERV-H) LTR7A (441bp), LTR7B (445bp), LTR7C (471bp) specific regions of IRAP-PCR and the comparative examination? Is there a variation in these integration regions in the normal human population? Is there any variation in gene expression in these regions in the normal human population?
Human blood cells were used as material in this study. Blood samples were taken from 20 volunteer individuals with 10 women and 10 men. Asia (Turkey, Azerbaijan, Kyrgyzstan, Indonesia, China) and individuals representing African populations are healthy, they randomly selected age range 18 - 30 range varies. Blood collection was carried out by Istanbul University, Health, Culture and Sports Department, Mediko Social Center.
In the 20 human genomes with the HERV-H LTR7A primer, ERVH-3s were targeted to the LTR regions of the ERVH-3s and the regions between the ERVH-3s were amplified by IRAP-PCR and the polymorphic and monomorphic bands were obtained in 2% agarose gel. HERV-H integration polymorphism was observed in all 20 individuals with this primer. Kodak Molecular Imaging Software analysis revealed polymorphism values ranging from 0.88 to 9.73%.
Moleculardimensions of PCR productsamplifiedusingthe HERV-H LTR7A primerwith molekül Kodak MolecularImaging Software (1994-2007 CarestreamHealth, INC.) “ “Kodak MolecularImaging Software (1994-2007 CarestreamHealth, INC.)” programı ile HERV-H LTR7A primeri kullanılarak çoğaltılan PCR ürünlerinin molekül boyutları
In 20 human DNA samples with HERV-HLTR7B primers, ERVH-3’s regions were replicated with IRAP-PCR by targeting the LTR regions of ERVH-3s, and polymorphic and monomorphic bands were obtained in 2% agarose gels. HERV-H integration polymorphism was observed in all 20 individuals with this primer. The results of the Kodak Molecular Imaging Software analysis revealed polymorphism values ranging from 1.4 to 14.08%.
Moleculardimensions of PCR productsamplifiedusingthe HERV-H LTR7B primerwiththe molekül Kodak MolecularImaging Software (1994-2007 CarestreamHealth, INC.) “Program
Polymorphism calculation using HERV-H LTR7A and HERV-H LTR7B primers by Jaccard similarity coefficient.
In this study, the comparison of the virus integration profile of the human endogenous viruses HERV-H, LTR7A (450 bp), LTR7B (465 bp) and LTR7C (471 bp) between healthy individuals was performed by ç Inter-retrotransposon amplified polymorphism HER (IRAP) method. Blood samples obtained from 20 different ethnic individuals were selected as material for the determination of variations in the genome and genomic DNAs obtained from these samples were examined by using the IRAP method with 3 primer pairs covering the LTR regions of the HERV-H gene. Although very few samples were studied, 0-87% polymorphism patterns were observed in all individuals. The findings are expected to contribute to the understanding of HERV-H's changes in human populations and their effects on genome. This is the first study to examine the integration polymorphism of HERV-H from human endogenous viruses. • Guliev M., Yilmaz S., Sahin K., Marakli S. andGozukirmizi N. (2013) Human endogenousretrovirus H (Herv-H) genomeinsertionvariations in humans. MolecularMedicineReports 7:1305-1309.
MaterialsandMethods • Individualsdiffer in differentagegroups(10-79) andgenders.
IRAP PCR ContentsandConditions • ReactioncontentsandconditionsarethesameforSukkula, Nikita, BAGY2andSIRE1. • PCR productsresolvedwithagarose gel electrophoresisfor 120 min at 150 V. • Annealingtemperature is 55 °C forSukkula; 51 °C forNikita, BAGY2andSIRE1. * SapphireAmp Fast PCR Master Mix (Takara, RR350A)
HERV-K6 / HERV-K11 IRAP-PCR Analizi • Reactioncontentsandconditionsarethesamefor HERV-K6 and HERV-K11. • PCR productsresolvedwithagarose gel electrophoresisfor 120 min at 150 V. • Annealingtemperature is 53 °C for HERV-K6; 56 °C for HERV-K11.
SIRE1 Internal Domain and LTR Analysis Weselectedtwoindividualsamongeachagegroupsfortheanalysis.
SIRE1 INTERNAL DOMAIN ANALYSIS Reactioncontentsandconditionsarethesamefor gag, rtandenv. * Annealingtemperature is 52oC for gag ve env; 50 oCfor RT.
UPGMA Result • 1, 11, 12, 22, 23, 24 numberedindividualsbelongonegorup; othersbelongsecondgroup.
UPGMA Result 1, 23 and 24 numberedindividualsbelongtoonegroupandothersbelongtosecondgroup.
UPGMA Result 1, 2, 3, 4, 5, 14, 15, 16, 17, 19 numberedindividualsbelongonegroup; othersbelongsecondgroup.
UPGMA Result • 1, 23 ve 24 numberedindividualsbelongonegroup; othersbelongsecondgroup.
UPGMAResult 2, 14, 16, 17, 18, 19, 21, 22, 23, 24 numberedindividualsbelongonegroup; 3, 4, 5, 7, 8, 9, 11, 13 numberedindividualsbelongothergroup.