MITOCHONDRIAL DNA POLYMORPHISMS AND FERTILITY IN BEEF CATTLE

Two regions of mitochondrial DNA, D-loop and ND-5 were characterized using polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) involving 422 beef cattle of Hereford and composite breeds from Wokalup’s research station. ANOVA models (model I, II) were used to estimate associations between molecular haplotypes and quantitative traits. The phenotypic data used were records on calving rate, defined as the mean number of live calves born over four years, while the genotypic data used were the result of PCR-RFLP analysis in both regions of mitochondrial DNA using 7 restriction enzymes. The results of the present study have provided evidence that mitochondrial polymorphisms in the D-loop and ND-5 regions are associated significantly with fertility. This is the first report of a correlation between mitochondrial polymorphism in D-loop and ND-5 on fertility in beef cattle. Key words: PCR-RFLP, bovine mitochondrial DNA, D-loop, ND-5

Increase in Mitochondrial D-Loop Region Methylation Levels in Mild Cognitive Impairment Individuals

Methylation levels of the mitochondrial displacement loop (D-loop) region have been reported to be altered in the brain and blood of Alzheimer's disease (AD) patients. Moreover, a dynamic D-loop methylation pattern was observed in the brain of transgenic AD mice along with disease progression. However, investigations on the blood cells of AD patients in the prodromal phases of the disease have not been performed so far. The aim of this study was to analyze D-loop methylation levels by means of the MS-HRM technique in the peripheral blood cells of 14 mild cognitive impairment (MCI) patients, 18 early stage AD patients, 70 advanced stage AD patients, and 105 healthy control subjects. We found higher D-loop methylation levels in MCI patients than in control subjects and AD patients. Moreover, higher D-loop methylation levels were observed in control subjects than in AD patients in advanced stages of the disease, but not in those at early stages. The present pilot study shows that peripheral D-loop methylation levels differ in patients at different stages of AD pathology, suggesting that further studies deserve to be performed in order to validate the usefulness of D-loop methylation analysis as a peripheral biomarker for the early detection of AD

Reduced mitochondrial D-loop methylation levels in sporadic amyotrophic lateral sclerosis

Background: Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms. Results: In the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females. Conclusions: Present data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms

Kajian Penanda Genetik Tarsius bancanus dan Tarsius spectrum dengan Sekuen D-Loop Parsial DNA Mitokondria

The objective of this research was to study the specific genetic marker on D-loop region of Tarsius bancanus and Tarsius spectrum. The sequencing of PCR product using primer DLTARPROF on D-loop resulted in base sequence of 270 nts. Result of D-loop fragments sequencing was put on multiple alignment with other primates from Genbank with the aid of software Genetyc-Win Version 3.0 and Clustal W, and was analyzed using MEGA program version 3.1. The genetic distance was based on nucleotide D-loop, the smallest genetic distance was 0% and the biggest was 11.8% and the average was 2.3%. The phylogenetic tree using neighbor Joining Method based on some nucleotide sequence on D-loop region could not be used to differentiate between Tarsius bancanus and Tarsius spectrum

First molecular data of the Borneo Banteng Bos Javanicus lowi from Sabah, Borneo

Phylogenetic relationships among three subspecies of banteng, Burma banteng Bos javanicus birmanicus in mainland Southeast Asia, Javan banteng Bos javanicus javanicus in Java, and Bornean banteng Bos javanicus lowi in Borneo, and the presence/absence of interbreeding between wild Bornean banteng and domestic cattle in Sabah, Malaysia, were investigated by partial sequences of cytochrome b and D-loop of mitochondrial DNA. The results show that genetic distance of the Bornean banteng are relatively close to the gaur Bos gaurus/gayal Bos frontalis (the cytochrome b, 0.004–0.025; the D-loop, 0.012–0.021) followed by Burma banteng (the cytochrome b, 0.027–0.035; the D-loop, 0.040–0.045), and kouprey Bos sauveli (the cytochrome b, 0.031–0.035; the D-loop, 0.037–0.042). There are much greater distances between Bornean banteng and domestic cattle, Bos taurus and Bos indicus (the cytochrome b, 0.059–0.076; the D-loop, 0.081–0.090). These results suggest that the Bornean banteng diverged genetically from other banteng subspecies and that the wild Bornean banteng from this study are pure strain and have high conservation value

CAPABILITY OF MITOCHONDRIA DNA D-LOOP MARKERS FOR SHARK SPECIES IDENTIFICATION

ldentification of dry-fin shark was conducted by mitochondria DNA (mtDNA) D-loop markers. Eighteen of thiity samples have been successfully amplified the mtDNA D-loop region

Estimation of genetic variability among traditional hungarian horses by mitochondrial DNA analysis

Munkám fő célja a gidrán és hucul kancacsaládok genetikai diverzitásának feltérképezése volt két mtDNS marker segítségével. A lovak genetikai struktúrájának meghatározása mellett, a mtDNS markerek gyakorlati alkalmazását is vizsgáltam. Célom volt a gidrán, illetve hucul méneskönyvekben esetlegesen előforduló hibák/elírások felderítése, illetve a módszer hatásosságának vizsgálata kancacsaládok haplotípus alapján való elkülönítésében. Vizsgálataim során összesen több mint ötszáz (250 gidrán és 267 hucul) különböző magyarországi ménesekből származó kancától vett szőrminta analízisét végeztem el. Első lépésben primereket terveztem a CYTB és a D-loop szakaszaira, majd specifitásukat gradiens PCR segítségével teszteltem. A hét primerpárt (A-G) tíz hibridizációs hőmérsékleten vizsgáltuk meg, majd kiválasztottam a legoptimálisabb párokat. A bioinformatikai ellenőrzés után a CYTB szekvencián belül 686 nukleotidnak, a D-loop régión belül pedig 202 nukleotidnak a vizsgálata valósult meg. A begyűjtött minták közül a CYTB vizsgálatánál (gidrán: 250; hucul: 265), a D-loop régiónál pedig (gidrán: 246; hucul: 267) minta került statisztikai értékelésre. A mtDNS markerek szekvenciaanalízise mindkét fajtánál megerősítette genetikai variabilitásukat. Ezt jól mutatják a kapott haplotípus számok (gidrán: CYTB=24, D-loop=32 haplotípus; hucul: CYTB=13, D-loop=22 haplotípus) és a magas haplotípus diverzitás mutatók (gidrán: CYTB=0,874, D-loop=0,914; hucul: CYTB=0,835, D-loop=0,878) is. A kapott haplotípusokat a GenBank adatbázisban publikált adatokkal összehasonlítva a gidránnál hat egyedi CYTB és öt D-loop, míg a huculnál két egyedi D-loop haplotípust azonosítottunk. A filogenetikai analíziseket mindkét lófajtánál a két marker együttes használatával is elvégeztem. A hucul kancák vizsgálata során kapott haplotípusokat különböző földrajzi területen élő póni, illetve kisló fajtákkal hasonlítottam össze. Összesen 35 minta került kiválasztásra, amelyeknek elérhető volt mind a CYTB, mind a D-loop szekvenciája Hat hucul CYTB haplotípus – a debao és a welsh pónikat kivéve – a yakut, shetlandi, przewalski, mongol, kaszpi, chincoteague, exmoor, konik, noriker, fjord, izlandi, német lovagló pónival alkotott közös csoportot, míg hét haplotípus egyedi csoportot alkotott. Nyolc D-loop haplotípus pedig a yakut, mongol, kaszpi, welsh, chincoteague, exmoor, konik, német lovagló pónival alkotott közös csoportot, míg 14 esetben egyedi csoportokat formáltak. A kapott eredmények alapján a mtDNS markerek nem megfelelőek az állatok földrajzi élőhelye szerinti elkülönítésére, míg a kancacsaládok, leszármazottsági vonalak vizsgálatánál sikeresnek bizonyultak. Dolgozatom másik célja az egyes kancacsaládok között átfedő haplotípusok azonosítása és a méneskönyvben előforduló esetleges elírások felkutatása volt. A gidrán kancáknál a molekuláris vizsgálat alapján 227 egyednél (91,9%) sikerült a korreláció. Az összes egyedszámot tekintve kilenc esetben kerültek olyan haplotípusba, melyek más kancacsaládokra jellemzőek, így valamilyen eltérésről lehet szó. A hucul lovaknál a kancacsalád és haplotípusok között elvégzett mtDNS analízis 187 (78%) egyednél egyértelműen a méneskönyvvel egyező eredményt mutatott. Méneskönyvi eltérésről 30 kancánál (12,5%) beszélhetünk. Hét egyednél a méneskönyvi adatok nem voltak elérhetőek, így a kancacsaládokat csak a mtDNS markerek alapján tudtam meghatározni. Külön megemlítendő, hogy az Aspiráns családnál nem találtuk eltérést a hagyományos és a mtDNS alapú csoportosítás között. A vizsgált CYTB és D-loop mtDNS markereket megfelelőnek találtam a hagyományos lófajták genetikai struktúrájának feltárása. Mind a gidrán, mind a hucul kancavonalak diverz genetikai állománnyal rendelkeznek. A filogenetikai vizsgálatokon túl, a két marker – különösen kombinálva – alkalmazható a kancacsaládok haplotípusok szerinti elkülönítésére is. A kapott eredmények alapján a vizsgált lófajták méneskönyvének vezetése – a néhány eltérés ellenére is – pontosnak mondható. The main aim of my study was to map the genetic diversity of Gidrán and Hucul horses using two mitochondrial markers. Besides the characterization of horses’ genetic structure, the practical usage of the mtDNA markers was also evaluated. My additional purpose was to test the efficiency of mtDNA analysis for the classification of mare families, according to their haplotypes, thereby reveal the possible inconsistencies in stud books. My analysis involved more than 500 (250 Gidrán & 267 Hucul) hair samples of mares originated from different Hungarian breeds. In the first step of my work, primers targeting CYTB and D-loop sequences were designed and their specificity was also tested using gradient PCR. The seven primer pairs (named from A to G) were analysed at ten different hybridization temperatures. Finally, the optimal primer pair was selected for further studies. After bioinformatic normalization of sequencing data, 686 bps length PCR products of CYTB and 202 bps fragments of D-loop sequences were involved in further analyses. Among the collected samples 250 Gidrán & 265 Hucul in case of CYTB and 246 Gidrán & 267 Hucul in case D-loop were involved in the final statistical calculations. The sequence analysis of the mtDNA markers confirmed that both breeds assessed significant genetic variability. These results are also supported by the observed haplotype numbers (Gidrán: CYTB=24, D-loop=32 haplotípus; Hucul: CYTB=13, D-loop=22 haplotípus), as well as the high haplotype diversity values (Gidrán: CYTB=0.874, D-loop=0.914; Hucul: CYTB=0.835, D-loop=0.878). Comparing haplotypes with sequence data in GenBank, six new CYTB and five D-loop, while two novel D-loop haplotypes were detected in Gidrán and Hucul horses, respectively. All phylogenetic analysis were performed with CYTB and D-loop markers separately, and with the combination of the markers. Haplotypes of hucul mares were compared with additional pony breeds originated from different geographical areas. Altogether 35 samples with available whole mtDNS genome sequences were involved to the analysis. Six hucul haplotypes shared groups with Yakut, Shetland, Przewalski, Mongolian, Caspian, Chincoteague, Exmoor, Konik, Noriker, Fjord, Icelandic, German riding pony (except only Debao and Welsh ponies), while seven haplotypes formed unique groups. On the other hand, eight D-loop haplotypes shared groups Yakut, Mongol, Caspian, Welsh, Chincoteague, Exmoor, Konik, German riding pony, while fourteen haplotypes formed unique groups. Although, according to these results the selected mtDNA markers are not sufficient to separate horses by their geographical origin, but they are useful for the analysis of mare families and genealogy. The additional goal of my dissertation was to identify overlapping mtDNA haplotypes among mare families, and to discern eventually errors in the studbook. The association between Gidrán mares and the molecular results was complete in case of 227 horses (91.9%). Considering the maximum case number, only nine individuals shared haplotypes with horses of other mare families implying discrepancies in the management of studbook. The mtDNA analysis of Hucul horses showed a clear association between haplotypes and mare families in 187 cases (78%). Possible discrepancy was observed in case of 30 mares (12.5%). Mare family records of seven horses were unknown, therefore their mare family classification was conducted by only mtDNA markers. In particular, no error was found between classical and mtDNA based classification in case of Aspiráns family. The analysed CYTB and D-loop mtDNA markers were found to be appropriate for mapping the genetic structure of traditional horses. Both Gidrán and Hucul mares assess genetically diverse populations. Besides their phylogenetic usefulness, the markers are also adaptable for the classification of mare families per mtDNA haplotypes. Based on these results, the management of the studbooks is rather adequate.N

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site