Isolation of genomic DNA from the nose-horned viper and initial analysis of metalloproteinase genes

Rubil, Tihomir (avtor), Pungerčar, Jože (mentor)

Snake venoms, including that of the nose-horned viper (Vipera ammodytes ammodytes, Vaa), are a valuable source of pharmacologically interesting substances. Vaa venom contains the following major groups of protein toxins: serine proteases, secreted phospholipases A2, snake C-type lectin-like proteins and metalloproteinases. The latter are mainly responsible for hemorrhagic and hemostatic effects of the venom. Of notable reference are metalloproteinases that belong to the P-III (MPIII) class. These metalloproteinases are diverse in both quaternary protein structure as well as in their function. The organization and features of their genes are yet largely unknown. The aim of this thesis was to isolate Vaa genomic DNA, and to amplify and analyze genomic sequences encoding two metalloproteinases, Vaa hemorrhagin 4-A and Vaa metalloproteinase III-like protein 3 (Vaa-MPIII-3). Their presumed nucleotide sequences were amplified by PCR and determined by the Sanger dideoxy-chain termination method. Results indicated that the specific amplification attempts for both genomic sequences were unsuccessful. In addition, a putative contig of 23,133 bp harboring the genomic sequence coding for Vaa-MPIII-3, obtained from the whole Vaa genome sequencing project, was acquired and its structure analyzed. The Vaa-MPIII-3 gene is composed of 10 exons and 9 introns. It has been confirmed that Vaa-MPIII-3 represents a new subtype of metalloproteinases lacking the entire metalloproteinase (enzymatic) domain and the first part of the succeeding disintegrin domain, reflecting a unique evolution of its gene, separated from that of enzymatically active snake venom MPIII.

• Jezik: Angleški jezik
• Ključne besede: Vipera ammodytes ammodytes, long-nosed viper, genomic DNA, gene structure, nucleotide sequencing, snake venom metalloproteinase, class III metalloproteinase, evolution
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: BF - Biotehniška fakulteta
• Leto izida: 2020
• PID: 20.500.12556/RUL-122031
• COBISS.SI-ID: 44392195
• Datum objave v RUL: 18.11.2020

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Izolacija modrasove genomske DNA in začetna analiza metaloproteinaznih genov

Izvleček

Kačji strupi, vključno s strupom modrasa (Vipera ammodytes ammodytes, Vaa), predstavljajo dragocen vir farmakološko zanimivih snovi. Strup Vaa vsebuje naslednje glavne skupine proteinskih toksinov: serinske proteaze, sekretorne fosfolipaze A2, kačji proteini, podobni lektinom tipa C, in metaloproteinaze. Metaloproteinaze so v glavnem odgovorne za hemoragične in hemostatske učinke strupa. Med pomembnejšemi so metaloproteinaze, ki spadajo v družino P-III (MPIII). Te metaloproteinaze so raznolike tako po kvartarni proteinski strukturi kot po svoji funkciji. Organizacija in značilnosti njihovih genov še vedno v veliki meri niso znani. Naš cilj je bil izolirati modrasovo genomsko DNA ter pomnožiti in analizirati genska zaporedja, ki bi vsebovala zapis za dve metaloproteinazi, Vaa-hemoragin 4-A in Vaa-metaloproteinazi III-podoben protein 3 (Vaa-MPIII-3). Njihova domnevna nukleotidna zaporedja smo tako pomnožili s PCR in določili s Sangerjevo dideoksi metodo. Rezultati so pokazali, da poskusi specifičnega pomnoževanja za obe genomski zaporedji niso bili uspešni. Dodatno smo na osnovi mednarodnega projekta določanja celotnega nukleotidnega zaporedja modrasovega genoma pridobili neprekinjeno nukleotidno genomsko zaporedje (soseska), dolžine 23.133 bp, ki vsebuje genski zapis za Vaa-MPIII-3, ter analizirali njegovo strukturo. Modrasov gen za MPIII-3 je sestavljen iz 10 eksonov in 9 intronov. Potrdili smo, da Vaa-MPIII-3 predstavlja nov podtip metaloproteinaz, ki nimajo celotne metaloproteinazne (encimske) domene in začetnega dela naslednje, disintegrinske domene, kar odraža edinstveno evolucijo tega gena, ločeno od evolucije encimsko aktivnih MPIII kačjih strupov.

• Ključne besede: Vipera ammodytes ammodytes, modras, genomska DNA, genska struktura, določanje nukleotidnega zaporedja, metaloproteinaza kačjega strupa, metaloproteinaza razreda III, evolucija