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L-lactate and 2, 3-butanediol are important chemicals used in many industries. Lactate is industrially produced by lactic acid bacteria, while Klebsiella bacteria are considered for the production of 2, 3-butanediol. However, these bioprocesses have certain disadvantages such as reliance on raw materials that could be used for human consumption or the use of potentially pathogenic bacteria. These disadvantages can be avoided by using a suitable microorganism and therefore the possibility of using the yeast Kluyveromyces marxianus for the production of L- lactate and 2, 3-butanediol was investigated in this study. K. marxianus is a non-conventional yeast that has some industrially important characteristics such as rapid growth rates, thermotolerance, ability to grow on xylose, and due to its GRAS status, it is also suitable for use in the food industry. To examine the potential of K. marxianus yeast for L-lactate production, a strain containing optimized genes encoding L- lactate dehydrogenase (ldhL) isolated from Lactobacillus gasseri was constructed. To study the production of 2, 3-butanediol, optimized genes encoding α-acetolactate synthase (alsS) and α- acetolactate decarboxylase (alsD) isolated from Bacillus subtilis were expressed in K. marxianus, and the effect of overexpression of native gene encoding 2, 3-butanediol dehydrogenase (BDH1) was examined. Wildtype yeast K. marxianus produces neither L-lactate nor 2, 3-butanediol when grown on glucose, while genetically modified strains produce up to 2, 5 g/L of L-lactate and about 4 g/L of 2, 3-butanediol.

The term “non-conventional yeasts” refers to all yeast species other than Saccharomyces cerevisiae and Shizosaccharomyces pombe, the two species which are exceptionally well studied and widely utilized in biotechnological processes. In recent years the interest of researchers and industry steers towards non-conventional yeasts harbouring new and potentially exploitable metabolic pathways or other features favourable in biotechnological processes such as thermotolerance, halotolerance, resistance to various growth inhibitors etc. These yeasts are being investigated both as sources of interesting genes which can be transferred into conventional systems but also as the industrial work organisms themselves. In the latter case it is desirable that the work organism can be genetically transformed and even precisely genetically modified allowing construction of improved and more versatile industrial strains. The Schwanniomyces genus is comprised of seven species of budding yeasts which are fairly poorly explored but could have interesting biotechnological properties. Notably, most species form this genus can grow on D-xylose and cellobiose making them worth exploring in the context of production of valuable biochemicals on waste lignocellulose materials as a substrate. Furthermore, the most prominent member of the genus, S. occidentalis var. occidentalis, is potentially interesting producer of heterologous proteins. The aim of this work was to establish a set of basic genetic tools allowing genetic transformation and construction of replicative plasmids for yeasts belonging to the Schwanniomyces genus, thus facilitating further research on these yeasts as well as their industrial application. Using an electroporation protocol, we developed earlier for Brettamomyces bruxellensis several species form the Schwanniomyces genus were successfully transformed with fairly high transformation efficiency. Moreover, several plasmid replication origins belonging to various budding yeast species were tested and origin belonging to a closely related species Scheffersomyces stipitis was identified to be active in Schwanniomyces yeasts, allowing construction of completely stable replicative plasmid in certain Schwanniomyces species.

Palindromes in DNA are symmetrical motifs consisting of two inverted repeats, i.e. having the same nucleotide sequence in the two complementary strands when read in the same direction of polarity. The inverted repeats can be completely identical and adjacent to each other which is referred to as a perfect palindrome, or have mismatches and/or a spacer region in between. If a palindrome is long enough, this symmetry allows for intrastrand base pairing to occur and secondary structures can form in dsDNA. Although palindromes are often found as important functional elements of cis-acting regions and protein binding sites in the genomes, they are also known as potentially dangerous elements which can lead to double strand breaks and thus instigate genetic recombination. In turn, this can lead to various rearrangements - deletions, duplications, translocations or amplifications which are all common features of cancer cells. In particular, palindromic amplifications which alter expression levels of certain genes are linked to poor disease outcome since they can make cancer cells more proliferative and more resistant to drugs. The aim of our research effort is to elucidate molecular mechanisms of palindrome recombinogenicity in the eukaryotic genome using yeast Saccharomyces cerevisiae as a model organism. Within this aim, we are pursuing several areas of research: first one is focused on the features of the palindrome sequence (e.g. the relationship between palindrome recombinogenicity and its length as well as the spacer length) ; second, on the physical conditions of the environment (e.g. the influence of growth temperature on palindrome recombinogenicity) ; and third, on the proteins and pathways involved in occurrence and repair of palindrome-initiated double strand break and its potential consequences for the genome stability. Taken together, the knowledge collected in S. cerevisiae can help better understand the underlying causes of palindrome-initated genetic instability in cancer cells.

Gene targeting is a genetic technique that allows the modification of an endogenous gene by homologous recombination. It is achieved by the introduction of linear, non-replicative DNA fragments that should replace the homologous (targeted) sequence in the genome. It is generally assumed that gene targeting in the conventional yeast Saccharomyces cerevisiae almost exclusively occurs by homologous recombination and that it is usually higher than 90 %. Non-conventional yeast species are isolated from different niches and have interesting properties such as utilization of different carbon sources, fermentation potential, tolerance to different growth inhibitors and they are emerging as potential producers in different biotechnological processes. However, the efficiency of gene targeting in most of those species is still unknown. Using linear transformation cassette, a DNA that contains antibiotic resistance gene in the middle flanked by regions homologous to the target sequence in the genome, we have determined the efficiency of gene targeting in Brettanomyces/Dekkera bruxellensis, Kluyveromyces marxianus and Scheffersomyces stipitis. Depending on the yeast species gene targeting efficiency, obtained using transformation cassettes with flanking homologies longer than 1 kb, varied from less than 0, 1 % to 75 %. Our results suggest that in some species (e. g. K. marxianus) use of flanking homologies, longer than 1 kb, allows the introduction of targeted modifications in the genome, while in some other yeast species, advanced gene-editing techniques e.g. CRISPR/Cas system might be mandatory to allow precise modifications of the genome.

Lignocelulozna otpadna biomasa je široko dostupna i jeftina, a sadrži velike količine spojeva koji se potencijalno mogu koristiti u različitim biotehnološkim procesima. Intenzivno se istražuje potencijal nekonvencionalnih kvasaca, poput kvasaca iz roda Schwanniomyces, da efikasno prevode supstrate u vrijedne produkte. Međutim, biotehnološki potencijal nekog mikroorganizma se može u potpunosti iskoristiti samo ako se taj mikroorganizam može dodatno genetički modificirati. U ovom radu su, korištenjem protokola razvijenog za transformaciju nekonvencionalnog kvasca Brettanomyces bruxellensis, po prvi puta uspješno transformirani kvasci S. pseudopolymorphus i S. polymorphus var. africanus, kao i prethodno uspješno transformirani kvasac S. polymorphus var. polymorphus. Svi spomenuti kvasci transformirani su linearnim, nereplikativnim, heterolognim fragmentima s efikasnošću 2⋅10² - 8⋅10³ μg⁻¹. U radu su korišteni i replikativni plazmidi koji sadržavaju ishodišta replikacije izolirana iz drugih vrsta kvasaca, a upotrebom nekih od ovih plazmida postignuta je visoka efikasnost transformacije (do 5⋅10⁵ μg⁻¹). Ovaj rad financiran je od strane Hrvatske zaklade za znanost u sklopu projekta IP- 2018-01-9717.

Divlji sojevi mikroorganizama sadrže niske koncentracije enzima te ih nije moguće koristiti kao primarni izvor enzima za industrijsku primjenu. Sama izolacija enzima iz divljih sojeva mikroorganizama predstavlja mukotrpan posao koji obično rezultira slabim iskorištenjem. Primjena metoda genetičkog inženjerstva (tehnologije rekombinantne DNA) omogućuje značajno povećanje koncentracije postojećeg enzima u stanici, ali i proizvodnju enzima kojeg divlji soj uopće ne proizvodi. Pritom se najčešće provodi prekomjerna ekspresija gena koji kodira za željeni enzim, a istovremeno je i moguće olakšati izolaciju i pročišćavanje enzima. Osim toga, metodama genetičkog inženjerstva moguće je željeni enzim učiniti pogodnijim za određeni industrijski proces, primjerice povećanjem njegove stabilnosti, aktivnosti, enantioselektivnosti i specifičnosti za određeni supstrat. Stoga genetičko inženjerstvo značajno skraćuje vrijeme razvoja industrijskog katalizatora, te omogućava da se enzim prilagodi industrijskim zahtjevima, umjesto da se industrijski proces prilagođava enzimu. Standardne tehnike molekularne biologije, koje će biti prezentirane u ovom radu, bile su korištene za pripremu novih biokatalizatora: halogenhidrin- dehalogenaze i Dfruktoza-6-fosfat aldolaze.

It is generally assumed that, in contrast to higher eukaryotes, gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, we found that even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out (gene replacement) and ends-in (plasmid integration) transformation assays. It was determined that ends-out gene targeting can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, ends-in gene targeting is often associated with multiple targeted integration events but illegitimate integration is extremely rare while, so far targeted chromosome duplication has not been reported. Here we systematically investigated the influence of the design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. We have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1 sgs1 double mutant suggesting that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.

Brojne vrste nekonvencionalnih kvasaca pokazuju biotehnološki zanimljiva svojstva, poput termotolerantnosti, halotolerantnosti, proizvodnje zanimljivih spojeva na jeftinim sirovinama koje su otpad drugih industrija (poput poljoprivredne i drvne industrije) ili rezistentnosti na inhibitore rasta koji se tijekom pripreme takvih sirovina pojavljuju. Stoga se sve intenzivnije istražuje mogućnost primjene raznih nekonvencionalnih vrsta kvasaca kao radnih mikroorganizama u biotehnologii. Međutim, da bi se biotehnološki potencijal nekog mikroorganizma mogao u potpunosti iskoristiti, neophodno je na raspolaganju imati efikasnu metodu za njegovu genetičku transformaciju. U ovom radu opisuje se prva genetička transformacije kvasaca Schwanniomyces polymorphus var. africanus i Schwanniomyces pseudopolymorphus, a intenzivno se radi i na transformaciji preostalih do sada netransformiranih vrsta iz roda Schwanniomyces.

Due to the constant increase in energy consumption, the production of biofuels from renewable sources, such as lignocellulosic waste, has become more frequent in the last several decades. Lignocellulosic waste consists of cellulose, hemicellulose and lignin and the yeast S. cerevisiae is not able to ferment it directly. Therefore, lignocellulosic waste is usually hydrolysed to release compounds that can be used for fermentation. However, the pretreatment also generates compounds that act as growth and fermentation inhibitors such as acetic and levulinic acid and 2-furaldehyde. In this study, newly constructed intraspecies hybrid diploids, obtained by crossing natural S. cerevisiae isolates, showed higher resistance to acetic and levulinic acid. Selected strains were also transformed with replicative vectors overexpressing yeast genes (e.g. ATR1, CTA1, FLR1, YAP1, ZWF1) encoding proteins involved in a stress response and the overexpression of YAP1 gene further increased the resistance to inhibitors. Therefore, our results suggest that the construction of intraspecies hybrids coupled with genetic engineering is a promising approach for construction of new biotechnologically relevant yeast strains.

The first isolated and most commonly used fluorescent protein is the green fluorescent protein (GFP). However, fluorescent proteins that cover the whole visible spectrum have been available and used as indispensable tools in a large spectrum of bioscience research. Fluorescent microscopes illuminate samples with the light of one colour, but detect only the light emitted by the fluorophores in the sample. Microscopes have been constantly developing, and one of the major advancements was the use of light emitting diodes (LED) as the source of light. The advantages of LEDs are uniform illumination, less dissipated heat and an extremely long life time. However, LEDs are limited to certain fixed wavelengths unlike the previously used arch lamps that emit white light. Therefore, the aim of this study was to determine the compatibility of ten commonly used fluorescent proteins (mTagBFP2, mTFP1, mEGFP, mCitrine, tdTomato, mTagRFP, mCherry, mKate, mPlum, and E2Crimson) with the EVOS Floid Imagining Station fluorescence microscope which uses blue, red and green light emitting diodes (LEDs) as a light source. We found out that, although the EVOS Floid Imagining Station fluorescence microscope has only three LEDs emitting specific wavelengths of the spectrum, it can detect a wide range of fluorescent proteins. Out of ten tested proteins only the mTagRFP was not detected. Furthermore, it is possible to distinguish five proteins in the same sample, one blue (mTagBFP2), one turquoise (mTFP1), one orange (mKate), one of three green (mEGFP, mCitrine or tdTomato) and one of three red (mCherry, mPlum or E2 Crimson) fluorescent proteins.

A palindromic sequence in DNA with two identical inverted repeats and no central spacer region is called a perfect palindrome. Otherwise the palindrome is referred to as a quasipalindrome. Both perfect palindromes and quasipalindromes can constitute fragile sites in the genome prone to chromosome breakage which can result in potentially dangerous mutations and rearrangements. Several conditions in humans (such as the Emanuel syndrome) are caused by recombinogenic palindromic sequences in the genome. Palindrome recombinogenicity is caused by their ability to form secondary structures – hairpins in ssDNA and cruciforms in dsDNA. Using yeast Saccharomyces cerevisiae as a model, we compared the recombinogenicity of a perfect palindrome and quasipalindromes with short central spacers (1-10 bp) in vivo. We determined that a spacer as short as 7 bp significantly reduces the recombinogenicity, while a 10 bp spacer can completely stabilize palindromes up to 150 bp long, most likely by disabling cruciform formation. Furthermore, we found that in the absence of Sgs1 helicase the recombinogenicity of a perfect palindrome and of otherwise stable quasipalindrome with a 10 bp spacer is increased by the same factor, indicating that these two sequences have the same probability for hairpin formation in the lagging strand during replication. On the other hand, the absence of Rad27 nuclease (involved in Okazaki fragment maturation) dramatically increases the recombinogenicity of a perfect palindrome, but not a quasipalindrome with a 10 bp spacer. This result indicates that in the case of Rad27 absence the hairpin might form in the relatively short 5’ flap displaced by Pol  during Okazaki fragment synthesis.

Gene targeting is one of the basic tools in genetic engineering allowing deletion, precise modification or insertion of genes thus facilitating new strain construction for research and industry. Gene targeting is based on transformation of cells with a linear DNA fragment carrying ends homologous to the targeted region. The desired result of such transformation is replacement of the targeted region of the genome with the transforming DNA fragment. Yeast Saccharomyces cerevisiae is known for extraordinarily efficient gene targeting, i.e. the occurrence of unwanted, aberrant genetic events during gene targeting in this organism is generally considered to be fairly low. The most frequent aberrant genetic event in yeast is the targeted chromosome duplication, where one chromosome copy carries the transforming DNA in the targeted region while the other chromosome copy retains the original allele. In recent study we used qPCR to analyse the targeted chromosome duplication events in more detail. We used telomere-proximal primer pairs and probes to confirm that indeed the entire length of the targeted chromosome is duplicated. Moreover, we determined not only the targeted chromosome copy number, but also the copy number of all other (non-targeted) chromosomes. Interestingly, only two out of eight analysed transformants were aneuploids carrying targeted chromosome duplication and a single copy of non-targeted chromosomes, while other six out of eight transformants were diploids. Based on this results, we proposed a two-step mechanism for genome duplication instigated by gene targeting. After the duplication of the targeted chromosome (by break-induced replication mechanism), a protein content imbalance occurs. Since the transformants were kept under selective pressure to maintain both targeted chromosome copies, the imbalance could not be relieved by loss of an extra chromosome copy. Therefore, the duplication of non-targeted chromosomes leading to diploidisation and restoration of protein content balance would be favoured in yeast cell culture.

Bioethanol production from raw lignocellulosic material requires a producer strain which tolerates high ethanol concentrations and presence of growth and fermentation inhibitors. Therefore we constructed hybrid diploid S. cerevisiae strains by mating two haploids having desirable traits. One haploid was isolated from wine thus tolerating high ethanol concentration while the other has been shown to be resistant to several growth and fermentation inhibitors. These haploids were mated and resulting homozygous (control) and heterozygous (hybrid) diploids were tested for their ability to grow in the presence of several inhibitors frequently found in different lignocellulosic hydrolysates (acetic and levulinic acid, 2- furaldehyde) and to produce ethanol. The results showed that some hybrid diploids were more resistant to inhibitors than parental haploid strains or homozygous diploids. Interestingly, constructed hybrids showed different fermentation ability in CO2 production test, suggesting genetic variability of constructed strains. All together, these results suggest that the approach used in this study may help to construct new strains as well as to improve traits of biotechnologically interesting strains.

Yeast Dekkera/Brettanomyces bruxellensis is long known as a spoilage microorganism in wine where it produces volatile phenols giving the wine a distinct offflavor described as horse sweat, mice or burnt plastic. This yeast produces and tolerates high ethanol concentrations which enables it to spoil the wine at late stages of its production, making the economic losses even greater. But besides being able to produce high ethanol concentrations (around 14%), it was shown that B. bruxellensis can ferment cellobiose and be adapted to growth inhibitors typically present in lignocellulose hydrolasate. Therefore, it is probably one of the most promising non- Saccharomyces yeast species for second- generation bioethanol production. In hand with growing scientific interest in physiological features and industrial potential of D. bruxellensis, there is also a growing need for development of molecular-genetic tools. The aim of our research is to develop a set of genetic markers, vectors, yeast strains and methods which will ultimately enable routine genetic modifications and protein expressions in D. bruxellensis. We showed that D. bruxellensis transformants can be obtained by transformation with linear DNA fragment containing KanMX4 selection marker, but initially transformation efficiencies achieved by standard LiAc/PEG or electroporation procedures were very low and yielded only several transformants per sample. By optimizing a series of parameters (growth phase at which the cells are harvested for transformation, pretreatment of cells, the final cell density in the transformation sample, pulse strength and DNA mass) we developed the electroporation procedure for D. bruxellensis. The optimized procedure consistently yields 300-500 transformants per sample and results in transformation efficiencies of 2.8x10E3 up to maximum 2.7x10E4 transformants per microgram of DNA. Establishing a reliable and efficient transformation procedure presents the first critical step toward developing full spectra of genome-editing tools for D. bruxellensis. Our current research is focused on assessing possibilities of homologous recombination in D. bruxellensis, as well as isolation of auxotrophic strains and development of other dominant selection markers. Taken together, these results will facilitate both fundamental and applied research of this interesting yeast species.

Lactic acid bacteria are a ubiquitous group of Gram-positive nonsporulating cocci and bacilli that produce lactic acid as an end product of carbohydrate fermentation. They are closely associated with our everyday life as some ferment food or have health promoting effect while others spoil food or even cause diseases. Their relevance motivated whole genome sequencing of many isolates belonging to this clade. As a result, numerous sequencing reads are publicly available but it is unclear which bioinformatics tools can organize them into high-quality genome assemblies resulting in time-consuming trial-and- error based strategies. To ameliorate this problem, we tested 13 state- of-the-art assemblers using four specialized quality control tools. Assemblers were tasked with inferring genome sequence of a representative species of lactic acid bacteria, Lactobacillus amylovorus DSM 20531T, using Illumina-generated reads. Produced genome sequences varied considerably regarding total length, continuity and accuracy. Several scaffolding and assembly merging tools were tried to improve the quality of the assemblies further but had limited success. Visualization of final stage de Bruijn graph showed that many “bulges” limit continuity while graph’s shape indicated that the genome contains small plasmid. From these insights, we derive a set of guidelines which streamline the path to a high-quality genome assembly of lactic acid bacteria.

Gene targeting is a genetic technique that allows modification of an endogenous gene by homologous recombination. This is usually achieved by introduction of linear, non- replicative DNA fragments that should replace homologous (targeted) sequence in the genome. While this procedure is widely used for the construction of different strains it is also studied as a model for targeted genetic manipulations by the "ends-out" recombination. It is generally assumed that, in contrast to other yeasts and higher eukaryotes, transformation of the yeast Saccharomyces cerevisiae with non-replicative DNA almost exclusively occurs by homologous recombination. However, we found that even in S. cerevisiae ends-out recombination may result in off- targeting events. These events include different genome alterations such as illegitimate integration, integration of the transforming DNA fragment next to the target region and duplication of a targeted chromosome. Therefore, we systematically investigated the influence of a design of the ends-out assay and transformation method on the transformation efficiency, fidelity of gene targeting and spectra of all aberrant events. We show that the fidelity of gene targeting in S. cerevisiae is mainly influenced by the nature of alleles involved in recombination and we propose guidelines that increase the efficiency of gene targeting.

Nekonvencionalni kvasci Kluyveromyces marxianus i Scheffersomyces stipitis imaju značajan potencijal kao proizvodni organizmi u različitim bioprocesima. S. stipitis jedan je od kvasaca s najvišom sposobnošću fermentacije ksiloze u etanol, dok K. marxianus osim što je termofilan, brzo raste i može koristiti širok spektar različitih supstrata. Međutim u oba kvasca uvođenje ciljnih genetičkih modifikacija otežano je zbog česte ilegitimne (nehomologne) rekombinacije. Prvi cilj ovog rada bila je molekularno-genetička karakterizacija prethodno konstruiranih transformanata K. marxianus i S. stipites, koji proizvode L-laktat i 2,3-butanediol, metodom hibridizacije po Sothern-u. U kvascu S. stipitis istražena je mogućnost transformacije metodom elektroporacije i metodom protoplastiranja te utjecaj koncentracije ribonukleinskog kompleksa pri korištenju sustava CRISPR/Cas9. Dobiveni rezultati ukazuju na zaključak da prethodno razvijena metoda protoplastiranja nije pogodna za transformaciju kvasca S. stipitis te da koncentracija ribonukleinskog kompleksa značajno utječe na uspješnost transformacije elektroporacijom. Non-conventional yeasts Kluyveromyces marxianus and Scheffersomyces stipitis have significant potential as production organisms in various bioprocesses. S. stipitis is one of the yeasts with the highest ability to ferment xylose into ethanol, while K. marxianus, in addition to being thermophilic, grows quickly and can use a wide range of different substrates. However, in both yeasts, the introduction of target genetic modifications is difficult due to frequent illegitimate (non-homologous) recombination. The first goal of this work was the molecular-genetic characterization of previously constructed transformants of K. marxianus and S. stipites, which produce L-lactate and 2,3-butanediol, using the Southern blotting hybridization method. In the yeast S. stipitis, the possibility of transformation by the electroporation method and the protoplasting method and the influence of the ribonucleic complex concentration when using the CRISPR/Cas9 system were investigated. The obtained results indicate the conclusion that the previously developed protoplasting method is not suitable for the transformation of the yeast S. stipitis and that the concentration of the ribonucleic complex significantly affects the success of the transformation by electroporation.

Zaraza virusom SARS-CoV-2, nazvana COVID-19, obilježila je posljednje 3 godine. U tom razdoblju novootkriveni korona virus intenzivno se istražuje kako bi se bolje razumjela njegova svojstva i način prenošenja, te pronašle pogodne mete za razvoj učinkovitih antivrusnih lijekova. Genom virusa SARS-CoV-2 koji je kodirajući (+) lanac jednolančane pozitivne ribonukleinske kiseline, a zajedno s proteinima membrane, nukleokapside, omotnice i glikoproteinom šiljka čini virusnu česticu SARS-CoV-2. Genetički materijal u stanicu domaćina ulazi reakcijom glikoproteina šiljka i receptora domaćina angiotensin 1 konvertirajućeg enzima. Nakon ulaska u stanicu, dolazi do translacije RNA virusa prilikom čega nastaje 16 nestruktrurnih proteina koji sudjeluju u ostatku životnog ciklusa. Također, formiraju replikacijsko-transkripcijski kompleks koji provodi kontinuiranu i diskontinuiranu transkripciju. Diskontinuirana transkripcija je jedinstveno obilježje korona virusa te se odvija mehanizmom zamjene kalupa, a rezultira subgenomskom RNA čijom ekspresijom nastaju strukturni proteini. Unatoč lektorirajućoj aktivnost nestrukturnog proteina 14 koji sudjeluje u transkripciji RNA, SARS-CoV-2 karakterizira velik broj mutacija, a time i novih sojeva, među kojima su oni koji izazivaju zabrinutost: alfa, beta, gama, delta i najnoviji, omikron. SARS-CoV-2 infection, or COVID-19, has strongly affected the last three years. During this period, the newly discovered coronavirus wasintensively researched in order to better understand its properties and mode of transmission, and to find suitable targets for the development of effective antiviral drugs. SARS-CoV-2 virus has a single-stranded, positive-sense(+)RNA genome which together with membrane, nucleocapsid, envelope, and spike protein, makes viral particles. Interaction of host-cell receptor angiotensin 1 converting enzyme 2 (ACE2) with spike protein leads to the release of virus genetic material into the host cell. After entering the cell, RNA translation occurs, giving rise to 16 nonstructural proteins with an essential role in the rest of the virus life cycle. Also, they form a replication-transcription complex that carries out continuous and discontinuous transcription. Discontinuous transcription is a unique feature of coronaviruses that functions by template switching. It yields a set of subgenomic RNAs used to express structural proteins. Despite the proofreading activity of nonstructural protein 14, SARS-CoV-2 is characterized by many mutations, resulting with new variants, some of which are variants of concern (VOC). These variants are alpha, beta, gamma, delta, and most recent, omicron.

Kako bi se ostvarila maksimalna efikasnost industrijskog bioprocesa, poželjno je da radni organizam bude otporan na širok raspon parametara procesnih uvjeta te da efikasno proizvodi željeni proizvod. Nekonvencionalni kvasci predstavljaju raznoliku skupinu organizama od kojih pojedine vrste prirodno posjeduju razne biotehnološki interesantne karakteristike te se sve češće koriste u proizvodnji. U skupinu nekonvencionalnih kvasaca spadaju i vrste iz roda Schwanniomyces, koje osim mogućnosti rasta na različitim izvorima ugljika pokazuju i efikasnu sekreciju proteina velike molekulske mase u podlogu. Unatoč navedenim prednostima, mnoge vrste ovog roda još uvijek nisu detaljno istražene zbog nedostatka prikladnih metoda za genetičku transformaciju i ciljane genetičke modifikacije. Stoga je cilj ovog rada bio transformirati kvasce Schwanniomyces occidentalis var. occidentalis, Schwanniomyces occidentalis var. persoonii, Schwanniomyces capriottii i Schwanniomyces vanrijiae var. yarrowii, te ispitati aktivnost ishodišta replikacije iz različitih vrsta kvasaca korištenjem plazmidnih vektora. In order to achieve the maximum efficiency of industrial bioprocesses, it is desirable for the working organism to be resistant to a wide range of process conditions and to efficiently produce the desired product. Non-conventional yeasts represent a diverse group of organisms, some species of which naturally possess various biotechnologically interesting characteristics and are increasingly used in production. The group of non-conventional yeasts also includes species from the genus Schwanniomyces, which, in addition to the ability to grow on different carbon sources, also show efficient secretion of high molecular weight proteins into the substrate. Despite the mentioned advantages, many species of this genus have not yet been investigated in detail due to the lack of suitable methods for genetic transformation and targeted genetic modification. Therefore, the aim of this work was to transform the yeast Schwanniomyces occidentalis var. occidentalis, Schwanniomyces occidentalis var. persoonii, Schwanniomyces capriottii and Schwanniomyces vanrijiae var. yarrowii, and to examine the activity of origins of replication from different yeast species using plasmid vectors.

The RecA protein is a key recombinase in bacteria that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, the RecBCD and RecFOR protein complexes mediate RecA assembly on DNA. Thus, two recombination pathways, RecBCD and RecFOR, are distinguished in E. coli. Inactivation of both recombination pathways by recB(CD) recF(OR) mutations results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF mutants. In transductional crosses, these mutants show only three-fold decrease of recombination frequency relative to the wild-type strain. At the same time, they recombine 40- to 90-fold better than their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, moderately depends on recG and ruvABC functions, whereas inactivation of dinI, recX, recN, radA, and uvrD genes has a negligible effect. After exposure to UV and gamma- irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB+sbcC+ and ΔsbcB sbcC counterparts. Inactivation of 3ˈ-5ˈ exonucleases ExoVII, ExoIX and ExoX does not mimic the effect of the sbcB15 mutation suggesting that protection of 3’ overhangs itself is not sufficient to enable RecBFI pathway. Rather, our results suggest that SbcB15 protein plays an active role in formation of the RecA filament.

Garlic (Allium sativum L.) is a vegetatively propagated vegetable extensively studied due to its beneficial health effects. The cultivation and use of garlic is a part of Croatian tradition and each region or even village has their own landraces preserved for generations. In a relatively small area, a high phenotypic diversity could be observed and therefore the aim of this study was to assess a genetic diversity of collected Croatian garlic accessions. We genotyped 43 accessions from three Croatian regions (Istria, Dalmatia and continental Croatia) along with 14 foreign landraces using six polymorphic SSR markers. The number of distinct genotypes has been determined and the genetic structure and relationships among the accessions have been accessed. This is the first attempt to evaluate a large set of garlic accessions maintained by Croatian institutions as a part of the National program for conservation and sustainable use of plant genetic resources for food and agriculture. Accompanied by morphological and biochemical description, the results of this study will contribute to better knowledge on garlic genetic resources and foster effective and sustainable management of Croatian collections.

The amazing complexity of metazoan genomes originated early in the evolution of the animal kingdom. Rapid advances of next-generation sequencing (NGS) technologies and bioinformatic data analysis are enabling elucidation of early metazoan gene regulation and development. Sponges have a unique evolutionary position of being the simplest and arguably the earliest branching metazoans, and are therefore critical for understanding the genomic traits of the ancestor of metazoa - the first multicellular animal. However, available genomic data from the phylum Porifera is still very scarce. We sequenced the genome of the freshwater sponge Eunapius subterraneus, Sket & Velikonja, 1984 using a combination of NGS methods (Illumina and Oxford Nanopore) and assembled a draft version of its genome. Eunapius subterraneus is the only cave-adapted sponge species, known from only a small region in Croatia. The sponge possess unique morphological and ecological features. Using the -omics methodologies in combination with experimental approaches we aim to advance our understanding of 1. basic processes involved in multicellular development and differentiation and 2. the process of adaptation to different environmental conditions. Financial support by the Croatian National Science Foundation (Project No. 6400) is gratefully acknowledged.

In spite of being a small country, Croatia presents an important source of grapevine biodiversity which can be interesting for wine industry and breeding. In last two decades our research activities are focused on preservation, genotyping and evaluation of production characteristics of native varieties. To define full potential of native varieties there is a need to define the level of their susceptibility to fungal diseases. The aim of this research was to define the level of susceptibility/resistance of Croatian grapevine varieties to downey mildew (Plasmopara viticola) using modified field screening method. Altogether 57 genotypes/varieties were included in the research: 52 Croatian native varieties, two susceptible international V. vinifera L. cultivars (Cabernet Sauvignon, Riesling), 2 resistant cultivars (Solaris, Regent) and 2 Vitis species (Vitis ripara, Vitis amurensis). Own-rooted cuttings were planted in drip irrigated pots in field conditions close to the production vineyard at experimental station “Jazbina”, Department of Viticulture and Enology, Faculty of Agriculture Zagreb. After initial growing of shoots to the certain stage the monitoring of disease symptoms was carried out according to OIV descriptor 452 (degree of resistance on leaves). Results showed small difference among native varieties and majority of them have very low and low level of resistance to downy mildew, as expected.

Oxygenic photosynthesis, performed by plants, algae, and cyanobacteria, is arguably the most important autotrophic process on Earth. Photosynthetic light-to-chemical energy conversion involves numerous electron transfer steps that require rapid and streamlined reactions in order to maintain efficacy and prevent energy leaks to undesignated acceptors, mainly oxygen. Flavoenzyme FNR (ferredoxin:NADP+ oxidoreductase) catalyzes the last step of energy transfer in the linear photosynthetic chain, namely electron transfer from ferredoxin to NADP+. FNR utilizes two reduced ferredoxins to produce one molecule of NADPH. This reaction must be performed with high fidelity in order to prevent ferredoxin from passing electrons to other molecular targets or pathways. FNR is tethered to photosynthetic membranes of vascular plants via an integral membrane protein TROL (thylakoid rhodanaselike protein). Another soluble chloroplast protein Tic62 also participates in FNR sequestration. TROL binds FNR at the vicinity of photosystem I and prioritizes linear electron transfer. When FNR is released from the TROL, highly efficient electron sink is activated. This pathway is faster than methyl viologen-mediated ROS propagation and represents so far undescribed mechanism of photosynthesis regulation.

Recent evidence indicates that invertebrates are capable of an unexpected degree of immune memory, i.e. the immune priming. However, the underpinnings of this phenomenon are still largely unknown and the mechanisms may differ from case to case. As microbiota has been suggested to shape immunological responses of various hosts, the main aim of this study was to elucidate the mechanisms behind the already observed phenomenon of oral priming. Our hypothesis was that the eradication of commensal bacteria from insect larvae might lead to loss of the priming effect. As a model we used a well-established host-parasite system, the red flour beetle Tribolium castaneum and its natural entomopathogen, Bacillus thuringiensis. Our results confirmed our hypothesis and undoubtedly proved the essential role gut microbes play in the oral immune priming in T. castaneum, although the molecular and mechanistic underpinnings behind this phenomenon need further research. Furthermore, we showed that the oral priming in our host-pathogen model comes with a certain level of specificity. Our results represent a significant contribution to the growing body of knowledge on the topic of insect immunity and immune memory through the aspect of insect microbiota.

A number of bacterial, archaeal, and eukaryotic species are known for their resistance to ionizing radiation. One of the challenges these species face is a potent environmental source of DNA double-strand breaks, potential drivers of genome structure evolution. Efficient and accurate DNA double-strand break repair systems have been demonstrated in several unrelated radiation- resistant species and are putative adaptations to the DNA damaging environment. Such adaptations are expected to compensate for the genome- destabilizing effect of environmental DNA damage and may be expected to result in a more conserved gene order in radiation-resistant species. However, we show that rates of genome rearrangements, measured as loss of gene order conservation with time, are higher in radiation- resistant species in multiple, phylogenetically independent groups of bacteria. Furthermore, we explore whether detected high rearrangement rates in radiation-resistant species constitute a survival strategy or are a passive consequence of environmental DNA damage.

Croatia excels in a valuable and rich, but insufficiently investigated olive biodiversity. Significant efforts have been made in the last decade to collect national genetic resources to ensure their long-term conservation in ex situ collections, and thus, their sustainable management. In Croatia there are four ex situ olive germplasm collections, with 88 accessions officially registered in the Croatian Plant Genetic Resources Database. In all countries with centuries-long tradition of olive growing, there are numerous traditional cultivars, as well as a pronounced problem of homonymy, synonymy and intravarietal variability. The aim of this study, carried out within the National programme for conservation and sustainable use of plant genetic resources for food and agriculture, was to characterize the collections and to assess the genetic diversity of collected olive germplasm. We genotyped 69 accessions of 39 traditional cultivars, along with five introduced cultivars, using 14 polymorphic SSR markers. The number of distinct genotypes, duplicated accessions, and the cases of homonymy and synonymy have been determined and the genetic structure and relationships among Croatian traditional olive cultivars have been accessed. Results obtained in this study can be used for germplasm conservation, breeding programs, nursery production improvement and for optimizing a national strategy for the management of olive genetic resources.

Research into the molecular diversity was conducted in two clonal seed orchards (Čazma, Nova Gradiška) on 92 plus trees belonging to the two seed regions (seed region upper Sava river, middle Croatia and Kupa river and seed region middle part of the Sava river). Genetic analysis was revealed with eight microsatellite markers (Femsatl 4, Femsatl 8, Femsatl 10, Femsatl 11, Femsatl 16, M2-30, FR639485, FR646655). The aim of the study was to determine the level of molecular diversity of narrow-leaved ash from this two seed regions. Since the plus trees go through a very narrow phenotypic selection the comparison of the obtained genetic parameters from this study and with other research in natural stands concluded that there were no significant interpopulation variability. The highest genetic variability belongs to the intrapopulation level of genetic structure. Bayesian analysis of the population structure of selected genotypes with consideration to the genomic share can not be clearly separated into two seed regions. They are artificially demarcated based on the ecological conditions in which the narrow-leaved ash populations exists, but the obtained genetic structure and variability of selected plus trees indicate their individual variability.

Environmental contaminants often occur in the nature at low concentrations that are not considered to be toxic. Anyhow, their presence and, in some cases, persistence, can cause significant side effects on members of affected ecosystem. Cadmium and phthalates are often found in the environment in nanomolar concentrations. Cadmium is classified to be human carcinogen and phthalates are not directly acting genotoxic compounds. If there are indices of their tumorigenicity it is supposed that it is a result of nongenotoxic mechanism of action. Bis-2 ethylhexyl phthalate is widely used phthalate, and there are no experimental data concerning genotoxicity of environmentally significant concentrations. In this work, genotoxic potential of environmentally significant concentrations of both compounds and their mixtures were investigated on different human cell lines ; tongue carcinoma cell line, human colon carcinoma cells and human hepatocellular carcinoma cells during 2 and 4 hours of incubation. Expectedly, results of testing for cytotoxicity were negative, but mixtures of cadmium and DHEP caused cell proliferation. Also, it was noticed that mixtures caused formation of free radicals and certain DNA damage after different times of treatment, so it can be concluded that mixtures of nontoxic concentrations of contaminants, cadmium and DHEP, caused genotoxic effect.

Most epithelial ovarian cancer patients are diagnosed with advanced-stage disease due to the late appearance of symptoms and lack of early diagnostic methods/markers. The major problem for successful therapy is the development of tumour drug resistance during carcinogenesis (20-30%) and upon exposure to chemotherapy. The ovarian cancer cell line OVCAR-3/CBP was established by treatment of the ovarian adenocarcinoma cell line OVCAR-3 with long-term, stepwise selection in carboplatin (CBP) up to 25 μM. The variant is ~1.5-2-fold resistant to CBP, with cross-resistance to paclitaxel (TAX, ~4-fold), and presents with a mesenchymal-like phenotype. The increased expression of NHE-1, ATP7-B and decreased expression of ABCC2 and CTR-1 implied decreased total cell platination as a possible mode of CBP resistance, which was confirmed by flame atomic absorption spectrometry. Despite the increased level of ABCB1 transcripts, OVCAR-3/CBP did not efflux [3H]-docetaxel differently compared to parental cells, and the P-glycoprotein inhibitor PSC-833 did not alter these drug accumulation profiles. This indicates that the TAX resistance in OVCAR-3/CBP is non-MDR, but is associated with elevated TUBB3 (class III beta-tubulin) content along with total α- and β-tubulin relative to parental OVCAR-3 cells. In summary, drug selection with carboplatin in an ovarian cancer cell line resulted in non-MDR cross-resistance to paclitaxel. Experiments investigating the functional significance of altered tubulin content and microtubule dynamicity in response to drug exposure in OVCAR-3/CBP are on-going.

The european wildcat Felis silvestris silvestris inhabits wide areas in Europe and is considered a subspecies genetically close to the domestic cat Felis silvestris catus. The two subspecies have different habitat preference, but as human activity causes undisturbed areas to disappear the two subspecies come increasingly into contact, allowing for hybridization and the disappearence of the genetically pure wildcat populations. While hybridization has been confirmed throughout Europe, the genetic structure of wildcats in Croatia has not been previosly studied. With relatively conserved forest habitats, Croatia might contain a wildcat population largely unaffected by hybridization. If present, such population is important in maintaining of the wildcat genetic structure for both conservation of the existing wildcat populations and reintroduction of the genetically pure wildcats into areas from which they have disappeared. Muscle, blood and oral mucosa samples have been obtained from both wildcats and domestic cats. After DNA extraction, microsatellite markers have been amplified by high fidelity DNA polymerase using multiplex touchdown PCR and analysed by capilary electrophoresis. Bayesian clustering using Structure 2.3.4 clearly separated the wildcat, domestic and hybrid cat populations and the genetic variability of each population has been analyzed using Arlequin 3.5.2.2. and CLUMPAK software. The results show low incidence of hybrids, and the existance of subpopulations in both wildcats and domestic cats. These results indicate the need for further wildcat habitat preservation, and might be used as a basis in tracking, monitoring and conservation of the wildcat population in Croatia.

Satellite DNAs (satDNAs) are tandemly repeated non-coding DNA sequences, and the most common constituents of every functional centromere. Holocentric chromosomes and multiple genome copies establish nematodes of the genus Meloidogyne as an attractive model for studying centromere- associated DNA sequences. In order to identify a pool of satDNAs that may be involved in genomics of the holocentromere, we have analyzed the satellitome (whole-genome complement of satDNAs) in four different publicly available Meloidogyne genomes using RepeatExplorer computational tool. Our results of genome-wide analyses revealed the presence of a remarkable collection of 38-80 different satDNAs in each examined genome. In addition, comparative analyses of satellitomes show three main groups of satDNAs ; common for all four studied species, and shared by subsets of three or only two species. This distribution of satDNAs supports a recent hypothesis based on comparative analyses of coding regions, that speciation processes in Meloidogyne may be a result of additive interspecific hybridizations. Our preliminary analysis of satDNA distribution on chromosomes of studied species also speaks in favor of such unusual genome evolution in Meloidogyne.

The ubiquitous and evolutionary conserved RecA protein binds to single-stranded DNA (ssDNA), producing a nucleoprotein filament. RecA has three known roles in E. coli: a) RecA nucleoprotein filament enables repair of DNA double-strand breaks (DSBs) and exchange of similar DNA molecules by promoting homologous recombination ; b) RecA nucleofilament facilitates autocleavage of LexA repressor, hence enabling induction of SOS response ; c) during SOS induction, RecA activates a mutagenic DNA polymerase V. Another role of RecA exists in E. coli, namely RecA inhibits DNA degradation. However, no relation of the latter RecA function to the three established ones has been explored previously. Here, we introduced DSBs (by gamma-irradiation) into E. coli chromosome labelled by [3H]thymidine and monitored its degradation. We have shown that RecA inhibition of DNA degradation depends on RecA’s concentration and its rate of association with ssDNA. Also, our results indicate that RecA suppression of DNA degradation is separable from the other three roles of RecA, thus suggesting it represents a novel RecA function in E. coli, namely regulation of DSB processing.

Obesity is, in most cases, a multifunctional disease resulting from the interaction of changes in the human genome and environmental factors affecting the development of the disease throughout one’s life. Single nucleotide polymorphisms (SNPs) are genetic variants that occur with a different frequency in the human genome. Fat mass and obesity associated (FTO) gene and genetic association of FTO SNPs are related to obesity and genetic aging. In this work, we investigated three common SNPs in the FTO gene in women from Zagreb County. In cases with obesity present, the frequencies of the risk genotypes AA rs9939609, CC rs1421085 and GG rs17817449 of the FTO gene were significantly higher. Interestingly, 60.87% of the obese women are not homozygous carriers of even one risk allele opposite the rest 39.13% of the obese women who are triple- homozygous carriers of the risk alleles for all three studied SNPs. Considering the bimodal distribution of the investigated SNPs in obese women, the other genetic, metabolic, physiological, behavioural, sociocultural and / or environmental factors that can contribute to female obesity are a matter of great interest. Furthermore, it is a long way to go before we may answer questions about which factors affect the occurrence and evolution of SNPs in the complex genetics of human diseases.

Root-knot nematodes (genus Meloidogyne) are established as important model organisms of plant parasites. Their characteristics, such as holocentric chromosomes, polyploidy and parthenogenesis make them an ideal system to study genome evolution, speciation processes, and centromere structure and function.In this regard, our analysis of centromere specific histone H3 (CENH3) in the genome of M. incognitareveals three divergent CENH3 variants instead of the commonly present only one. All CENH3 variants are completely conserved in related species, and transcriptome data show their developmental stage- specific expression. Immunostaining of chromosomes with antibodies specific for CENH3 variants reveals holocentric distribution pattern for each of them. Given that the centromere tends to be established upon satellite DNA arrays wefurther focus on their characterization in related Meloidogyne genomes. Using next-generation sequencing combined with novel bioinformatics tools we detected a remarkable collection of satellite DNAs (satellitome) in each genome. Comparative analyses of satellitomes indicate the species evolutionary history in accordance with a recent hypothesis which suggests a multiple hybridization events in species origin. All results will be discussed in terms of coexistence and possible roles of different CENH3 variants in holocentric centromeres, and correlated with trends in satellitome evolution

Telomeres are the main guardians of genome stability. During DNA replication, progressive shortening of telomeres takes place with each cell division. Critically short telomeres induce cell cycle arrest or apoptosis. Oxidative stress also plays an important role in telomere dynamics. Recent evidence highlights direct molecular connection between telomere attrition and mitochondrial dysfunction. Although reactive oxygen species (ROS) are normal products in intermediary metabolism, and are crucial for different intracellular signalling pathways, a disbalance resulting in ROS excess induces oxidative stress and accelerates telomere shortening. It is assumed that with ageing, production of ROS may be increased causing faster telomere shortening, and that both may be ameliorated by antioxidants. To test this hypothesis, we treated male and female Wistar rats for 9 or 21 months with melatonin and resveratrol and investigated their effect on telomere attrition rate in liver and kidney (various zones) tissues. Telomere length was assessed by Southern blotting of genomic DNA. The results in 1- and 2- year-old male and female rats showed an absence of significant differences in telomere length in the tissues from both organs after treatment with antioxidants compared to control (vehicle-treated) animals (N=4 per group). Based on obtained results, we did not observe beneficial properties of tested antioxidants towards age-dependent telomere shortening.

To understand the role of epigenetic processes in plant adaptation, existing epigenetic variation of natural plant populations has been assessed and compared to genetic variation. Twenty-five Dalmatian sage (Salvia officinalis L.) populations, each consisting of 20 to 25 plants, were analysed using epigenetic Methylation-Sensitive Amplification Polymorphism markers (MSAP) and genetic AFLP markers. Epigenetic and genetic diversity was compared by analysing within-population diversities and pairwise genetic differentiation between populations. The importance of epigenetic processes in divergence and adaptation of Dalmatian sage was analysed by comparing the number of outlier epiloci/loci under divergent selection and by the assessment of the association between epiloci/loci and bioclimatic variables. The identification of FST outlier epiloci/loci was carried out using a Bayesian method while the spatial analysis was used to test the probability of presence of an epiallelic/allelic variant given the environmental conditions of the sampling locations.

Common bean (Phaseolus vulgaris L.) is economically the most important among five domesticated species from genus Phaseolus. In Croatia, common bean is a traditionally grown in low input production systems. The production is based on landraces displaying high levels of diversity. In order to assess the genetic diversity of Croatian common bean landraces, 174 accessions were analysed using a DArTseq method of SNP genotyping. Out of 17, 514 polymorphic markers, 8, 092 (46%) had high scoring reproducibility (>0.95), high call-rate (>0.90) and the minor-allele frequency higher than 5%. Genomic regions flanking SNPs were than aligned against the reference genome of P. vulgaris. From the 8, 092 SNP sequences, 6, 599 (82%) were aligned to 11 chromosomes of common bean. The average number of SNPs per chromosome was 599.91, ranging from 403 on chromosome 4 to 834 on chromosome 2. Mean number of SNPs per Mbp was 12.85 or on average one SNP every 77, 828 base pairs. The average expected heterozyogsity was 0.373, ranging from 0.096 to 0.500 while the polymorphism information content ranged from 0.091 to 0.373 with an average of 0.298. The model-based analysis of population structure was carried out using 923 SNPs in linkage equilibrium. The results were compared to those obtained by microsatellite markers. The SNP set will be used in genome- wide association studies (GWAS) concerning bioactive nutrient content.

Croatian Adriatic coast is rich in germplasm of local olive (Olea europaea L. subsp. europaea var. europaea) varieties that have not been fully explored yet. One of these varieties, cv. ‘Piculja’, is grown only in the south Dalmatian region (the Dubrovnik-Neretva County in the south of Croatia). The largest concentration of cv. ‘Piculja’ population is on the Island Lastovo. According to Bulić (1921), there is a mention of this cultivar in Dubrovnik area in 1869, and at that time, the Island Lastovo was part of the Dubrovnik Republic. It is a variety characterized by later ripening and extremely lush vertical growth, and its fruits remain on the tree even until the next flowering. Compared to the cv. 'Oblica' it has a greater tolerance on Olive Fruit Curculio (Rhynchites Cribripennis). It is not among the leading cultivars, but besides its significant role in the production of high-quality olive oil, it has also important economic and technological role as a pollinator for other olive varieties. The aim of ‘pilot’ research was to get insight into genetic variability within limited, but hypothetically diverse set of four old trees of ‘Piculja’. Genetic variability within the cultivar may have an important role as a basis for clonal selection, production of reproductive material and clonal recognition. The insight into genetic variability may also contribute to the knowledge of cultivar evolution and development.

The loss of pigmentation is one of specialized traits which evolved as an adaptation to subterranean habitats. It appeared in almost all groups with cave-adapted species, but not much is known about the molecular and evolutionary mechanisms of its appearance. The exception is the teleost model system Astyanax mexicanus, in which a deletion in the oca2 gene underlies albinism in several independently evolved cavefish populations. We have investigated albinism in diverse cave species and discovered that 1. pigment cells are invariably present and 2. there is a lesion in the first step of melanin synthesis pathway. The preservation of pigment cells is suggestive of their function in processes other than pigmentation. Further examination confirmed the role of melanocytes in vertebrate immunity and maintenance of melanin synthesis capacity as a part of the innate immune response in albino cave arthropods. The non-random inactivation of melanin synthesis suggests that there must be an adaptive benefit. The cessation of pigment production could be selected for as a result of minimizing energy expenditure for production of unnecessary substances, or as an indirect effect since many pleiotropic phenotypes are intertwined with pigmentation. We found evidence for both of these hypotheses implicating that natural selection may be involved in the evolution of albinism, contrary to traditional views of pigmentation being a trait that is simply discarded in the absence of light.