Comparative genomic analysis of prion genes

BACKGROUND: The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far. RESULTS: While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRNs harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTRs (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTRs, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18–25% identity and 28–34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold. CONCLUSION: It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems

Evaluation of DNA Damage in Radiotherapy-Treated Cancer Patients Using the Alkaline Comet Assay

The aim of this study was to evaluate primary DNA damage and the dynamics of the repair of radiotherapy-induced DNA lesions in non-target cells of cancer patients. This study included patients diagnosed with different solid tumors who received radiotherapy. The levels of DNA damage were evaluated using the alkaline comet assay on peripheral blood leukocytes. Altogether four blood samples per patient were collected: before and after receiving the first dose of radiotherapy, in the middle of radiotherapy cycle, and after the last dose of radiotherapy. The results indicate that after the first radiation dose significantly increased levels of DNA damage were recorded in almost all cancer patients compared to their baseline values. Specific patterns of DNA damage were recorded in samples analyzed in the middle of radiotherapy and after receiving the last dose, indicating the possibility of adaptive response in some patients. Our results indicate that persistence of post-irradiation damage in peripheral blood leukocytes (and possibly in other non-target cells) of cancer patients that are strong determinants for the secondary cancer risk. Moreover, the alkaline comet assay was confirmed as a rapid and sensitive assay for the assessment of genome damage after in vivo irradiation

Protein SRP54 iz morske spužve Geodia cydonium

In the systematic search for phylogenetically conserved proteins in the simplest and most ancient extant metazoan phylum – Porifera, we have identified and analyzed a cDNA encoding the signal recognition particle 54 kD protein (SRP54) from the marine sponge Geodia cydonium (Demospongiae). The signal recognition particle (SRP) is a universally conserved ribonucleoprotein complex of a very ancient origin, comprising SRP RNA and several proteins (six in mammals). The nucleotide sequence of the sponge cDNA predicts a protein of 499 amino acid residues with a calculated Mr of 55175. G. cydonium SRP54 displays unusually high overall similarity (90 %) with human/mammalian SRP54 proteins, higher than with Drosophila melanogaster (88 %), or Caenorhabditis elegans (82 %). The same was found for the majority of known and phylogenetically conserved proteins from sponges, indicating that the molecular evolutionary rates in protein coding genes in Porifera as well as in highly developed mammals (vertebrates) are slower, when compared with the rates in homologous genes from invertebrates (insects, nematodes). Therefore, genes/proteins from sponges might be the best candidates for the reconstruction of ancient structures of proteins and genome/proteome complexity in the ancestral organism, common to all multicellular animals.U sistematskoj potrazi za filogenetski sačuvanim proteinima u spužava, najjednostavnijih i najstarijih živućih Metazoa, autori su identificirali i analizirali cDNA koja kodira protein SRP54 u morske spužve Geodia cydonium. SRP54 je evolucijski najsačuvaniji protein ribonukleoproteinskoga kompleksa SRP (signal recognition particle), odgovornog za translokaciju sekretornih i transmembranskih proteina. SRP je nastao vrlo rano u evoluciji i svugdje je prisutan u živom svijetu, od bakterija do čovjeka. cDNA spužve G. cydonium kodira protein SRP54 dug 499 aminokiselina, izračunate molekularne mase 55175, koji pokazuje najviši stupanj sličnosti (90 %) s ljudskim proteinom SRP54, više nego sa SRP54 iz kukca Drosophila melanogaster (88 %) ili oblića Caenorhabditis elegans (82 %). Velika sličnost proteina spužava s homolozima u sisavaca uočena u ovom radu, kao i u našim prijašnjim istraživanjima, upućuje na sporije evolucijske promjene u genima spužava i sisavaca (kralježnjaka) u usporedbi s ubrzanijim promjenama u genima kukaca i oblića (beskralježnjaka). Geni/proteini spužava su stoga posebno korisni za rekonstrukciju strukture proteina i kompleksnosti genoma/proteoma u ancestralnom organizmu koji je bio zajednički predak svih višestaničnih životinja

Dvije nukleozid-difosfat kinaze (NDPK/Nm23) iz morske spužve Suberites domuncula

Suberites domuncula is a member of the most ancient and simplest extant phylum of multicellular animals – sponges (Porifera). A database of S. domuncula expressed sequence tags (ESTs) was recently constructed by random cDNA sequencing. Two NDPK/Nm23 proteins from the sponge Suberites domuncula are reported here. Sponge proteins were named Nm23-SD1 and Nm23-SD6, because they display the highest sequence similarity with human Nm23-H1 and -H6 proteins. Overall sequence conservation of Nm23-SD1 with human Nm23-H1 is very high – 79 % (71 % identical amino acids). Nm23-SD6 possesses an insertion at the C-terminus and displays 55 % overall homology (40 % identical amino acids) with human Nm23 H6. Secondary structure predictions for both sponge and human Nm23 protein pairs are almost identical. S. domuncula Nm23 proteins display high similarity to homologues from mammals/humans, higher than to e.g. NDPK/Nm23 proteins from Drosophila or other invertebrates. Sponge Nm23 proteins are more similar to mammalian/human Nm23 proteins than most known Nm23 proteins of invertebrates.Suberites domuncula je pripadnik najstarije i najjednostavnije postojeće skupine (koljena) višestaničnih životinja – spužvi (Porifera). Nasumičnim sekvenciranjem cDNA S. domuncula nedavno je konstruirana baza EST-ova (expressed sequence tags). U ovom radu opisana su dva NDPK/Nm23 proteina iz spužve Suberites domuncula. Spužvini proteini nazvani su Nm23-SD1 i Nm23-SD6 jer su po aminokiselinskim sljedovima najslič niji ljudskim Nm23-H1 i -H6 proteinima. Ukupna sačuvanost sekvence izme|u Nm23-SD1 i Nm23-H1 vrlo je visoka i iznosi 79 % (71 % je identična). Nm23-SD6 ima inserciju na C-kraju i pokazuje ukupnu homologiju od 55 % (40 % identičnosti) s ljudskim Nm23-H6. Predikcija sekundarne strukture pokazuje da su oba proteinska para gotovo jednaka. Nm23 proteini iz spužve S. domuncula pokazuju visoku sličnost s homolozima iz sisavaca/čovjeka, višu nego s npr. NDPK/Nm23 proteinima iz vinske mušice ili drugih beskralješnjaka. Osim toga, sličniji su proteinima iz sisavaca/čovjeka od većine poznatih Nm23 proteina iz beskralješnjak

Environmental Exposure to Metals, Parameters of Oxidative Stress in Blood and Prostate Cancer: Results from Two Cohorts

We studied the potential role of exposure to various metal(oid)s (As, Cd, Cr, Hg, Ni, and Pb) in prostate cancer. Two cohorts were established: the Croatian cohort, consisting of 62 cases and 30 controls, and the Serbian cohort, consisting of 41 cases and 61 controls. Blood/serum samples were collected. Levels of investigated metal(oid)s, various parameters of oxidative stress, and prostate-specific antigen (PSA) were determined in collected samples. A comparison of the measured parameters between 103 prostate cancer patients and 91 control men from both Croatian and Serbian cohorts showed significantly higher blood Hg, SOD, and GPx levels and significantly lower serum SH levels in prostate cancer patients than in controls. Correlation analyses revealed the significant relationship between certain parameters of oxidative stress and the concentrations of the measured metal(loid)s, pointing to the possible role of metal(oid)-induced oxidative stress imbalance. Furthermore, a significant inverse relationship was found between the blood Pb and the serum PSA in prostate cancer patients, but when the model was adjusted for the impacts of remaining parameters, no significant association between the serum PSA and the measured parameters was found. The results of the overall study indicate a substantial contribution of the measured metal(loid)s to the imbalance of the oxidant/antioxidant system. Although somewhat conflicting, the results of the present study point to the possible role of investigated metal(oid)s in prostate cancer, especially for Hg, since the obtained relationship was observed for both cohorts, followed by the disturbances in oxidative stress status, which were found to be correlated with Hg levels. Nevertheless, further studies in larger cohorts are warranted to explain and confirm the obtained results

Low Genetic Diversity of the Turopolje Pig Breed

We have performed a genetic diversity study of the Turopolje pig breed. Microsatellite genotyping on ten loci recommended by International Society for Animal Genetics (ISAG) and Food and Agriculture Organization (FAO) as well as mitochondrial D-loop sequencing were chosen as methods. Allele numbers, effective allele numbers (Ne), polymorphism information content (PIC), observed and expected heterozygosities were calculated. Mitochondrial sequences were compared to the sequences in the National Center for Biotechnology Information (NCBI) database. We have found relatively low genetic diversity at ten microsatellite loci and no differences in the partial D-loop sequence. Reasons for and consequences of low genetic diversity are discussed

Genetička raznolikost pasmine Turopoljska svinja

We have performed a genetic diversity study of the Turopolje pig breed. Microsatellite genotyping on ten loci recommended by International Society for Animal Genetics (ISAG) and Food and Agriculture Organization (FAO) as well as mitochondrial D-loop sequencing were chosen as methods. Allele numbers, effective allele numbers (Ne), polymorphism information content (PIC), observed and expected heterozygosities were calculated. Mitochondrial sequences were compared to the sequences in the National Center for Biotechnology Information (NCBI) database. We have found relatively low genetic diversity at ten microsatellite loci and no differences in the partial D-loop sequence. Reasons for and consequences of low genetic diversity are discussed.U ovom je radu provedeno istraživanje genetičke raznolikosti Turopoljske svinje. Genotipizacija je provedena analizom mikrosatelita i sekvencioniranjem mitohondrijske D-petlje. Odabrano je deset mikrosatelitskih lokusa prema preporuci International Society for Animal Genetics (ISAG) i The Food and Agriculture Organization (FAO). Izračunat je broj alela, efektivni broj alela (Ne), informacijski sadržaj polimorfizma, te očekivana i opažena heterozigotnost. Mitohondrijske sekvencije uspoređene su sa sekvencijama pohranjenim u bazi National Center for Biotechnology Information (NCBI). Na osnovi analize deset mikrosatelitskih lokusa ustanovljena je razmjerno niska razina genetičke raznolikosti. Također je utvrđeno da sve jedinke u uzorku imaju identičnu sekvenciju mitohondrijske D-petlje. U radu su komentirani mogući uzroci i posljedice niske razine genetičke raznolikosti ove pasmine