Intramolecular recombination in bacteria Escherichia coli and Agrobacterium tumefaciens

Uzastopno ponovljeni sljedovi nalaze se u genomima mnogih organizama i podložni su delecijama i amplifikacijama, te su velik uzrok genomske nestabilnosti. Genetička analiza rearanžmana ponovljenih sljedova u bakteriji Escherichia coli je pokazala da je u njihov nastanak uključeno nekoliko mehanizama. Mogu se ugrubo podijeliti na RecA-ovisne i RecA-neovisne mehanizme. Ovim radom željeli smo ispitati efikasnost intramolekularne rekombinacije u bakteriji Agrobacterium tumefaciens, te je usporediti s onom u bakterije Escherichia coli. U tu svrhu napravili smo plazmid koji sadrži direktno ponovljene nepotpune gene za otpornost na spektinomicin razmaknute genom za otpornost na ampicilin. Otpornost na spektinomicin može nastati ili RecA-ovisnom homolognom rekombinacijom, pri čemu dolazi do delecije gena za otpornost na ampicilin ili RecA-neovisno pri čemu se zadržava gen za otpornost na ampicilin i amplificiraju geni za otpornost na spektinomicin. U ovom radu smo pokazali da je intramolekularna rekombinacija u A. tumefaciens uglavnom RecA-neovisna.Tandemly repeated DNA sequences are common in the genomes of many organisma and are susceptive to deletions and amplifications. That way they are a major cause of genome instability. Genetic analysis of repeated sequence rearrangements in Escherichia coli has revealed that multiple mechanisms participate in the process. They can be roughly divided into RecA-dependant and RecA-independent mechanisms. In this graduation thesis we wanted to test intramolecular recombination efficiency of bacterium Agrobacterium tumefaciens and compare it to that in Escherichia coli. For that purpose we have designed a plasmid which has tandem repeats of incomplete spectinomycin genes separated with ampicillin resistance gene. Spectinomycin resistance can be achieved either by RecA-dependant homologous recombination between direct repeats, while simultaneously deleting ampicillin resistance gene, or by RecAindependent mechanisms, which yields both ampicillin resistance gene and spectinomycin resistance genes amplification as a product. In this graduation thesis we have shown that intramolecular recombination in A. tumefaciens is mostly RecAindependent

Intramolecular recombination in bacteria Escherichia coli and Agrobacterium tumefaciens

Uzastopno ponovljeni sljedovi nalaze se u genomima mnogih organizama i podložni su delecijama i amplifikacijama, te su velik uzrok genomske nestabilnosti. Genetička analiza rearanžmana ponovljenih sljedova u bakteriji Escherichia coli je pokazala da je u njihov nastanak uključeno nekoliko mehanizama. Mogu se ugrubo podijeliti na RecA-ovisne i RecA-neovisne mehanizme. Ovim radom željeli smo ispitati efikasnost intramolekularne rekombinacije u bakteriji Agrobacterium tumefaciens, te je usporediti s onom u bakterije Escherichia coli. U tu svrhu napravili smo plazmid koji sadrži direktno ponovljene nepotpune gene za otpornost na spektinomicin razmaknute genom za otpornost na ampicilin. Otpornost na spektinomicin može nastati ili RecA-ovisnom homolognom rekombinacijom, pri čemu dolazi do delecije gena za otpornost na ampicilin ili RecA-neovisno pri čemu se zadržava gen za otpornost na ampicilin i amplificiraju geni za otpornost na spektinomicin. U ovom radu smo pokazali da je intramolekularna rekombinacija u A. tumefaciens uglavnom RecA-neovisna.Tandemly repeated DNA sequences are common in the genomes of many organisma and are susceptive to deletions and amplifications. That way they are a major cause of genome instability. Genetic analysis of repeated sequence rearrangements in Escherichia coli has revealed that multiple mechanisms participate in the process. They can be roughly divided into RecA-dependant and RecA-independent mechanisms. In this graduation thesis we wanted to test intramolecular recombination efficiency of bacterium Agrobacterium tumefaciens and compare it to that in Escherichia coli. For that purpose we have designed a plasmid which has tandem repeats of incomplete spectinomycin genes separated with ampicillin resistance gene. Spectinomycin resistance can be achieved either by RecA-dependant homologous recombination between direct repeats, while simultaneously deleting ampicillin resistance gene, or by RecAindependent mechanisms, which yields both ampicillin resistance gene and spectinomycin resistance genes amplification as a product. In this graduation thesis we have shown that intramolecular recombination in A. tumefaciens is mostly RecAindependent

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR-associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double-stranded breaks (DSBs) into a DNA molecule in a sequence-specific manner, thereby stimulating the error-prone non-homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9-based construct targeting the IL6ST gene was shown to be active, while the two TALEN-based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose-dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies. This work is licensed under a Creative Commons Attribution 4.0 International License

Usporedba intraplazmidne rekombinacije u bakterijama Agrobacterium tumefaciens i Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism.U ovom smo radu konstruirali plazmid koji nam je omogućio da usporedimo intraplazmidnu rekombinaciju u bakterijama Agrobacterium tumefaciens i Escherichia coli. Plazmid sadržava dvije istosmjerno ponovljene kopije gena odgovornog za rezistenciju na spektinomicin, pri čemu jednoj kopiji nedostaje 5\u27, a drugoj 3\u27 kraj gena, a međusobno su homologne u duljini od 570 pb. Osim toga, DNA koja se nalazi između ove dvije istosmjerno ponovljene sekvencije sadržava gen koji daje rezistenciju na antibiotik ampicilin. Homolognom rekombinacijom između nepotpunih gena za rezistenciju na spektinomicin nastaje funkcionalni gen, odgovoran za pojavu rezistencije. Pritom može doći do delecije gena za rezistenciju na ampicilin ili njegovog umnožavanja, zajedno s nepotpunim genima za otpornost na spektinomicin. Ovaj eksperimentalni sustav omogućio nam je da po prvi put pratimo pojavu delecija i amplifikacija tijekom intraplazmidne rekombinacije u bakteriji A. tumefaciens. Pokazali smo da delecije i amplifikacije u bakterijama Agrobacterium tumefaciens i Escherichia coli nastaju prvenstveno RecA-neovisnim mehanizmom. Osim toga, ustanovili smo da se delecije i amplifikacije pojavljuju s podjednakom učestalošću, što upućuje na to da je mehanizam oba rekombinacijska događaja sličan

Maternal Physical Activity in Pregnancy and Newborns` Anthropometry-The Preface From the CRIBS Study

Health benefits of physical activity during pregnancy include reduced risk of excessive gestational weight gain and conditions such as gestational diabetes, preeclampsia and preterm birth. The ongoing CRoatian Islands Birth Cohort Study (CRIBS) is the first cohort study in the South-Eastern Europe with an aim to assess the prevalence of risk factors (biological, environmental and behavioral) for the Metabolic Syndrome in populations from Dalmatian islands of Hvar and Brač and coastal Split city with its surroundings. At the time of writing, Over 350 pregnant women and 220 of their newborns have been involved in the study. Here we present the preliminary results of testing the association of mothers` self-estimated physical activity during pregnancy with newborns` anthropometric characteristics (birth weight, length and head circumference) using the data from questionnaires and obstetric records of 116 mother – newborn pairs. The difference in weight-at-birth was detected between newborn girls whose mothers were from Low vs. Intensive physical activity categories, as well as from Moderate vs. Intensive physical activity categories. In addition to that, the significant difference in weight-at-birth and height/length-at-birth was detected between newborn boys whose mothers were from Moderate vs. Intensive physical activity categories (p<0.01). No association between self-estimated level of physical activity and mothers’ body mass index was found. For women with normal pregnancies, light occupational activities do not cause problems with the fetal growth rate, but the same was not reported for women who maintained high-intensity activities

Intramolecular recombination in bacteria Escherichia coli and Agrobacterium tumefaciens

Uzastopno ponovljeni sljedovi nalaze se u genomima mnogih organizama i podložni su delecijama i amplifikacijama, te su velik uzrok genomske nestabilnosti. Genetička analiza rearanžmana ponovljenih sljedova u bakteriji Escherichia coli je pokazala da je u njihov nastanak uključeno nekoliko mehanizama. Mogu se ugrubo podijeliti na RecA-ovisne i RecA-neovisne mehanizme. Ovim radom željeli smo ispitati efikasnost intramolekularne rekombinacije u bakteriji Agrobacterium tumefaciens, te je usporediti s onom u bakterije Escherichia coli. U tu svrhu napravili smo plazmid koji sadrži direktno ponovljene nepotpune gene za otpornost na spektinomicin razmaknute genom za otpornost na ampicilin. Otpornost na spektinomicin može nastati ili RecA-ovisnom homolognom rekombinacijom, pri čemu dolazi do delecije gena za otpornost na ampicilin ili RecA-neovisno pri čemu se zadržava gen za otpornost na ampicilin i amplificiraju geni za otpornost na spektinomicin. U ovom radu smo pokazali da je intramolekularna rekombinacija u A. tumefaciens uglavnom RecA-neovisna.Tandemly repeated DNA sequences are common in the genomes of many organisma and are susceptive to deletions and amplifications. That way they are a major cause of genome instability. Genetic analysis of repeated sequence rearrangements in Escherichia coli has revealed that multiple mechanisms participate in the process. They can be roughly divided into RecA-dependant and RecA-independent mechanisms. In this graduation thesis we wanted to test intramolecular recombination efficiency of bacterium Agrobacterium tumefaciens and compare it to that in Escherichia coli. For that purpose we have designed a plasmid which has tandem repeats of incomplete spectinomycin genes separated with ampicillin resistance gene. Spectinomycin resistance can be achieved either by RecA-dependant homologous recombination between direct repeats, while simultaneously deleting ampicillin resistance gene, or by RecAindependent mechanisms, which yields both ampicillin resistance gene and spectinomycin resistance genes amplification as a product. In this graduation thesis we have shown that intramolecular recombination in A. tumefaciens is mostly RecAindependent

Comparison of Intraplasmid Rearrangements in Agrobacterium tumefaciens and Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism

METABOLIC SYNDROME AND GUT MICROBIOTA - A REVIEW

The relationship between gut microbiota and human health is complex, and the role of gut microbiota in pathogenesis of various diseases has been in the focus during the last decade. There is accumulating evidence that dysbiosis can be linked to different diseases, such as metabolic syndrome (MetS). Still, there is no consensus on the most appropriate tools and approaches for microbiota analyses. Numerous factors - diet, lifestyle, chemical microenvironment etc. influence the composition of gut microbiota. We aimed to analyze the current state of the knowledge on complex interplay between gut microbiota and development of MetS, as a basis for future research. The permanent interplay between immune system, metabolism, and gut microbiota plays a significant role in the homeostasis control and potential obesity development. Increased energy harvest from the diet, changes in gene expression, energy expenditure and storage are mentioned to lead to inflammation, insulin resistance and MetS. Most of the data on its mechanisms were from mouse models, so the question of their informativeness for human microbiota research arose. Current state of the literature (using PubMed database), including GWAS studies of obesity in mice, suggests that they are relevant for human studies of microbiota composition change in response to diet. Besides the role of Firmicutes/Bacteroidetes ratio in predisposition to obesity, its difference in obese and lean humans and its decrease with weight loss, confirms the dominating role of nutrition in shaping gut microbiota composition and functions. There is an increasing evidence that microbiota can inflict their reach on physiological functions outside the gastrointestinal tract, and can therefore possibly manipulate our eating behaviour, using metabolic, neural, immune and endocrine pathways. Our findings implicate a deeper host-microbiota relationship than previously realized, which may contribute to broader and multilayer approaches in future research of gut microbiota and shaping of prevention strategies for tackling MetS