Faculty of Medicine
6 credit points, terminal examination, Miklós Csala; AOKMBT795_1A
Faculty of Dentistry
4 credit points, terminal examination, Miklós Csala; FOKOOVM205_1A
Teaching secretary: Gergely Keszler
Description of the curriculum
The principal aim of the course is to provide an insight into the storage and expression of genetic information throughout replication, transcription and translation. Our current understanding of the multilevel regulation of gene expression will also be discussed, followed by an up-to-date summary of methods applied in molecular biotechnology.
Module I. DNA, RNA and protein synthesis (storage and expression of genetic information)
Nucleic acids – structure and function. Bases, nucleosides, nucleotides, DNA structure, DNA denaturation, hybridization. DNA replication. Replication in prokaryotes, leading and lagging strand. Okazaki fragments. DNA-dependent DNA polymerases. DNA ligase. Telomerase. topoizomerases. Replication in eukaryotes. Structure of eukaryotic chromosomes. Mitochondrial DNA. Nucleosome structure. DNA repair. Types of DNA damage; mutations, frameshift, missense/nonsense/samesense mutations, mismatch repair. Coordination of repair and replication. Transcription in prokaryotes. Structure of RNA; t-RNA, r-RNA, m-RNA, differences between the prokaryotic and eukaryotic genomes. Transcription complexes, initiation, elongation, termination in prokaryotes. Transcription in eukaryotes, RNA polymerases, promoters, enhancers, silencers. Transcription factors. Processing of mRNA, mechanism of splicing. Alternative splicing, mRNA editing. The genetic code. Activation of tRNA. Mechanism of translation initiation, elongation, termination. Antibiotics. Posttranslational modification of proteins. Protein folding, sorting, quality control and transport into intracellular compartments. Ubiquitination and intracellular proteolysis. Epigenetic regulation of gene expression: DNA methylation, histone modifications, miRNA. Mobile genetic elements, genome evolution and the molecular biology of viruses.
Regulation of gene expression in prokaryotes. The operon model. Positive and negative regulation in the lac operon. Regulation of gene expression in eukaryotes at the transcriptional level. Role of chromatin structure; covalent and non-covalent chromatin modifying activities and DNA methylation (epigenetics). Post-transcriptional regulation in eukaryotes. Regulation of mRNA stability. Translational regulation.
Module II. Methods in molecular biology and gene technology
Principles of recombinant DNA technology: molecular cloning, restriction endonucleases. Genomic and cDNA libraries. Blotting techniques (Southern, Northern, Western) and their utilization. DNA microarrays. PCR, real-time PCR and its application in molecular biology. Recombinant vectors (reporter and expression vectors); synthesis of recombinant proteins. Transgenic, knock-out and knock-in animals in medical research. Human gene therapy (ex vivo vs in vivo; genome editing by the CRISPR/Cas9 technology). The Human Genome Project and its results: organization and polymorphic nature of the human genome; implications for human traits and diseases. Genotyping methods (PCR-RFLP, PCR-ASA, primer extension, (next generation) sequencing). Online databases.
Requirements for acknowledgement of the semester
- Participation in the laboratory lessons is obligatory; students have to sign the attendance sheets at the end of each lesson. In case of more than three absences from the lab lessons (including the oral midterm and the written test) for any reason, the semester will not be acknowledged and the student will be not allowed to sit for the semifinal exam. Missed practicals can be made up in the same week at another group; certificate from the host teacher should be presented by the student to his/her own teacher. The schedule of practical lessons can be downloaded from the official homepage of the Department.
- The oral midterm examination has to be passed before the commencement of the examination period. The material of the midterm examination corresponds to that of lectures of weeks 1-8. Failed midterms can be retaken at most twice. Importantly, students cannot get a better mark than 2 on the retake (this rule does not pertain to students who missed the midterm due to an illness proven by a medical certificate). Dates of retakes will be disclosed in due course.
Lab test and exam bonus
A written test will be held in the semester. Students are supposed to answer a lab topic from the corresponding topic list („Lab test”). Topics will be selected and marked by your own lab teacher. As it is not compulsory to pass the lab test, it cannot be taken again.
If the average of the marks obtained on the oral midterm and the written lab test is at least 4.0 or better, one will be exempted from answering lab topics (group IV) on the semifinal exam.
The course is finished with an oral exam. Students will be examined by a two-member examination committee. 4 topics taken from the topic list have to be answered.
Students are not allowed to leave the examination room between taking exam topics and getting their grades. Importantly, you fail the whole exam if you fail even on just one of your topics.
Lodish: Molecular Cell Biology (8th edition)
Hrabák: Laboratory Manual – Medical Chemistry and Biochemistry
Topics - YEAR 2019/20
I. DNA structure, replication and repair
- Comparison of pro- and eukaryotic cells: compartmentation and the role of the most important subcellular organelles
- The chemical structure of nucleotides. The primary structure of nucleic acids (DNA and RNAs)
- Condensation of the genetic material in pro- and eukaryotic cells. The role of topoisomerases and chromatin proteins
- The structure of human chromosomes and its alterations during the cell cycle
- The structure of the human genome: coding and gene regulatory sequences. Non-coding genomic sequences: introns, pseudogenes, repetitive sequences
- Principles of the semiconservative DNA replication: replication fork, leading and lagging strand synthesis
- Comparison of DNA replication in pro- and eukaryotic cells: principles, enzymes, proteins
- Telomeric repeat sequences and their maintenance
- Classification of DNA damages and repair systems. Repair of base deamination
- Formation and repair of thymine dimers. Mismatch repair
- Classification of point mutations. The origin of spontaneous point mutations. DNA polymorphisms. Possible effects of DNA sequence alterations on the corresponding mRNA and proteins
- Genetic variations in the human genome and their role in the pathogenesis of diseases. Methods to study genetic factors
- The polymerase chain reaction and real-time PCR: principles and fields of application
- Genotyping of mutations and polymorphisms by RFLP, allele specific PCR, DNA sequencing and primer extension
- Genetic engineering and its biomedical importance (transgenic animals; knock-out, knock-in and knock-down techniques; cloning)
II. Transcription and its regulation
- Structure and function of the prokaryotic RNA polymerase holoenzyme. Transcriptional initiation and termination in bacteria; the prokaryotic transcription unit
- The different types of RNA and their biological importance. Synthesis and maturation of rRNAs and tRNAs
- Regulation of transcription in prokaryotes. Strong and weak promoters, constitutive genes, operons, positive and negative regulation
- Structure of eukaryotic genes. Transcriptional initiation and termination in eukaryotic cells
- Regulation of transcription in eukaryotes. Specific transcription factors, cis- and trans-acting regulatory sequences, coactivators, corepressors
- Processing of eukaryotic mRNAs
- Regulation of eukaryotic gene expression at post-transcriptional levels
- Regulation of eukaryotic gene expression through RNA interference (miRNA, siRNA)
- Epigenetic regulation of eukaryotic transcription: the role of DNA methylation and histone modifications
- Regulation of mRNA stability
- DNA-binding proteins and their characteristic structural elements in prokaryotes (helix-turn-helix) and eukaryotes (histone fold, helix-turn-helix, zinc finger, leucine zipper) with examples
- Structure and function of nuclear receptors (steroid, thyroid, aryl hydrocarbon receptor)
- Methods for studying gene expression (real-time PCR, DNA microarrays, reporter genes)
III. Translation, protein degradation and viruses
- The genetic code. Structure and function of tRNAs; aminoacyl-tRNA synthetases; the codon-anticodon hybridization
- The structure of prokaryotic and eukaryotic ribosomes. The ribosome cycle. tRNA binding to ribosomes
- Initiation of translation in pro- and eukaryotic cells. Regulation of eukaryotic translation via phosphorylation of eIF2α
- Elongation and termination of pro- and eukaryotic translation. Pharmacological inhibitors of translation
- Post-translational modifications of proteins
- Folding and quality control of proteins
- Intracellular protein degradation. Proteostasis
- The structure and function of proteasomes and the immunoproteasome. Proteasome inhibitors. Function of the TAP complex. The mechanism of ER-associated degradation (ERAD)
- The different types of autophagy. The role of lysosomes. Microautophagy, chaperone mediated autophagy
- Macroautophagy. Relationships between cellular metabolism and autophagy
- Lytic and lysogenic cycles of bacteriophages. The role of the phage repressor
- Classification of animal viruses according to replication mechanisms. Structure and replication of retroviruses
- Production of recombinant DNA by cloning. Fields of application
- Principles of human gene therapy (in vivo vs. ex vivo; viral vs. non-viral; gene augmentation; targeted genome editing with the CRISPR/Cas9 system)
IV. Lab topics
- Reversible and irreversible precipitation of proteins
- Color reactions of proteins: the xanthoprotein, Millon and Adamkiewicz reactions
- Quantitative determination of proteins: the biuret reaction. Determination of SH-groups: the Ellman’s reaction.
- Gel filtration: separation of proteins from low molecular weight substances
- Analysis of proteins by SDS-polyacrylamide gel electrophoresis. Principles of western blotting
- Induction of β-galactosidase in E. coli
- Production of green fluorescent protein by in vitro translation (For EM students only)
- Restriction digestion and gel electrophoresis of a reporter vector
- Biomedical application of bioinformatic methods (sequence databases, identification of genetic polymorphisms, primer design, restriction pattern analysis)
- Genotyping of a taste receptor SNP by PCR-RFLP
- Detection of marker enzymes in subcellular fractions