diplomsko delo
David Valte (Author), Janez Plavec (Mentor)

Abstract

Kratke tandemske ponovitve oz. mikrosateliti imajo lahko zaradi določenih mutacij, deaminacij ali povečanja števila kopij patogeno funkcijo. Ponovitve zaporedja ATTTC se pojavljajo kot razlog za nekatere vrste spinocerebelarnih ataksij (SCA) ter za več vrst družinske odrasle mioklonične epilepsije (FAME). Večkratne ponovitve ATTTC v intronskih regijah nekaterih genov vodijo v alternativno izrezovanje in nastanek proteinskih izooblik, ki so s svojo spremenjeno funkcijo lahko razlog za patogena stanja. Kratka tandemska ponavljajoča se zaporedja so zanimiva tudi zaradi njihove sposobnosti tvorbe nekanoničnih struktur. V sklopu diplomske naloge smo preučevali zvijanje oligonukleotida DNA d(ATTTC)3, imenovanega EP3, ki glede na preliminarne študije ob prisotnosti Ca2+ in Mg2+ ionov v raztopini tvori nekanonično dimerno strukturo. Znanje pridobljeno iz prejšnjih študij smo skušali razširiti s preverjanjem strukturnih lastnosti EP3 ob prisotnosti Zn2+ ionov, da bi poskusili bolje razumeti pomen in vlogo dvovalentnih ionov potrebnih na zvijanje DNA. S pomočjo 1D 1H NMR eksperimentov smo preučili zvijanje EP3 v odvisnosti od pH, temperature, prisotnosti pufra in vrste ter koncentracije dvovalentnih ionov. Naša študija je obsegala strukturno karakterizacijo EP3 v prisotnosti Zn2+ ionov, ki smo jo izvedli s pomočjo 1D 1H in 2D 1H-1H NOESY eksperimentov. Strukturne podatke smo primerjali z že znanimi podatki o strukturi, ki nastane v prisotnosti Mg2+ ter ugotovili, da je topologija zvitja EP3 ne glede na vrsto dvovalentnega iona identična vsaj v območju ionskih radijev 0,72–1,00 Å. Pokazali smo, da do zvitja pride vsaj v pH območju od 3,5 do 6,0 ter pri temperaturah do 10 °C. Z višanjem temperature nad 10 °C je EP3 predvsem v nezviti obliki v prisotnosti Zn2+ ionov. Prisotnost natrijevega fosfatnega pufra v raztopini na nastanek nekanonične strukture nima vpliva. Poznavanje strukture in pogojev njenega nastanka bi lahko nudila vpogled v nastanek patogenih stanj in identifikacijo potencialnih načinov zdravljenja nevrodegenerativnih obolenj, v katerih se pojavljajo ATTTC ponovitve.

Keywords

mikrosateliti;oligonukleotidi;dvovalentni kationi;tekočinska kromatografija za hitro ločevanje proteinov;FPLC;2D NOESY spektri;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [D. Valte]
UDC: 577.113:544.176(043.2)
COBISS: 171443459 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Study of the roles of divalent cations on the structural properties of pentanucleotide DNA repeats d(ATTTC)3
Secondary abstract: Short tandem repeats or microsatellites can present a pathogenic function in the presence of certain mutations, deaminations or copy number increases. Repeats of the sequence ATTTC have been shown to cause certain types of spinocerebellar ataxia (SCA) and familial adult myoclonic epilepsy (FAME). Multiple repeats of ATTTC in intronic regions of some genes lead to alternative splicing and the formation of protein isoforms, which can cause pathogenic activity due to their change in function. Short tandem repeats are of interest because their ability to form noncanonical structures. During the diploma thesis our research interests were focused in sequence d(ATTTC)3, DNA oligonucleotide denominated EP3, for which preliminary studies have shown that it forms a noncanonical structure in the presence of Ca2+ or Mg2+ ions in solution. We expanded on the knowledge gathered from previous studies by testing whether the same structure is formed due to the presence of Zn2+ ions to try to better understand the meaning and role of the divalent ions needed for the formation of the structure. Using 1D 1H NMR experiments we determined the effect of pH, temperature, and the presence of buffer along with the type and concentration of the divalent ions. We performed structural characterization of EP3 structure formed in the presence of Zn2+ with the use of 1D 1H and 2D 1H-1H NOESY experiments and compared it with the already known structure which is formed in the presence of Mg2+ ions and concluded that the structure is identical at least in the range of ionic radii 0,72–1,00 Å. We showed that the structure forms at least in the pH range from 3,5 to 6,0 and at temperatures of up to 10 °C. With further increases in temperature EP3 is predominantly in the unfolded form. The presence of potassium phosphate buffer in solution also doesn't affect the formation of the noncanonical structure. Knowing the structure and the conditions of its formation could offer an insight into the formation of certain pathogenic states and the identification of potential ways of treatment for neuredegenerative diseases in which ATTTC repeats are present.
Secondary keywords: NMR-spectroscopy;microsatellites;divalent cations;DNA;Jedrska magnetna resonančna spektroskopija;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 42 str.
ID: 19912999