Variability of major histocompatibility complex class II genes in the golden jackals (Canis aureus) from Israel

Geni glavnog sustava tkivne podudarnosti (MHC) ključna su komponenta imunološkog sustava kod sisavaca te su i važan molekularni marker adaptivne genetičke raznolikosti u divljim populacijama životinja. Veća raznolikost MHC gena unutar populacije povećava izglede populacije za obranu od patogena i parazita, a time i izglede za dugoročno preživljavanje. Čagalj (Canis aureus) pripada porodici Canidae (psi), podred Caniformia, redu Carnivora (mesojedi). Do sada nema objavljenih istraživanja raznolikosti MHC gena ove vrste. Cilj ovog diplomskog rada je odrediti razinu raznolikosti DQA, DQB i DRB gena skupine II MHC sustava u čagljeva iz Izraela. Iz 30 uzoraka uške čaglja, postupcima izolacije DNA, PCR metodom, elektroforezom, sekvenciranjem i molekularnim kloniranjem, pronađena su tri alela na DQA lokusu, pet alela na DQB lokusu i četiri alela na DRB lokusu. Našli smo jedan novi jedinstveni alel na DRB lokusu (DRB1*04802var). Ostali pronađeni aleli poznati su iz prethodnih istraživanja na ostalim vrstama porodice pasa, što potvrđuje postojanje trans-specijskog polimorfizma. DQB i DRB lokusi pokazuju veliku raznolikost alela i velike evolucijske udaljenosti te visok omjer nesinonimnih supstitucija naspram sinonimnih, što potvrđuje pretpostavku djelovanja pozitivne selekcije na te lokuse.The genes of major histocompatibility (MHC) system are a key component of the immune system in mammals and are important molecular markers of adaptive genetic diversity in wild animal populations. Greater diversity of MHC genes within the population increases the chances of defence against pathogens and parasites, and thus the prospects for long-term survival. Golden jackal (Canis aureus) belongs to the family Canidae (dogs), suborder Caniformia, order Carnivora (carnivores). So far there are no published studies of MHC gene diversity on this species. The aim of this graduate thesis is to determine the level of diversity of the MHC system class II DQA, DQB and DRB genes in golden jackals from Israel. Three alleles at DQA locus, five alleles at DQB locus and four alleles at the DRB locus were found in 30 samples of jackal's ear. Methods used were DNA isolation, PCR method, electrophoresis, sequencing and molecular cloning. We found one new unique allele on DRB locus (DRB1*04802var). The rest of the alleles found are known from previous research on the other species of the dog family, which confirms the existance of a trans-species polymorphism. DQB and DRB loci show great diversity of alleles, large evolutionary distances, high ratio of non-synonymous versus synonymous substitutions, confirming the assumption that loci are influenced by positive selection

Lethal alleles in evolution

Letalni aleli uzrokuju smrt organizma koji ih nosi, no ponekad smrt nije trenutačna - mogu proći i godine prije nego smrt nastupi, ovisno o utjecaju letalnog gena na organizam odnosno njegovoj funkciji. Ako mutacija završava trenutnom smrću, tada je zahvaćeni gen esencijalan za rast, razvoj i preživljavanje organizma. Prvi koji je primijetio letalne alele 1905. godine bio je Lucien Cuénot, a nakon njega su mnogi istraživači započeli rad u tom području. Letalni aleli ne izazivaju smrt uvijek u istim uvjetima, pa stoga postoji nekoliko vrsta letalnih alela (dominantni, recesivni, subletalni, uvjetni i sintetički letalni aleli). Najopsežnija istraživanja broja i učinka letalnih alela su napravljena u prirodnim populacijama vrsta Drosophila melanogaster M. i Mus musculus L., te u novije vrijeme u vrsta riba Lucania goodei G. i Danio rerio H. Usporedbama rezultata dobivenih na tim vrstama dobivene su bitne činjenice o evoluciji letalnih alela, no još je uvijek vrlo teško sa sigurnošću zaključiti što sve utječe na njihovu distribuciju u različitim populacijama. Otkriven je i učinak nekih letalnih alela na spolne kromosome koji uzrokuju brojne bolesti i mutacije. Saznanja o učestalosti letalnih alela u populaciji su važna za razumijevanje populacijske genetike i evolucije. Letalne mutacije mogu objasniti mnoge fenomene u evolucijskoj biologiji, poput usrođenosti i njezinog utjecaja na populacije, ili pak uzroka nekih nasljednih bolesti koje imaju letalan učinak, a povezane su s letalnim alelima. Stoga je vrlo važno nastaviti s istraživanjem letalnih alela kako bi se eventualno otkrio njihov utjecaj na razvoj i život jedinki.Lethal alleles cause the death of an organism that carry them, but sometimes, death is not immediate - it may take years before death occures, depending on the function of a lethal gene and its impact on an organism. If the mutation results in lethality then the affected gene is essential for growth, development, and survival of an organism. Lucien Cuénot was the first researcher who noticed the effects of lethal genes on mice in 1905, thereafter many researchers started to explore lethal genes. There are several types of lethal alleles due to the fact that they cause death under the different conditions (dominant, recessive, sublethal, conditionally lethal and synthetic lethal alleles). The most aboundant research of the number and the effect of lethal alleles was done on Drosophila melanogaster M. and Mus musculus L., as well as on fish species Lucania goodei G. and Danio rerio H. Comparisons of the data obtained on the different species have provided important conceptions about evolution of lethal alleles, neverthelles the mechanisms of their distribution in different wild populations are still vaguely understood. Effect of some lethal alleles on sex chromosomes, which causes numerous diseases and mutations, was also discovered. Informations about the frequency of lethal alleles in populations are important for understanding population genetics and evolution. Lethal mutations could explain a number of phenomens in evolutionary biology, such as inbreeding and it`s effect on populations. Also, determination of frequency of lethal alleles would enable certain lethal alleles to be correlated with some common hereditary diseases which have a lethal effect. It is therefore very important to continue researching in the area of lethal alleles, in order to get a better insight in their impact on development and life of organisms

Characterization and diversity of major histocompatibility complex class II DQB gene in brown bear (Ursus arctos)

Glavni sustav tkivne podudarnosti (engl. major histocompatibility complex, MHC) ključan je u pokretanju obrambenih mehanizama kralježnjaka, a smatra se da su neki od lokusa MHC među najpolimorfnijim u kralježnjaka. Raznolikost gena MHC utječe na sposobnost populacije da se obrani od različitih patogena. Varijabilnost lokusa MHC održava ravnotežna selekcija, koju karakterizira veći broj raznolikih alela u populaciji. Geni MHC pokazali su se kao dobar marker za proučavanje adaptivne evolucije vrsta i populacija. Mrki medvjed (Ursus arctos) pripada redu Carnivora (zvijeri), porodici Ursidae (medvjedi) i rodu Ursus. Ovo je prvo ovakvo istraživanje u Hrvatskoj. Kod 30 uzoraka tkiva medvjeda pronađeno je šest alela lokusa DQB. Od njih su tri alela bila nova: RH102_7_M13F, Urth DQB*0401var i Urth DQB*0401vv. Ostali pronađeni aleli poznati su iz prethodnih istraživanja mrkih medvjeda. U osam jedinki sam identificirala po tri alela DQB lokusa, dok sam u 22 jedinke identificirala po dva alela, što ukazuje na to da je lokus DQB dupliciran barem u dijelu jedinki populacije mrkog medvjeda iz Hrvatske.Major histocompatibility complex (MHC) plays a major role in initiating defense mechanisms of vertebrates. It is believed that some of the MHC loci are the most polymorphic invertebrates. The variability of MHC genes affects population`s ability to defend itself fromvarious pathogens.The variability of the MHC loci is maintained by the balancing selection, which is characterized by a larger number of divergent alleles in the population. MHC genes proved to be a good marker for the study of adaptive evolution of the species and populations. Brown bear (Ursus arctos) belongs to the order Carnivora (carnivores), family Ursidae (bears) and gene Ursus. From 30 samples of bear tissue I found six aleles of the gene DQB. Three alele were new: RH102_7_M13F, Urth DQB*0401var and Urth DQB*0401vv. Other three were known from the previous research. In eight individuals I identified three alleles of DQB locus, while in 22 individuals I identified two alleles, indicating that the DQB locus was duplicated at least in the part of the brown bear populations from Croatia

Polymorphism of HLA-DRB4 gene in Croatia

U ovom istraživanju analiziran je polimorfizam gena HLA-DRB4 i haplotipova HLA-DRB1-DRB4 u Hrvatskoj među kadaveričnim davateljima organa (N=144). Svi uzorci bili su odabrani na temelju prisustva jednog od alela skupine HLA-DRB53 (aleli DRB1*04, DRB1*07 i DRB1*09). Određivanje alela lokusa HLA-DRB4 provedeno je metodom lančane reakcije polimerazom i početnicama specifičnim za jedan ili više alela HLA (engl. Polymerase Chain Reaction–Sequence Specific Primers, PCR-SSP). Aleli lokusa HLA-DRB1 i -DQB1 određeni su metodom lančane reakcije polimerazom i probama specifičnim za jedan ili grupu alela HLA (engl. Polymerase Chain Reaction–Sequence Specific Oligos, PCR-SSO). Na lokusu HLA-DRB4 otkriveno je sedam različitih alela, 5 funkcionalnih alela (DRB4*01:01:01:01, DRB4*01:02, DRB4*01:03, DRB4*01:05 i DRB4*01:08) i dva nul alela (DRB4*01:03:01:02N i HLA-DRB4*03:01N). Među ispitanicim pozitivnim za jedan od alela skupine HLA-DRB1*04 nije otkrivena statistički značajna razlika u raspodjeli alela DRB4 s obzirom na prisustvo alela DQB1. U haplotipovima HLA-DRB1*04-DQB1 najčešći prisutni alel je bio DRB4*01:03 (86,7%). Među HLA-DRB1*07:01 pozitivnim haplotipovima uočena je statistički značajna razlika (p<0,05) u raspodjeli alela HLA-DRB4 s obziroma na prisustvo alela HLA-DQB1. Haplotipovi: HLA-DRB1*07:01-DQB1*03:03 na lokusu HLA-DRB4 u 98,0% slučajeva imali su nul alel DRB4*01:03:01:02N, dok su haplotipovi DRB1*07:01-DQB1*02:02 na lokusu DRB4 u 65,5% slučajeva imali prisutan alel DRB4*01:03. Unutar skupine HLA-DRB1*04 pozitivnih haplotipova nije uočena statistički značajna razlika u raspodjeli alela HLA-DRB4 s obzirom na prisustvo alela HLA-DQB1. Alel HLA-DRB1*09:01 je kod svih ispitanika uočen u kombinaciji s alelima HLA-DRB4*01:03-DQB1*03:03.In this study the polymorphism of HLA-DRB4 alleles and HLA-DRB1-DRB4 haplotypes among Croatian cadaveric organ donors (N = 144) was analyzed. All samples were selected based on the presence of one of the alleles of HLA-DRB53 group (DRB1*04, DRB1*07, and DRB1*09 alleles). Typing of HLA-DRB4 alleles was carried out by polymerase chain reaction sequence specific primers for one or more HLA alleles (method: PCR-SSP). HLA-DRB1 and -DQB1 alleles were determined by polymerase chain reaction sequence specific oligos for one allele or a group of HLA alleles (method: PCR-SSO). On the HLA-DRB4 locus seven different alleles were discovered; 5 functional alleles (DRB4*01:01:01:01, DRB4*01:02, DRB4*01:03, DRB4*01:05, and DRB4*01:08) and two null alleles (DRB4*01:03:01:02N and HLA-DRB4*03:01N). Among individuals positive for one of the HLA-DRB1*04 alleles no statistically significant difference was detected in the distribution of the HLA-DRB4 alleles given the presence of the DQB1 alleles. Among HLA-DRB1*04-DQB1 haplotypes the most present was DRB4*01:03 allele (86.7%). Among HLA-DRB1*07:01 positive haplotypes a statistically significant difference (p<0.05) in the distribution of HLA-DRB4 alleles due to the presence of different HLA-DQB1 alleles was observed. The HLA-DRB1*07:01-DQB1*03:03 haplotype in 98.0% of cases had the DRB4*01:03:01:02N allele on the HLA-DRB4 locus, while DRB1*07:01-DQB1*02:02 haplotype on the HLA-DRB4 locus in 65.5% of cases had the DRB4*01:03 allele (65.5%). Within the group of the HLA-DRB1*04 positive haplotype, there was no statistically significant difference in the distribution of HLA-DRB4 alleles due to the presence of different HLA-DQB1 alleles. The HLA-DRB1*09:01 allele was observed in combination: HLA-DRB4*01:03-DQB1*03:03 in all the cases

Variability of major histocompatibility complex class II DQA/DQB/DRB haplotypes in the golden jackal (Canis aureus) from Serbia

Glavni sustav tkivne podudarnosti (MHC) sudjeluje u adaptivnom imunosnom odgovoru sisavaca. Veća raznolikost gena MHC omogućuje limfocitima T da prepoznaju širi spektar antigena, što u konačnici daje jedinci, ali i populaciji veću šansu za preživljavanje napada patogena. Dobar su molekularni marker za proučavanje genetičke raznolikosti vrsta i populacija zbog svoje poligenosti i polimorfnosti. Čagalj (Canis aureus) je zvijer (red Carnivora) koja pripada porodici pasa (Canidae). Istraživanja raznolikosti gena MHC kod čaglja tek su nedavno započela unatoč njegovoj širokoj rasprostranjenosti na euroazijskom i afričkom kontinentu. Cilj ovog istraživanja je određivanje raznolikosti tro-lokusnih haplotipova DQA/DQB/DRB te pojedinih lokusa skupine II sustava MHC u populaciji čagljeva iz Srbije. U ovom istraživanju na 29 uzoraka mišića čagljeva pronađeno je tri alela lokusa DQA i DQB, četiri alela lokusa DRB te šest haplotipova. Sedam je alela dosad pronađeno samo kod čaglja, a tri su trans-specijski polimorfizmi. Tri su haplotipa pronađena samo u srpskoj populaciji, a tri haplotipa su zajednička s istočnoeuropskom populacijom. Potvrđena je postavljena hipoteza djelovanja pozitivne selekcije na svim lokusima koristeći dN/dS test, odnosno omjer nesinonimnih i sinonsimnih supstitucija na nukleotidnom slijedu alela.The genes of the major histocompatibility (MHC) system takes part in the adaptive immune response in mammals. Greater diversity of MHC genes enables T lymphocytes to recognise a wider spectrum of antigens, which in turn gives the individual a greater chance of surviving a pathogen attack. They are a good molecular marker for studying genetic diversity of both species and populations because they are polygenic and polymorphic. The golden jackal (Canis aureus) is a carnivore from the dog family (Canidae). Studies of MHC gene diversity of golden jackals have only recently emerged despite its wide distribution on the Eurasian and African continent. The aim of this graduate thesis is to determine variability of three-locus DQA/DQB/DRB haplotypes and individual Class II MHC loci in golden jackals from Serbia. In this study of 29 jackal muscle samples we have found three DQA and DQB alleles, four DRB alleles and six haplotypes. Seven alleles have only been found in jackals so far, while three alleles are trans-species polymorphisms. Three haplotypes are private to the serbian populations, while three haplotypes are shared with the Eastern European population. We have confirmed the proposed hypothesis of existing positive selection on all loci using the dN/dS test, i.e. the ratio of non-synonymous and synonymous substitutions on the allelic nucleotide sequences

The role of HLA-DPB1 alleles on outcome of hematopoietic stem cell transplantation from unrelated donor

U ovom radu analizirali smo podudarnost HLA među parovima primatelj-nesrodni davatelj u programu nesrodne transplantacije krvotvornih matičnih stanica (TKMS). Unutar ispitivane skupine odredili smo raspodjelu alela HLA-DPB1 među bolesnicima, njihovim nesrodnim davateljima i kontrolama, broj nepodudarnosti na lokusima HLA-A, -B, -C, -DRB1, -DQB1 i –DPB1 između primatelja i nesrodnog davatelja te ispitali povezanost dopuštenih i nedopuštenih nepodudarnosti HLA-DPB1 s ishodom TKMS. Skupina je obuhvatila 57 parova primatelj-davatelj kojima su određeni aleli HLA-A, -B, -C, -DRB1, -DQB1 i –DPB1 metodom lančane reakcije polimerazom i specifičnim početnicama ili probama za alele HLA (metode: PCR-SSP i PCR-SSO). Među bolesnicima najčešći alel bio je HLA-DPB1*04:01 (42,1%) što je u skladu s rezultatima za kontrolnu skupinu. Unutar cjelokupne skupine bolesnika nije otkrivena statistički značajna razlika u preživljavanju između bolesnika s 0, 1 ili 2 nepodudarnosti DPB1. Statistički značajno bolje preživljavanje bolesnika s 2 nepodudarnosti za alele DPB1 uočeno je samo u skupini bolesnika koji su imali davatelja 10/10. Nije uočena statistički značajna razlika u preživljavanju između bolesnika s dopuštenim i onih s nedopuštenim nepodudarnostima DPB1. Za donošenje konačnog zaključka o nepodudarnosti za alele HLA-DPB1 u ishodu TKMS neophodno je provesti veći broj istraživanja.In the present study the correlation between HLA mismatches among recipient-unrelated donor in hematopoietic stem cell transplantation (HSCT) were analyzed. Tested group comprised of 57 pairs. The distribution of DPB1 alleles in the group of patients, unrelated donors and healthy controls was compared. The next goal was to analyze the number of mismatches at HLA-A,-B,-C, -DRB1, -DQB1 and -DPB1 loci between recipient-donor pairs and their correlation on the outcome of HSCT. HLA alleles were tested by Polymerase Chain Reaction and specific primers or probes (PCR-SSP and PCR-SSO methods). Among all three tested groups DPB1*04:01 allele was the most frequent (42.1%). No statistically significant difference in survival after HSCT was observed between patients with 0, 1, or 2 mismatches at DPB1 locus. In the group of 10/10 matched pairs significantly better survival was observed for patients with 2 DPB1 mismatches. There was no statistically significant difference in survival between patients with permissive and non-permissive DPB1 mismatches. For the final conclusion about the necessity of matching for HLA-DPB1 alleles it is required to enlarge the number of studies

Estabilishment of SSCP profile reference panel for analysis of the major histocompatibility complex class II DRB gene in the red deer (Cervus elaphus)

Imunološko prepoznavanje vlastitog od stranog djelomično je kontrolirano setom gena unutar glavnog sustava tkivne podudarnosti (MHC). Ti geni kodiraju sustav membranskih glikoproteinskih receptora čija je glavna uloga prepoznati strane proteine, prezentirati ih specijaliziranim imunosnim stanicama, te pokrenuti imunološki odgovor. Veća varijabilnost genskih lokusa MHC omogućuje prepoznavanje šireg spektra antigena, pa posljedično i bolju obranu od patogena. Istraživanja varijabilnosti lokusa MHC analizom sekvenci fragmenata DNA umnoženih PCR reakcijom, obično uključuju i molekularno kloniranje. Uvođenjem metode analize polimorfizma konformacije jednolančane DNA (SSCP) postupak utvrđivanja genske varijabilnosti bio bi jednostavniji, brži i jeftiniji smanjujući potrebu za molekularnim kloniranjem i dodatnim sekvenciranjem. U ovom istraživanju, po prvi puta u Hrvatskoj, uvela sam metodu SSCP u istraživanje raznolikosti alela lokusa DRB, te uspostavila referentni panel SSCP profila spomenutih alela u jelena (Cervus elaphus). U ispitivanim uzorcima utvrdila sam postojanje ukupno sedam različitih alela, te postojanje dupliciranog lokusa DRB. Na referentnom panelu, svaki od pojedinačnih alela pokazuje jedinstven SSCP profil, ali utvrđeno je i preklapanje pojedinih vrpci različitih alela. Kod nekoliko odabranih uzoraka, analiza SSCP izravno umnoženih fragmenata alela potvrdila je rezultate dobivene molekularnim kloniranjem i sekvenciranjem. No, za rutinsku uporabu SSCP panela u procjeni imunogenetičke varijabilnosti jelena u Hrvatskoj, trebalo bi metodu dodatno optimizirati i unaprijediti.Immunological self/nonself recognition is partly controlled by a set of genes in the major histocompatbility complex (MHC). These genes encode complex of membrane glicoproteine receptors which primary role is to recognize foreign proteins, present them to specialist immune cells and initiate an immune response. Increased variability of MHC gene loci enables identification of a broader range of antigens, and consequently a better defense against pathogens. Investigation of MHC variability by analyses of sequence fragments multiplied by PCR reaction, usually includes molecular cloning. By introducing SSCP (single strand conformation polymorphism) method, the process of determining gene variability would be simpler, faster and cheaper and reduce the need for molecular cloning and additional sequencing step. In this experment, for the first time in Croatia, I introduced the SSCP method to explore the diversity of alleles of the DRB locus and estabilished a reference panel of the SSCP profile of the mentioned alleles in the red deer (Cervus elaphus). In analyzed samples, I determined the presence of seven unique alleles in total and a duplication of DRB locus. On the reference panel, each of the alleles showed a unique SSCP profile; however, the overlapping of some indivudual bands of different alleles was found. In chosen representative samples, SSCP analysis of directly amplified allele fragments confirmed the results obtained by molecular cloning and sequencing. Nonetheless, the use of SSCP panel in routine determination of immunigenetic variability of deer in Croatia should be further optimized and improved