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Molecular Cell Biology I

Faculty of Medicine
6 credit points, terminal examination, Gábor Bánhegyi; AOKOVM464_1A

2016/2017 Spring semester

Teaching secretary: Gergely Keszler

Intro


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 as well as our current understanding of the regulation of gene expression, followed by an up-to-date summary of current methods 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, nonsense mutations, mismatch repair. Coordination of repair and replication. Transcription in procaryotes. Structure of RNA; t-RNA, r-RNA, m-RNA, differences between the procaryotic and eucaryotic 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. Mobile genetic elements 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 chromatine structure; covalent and non-covalent chromatin modifying activities and DNA methylation (epigenetics).  Post-transcriptional regulation in eukaryotes. Regulation of  mRNA stability; microRNAs. 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 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. 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). Application of bioinformatics and systems biology in biological and medical research.

Requirements for acknowledgement of the semester

  1. Participation in the laboratory practicals, consultations and seminars is obligatory; students have to sign the attendance sheets at the end of every lesson. In case of more than three absences from the practical lessons 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, consultations and seminars can be downloaded from the official homepage of the Institute.
  2. The oral midterm examination (week 8) has to be passed before the commencement of the examination period. The material of the midterm examination corresponds to that of lectures of weeks 1-7 (see the Midterm topic list on the website). Failed midterms can be retaken twice. Dates of retakes will be disclosed in due course.

Lab test and exam bonus
A written test will be held in week 12 or 13 of the semester (see the schedule of labs for details). Students are supposed to answer two lab topics 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 (semifinal topic list: group 4, # 41–50) on the semifinal exam.

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.

Recommended textbooks
Lodish: Molecular Cell Biology (8th edition)
Hrabák: Laboratory Manual – Medical Chemistry and Biochemistry

Lectures


Location: EOK (Tűzoltó u. 37-47.) Szent-Györgyi Lecture Hall
Duration: 55/60 min (Wednesdays 16:10-17:05 & Thursdays 14:50-15:50)
WeekDateTopicLecturer
1.30 Jan - 3 FebEucaryotic and procaryotic cell, the genetic informationKeszler
Basic concepts of molecular cell biology, structure and function of nucleic acidsKeszler
2.6-10 FebThe chromosomes and the organization of DNA IKeszler
The chromosomes and the organization of DNA IIKeszler
3.13-17 FebThe replication, repair and recombination of DNA IRónai
The replication, repair and recombination of DNA IIRónai
4.20-24 FebTranscription ICsala
Transcription IICsala
5.27 Feb - 3 MarTranscription IIICsala
The regulation of gene expression, nuclear receptorsSipeki
6.6-10 MarTranscriptional factors, DNA-binding domainsSipeki
The genetic code and translationMészáros T.
7.13-17 MarPosttranslational modification of proteinsMészáros T.
8.20-24 MarFolding and quality controlMészáros T.
Proteostasis, the ubiquitin-proteasome systemSőti
9.27-31 MarThe types of autophagySőti
Viruses ICsala
10.3-7 AprViruses IICsala
Evolution of genes and the genomeNemoda
11.18-21 AprEpigeneticsNemoda
Methods in molecular biology IRónai
12.24-28 AprMethods in molecular biology IIRónai
13.2-5 MayMethods in molecular biology IIIRónai
Methods in molecular biology IVRónai
14.8-12 MayBioinformatics, systems biology IKapuy/Csermely
Bioinformatics, systems biology IIKapuy/Csermely

Labs/Seminars


Location: EOK (Tűzoltó u. 37-47.) 1st floor, corridor ‘D’
WeekDateLab / Consultation
1.30 Jan - 3 FebProteins I
2.6-10 FebProteins II
3.13-17 FebPaper and thin layer chromatography*
4.20-24 FebGel electrophoresis*
5.27 Feb - 3 MarDetermination of urease activity
6.6-10 Mar
(Friday between 11.00-13.00 is off)
β-Galactosidase
7.13-17 Mar
(Wednesday: holiday)
Molecular biology (Consultation)
8.20-24 MarMidterm (oral)
9.27-31 MarPlasmid digestion*
10.3-7 AprPCR-based genotyping I*
11.18-21 Apr
(Monday is off)
PCR-based genotyping II*
12.24-28 Apr
(Wednesday is off)
Biotechnology (Consultation)
13.2-5 May
(Mondayis off)
Written test
14.8-12 MayGel filtration

*The order of marked labs varies from group to group. See the detailed schedule.

Groups


GroupTeacherDayTime
EM/1SipekiTuesday10:45-14:00
EM/2VereczkeiTuesday10:45-14:00
EM/3Mészáros T.Tuesday10:45-14:00
EM/4SasváriTuesday10:45-14:00
EM/5SzeitnerMonday15:15-18:30
EM/6KeszlerMonday11:45-15:00
EM/7BőgelMonday11:45-15:00
EM/8TányaMonday15:15-18:30
EM/9KardonMonday11:45-15:00
EM/10LizákMonday11:45-15:00
EM/11MargittaiMonday15:15-18:30
EM/12HrabákFriday11:30-14:45
EM/13CsalaMonday13:20-16:35
EM/14StroeMonday15:15-18:30

Topics


I. DNA structure, replication and repair

  1. Comparison of pro- and eukaryotic cells: compartmentation and the role of the most important subcellular organelles
  2. The chemical structure of nucleotides. The primary structure of nucleic acids (DNA and RNAs)
  3. Condensation of the genetic material in pro- and eukaryotic cells. The role of topoisomerases and chromatin proteins
  4. The structure of human chromosomes and its alterations during the cell cycle
  5. The structure of the human genome: coding and gene regulatory sequences. Non-coding genomic sequences: introns, pseudogenes, repetitive sequences
  6. Principles of the semiconservative DNA replication: replication fork, leading and lagging strand synthesis
  7. Comparison of DNA replication in pro- and eukaryotic cells: principles, enzymes, proteins
  8. Telomeric repeat sequences and their maintenance
  9. Classification of DNA damages and repair systems. Repair of base deamination
  10. Formation and repair of thymine dimers. Mismatch repair
  11. Classification of point mutations. The origin of spontaneous point mutations. DNA polymorphisms. Possible effects of DNA sequence alterations on the corresponding mRNA and proteins 

II. Transcription and its regulation

  1. Structure and function of the prokaryotic RNA polymerase holoenzyme. Transcriptional initiation and termination in bacteria; the prokaryotic transcription unit
  2. The different types of RNA and their biological importance. Synthesis and maturation of rRNAs and tRNAs
  3. Regulation of transcription in prokaryotes. Strong and weak promoters, constitutive genes, operons, positive and negative regulation
  4. Structure of eukaryotic genes. Transcriptional initiation and termination in eukaryotic cells
  5. Regulation of transcription in eukaryotes. Specific transcription factors, cis- and trans-acting regulatory sequences, coactivators, corepressors
  6. Processing of eukaryotic mRNAs
  7. Regulation of eukaryotic gene expression at post-transcriptional levels
  8. Regulation of eukaryotic gene expression through RNA interference (miRNA, siRNA)
  9. Epigenetic regulation of eukaryotic transcription: the role of DNA methylation and histone modifications
  10. Regulation of mRNA stability
  11. 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
  12. Structure and function of nuclear receptors (steroid, thyroid, aryl hydrocarbon receptor)

III. Translation, protein degradation and viruses

  1. The genetic code. Structure and function of tRNAs; aminoacyl-tRNA synthetases; the codon-anticodon hybridization
  2. The structure of prokaryotic and eukaryotic ribosomes. The ribosome cycle. tRNA binding to ribosomes
  3. Initiation of translation in pro- and eukaryotic cells. Regulation of eukaryotic translation via phosphorylation of eIF2α
  4. Elongation and termination of pro- and eukaryotic translation. Pharmacological inhibitors of translation
  5. Post-translational modifications of proteins
  6. Intracellular protein degradation. Proteostasis
  7. The structure and function of proteasomes and the immunoproteasome. Proteasome inhibitors. Function of the TAP complex. The mechanism of ER-associated degradation (ERAD)
  8. The different types of autophagy. The role of lysosomes. Microautophagy, chaperone mediated autophagy
  9. Macroautophagy. Relationships between cellular metabolism and autophagy
  10. Lytic and lysogenic cycles of bacteriophages. The role of the phage repressor
  11. Classification of animal viruses according to replication mechanisms. Structure and replication of retroviruses

IV. Recombinant DNA technology and labs

  1. Genetic variations in the human genome and their role in the pathogenesis of diseases. Methods to study genetic factors.
  2. The polymerase chain reaction and real-time PCR: principles and fields of application
  3. Genotyping of mutations and polymorphisms by RFLP, allele specific PCR, DNA sequencing and primer extension
  4. Methods for studying gene expression (real-time PCR, DNA microarrays, reporter genes)
  5. Production of recombinant DNA by cloning. Fields of application
  6. Systems biology and biological networks. The role of nodes and network connections in protein-protein interactions as well as in metabolic, signaling and gene expression networks of cells
  7. Reversible and irreversible precipitation of proteins
  8. Color reactions of proteins: xanthoprotein, Millon and Adamkiewicz reactions
  9. Quantitative determination of proteins: the biuret reaction. Determination of SH-groups: Ellman’s reaction.
  10. Gel filtration: separation of proteins from low molecular weight substances
  11. Separation of amino acids by means of paper and thin layer chromatography
  12. Analysis of serum proteins by SDS-polyacrylamide gel electrophoresis. Principles of western blotting
  13. Determination of the kinetic parameters (KM and vmax) of urease. Reversible and irreversible inhibition of the enzyme
  14. Induction of β-galactosidase in E. coli
  15. Restriction digestion and gel electrophoresis of the pGL3-Basic vector
  16. Genotyping of a taste receptor SNP by PCR-RFLP

Midterm


Topics for the midterm examination

  1. Comparison of pro- and eukaryotic cells: compartmentation and the role of the most important subcellular organelles
  2. The chemical structure of nucleotides. The primary structure of nucleic acids (DNA and RNAs)
  3. Condensation of the genetic material in pro- and eukaryotic cells. The role of topoisomerases and chromatin proteins
  4. The structure of human chromosomes and its alterations during the cell cycle
  5. The structure of the human genome: coding and gene regulatory sequences. Non-coding genomic sequences: introns, pseudogenes, repetitive sequences
  6. Principles of the semiconservative DNA replication: replication fork, leading and lagging strand synthesis
  7. Comparison of DNA replication in pro- and eukaryotic cells: principles, enzymes, proteins
  8. Telomeric repeat sequences and their maintenance
  9. Classification of DNA damages and repair systems. Repair of base deamination
  10. Formation and repair of thymine dimers. Mismatch repair
  11. Classification of point mutations. The origin of spontaneous point mutations. DNA polymorphisms. Possible effects of DNA sequence alterations on the corresponding mRNA and proteins 
  12. Structure and function of the prokaryotic RNA polymerase holoenzyme. Transcriptional initiation and termination in bacteria; the prokaryotic transcription unit
  13. Types of RNA and their biological importance. Synthesis and maturation of rRNAs and tRNAs
  14. Regulation of transcription in prokaryotes. Strong and weak promoters, constitutive genes, operons, positive and negative regulation
  15. Structure of eukaryotic genes. Transcriptional initiation and termination in eukaryotic cells
  16. Regulation of transcription in eukaryotes. Specific transcription factors, cis- and trans-acting regulatory sequences, coactivators, corepressors
  17. Processing of eukaryotic mRNAs
  18. Regulation of eukaryotic gene expression at post-transcriptional levels
  19. Post-transcriptional modification of eukaryotic mRNAs and regulation of their stability
  20. 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
  21. Structure and function of nuclear receptors (steroid, thyroid, aryl hydrocarbon receptor)
  22. The genetic code. Structure and function of tRNAs, aminoacyl-tRNA synthetases, the codon-anticodon hybridisation
  23. The structure of prokaryotic and eukaryotic ribosomes, the ribosome cycle, tRNA binding to ribosomes
  24. Initiation of translation in pro- and eukaryotic cells. Regulation of eukaryotic translation via phosphorylation of eIF2
  25. Elongation and termination of pro- and eukaryotic translation. Pharmacological inhibitors of translation

Lab Test


Topics for the written lab test

  1. Reversible precipitation of proteins
  2. Irreversible precipitation of proteins
  3. Color reactions of proteins: xanthoprotein, Millon and Adamkiewicz reactions
  4. Quantitative colorimetric determination of proteins: the biuret reaction
  5. Quantitative colorimetric determination of SH-groups in proteins: Ellman’s reaction
  6. Paper and thin layer chromatography of amino acids
  7. Principles of denaturing polyacrylamide gel electrophoresis of proteins
  8. Separation of plasma proteins by SDS-PAGE gel electrophoresis; in-gel protein staining and electroblotting
  9. Determination of kinetic parameters of urease
  10. Reversible and irreversible inhibition of urease activity
  11. Induction of β-galactosidase in E. coli and its inhibition by antibiotics
  12. Restriction digestion and gel electrophoresis of the pGL3-Basic vector
  13. Genotyping of a taste receptor SNP by PCR-RFLP I: isolation of genomic DNA and setting up the PCR reaction
  14. Genotyping of a taste receptor SNP by PCR-RFLP II: restriction digestion and gel electrophoresis of PCR products

Download


Lecture slides, seminar handouts, laboratory manuals are available here.
The password to download pages can be obtained from the laboratory teachers personally.