Molecular Biology 1400-215BM

Lecture.
DNA structure. DNA replication in eukaryotic organisms. Details of Okazaki fragment structure and role in DNA replication and issues regarding its termination. The role and structure of telomere. DNA damage types and their repair mechanisms. Non-homologous end joining and homologous recombination.
Tools in molecular biology and DNA sequencing. Chemical synthesis of oligonucleotides and their application in PCR. Enzymes utilised in molecular biology laboratory procedures. Methods for genome sequencing: Sanger’s method and next generation sequencing. The history of human genome sequencing. DNA data storage.
Genome structure, its elements and evolution. The first sequenced genomes and their characteristics. Hypotheses regarding the evolutionary origin of genes. Key structural elements of genes. Genome organisation. Transposable elements, their structures and mechanisms of transposition.

Examples of different regulatory mechanisms of gene expression in prokaryotic organisms at the level of initiation and termination of transcription (e.g. Lac operon, arabinose operon, tryptophan operon. attenuation). Expression of prokaryotic genes: regulatory mechanisms. Regulation at the following levels (i) post-transriptional level with the participation of proteins and regulatory RNA (including riboregulation, riboswitches), (ii) translation and (iii) post-translational level. Operon, regulon, modulon. Global regulation at the cellular (including a two-component regulatory system) and the entire population level (including quorum sensing). DNA replication and recombination in bacteria.

Eukaryotic replication, recombination and DNA repair. Chromatin. Structure of genetic material of eukaryotes: histones, nucleosomes, higher order structures of chromatin. RNA: transcription and processing. Regulation of eukaryotic transcription: RNA polymerases, transcription factors, chromatin remodeling, histone modifications. Interference RNA: microRNA and RNAi.DNA methylation and epigenetic mechanisms.

Eukaryotic translation. Amino acids and polypeptide chain. Primary, secondary, tertiary and quarternary protein structure. Protein folding. Chaperones, chaperonins. Posttranslational modifications of proteins.

Laboratory.
Restriction endonucleases and enzymes used to modify DNA. DNA recombination and cloning. Methods applied to introduce DNA to bacterial cells. Competent cells and bacterial transformation. Plasmid vectors. Antibiotics applied in molecular biology. Isolation of plasmid DNA, DNA electrophoresis, estimation of its purity and quality. Application of Escherichia coli lac operon in DNA cloning. Methods applied to the selection of recombinant DNA, alfa-complementation. Catabolic repression.
Purification of the recombinant enzymatic protein expressed in E. coli cells by the means of immobilized-metal affinity chromatography (IMAC). Evaluation of the quality of protein preparations, what includes: i) evaluation of catalytic activity in the protein preparation, ii) analysis of protein purity by SDS polyacrylamide gel electrophoresis (SDS PAGE) (various gel staining techniques, Coomassie Brilliant Blue staining, silver staining and staining with fluorescent dye, will be compared), iii) immunological identification of the protein by Western blotting (secondary antibodies conjugated with alkaline phosphatase or horseradish peroxidase will be used and various detection methods, including enhanced chemiluminescence, will be applied).DNA isolation from plant cells.
Transgenic plants as examples of genetically modified organisms - generation, application and analysis. Transformation of tobacco plants - leaf infiltration with the Agrobacterium tumefaciens bacterial culture carrying the genetic construct with the marker gene.
Reporter genes, like green fluorescent protein (GFP), luciferase, glucouronidase (GUS), etc, often used to indicate of whether a certain gene has been taken up by or expressed in the plant . The methods of testing protein interactions, such as yeast two-hybrid system, FRET (Föster Resonance Energy Transfer), and BiFC (Bimolecular fluorescence complementation) will also be discussed and presented. The transient transformation of plants will be performed and the introduced gene expression will be analysed by observing the green fluorescence protein under the fluorescence microscope. The DNA constructs used during the experiment contain genes encoding GFP translational fusions with proteins from various parts of the cell. This will allow the localization of these proteins to be analyzed. Observations of plants expressing the glucuronidase gene under the control of various tissue-specific promoters will be carried out.
The course will discuss the real-time PCR technique, principles of operation, including specific and non-specific detection of nucleic acids, and issues related to experiment planning, such as the selection of reference genes and the design of oligonucleotides. Variants of the method and possible applications will be presented.
Molecular biology methods used in analyses of Arabidopsis mutants and transgenic lines. Plant genetics - forward and reverse genetics approaches for Arabidopssi thaliana model; mutagenesis and forward genetic screen of Arabidopsis mutants; mapping of point and insertional mutations, PCR analysis of polymorphic sequences in two Arabidopsis ecotypes.