THE IMPACT OF PROMOTERS AND CELL-LINE TYPES ON ALTERNATIVE SPLICING OF RECOMBINANT CONSTRUCTS

Alternative intron splicing is a process where introns are excised from pre-mRNA following different patterns. The consequence of it is a presence of several different forms of mature mRNAs differing in length and in content of the whole exons or their parts. Studies in the past decade lead to the view that a complex and extensively coupled network has evolved to coordinate the activities of gene expression pathway, which includes transcription, several pre-mRNA processing steps and the export of mature mRNA to the cytoplasm. Coupling of transcription to splicing was revealed in studies showing that transcription of pre-mRNA by different promoters can generate different alternatively spliced mRNAs. The main goal of this study was to determine the impact of different milk gene promoters on alternative splicing under in vitro conditions. Besides, we also investigated how different cell types influence the splice site selection. With this experiment, we confirmed our predictions that promoter structure and cell-type have an impact on alternative splicing. Differences were observed in quantity of transcripts, as well as in splice-site selection

MILK PROTEIN GENES: A MOLECULAR APPROACH

Ker se delež mleka, ki ga v svetu porabijo v tekoči obliki, stalno zmanjšuje, težijo sodobni trendi v selekciji mlečnega goveda k višji vsebnosti suhe snovi v mleku in izboljšanju tehnoloških lastnosti mleka. Vsebnost beljakovin v mleku je kvantitativna lastnost s kompleksnim genetskim ozadjem, ki uravnava kakovost in količino mlečnih beljakovin. Sklop štirih kazeinskih genov in genov za dva najpomembnejša proteina sirotke (a -laktalbumin in b -laktoglobulin) predstavljajo regije genoma, ki kodirajo najpomembnejše beljakovine mleka. Vseh šest genov nastopa v večih alelnih variantah, ki vplivajo tako na kvaliteto, kot tudi na količino beljakovin v mleku. Alelne variante laktoproteinskih genov se med seboj razlikujejo v kodogenih in regulatornih predelih, kar vpliva na uravnavanje genske ekspresije na transkripcijski in post-transkripcijski ravni. Do sedaj je uspelo dokazati kvantitativno različno ekspresijo alelnih variant genov, ki kodirajo k -kazein in b -laktoglobulin. Študij promoterskih regij genov za proteine mleka nam omogoča razumevanje molekularnih mehanizmov, ki uravnavajo sintezo proteinov mleka. Plod iskanja najpomembnejših lokusov, ki kvantitativno vplivajo na proizvodnjo mleka so ozko omejeni deli genoma, ki imajo vpliv na to lastnost. Tovrstne informacije lahko uporabimo za selekcijo s pomočjo genetskih markerjev in za biotehnološke modifikacije komponent mleka.Modern trends in selection of dairy cattle are geared towards higher solids content and better technological properties of milk because of the increasing proportion of milk being used for manufacturing. Protein content in milk is a quantitative trait with a very complex genetic mechanisms involved in it’ s regulation. Cluster of four casein genes and genes for two major whey proteins (a -lactalbumin and b -lactoglobulin) represent genomic coding regions for the major bovine lactoproteins. All are present as allelic variants, which have a qualitative but also quantitative effect on the protein content of milk. Polymorphisms between allelic variants are present within coding regions and also within regulatory regions of milk protein genes, having a regulatory effect on gene expression at the transcriptional and post transcriptional level. Differential allelic production has been demonstrated for k - casein and b -lactoglobulin genes. Study of different promoter regions from milk protein genes offers information about the molecular mechanisms regulating milk protein synthesis. The search for the major loci involved in the regulation of milk production revealed some regions within the genome related with the milk yield. This molecular data can be used for marker assisted selection and biotechnological modifications of milk components

VARIABLE DNA SEQUENCE OF THE pMGA GENE IN Mycoplasma gallisepticum

Specific fragments of Mycoplasma gallisepticum genomic DNA, representing different regions of pMGA1.1 and pMGA1.2 genes, were amplified by Polymerase Chain Reaction (PCR). Using sequence analysis of pMGA terminal domains, differences in nucleotide sequences among strains of M. gallisepticum were identified. Restriction analysis revealed polymorphism at the 5? -end of the pMGA coding region. By means of sequence analysis it was established that the most variable part of pMGA sequence was the 5? -end of the pMGA gene, the region coding amino-terminal part of pMGA protein, while the 3? -end of the gene was conserved among various M. gallisepticum strains analysed. In some strains insertions or deletions were found that indicate the diversity of the pMGA gene family and might be related to the variable expression of the pMGA gene family. Changes in nucleotide sequences of the pMGA genes were also found within isogenic lineages. Genotypic variability is also confirmed by results of the study of the gene product expression

Structural differences among pig genomes illustrate genetic uniqueness of breeds

The availability of high-throughput whole-genome sequencing (WGS) data illustrating differences among different pig breed genomes opened a new area of genomic research focused on variation caused by single nucleotide polymorphisms (SNP), small scale variation and structural variants which may all contribute to phenotypic variation among pig breeds. In our study (performed within TREASURE project) we re-analysed WGS-based data sets from more than 20 breeds, including commercial and local breeds as well as some wild boar genomes, deposited in publicly available databases. This bioinformatics tool enables discovery of new SNPs, estimation of allele frequencies (genotyping by sequencing) at candidate loci and identification of structural variation in a wide range of pig breeds. The analysis underlined the relevance of structural differences at KIT and MC1R locus involved in colour pattern formation, as well as LEPR locus associated with fatness, fatty acid metabolism and intramuscular fat composition. This approach allows discovery of important genomic differences between commercial breeds and local breeds which are analysed in the frame of the TREASURE project. Extensive mining of publicly available genomic data can together with the newly generated genomic information from local breeds, significantly contribute to the detailed characterisation of animal genetic resources present in local pig breeds. Funded by European Union’s H2020 RIA program (grant agreement No. 634476)

MOLEKULARNA IDENTIFIKACIJA IN KARAKTERIZACIJA PTIČJIH MIKOPLAZEM

Serological techniques are recommended procedures for the identification of Mycoplasma species. They enable separation of mycoplasmas at the species level and are widely used for practical laboratory identification of the isolated microorganisms. However, complex growth media are required for the isolation of Mycoplasma species and some strains of pathogenic Mycoplasma species grow very fastidious and can not be cultured. Introduction of DNA based diagnostic methods in mycoplasma research enabled survey of the whole genome in a single assay and reveal also information about non-coding regions of the genome. Restriction enzyme analysis is a powerful method for differentiation of close related strains but requires high quality of DNA which can only be obtained from cultured mycoplasmas. The increasing number of cloned mycoplasma genomic fragments are successfully used for identification and differentiation of mycoplasma strains and species in hybridization studies. The polymerase chain reaction enables identification and characterization of mycoplasmas without culture directly from clinical material. Large genomic sequencing projects produced complete nucleotide sequence from two Mycoplasma species and several more are in progress. This data will open the possibility to understand better the biology of mycoplasmas.Tradicionalne metode za identifikacijo in diferenciacijo mikoplazem temeljijo na seroloških tehnikah. Omogočajo ločevanje mikoplazem na ravni vrst in jih uporabljamo za praktično laboratorijsko identifikacijo izoliranih mikroorganizmov. Zahteva po kompleksnih medijih za izolacijo mikoplazem, njihova počasna rast in dejstvo, da nekaterih sevov patogenih vrst mikoplazem ni moč gojiti v kulturi, so glavni razlogi za uvedbo molekularnih metod v diagnostiko mikoplazemskih infekcij. Z uporabo molekularnih metod lahko zajamemo celoten genom v enem samem testu, vključno z nekodogenimi regijami. Analiza genomske DNK z restrikcijskimi encimi je zelo informativna metoda, ki omogoča razlikovanje med sorodnimi sevi, zahteva pa visoko kvalitetno DNK, ki jo lahko izoliramo samo iz mikoplazem, ki jih gojimo v kulturi. Vedno večje število kloniranih genomskih odsekov mikoplazem lahko s pridom uporabljamo za identifikacijo in diferenciacijo sevov mikoplazem v hibridizacijskih študijah. Polimerazna verižna reakcija omogoča identifikacijo mikoplazem direktno v kliničnem materialu brez predhodne kultivacije. Veliki projekti sekvenciranja genoma mikoplazem odpirajo vpogled v ustroj celotnega genoma in obetajo boljše razumevanje biologije mikoplazem