Generation of long palindromes in ADE2 gene by dint of computer program Synonymous codon shuffler

Palindromi su sekvencije DNA u kojima se dva obrnuta ponavljanja nalaze neposredno jedan do drugog. Drugim riječima, prva polovina palindroma komplementarna je drugoj polovini te stoga palindromi u DNA mogu tvoriti sekundarne strukture ukosnice ili križne strukture. Cilj ovoga rada bio je predložiti eksperimentalni sustav u kojem bi se mogla testirati hipoteza prema kojoj pojava navedenih sekundarnih struktura u nukleinskim kiselinama može omesti ekspresiju gena. U tu svrhu najprije je zamjenom položaja sinonimnih kodona in silico generirano 4∙106 permutiranih varijanti reporter gena ADE2, koji su po duljini, GC-sastavu i najvažnije, upotrebi kodona, bili identični ishodnoj sekvenciji. Iz ove populacije odabrana je varijanta gena ADE2 sa najduljim palindromom, na temlju koje je zatim predložen niz postupaka za njenu konstrukciju in vitro kao i eksperimentalni sustav za procjenu utjecaja prisutnosti dugačkog palindroma u genu ADE2 na razinu njegove ekspresije u kvascu Saccharomyces cerevisiae.Palindromes are DNA sequences in which two inverted repeats are directly linked one to another. In other words, the first half of the palindrome is complementary to the other half and as a consequence palindromic sequences can form secondary structures known as hairpins and crucifroms. The goal of this work was to suggest an experimental system for testing the hypothesis according to which these secondary structures can have negative impact on gene expression. First we have generated 4∙106 permutated variants of the ADE2 reporter gene in silico by shuffling synonymous codons. These permuted variants had the same length, GC-content, and most importantly, codon usage, as the original ADE2 gene. Permuted variant with the longest palindrome was chosen for subsequent steps in which procedures for its in vitro construction were proposed, along with an experimental system for testing the effect of the presence of a long palindrome on the level of expression of the reporter gene ADE2 in yeast Saccharomyces cerevisiae