MOLECULAR CELL BIOLOGY (Syllabus)

University Medical School of Pécs 1999/2000

1. EDUCATIONAL OBJECTIVES Educational principles ∗ ∗ ∗ ∗

the course integrates molecular biology and cell biology medical aspects are emphasized experimantal approach is used to develop problem-solving skills continuous preparation for the classes is recommended

Forms of education lectures

∗ to emphasize the most important aspects ∗ lecture material is required for the exam ∗ not all topics are covered

seminars

∗ are compulsory (absences are recorded) ∗ seminar tests (every 3 weeks)

practicals

∗ are compulsory ∗ students are required to make up for missed lab programs ∗ 13 hours /of lab-seminar absences/ semester are tolerated

Examinations semester test

∗ on the last week of the semester ∗ multiple choice test: 40 traditional questions 40 application tests ∗ score: % of test + bonus points (max. 10)

semester exam

∗ successful test is accepted as exam ∗ oral exam: 3 questious from the list

final exam

∗ best students (3) are exempted ∗ complex exam: 2nd semester test (last week) MRT lab exam oral exam

Recommended books Cooper, G.M.: The Cell. A Molecular Approach Gelehrter, T.D., Collins, F.S.: Principles of Medical Genetics Szeberényi J.: Experiments in Molecular Cell Biology

Credit courses Szeberényi J.: Molecular Cell Biology Methods in Medicine (10 hours, 5 points, Semester 1) Komáromy L.: Pathology of Cell Nucleus (12 hours, 5 points, Semester 2) Kosztolányi Gy., Szeberényi J.: Molecular Medicine (28 hours, 12 points, Semester 2)

Office hours (J. Szeberényi) Mondays, 2-3 p.m.

2. COMPARISON OF PROKARYOTIC AND EUKARYOTIC CELLS (Cooper: pages 4-15) Cellular organisation prokaryotes no nucleus bacteria, cyanobacteria

eukaryotes contain nucleus unicellular or multicellular organisms

Genetic apparatus prokaryotes single circular chromosomal DNA molecule mostly coding regions coupled transcription-translation (chromosome-polysome complex) extrachromosomal DNA: plasmids F-factor (F+, F- cells) conjugation

eukaryotes several linear chromosomal DNA molecules in the form of chromatin mostly non-coding regions extrachromosomal DNA: mitochondrial DNA chloroplast DNA (plants)

Cellular organelles prokaryotes cell wall (peptidoglycan) cell membrane ribosomes no membrane-bounded organelles

eukaryotes membrane-bounded organelles (compartmentalization): nucleus, endoplasmic reticulum, Golgi complex, lysosomes, mitochondria, peroxisomes

Cytoskeleton, cell division prokaryotes no cytoskeleton no endocytosis, exocytosis no mitotic spindle cell division by fission

eukaryotes microfilaments, intermediate filaments, microtubules endocytosis, exocytosis, organelle movement mitotic spinde cell division: mitosis, meiosis

The origin of eukaryotic cells endosymbosis theory origin of peroxisomes, mitochondria, chloroplasts

3. NUCLEIC ACIDS (Cooper: pages 45-47, 87-94) General features linear macromolecules consist of nucleotides are capable to store and express genetic information nucleotides (= base + pentose + phosphate/s/) heterocyclic bases purines: adenine (A), guanine (G) pyrimidines: cytosine (C), thymine (T), uracil (U) pentose ribose, deoxyribose phosphoric acid nucleosides (= base + pentose) 3'5'-phosphodiester bond oligonucleotides, polynucleotides

DNA (= deoxyribonucleic acid) DNA is the genetic material bacterium transformation pneumococus: S variant, R variant phage infection transfection structure Watson-Crick model double helix sugar-phosphate backbone complementary base pairing antiparallel orientation denaturation, renaturation hyperchromicity, melting point (Tm ) B form, Z form circular, linear DNA superhelix DNP (= deoxyribonucleoprotein)

RNA (= ribonucleic acid) usually single-stranded selfcomplementary hairpins, stem-and-loop structures RNP (= ribonucleoprotein)

types of RNA mRNA (= messenger RNA) template of protein synthesis rRNAs (= ribosomal RNAs) components of ribosomes prokaryotes: 5S, 16S, 23S rRNAs higher eukaryotes: 5S, 5.8S,18S, 28S rRNAs tRNAs (= transfer RNAs) carry amino acids

pre-mRNA, pre-rRNA RNA precursors

Ribozymes catalytic RNAs functions: auto-splicing; RNA cleavage; peptide bond formation etc.

4. PROTEINS (Cooper: pages 48-61) General features linear, non-branching polypeptide chains consist of amino acids functional categories - enzymes - structural proteins (e.g. cytoskeleton proteins) - regulatory proteins (e.g. transcription factors) - transport proteins (e.g. importin) - signaling proteins (e.g. polipeptide hormones, adapter proteins) - proteins with defense function (e.g. antibodies, stress proteins) - receptor proteins (e.g. hormone receptors)

Amino acids general structure (Cα-atom, amino group, carboxyl group, R group) functional categories basic amino acids (e.g. arginine, lyine) acidic amino acids (e.g. aspartic acid, glutamic acid) uncharged polar amino acids (e.g. serine, tyrosine) nonpolar amino acids (e.g. leucine, valine) peptide bond formed between an amino and a carboxyl group (-CO-NH-) ends of a polypeptide chain: N-terminus, C-terminus

Protein structure primary structure = sequence of amino acids secondary structure α helix, β sheet stabilized by H bonds tertiery structure stabilized by ionic bonds, nonpolar bonds, van der Waals interactions, H bonds, disulfide bonds conformation of proteins structural domains chaperone proteins quaternery structure = subunit structure multimer complexes (e.g. replisome, transcriptosome, spliceosome etc.)

Enzymes = biocatalysts activation energy active center lock-and-key mechanism induced fit mechanism coenzymes

5. CARBOHYDRATES AND LIPIDS (Cooper: pages 39-45) Carbohydrates general features general formula: (CH2O)n polyhydroxi aldehydes or ketones

classification monosaccharides trioses: e.g. glyceraldehyde, dioxyacetone pentoses: e.g. ribose, deoxyribose hexoses: e.g. glucose, fructose, mannose, galactose disaccharides: e.g. sucrose (glucose + fructose), maltose (glucose + glucose), lactose (glucose + galactose) oligosaccharides polysaccharides cellulose linear glucose polysaccharide chaines component of plant cell wall starch amylose + amylopectin glucose polysaccharide storage form of glucose in plants glycogene glucose polysaccharide storage form of glucose in animals (mainly in liver and muscle) glycosaminoglycans consist of repeated disaccharide units contain hexose derivatives present in the extracellular matrix bind to proteins (proteoglycans) e.g. chondroitin sulfate, heparin, dermatane sulfate

Lipids general features highly hydrophobic compounds are souble in organic solvents

classification triglycerides glycerol + 3 fatty acids stored as cytoplasmic lipid droplets significance: energy storage phospholipids contain phosphoric acid amphipathic lipids: contain hydrophobic and hydrophylic regions significance: membrane components signal transduction sphyngomyelins, glycerophospholipids glycolipids contain sugar moieties → increased water-solubility present on the exoplasmic surface of membranes significance: serve as markers e.g. cerebrosides, gangliosides steroids sterane structure e.g. cholesterol (membrane component), steroid hormones, bile acids, vitmin D3 carotenoids pigments (conjugated double-bonds) e.g. β-carotene, retinal, retinoic acid

6. IN VIVO AND IN VITRO AMPLIFICATION OF DNA FRAGMENTS (Cooper: pages 102-109, 113-120) Restriction endonucleases

infection of E. coli cells by bacteriophage λ restriction, modification restriction endonucleases recognition site: palindromic sequence direct cleavage → blunt ends staggered cleavage → cohesive (sticky) ends modification methylases

Cloning of DNA fragments recombinant DNA techniqes vectors: plasmids λ phage cosmids adenoviruses, retroviruses (in mammals) yeast artifical chromosome (YAC) DNA ligase

Genomic libraries

constructed in insertion vectors (e.g. λ phage) main steps: genomic restriction fragments →ligation with λ arms → packing → infection of E. coli cells → screening of library (by molecular hybridization)

Polymerase chain reaction (PCR) in vitro DNA amplification DNA synthesizing mixtures: primers, dNTPs, template, Taq polymerase steps: denaturation → primer-binding → DNA synthesis thermocycler

LIST OF TOPICS (1999/2000, 1st semester) /Page numbers refer to Cooper's book/ pages Biological macromolecules Light and electron microscopy Pro- and eukaryotic cells Separation techniques

39-62 87-94 20-27 +lab manual 4-15 27-30 +lab manual

Methods of molecular biology I (restriction enzymes, cloning, genomic libraries, PCR, hybridization, Southern blotting, sequencing of DNA)

102-117 118-120

Methods of molecular biology II (cDNA libraries, gene transfer, transgenic animals, gene targeting, antisense techniques, Northern blotting, immunocytochemistry, immunoprecipitation, Western blotting)

117-118 120-129

T he cell nucleus

315-331

Genome organisation

135-143 211-220

Chromatin

144-152

T he cell cycle

561-577

DNA replication

94-95 175-190

DNA repair

190-199

Mitosis

336-342 577-583

T ranscription

331-336 225-229 235-240

RNA processing

256-267

T he pathology of cell nucleus lecture T ranslation Regulation of gene expression

95-98 273-288 229-235 240-256 288-290 300-308

300-308 Rough endoplasmic reticulum

290-300 347-359

Golgi complex

365-373

Vesicular transport

373-379

Mitochondria

389-404

Lysosomes, smooth endoplasmic reticulum

359-365 379-384

Oxygene free radicals, membrane damage lecture

LIST OF TOPICS IN MOLECULAR CELL BIOLOGY (1999/2000, 2nd semester) Cooper T he cell membrane

467-476 509-513

T ransport

476-500

Extracellular matrix

504-509

Cytoskeleton

423-458

Signal transduction

521-553

Developmental biology T he tumor cell

553-555 589-592 + lecture 604-608

T umor viruses

608-612

Retroviral oncogenes, proto-oncogenes

612-616

Cellular oncogenes

616-622

T umor suppressor genes

623-629

Oncogenes and the cell cycle

568-577 + lecture

Apoptosis

592-593 + lecture

Multistage mechanism of carcinogenesis

599-603 + lecture Gelehrter-Collins (1990)

Cytogenetics

159-189

Inheritance in families

27-45 57-65 97-107 Cooper

Molecular diagnostics

629-630 + lecture

Gene therapy

630-632 + lecture

7. DNA SEQUENCE ANALYSIS (Cooper: pages 102-105,109-111, 115, 162-163) Molecular hybridization denaturation - renaturation labelled probe filter hybridization in situ hybridization fluorescence in situ hybridization (FISH)

Restriction mapping by

digestion of DNA with restriction endonucleases (RE) → fractionation of DNA fragments gel electronphoresis → size determination → mapping of RE-sites

Southern blotting to identify DNA fragments carrying specific sequences RE digestion of DNA → agarose gel electrophoresis → denaturation → blotting → hybridization of the membrane → autoradiography

DNA sequencing Maxam-Gilbert method (chemical sequencing)

base-specific cleavage of DNA → size determination by PAGE → DNA sequence Sanger method (dideoxy sequencing, chain termination method) in vitro DNA synthesizing mixture synthetic oligonucleotide primers dideoxyribonucleoside triphosphates (ddATP, ddTTP, ddCTP, ddGTP) are used to terminate reactions size determination by denaturing PAGE can be automated

DNA microchips = sequencing by hybridization microchip with oligonucleotides of known sequences → hybridization with fluorescent target nucleic acid → confocal microscope → computer → sequence is determined

Human genome project to sequence the entire human genome launched in 1990 expected to be finished in 15 years

8. EXPRESSION OF FOREIGN GENES (Cooper: pages 111-112, 120-125) cDNA cloning cDNA = DNA complementary to a specific RNA molecule synthesis of cDNA mRNA template + oligo(dT) primer + reverse transcriptase + dNTPs cDNA cloning double stranded cDNA → ligation of sticky ends → ligation into a cloning vector insertion vectors, expression vectors baculovirus vectors

Gene transfer into eukaryotic cells physical techniques microinjection electroporation chemical methods transfection liposome-mediated gene transfer biological methods = virus vectors retroviruses, adenoviruses etc. transient vs. stable expression

Transgenic organisms = carry the foreign gene (transgene) in all their diploid cells introduction of the transgene - microinjection of the transgene into fertilized eggs - gene transfer into embryonal stem cells → injection into blastocysts → chimera embryos → breeding → real transgenic animals

9. INHIBITION OF ENDOGENOUS GENE EXPRESSION (Cooper: pages 125-129) Targeted gene disruption = K.O. mutation K.O.vector neor gene - geneticin selection tk (= thymidine kinase) gene - gancyclovir selection random integration homologous recombination

Antisense oligonucleotides = complementary to a region of target RNA

Ribozymes = complementary to a region of target RNA + endonuclease activity

Inhibition of protein function - microinjection of specific antibody - expression of specific intracellular antibody - expression of dominant inhibitory protein - peptidomimetics - competitive inhibitors

10. IDENTIFICATION OF SPECIFIC GENE PRODUCTS (Cooper: pages 114-120) cDNA library

total mRNA (purified by oligo(dT)-cellulose chromatography) → cDNA synthesis → ligating into a cloning vector → transfer into host cells → screening of the library expression cDNA library immunoscreening

Northern blotting = identification of a specific RNA in a complex RNA mixture formaldehyde/agarose gel electrophoresis → blotting → hybridization → autoradiography

Immunological techniques = identify specific proteins using their antibodies

immunocytochemistry immunofluorescence microscopy antibody labelled with a fluorescent dye visualized in a fluorescence microscope (confocal microscope) immunogold technique for immune electron microscopy antibody labelled with gold particles

immunoprecipitation

cell extract → mixed with antibody-covered agarose beads → centrifugation → SDSPAGE → visualization of protein (e.g. autoradiography)

immunoblotting (Western blotting)

protein mixture → SDS-PAGE → blotting → treatment of membrane with specific antibody → visualization of antibody (e.g. with color reaction)

11. THE CELL NUCLEUS (Cooper: pages 114-120) Ultrastrastructure nuclear membrane chromatin nuclear matrix nucleolus

Nuclear membrane onter membrane, inner membrane, perinuclear space nuclear lamina lamin proteins

nuclear pore complex nucleoporins cytoplasmic filaments nuclear basket central plug

nucleocytoplasmic transport protein import nuclear proteins: nuclear localization signal shuttling carrier proteins e.g. importins Ran•GDP, Ran•GTP RNA export proteins with nuclear export signal

Chromatin types: heterochromatin -constitutive - facultative perinucleolar chromatin peripheral chromatin transcriptionally inactive euchromatin transcriptionally active

Nuclear matrix protein filament network

12. EUKARYOTIC GENOME ORGANISATION (Cooper: pages 135-143, 211-220) DNA renaturation experiment

genomic DNA → sheared by sonication → heat denaturation → slow cooling → renaturation → measuring the rate of renaturation E.coli DNA: mostly unique sequences mammalian DNA: ~ 60% unique sequences ~ 40% repetitive sequences

Satellite DNA highly repetitive DNA: short, tandemly repeated sequences can be separated from the bulk of the DNA by isopycnic gradient centrifugation present in centromeric, telomeric DNA it is not transcribed minisatellites: short repeats highly polymorphic → VNTR (variable number of tandem repeats) DNA fingerprinting microsatellites: very short repeats (e.g. trinucleotide repeats) genetic instability → dynamic mutation → trinucleotide repeat expansion e.g. fragile X syndrome Huntington disease

Interspersed repeats copies spread all over the genome e.g. Alu family

Tandemly repeated genes identical gene copies present in the same region separated by spacers e.g. 45S pre-rRNA genes histone genes gene amplification e.g. rRNA genes

Gene families similar, but not identical copies e.g. globin gene family (α- and β-globin genes) pseudogenes

Unique sequences present in 1 copy/haploid genome e.g. insulin gene

Mobile genetic elements move from one site to another in the genome

transposons transposition - replicative - non-replicative transposase

retrotransposons transposition via an RNA intermediate viral retrotransposons endogenous retroviruses nonviral retrotransposons e.g. Alu sequences

13. CHROMATIN (Cooper: pages 144-152) Chromatin organisation levels of morphological organisation - DNA double helix - beads-on-a-string nucleosomes histone octamer linker DNA H1 histone chromatosome = octamer + H1 - solenoid 6 nucleosomes/turn - looped domains stabilized by chromosome scaffold - metaphase chromosome highly condensed

Chemical composition DNA, histones, nonhistone proteins (RNA, inorganic ions)

histones small, basic proteins rich in lysine, arginine nucleosomal histones form the octamer H2A, H2B, H3, H4 are highly conserved H1 linker histone induces solenoid formation chemical modifications phosphorylation acetylation function: structural elements regulation of gene expression

nonhistone proteins structural heterogenity hundreds of species tissue-specific expression chemical modification phosphorylation e.g. lamins transcription factors functional diversity - structural proteins (e.g. lamins) - enzymes (e.g. DNA, RNA polymerases) - transcription factors - receptor proteins (e.g. steroid receptors) - transport proteins (e.g. importin) - chaperones (e.g. nucleoplasmin)

14. PHASES OF THE CELL CYCLE (Cooper: pages 561-564) Interphase and mitosis interphase G 1 phase diploid (2n) DNA content G0 phase: quiescent cell S phase DNA replication histone synthesis centriole duplication G 2 phase tetraploid (4n) DNA content

Synchronisation of cell cultures - serum starvation = withdrawal of growth factors - mitotic shake-off - colchicine inhibits microtubule polymerisation - 5-fluorodeoxyuridine inhibits DNA replication - FACS (= fluorescence-activated cell sorter) based on flow cytometry

Duration of phases generation time = time required for doubling of cell number S phase [3H]thymidine pulse labelling → autoradiography → % of cells in S phase → duration of S phase M phase % of mitotic cells (= mitotic index) → duration of M phase G 1 phase synchronisation by mitotic shake-off → [3H]thymidine labelling → autoradiography → first labelled nuclei G 2 phase nonsynchronized culture → [3H]thymidine labelling → first labelled chromosomes

15. REGULATION OF THE CELL CYCLE (Cooper: pages 564-577) Methods to analyze cell cycle regulation genetic studies in yeast heat-sensitive cdc-mutants (= cell division cycle) permissive and nonpermissive temperature microinjection of frog oocytes with: specific proteins, mRNAs, antisense oligos studies with frog oocyte extracts to induce division of added nuclei cell fusion experiments hybrid cells S + G1 cell → replication in G1 nucleus → S phase promotion factor (SPF) G 1 + G2 cell → no replication S + G2 cell → no replication in G2 cell → re-replication block M + interphase cell → chromatin condensation in interphase nucleus → M phase promotion factor (MPF)

Cyclin/Cdk complexes endogenous regulators heterodimers: cyclin - regulatory subunit Cdk (= cyclin-dependent kinase) - catalytic subunit : protein kinase G1/S transition restriction point SPF = G1 cyclin/Cdk complex re-replication block initiation of replication: origo recognition complex + licensing factor G2/M transition MPF = mitotic cyclin/Cdk complex degradation of cyclin in M phase → completion of mitosis regulation of Cdk activity - cyclin synthesis/degradation - Cdk phosphorylation/dephosphorylation - Cdk-inhibitors

Checkpoints in cell cycle regulation G 1 checkpoint DNA damage → p53 tumor suppressor protein ↑ → Cdk inhibitor ↑ → arrest in G1 G 2 checkpoint unreplicated DNA → no MPF activation → no entry into mitosis → arrest in G2 M checkpoint abnormal mitotic spindle → no cyclin degradation → arrest in M phase

16. MITOSIS (Cooper: pages 336-342, 577-583) Eukaryotic cell division chromosomes are formed mitosis: production of somatic cells meiosis: production of germ cells

Interphase G1-S-G2

mitotic apparatus microtubule organizing center (MTOC) or centrosome consists of 2 centrioles and pericentriolar material duplicates in the S phase produces the mitotic spindle

Phases of the mitotic division Prophase

MPF activation → H1 phosphorylation → chromatin condensation → lamin phosphorylation → breakdown of nuclear envelope → cytoskeletal protein phosphorylation → mitotic spindle disappearance of nucleolus fragmentation of endoplasmic reticulum, Golgi complex

Metaphase mitotic spindle - kinetochor microtubules - polar microtubules - astral microtubules

Anaphase chromatid segregation and migration anaphase A shortening of kinetochor microtubules → migration of chromatids anaphase B sliding of polar microtubules → elongation of the cell

Telophase

MPF degradation →chromosome decondensation → nuclear membrane reformation → reappearance of endoplasmic reticulum and Golgi complex karyokinesis

Cytokinesis = division of cytoplasm microfilaments → contractile ring

17. DNA REPLICATION GENERAL FEATURES (Cooper: pages 94-95) Methods to study replication in vitro methods cell-free replication (Kornberg system) - template DNA - DNA polymerase - dNTPs (at least one radioactively labelled) - ions, pH etc. TCA filter precipitation liquid scintillation counting in vivo methods - density labelling (e.g. 15NH 4Cl) - radioactive labelling (e.g. [3H]thymidine) - bromodeoxyuridine labelling → anti-BrdU cytometry

→

immunocytochemistry,

Replication is semiconservative Meselson-Stahl experiment density labelling with 15NH4Cl

Replication is template- and primer-dependent template-strand determines the nucleotide sequence of newly synthesized DNA by complementary basepairing primer complementary to template strand provides free 3'-OH group for DNA polymerase

DNA replication is bidirectional starts at ori ( = origine of replication) replication bubble, replication forks fiber autoradiography

Replication is semidiscontinuous antiparallel structure of template direction of elongation: 5'→ 3' leading strand, lagging strand Okazaki fragments

flow

18. THE MECHANISM OF DNA REPLICATION (Cooper: pages 175-190) DNA replication in E. coli is performed by a multienzyme complex (replisome) origo recognition complex ori contains short repeats binds specific proteins (e.g. initiator protein) topoisomerase I reversible endonuclease that cuts one DNA strand induces superhelix relaxation DNA helicase cleaves H-bonds → unwinding of double helix Ssb proteins = single-strand binding proteins prevent renaturation of template primase special RNA polymerase generates primers is present in primosomes with other protein (e.g. helicase) DNA polymerase III holoenzyme core enzyme dimer accessory proteins γ-subunit fix core polymerase on the leading strand β-subunit → high processivity 5' → 3' elongation activity 3' → 5' exonuclease activity - proofreading DNA polymerase I 5' → 3' exonuclease activity → removes primers 5' → 3' elongation activity → fills the gaps DNA ligase links Okazaki fragments to each other topoisomerase II reversible endonuclease → cuts both strands → - separation of daughter molecules - untangling - superspiralisation

Eukaryotic DNA replication multiple origins ~ 104/genome replicon DNA polimerases α, δ, ε - nuclear replicative ploymerases β - repair enzyme γ - mitochondrial DNA polymerase replication in chromatin histone synthesis in S phase telomer replication tandem repeats telomerase - RNA template reverse transcriptase

19. DNA REPAIR (Cooper: pages 190-199) DNA damage various types - replication errors → mismatch - base deamination → abnormal base - depurination → AP (apurin) -site - chemical mutagens → base-methylation, covalent adducts, crosslinking etc. - UV radiation → pyrimidine dimers - ionizing radiation → strandbreaks

Direct eliminiation of DNA damage e.g. photolyase cleaves pyrimidine dimers in bacteria

Excision repair = excision of the damaged region + substitution by newly synthesized DNA

base excision repair = removal and substitution of damaged bases enzymes involved - DNA glycosylase → removes damaged base → AP site - AP endonuclease → cleaves at AP site - DNA polymerase → fills gap - DNA ligase → seals nick

nucleotide excision repair = removal of larger damaged region (e.g. thymine dimer, covalent adducts) enzymes involved - damage recognition proteins - excinuclease → cleaves on both sides of the damage - helicase → removes damaged strand - DNA polymerase → fills gap - DNA ligase → seals nick xeroderma pigmentosum autosomal recessive inheritance mutation in one of the nucleotide excision repair genes (XPA-XPG)

mismatch repair = removal of non-complementary nucleotide enzymes involved - recognition proteins → bind to non-complementary base in nonmethylated strand - endonuclease → cuts DNA strand - helicase → unwinds double helix - exonuclease → removes region of abnormal nucleotide - DNA polymerase → fills gap - DNA ligase → seals nick hereditary nonpolyposis colon cancer (HNPCC) autosomal recessive disease mutation in a mismatch repair gene → increased rate of point muttaions, microsatellite instability → increased risk of colon cancer

20. GENERAL FEATURES OF TRANSCRIPTION AND RNA PROCESSING (Cooper: pages 225-229) DNA template → transcription → primary RNA transcript → RNA processing → mature RNA

Methods to study transcription in vitro methods cell-free RNA synthesizing mixture: template DNA 4 dNTPs (one labelled) RNA polymerase ions, pH etc. filter precipitation → radioactivity measurement in vivo methods 3 H-uridine labelling → autoradiography bromouridine labelling → cytochemistry with anti-BrU antibody

Transcription and RNA processing in prokaryotes general features of RNA synthesis promoter, terminator, transcription unit coupled transcription- translation (chromosome-polysome complex)

mechanism of prokaryotic transcription RNA polymerase holoenzyme = σ factor + core polymerase (α2ββ') steps: - initiation promoter: - 35 site; -10 site (Pribnow box) closed and open initiation complex - elongation core polymerase formation of phosphodiester bonds 5' → 3' direction triphosphate end - termination ζ-factor ζ-dependent or ζ-independent termination

RNA processing characteristic of tRNA and rRNA - nucleolytic cleavages (endonucleases, exonucleases) - nucleotide addition (e.g. tRNA) - nucleoside modification (e.g. methylation)

General features of transcription in eukaryotes - takes place in chromatin - RNA polymerases I product

pre-rRNA

location

nucleolus

α -amanitin sensitivity

none

II pre-mRNA snRNAs

extranucleolar high

- transcription and translation separated by nuclear membrane - nucleocytoplasmic transport of RNA

III 5S rRNA tRNAs snRNAs chromatin moderate

21. RIBOSOME BIOGENESIS IN EUKARYOTES (Cooper: pages 332-336) ribosomes: sites of protein synthesis are assembled in the nucleolus

Nucleolus is formed around the rDNA repeats (= tandem rRNA genes) electron microscopic structure - perinucleolar chromatin - fibrillar centers lightest regions nucleolar organizing regions sites of pre-rRNA synthesis - fibrillar component darkest regions contains pre-rRNP - granular component contains preribosomal particles

Ribosome biogenesis chromatin spreading

→ visualization of genes in action "Christmas trees" sites of initiation and termination transcribed and non-transcribed spacers RNA polymerase I

rRNA transcription units tandem repeats primary transcript: 45S pre-rRNA → processed to 18S, 5.8S and 28S rRNAs 5S rRNA genes outside the nucleolus transcribed by RNA polymerase III snoRNAs = small nucleolar RNAs bind to pre-rRNA → act as RNA chaperones

assembly of ribosomes 80S particle = 45S pre-rRNA + snoRNAs + proteins 30S preribosomal particle = immature large subunit 40S preribosomal particle = immature small subunit final maturation in the cytoplasm

22. SYNTHESIS OF PRE-mRNA, CAP FORMATION AND POLYADENYLATION (Cooper: pages 235-240, 256-259) pre-mRNA = hnRNA (heterogenous nuclear RNA) synthesis of pre-mRNA - initiation - elongation - termination posttranscriptional modifications - cap formation - polyadenylation - splicing

Initiation of pre-mRNA synthesis promoter: - core promoter elements TATA box initiator region - enhancer elements RNA polymerase II core polymerase + initiation factors = holoenzyme histone acetyltransferase helicase subunit

Elongation promoter clearance pol II elongation complex

Formation of 5'-cap at the triphosphate end enzymes involved: - nucleotide phosphohydrolase - guanyl transferase - methyl transferase

Termination and polyadenylation poly(A)-signal poly(A) polymerase poly(A) tail

23. PRE-mRNA SPLICING (Cooper: pages 259-266) many eukaryotic protein-coding genes are discontinuous: introns + exons splicing = removal of introns from pre-mRNA → joining of exons

Splicing of adenovirus late mRNAs adenoviruses linear, dsDNA genome early region → early proteins late region → late proteins (virion proteins) complex transcriptional unit single promoter alternative splicing 5 alternative poly(A) signals exons (leader sequences, body sequence) and introns

Mechanism of splicing 5`-splice site, 3`-splice site spliceosome pre-mRNA + snRNAs + proteins U1, U2, U4/U6, U5 snRNAs lariat formation

Abnormalities of splicing systemic lupus erythematosus (SLE) autoimmune disease anti-snRNP antibodies thalassaemia reduced α- or β-globin synthesis sometimes caused by splice site mutation

24. THE PATHOLOGY OF CELL NUCLEUS

Objects for investigations - pathologically altered tissues, cells from the body - experimental pathology: treatment with injuring substances, chemicals, etc. pathological tissues, cell models of pathological alterations and changes reversible - irreversible changes

Main types of pathological alterations 1. Chromatin - earliest change is a reversible clumping - chromatin aggregates attached * to the nuclear membrane (chromatin margination) * to the nucleolus degenerative changes karyopyknosis (nuclear pyknosis) - nucleus progressively shrinks LM: increased basophilic properties (basic dyes → basophilic, haematoxylin) EM: homogenous, dense chromatin karyolysis - LM: basophilic properties decreasing - EM: "empty nucleus" - hydrolytic action of DNAse (pH is acidic) nucleus disappears - karyorrhexis - chromatin (nucleus) breaks up into many clumps - chromatin fragments in the cytoplasm (final step - karyolysis)

2. Nucleolar changes a. nucleolar hypertrophy - enlargement of nucleolar volume - increased nucleolar RNA synthesis - in tumor cells - increased activity of protein synthesis - in active normal cells (e.g. regenerating liver after partial hepatectomy) b. nucleolar segregation - separation of nucleolar F and G components - experimental pathology: inhibition of RNA polymerase by actinomycin D: segregation of nucleolus - cytotoxic → cytostatic chemotherapy

3. Alterations of nuclear envelope 4. Inclusions in the nucleus

25. THE ROLE OF mRNA, tRNAs AND RIBOSOMES IN PROTEIN SYNTHESIS (Cooper: pages 273-279)

mRNA is the template of protein synthesis mRNA = messenger RNA determines the amino acid sequence of the protein

tRNAs as adapters tRNA = transfer RNA carries amino acids structure of tRNA cloverleaf model aminoacid binding site at 3`-end (CCA) TφCG arm ribosome binding anticodon loop codon binding D loop binding of aminoacyl-tRNA synthase synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthases step 1 - activation of amino acid amino acid + ATP → aminoacyl-AMP + pyrophosphate step 2 - formation of aminoacyl-tRNA aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP ribosomes and translation prokaryotes 30S small subunit + 50S large subunit = 70S monomer eukaryotes 40S small subunit + 60S large subunit = 80S monomer

Methods to study translation in vitro techniques cell-free system (Nirenberg system) cell extract (ribosomes, mRNA, tRNAs, protein factors) 20 amino acids (one labelled) ATP, GTP ions, pH detection: - filter precipitation - PAGE in vivo techniques - isolation of polysomes - radioactive labelling (e.g. 3H-leucine) - autoradiography - PAGE - immunoprecipitation

26. THE GENETIC CODE (Cooper: pages 96-99)

Deciphering the genetic code in vitro translation of synthetic polynucleotides monoton polynucleotides, copolymers → used as templates in Nirenberg-mixtures aminoacyl-tRNA binding assay Nirenberg-system → omission of GTP → aa-tRNA binding, but no protein synthesis → filter binding

Features of the genetic code triplet code 61 sense codons → amino acids 3 nonsense codons → stopcodons the code is redundant (degenerate) = one amino acid can be coded by more than one codons wobble = uncertainty at the third codon position the code is unambiguous = a codon codes for a single amino acid the code is continuous = no punctuations, no overlapping open reading frame (ORF) monocystronic and polycystronic mRNAs the code is universal = highly conserved in the living world exceptions (e.g. mitochondrial genetic apparatus)

27. THE MECHANISM OF PROTEIN SYNTHESIS (Cooper: pages 273-288)

Translation in prokaryotes initiation initiation codon (AUG or GUG) → fMet-tRNA Shine-Dalgarno sequence → ribosome binding initiation factors 30S initiation complex = mRNA + 30S subunit + fMet-tRNA + initiation factors 70S initiation complex = 30S initiation complex + 50S subunit P site: fMet-tRNA A site: empty elongation Aa-tRNA binding with the help of EF-Tu • GTP to A site peptide bond formation peptidyl transferase (ribozyme) translocation with the help of EF-G • GTP termination stop codon releasing factors

Unique features of eukaryotic translation 5`-cap

→ ribosome binding ribosomes - free ribosomes → proteins of cytosol, nucleus, mitochondria - bound ribosomes → secretory proteins, proteins of endoplasmic reticulum, Golgi complex, lysosomes, cell membrane

General features of translation

- direction: 5`end → 3`end N terminus → C terminus - polysomes are formed - one polypeptide chain/ribosome - requires 4 high energy bonds/peptide bonds (ATP → AMP, 2GTP → 2GDP)

Inhibitors of protein synthesis chloramphenicol ƒƒ¥ peptidyl transferase erythromycin ƒƒ¥ translocation tetracyclin ƒƒ¥ aa-tRNA binding streptomycin ƒƒ¥ 30S subunit puromycin → early termination

28. THE OPERON MODEL (Cooper: pages 229-235)

Gene expression - constitutive - regulated - induction - repression

Components of gene regulation DNA sequence elements cis-acting elements regulatory proteins bind to DNA repressors or activators trans-acting elements effector molecules small molecules bind to regulatory proteins inducers or corepressors

Inducible operons code for catabolic enzymes operon = promoter + operator + structural gene(s)

lactose operon - regulation by lactose no lactose → lac repressor binds to operator → blocks RNA polymerase function → no expression lactose → binds to repressor → displaces it from operator → expression of operon is possible (lactose acts as inducer) - regulation by glucose no glucose → high cAMP level → CAP (= catabolite activator protein) is activated → binds to lac promoter → helps the binding of RNA polymerase → expression of lac operon

Repressible operons code for biosynthetic enzymes

tryptophan operon polymerase

no tryptophan → operator is not blocked → operon is expressed tryptophan → binds to trp repressor → binds to operator → blocks RNA → no expression (tryptophan acts as corepressor)

29. MECHANISMS OF GENE REGULATION IN EUKARYOTES (Cooper: pages 240-246, 252-256, 288-290, 300-308)

Gene regulation in development Gurdon experiment

enucleated frog oocytes → microinjected with nuclei from tadpole intestine cells → some developed into normal frogs genome equivalence housekeeping genes tissue-specific genes

nuclear transplantation in mammals 1997: Dolly the lamb enucleated oocyte → transfer of the nucleus of a mature udder cell → Dolly

Levels of gene regulation in eukaryotes transcription - structure of chromatin H1 phosphorylation role of non-histone proteins histone acetylation - DNA methylation → gene inactivation genomic imprinting - transcription factors

pre-mRNA processing - alternative splicing = one pre-mRNA → several mRNAs - RNA editing = deletion, addition or substitution of one nucleotide in mRNA

RNA transport nucleocytoplasmic RNA export can be regulated

mRNA degradation role of - 5′-cap poly(A) tail 3′-nontranslated region

translation e.g. phosphorylation of initiation factors

protein degradation - lysosomal proteolysis autophagosomes → phagolysosomes - ubiquitin-proteasome pathway proteins with "destruction box" → ubiquitination → degradation by proteasomes

protein function - allosteric regulation by small molecules e.g. cAMP, GTP etc. - covalent modification e.g. phosphorylation protein kinases and phosphatases - protein-protein interactions e.g. cyclin/Cdk complex

30. TRANSCRIPTION FACTORS (Cooper: pages 246-252)

Transcription factors (TF)

= DNA-binding proteins that regulate transcription basal TFs → promoter elements regulatory TFs → enhancer elements

Transcription factor families domain structure: - DNA binding domain - activation domain

helix-turn-helix proteins e.g. homeotic proteins coded by homeotic genes contain a homeodomain (coded by a homeobox region)

zinc finger proteins bind as dimers e.g. steroid receptors

amphipathic helix proteins dimerization domain forms a leucine zipper leucine zipper proteins e.g. AP1 factor (= Fos/Jun dimer) helix-loop-helix (HLH) proteins e.g. MyoD protein

Steroid receptor superfamily steroids bind to intracellular receptors e.g. steroid hormones, vitamin D3, retinoic acid

glucorticoid action

glucocorticoid → binds to cytosolic receptor → inhibitory chaperone is displaced → hormone-receptor complex translocates to nucleus → binds to glucocorticoid response element → gene activation

Transcription factor diseases caused by TF-mutations

inborn errors of development combined pituitary hormone deficiency caused by mutation in the Pit-1 TF pituitary dwarfism, mental retardation

endocrine syndromes testicular feminisation no functional androgen-receptor → male characteristics do not develop vitamin D resistant rickets

tumors oncogenic TFs e.g. Myc, Fos, Jun tumor suppressor proteins e.g. p53, WT-1

31. SYNTHESIS OF LIPIDS AND PROTEINS IN THE ENDOPLASMIC RETICULUM

(Cooper: pages 347-365) Endoplasmic reticulum rough and smooth endoplasmic reticulum microsome fraction cytoplasmic and exoplasmic surface

Protein synthesis on the rough endoplasmic reticulum free and bound ribosomes cotranslational transport of proteins N-terminal signal sequence signal recognition particle (SRP) RNP particle SRP-receptor translocation channel signal peptidase posttranslational modifications - protein glycosylation - formation of disulfide bonds in the lumen of endoplasmic reticulum protein disulfide isomerase - stabilization of conformation chaperone proteins (e.g. BiP, calnexin) quality control pathological aspects (e.g. cystic fibrosis, familial hypercholesterolemia)

Lipid synthesis on the smooth endoplasmic reticulum synthesis of phospholipids in the cytoplasmic layer translocation by phospholipid flippase transport to other organelles: - vesicular transport - phospholipid exchange proteins synthesis of steroids

32. THE SECRETORY PATHWAY. PROTEIN GLYCOSYLATION AND SORTING (Cooper: pages 365-373) Secretory pathway vesicular transport transport of secretory, membrane and lysosomal proteins can be studied by pulse-chase labelling with an [3H]amino acid → autoradiography lumen of endoplasmic reticulum → transport vesicles→ cis-Golgi → median Golgi → transGolgi → trans-Golgi network → protein-processing → sorting sorting signals - signal sequences - signal patches - oligosaccharides (e.g. mannose-6-phosphate → lysosomes) secretion - regulated secretory granules processing (e.g. proinsulin → insulin) exocytosis e.g. insulin secretion - constitutive e.g. extracellular matrix proteins, antibodies targeted secretion in polarized cells (apical and basolateral membrane) vectorial transport (e.g. transepithelial transport)

Protein glycosylation = addition of oligosaccharides glycosyl transferases sugar-nucleotide complexes

N-linked glycosylation

dolichol → 2 N-acetylglucosamines + 9 mannoses + 3 glucoses → oligosaccharideprotein transferase → glycosylation on asparagine → oligosaccharide processing mannose rich, complex and hybrid oligosaccharides

O-linked glycosylation glycosyl transferases

33. THE ENDOCYTIC PATHWAY (Cooper: pages 373-379) Vesicular transport - secretory pathway - endocytic pathway

Endocytic pathway fate of engulfed material degradation endocytic vesicle → fusion with primary lysosomes → secondary lysosome → hydrolytic degradation storage in storage vesicles transcytosis endocytosis → exocytosis on the other side of the cell

types of endocytosis phagocytosis uptake of large particles, viruses, bacteria etc pinocytosis uptake of soluble molecules non-specific, non-regulated receptor-mediated endocytosis e.g. uptake of LDL particles LDL = low density lipoprotein cholesterol-ester + phospholipid monolyer + apolipoprotein B (apoB) LDL-receptors in clathrin-coated pits → LDL-binding → endocytosis → coated vesicle → shedding of clathrin → early endosome → dissociation of LDL from receptor (recycled to cell membrane) → transport vesicle → LDL transported to late endosome → fusion with primary lysosomes → secondary lysosome familial hypercholesterolemia hereditary defect of LDL receptor → increased serum LDL → atherosclerosis acquired hypercholesterolemia

The mechanism of vesicular transport bidirectional = autograde and retrograde sorting signals targeting proteins

components of transport

donor compartment → budding of coated transport vesicle (ARF protein, adaptin proteins) → shedding of coat → fusion with acceptor membrane (targeting proteins, fusion proteins, Rab proteins)

types of coated vesicles clathrin-coated vesicles e.g. receptor-mediated endocytosis formation of lysosomes other coated vesicles e.g. COP-coated vesicles = coat protein between endoplasmic reticulum and Golgi complex

34. CELL DEFENSE MECHANISMS (Cooper: pages 359-365, 379-384, lecture) Biotransformation of xenobiotics

the cell

xenobiotics = chemicals of nonbiological origin cytochrome P450 system heme proteins oxidative enzymes in the endoplasmic reticulum membranes coded by a gene family (CYP genes) biotransformation phase I: hydroxylation of molecules → increased water-solubility phase II: conjugation with glucuronic acid, sulfuric acid etc. → excretion epoxide formation → increased toxicity, mutagenicity → malignant transformation of e.g. polycyclic aromatic hydrocarbons induction of CYP genes e.g. phenobarbital hypertrophy of smooth endoplasmic reticulum

Lysosomes types of lysosomes primary lysosomes contain acid hydrolases (phosphatase, nuclease, protease etc.) active proton pump → pH 5 secondary lysosomes fusion of pr. lysosomes with vesicles

function of lysosomes heterolysis endo-, phagocytosis → phagosome → + pr. lysosomes → phagolysosome autolysis autophagolysosome → + pr. lysosomes → sec. lysosome

formation of lysosomes complex →

rough endoplasmic reticulum → lysosomal enzymes → N-linked glycolysation → Golgi

synthesis of mannose-6-phosphate (M6P) → binding to M6P-receptor → clathrincoated vesicles → primary lysosomes

lysosomal storage diseases specific lysosomal enzyme deficiencies e.g. Gaucher disease - cerebrosidosis Tay-Sachs disease - gangliosidosis I-cell disease = abnormality of M6P-synthesis or M6P receptor enzyme substitution therapy

Free radicals reactive oxygen species (ROS) e.g. superoxide, hydrogen peroxide, hydroxyl radical

phagocytosis

NADPH oxidase → generates superoxide radical Ran protein chronic granulomatous disease abnormal NADPH oxidase → recurring infections

ROS-induced macromolecular damage

DNA damage → mutations, carcinogenesis lipid peroxidation → membrane damage

antioxidants enzymes e.g. superoxide dismutases (SOD), catalase, peroxidase anyotrophic lateral sclerosis small molecules e.g. vitamin C, vitamin E, carotene, selen, etc.)

35. THE STRUCTURE AND FUNCTION OF MITOCHONDRIA (Cooper: pages 389-391, 398-404) Breakdown of glucose

C6H12O6 + 6O2 → 6CO2 + 6H2O 32 ATP molecules/glucose molecule are produced 3 steps: 1. glycolysis glucose → 2 pyruvate molecules in the cytosol 2. citric acid cycle   in the mitochondria 3. oxidative phosphorylation 

Structure of mitochondria 2 membranes, 2 compartments outer membrane porin → highly permeable intermembrane space inner membrane cristae mitochondriales high cardiolipin content → impermeability proteins: - transport proteins e.g. H+/pyruvate symporter, H +/phosphate symporter, ADP/ATP antiporter - respiratory chain = electron transport system - ATP synthase mitochondrial matrix high enzyme concentration citric acid cycle genetic apparatus

Energy metabolism in mitochondria

pyruvate → acetyl-coenzyme A citric acid cycle Ac-CoA + oxalacetate → citric acid → cycle results: GTP is produced reduced coenzymes (NADH, FADH2) are produced oxidative phosphorylaton chemiosmosis mechanism reduced coenzymes → electrons passed to respiratory chain → electrochemical proton gradient is generated (potential gradient + pH gradient) → ATP synthase (F0F1 complex) → ATP

36. MITOCHONDRIAL DNA (Cooper: pages 391-397) Extrachromosomal DNA in eukaryotes - mitochondrial DNA (mtDNA) - chloroplast DNA (in plants) endosymbiosis theory

The human mitochondrial genetic apparatus mtDNA small, circular, double-stranded DNA 2-10 copies/mitochondrion heavy (H) and light (L) chain mostly coding sequences coding sequences in both strands genes code for: 2 rRNAs 22 tRNAs 13 mRNAs (code for subunits of respiratory chain, ATP synthase) symmetric transcription, no splicing no RNA import or export protein import (no export) high mutation rate (free radicals, no histones, no repair)

Mitochondrial protein import posttranslational guided by signal sequences protein synthesis on free polysomes → binding of signal-specific chaperones → other cytosolic chaperones → binding to receptor in outer mitochondrial membrane → translocation channel → import into matrix → binding of mitochondrial chaperones → cleavage by matrix protease → targeting to specific compartment (matrix, inner membrane etc)

Mitochondrial diseases

mtDNA mutations → decrease in ATP production hereditary diseases germ-line mtDNA mutations → maternal inheritance homoplasmy and heteroplasmy e.g. Leber's hereditary optic neuropathy acquired diseases somatic mtDNA mutations → somatic heteroplasmy e.g. Parkinson disease Alzheimer disease diabetes mellitus physiological aging

37. CYTOSKELETON I: MICROFILAMENTS (Cooper: pages 423-434) Cytoskeleton functions: - determines cell shape - drives active cell movements - transport organelles - drives cell division components: - microfilaments (diameter: 7 nm) - intermediate filaments (diameter: 10 nm) - microtubules (diameter: 25 nm)

Microfilaments structure and assembly actin protein family monomer: G actin, filament: F actin actin polymerization: G actin → nucleation → elongation of F actin plus and minus end dynamic instability (treadmilling mechanism) growth at plus end - depolymerization at minus end actin • ATP complexes bind at plus end → ATP hydrolysis → actin • ADP complexes are released at minus end actin-binding proteins profilin - sequesters G actin gelsolin - cleaves microfilaments inhibitors of microfilament function (cytochalasin, phalloidin) myosins = actin-activated ATPases motor proteins type II myosin 2 heavy + 4 light chains ATP hydrolysis → myosin moves toward plus end of microfilament e.g. muscle contraction (sliding filament model) type I myosin vesicular transport along microfilaments

Organisation of microfilaments actin bundles parallel microfilaments held together by actin-binding proteins (e.g. fimbrin, α-actinin etc.) e.g. stress fibres, microvilli actin networks flexible crosslinking proteins (e.g. filamin) e.g. cortical network cell membrane - microfilament connections e.g. focal adhesion, belt desmosome Duchenne muscular dystrophy X-linked, recessive absence of dystrophin → progressive muscle degeneration → death dystrophin - anchores microfilaments to cell membrane special actin-based structures microvillus, pseudopodium, lamellipodium, filopodium

38. CYTOSKELETON II: INTERMEDIATE FILAMENTS AND MICROTUBULES (Cooper: pages 442-460)

Intermediate filaments provide mechanical scaffold to the cell monomer structure head - rodlike α-helical region - tail organisation monomer → dimer → tetramer → protofilament → intermediate filament types intermediate filament proteins are tissue-specific - keratins e.g. cytokeratins in epithelical cells - vimentin connective tissue, smooth muscle cells, leukocytes - desmin muscle - peripherin peripheral neurons - neurofilaments neurons of central nervous system - lamins nuclear lamins intermediate filament diseases epidermolysis bullosa simplex mutation in cytokeratin genes familiar cardiomyopathy mutation in neurofilament gene

Microtubules

consist of αβ-tubulin dimers → protofilament → microtubule plus end, minus end dynamic instability microtubule organizing center (MTOC) = centrosome = 2 centrioles + pericentriolar material nucleation → binding of dimer • GTP complexes at plus end → release of dimer •GDP complexes at minus end inhibitors of microtubule polymerization colchicin, vincristine, vinblastine, griseofulvin microtubule motor proteins driven by ATP-hydrolysis kinesins move toward plus end dyneins move toward minus end

39. THE STRUCTURE OF LIPOPROTEIN MEMBRANES. CELL-CELL JUNCTIONS (Cooper: pages 77-82, 467-476, 509-513) Cell membrane - selective filter - maintains ion gradients - transduces signals

Structure of membranes fluid mosaic model lipid bilayer + proteins lipid bilayer contains amphipathic lipids - phospholipids glycerol-phospholipids sphyngomyelins - glycolipids - cholesterol lateral movements, rotation, flip-flop (flippase) assymetric bilayer phase transition liposomes unilamellar, multilamellar applications: research therapy membrane proteins amphipathic proteins integral proteins transmembrane proteins peripheral proteins asymmetric structure

Cell junctions transient junctions e.g. leukocyte - endothel interaction selectins stable junctions occluding junctions tight junction (zonula occludens) e.g. between small intestinal cells occludin anchoring junctions actin filament-associated junctions belt desmosome (zonula adherens) cadherins, catenins focal adhesions linked to the extracellular matrix intermediate filament-associated junctions spot desmosome (macula adherens) cadherins, catenins, cytoplasmic plaque hemidesmosomes link cells to basal membrane pemphigus communicating junctions gap junction connexin channels e.g. between heart muscle cells

40. TRANSPORT ACROSS MEMBRANES (Cooper: pages 80-82, 476-491) Passive transport processes are driven by concentration gradients simple diffusion e.g. gases (O2, CO2), small nonpolar molecules (chloroform), small, polar, uncharged molecules (water, urea) osmosis e.g. osmotic haemolysis facilitated diffusion channel proteins voltage-gated channels ligand-gated channels membrane potential, action potential depolarization-repolarization CFTR protein = cystic fibrosis transmembrane conductance regulator aquaporins e.g. in lens in kidney tubules carrier proteins uniporter proteins e.g. glucose transporter contransporter proteins symporters (e.g. Na+/glucose symporter) antiporters (e.g. Na+/Ca++ antiporter)

Active transport processes generate concentration gradients

ATP-dependent transporters hydrolyze ATP P-type ATPases become phosphorylated during function e.g. Na+K+ ATPase α 2β 2 tetramer binding of 3Na+ → phosphorylation → pumping Na+ ions out → binding of 2K+→ dephosphorylation → pumping K+ in ++ Ca ATPase maintains low Ca++ level in cytosol V-type ATPases proton pumps (e.g. lysosomes, endosomes) F-type ATPases F 0F 1 complex (e.g. in mitochondria) function as ATP synthases ABC transporters e.g. multidrug transporter

Ion-dependent transporters cotransporters active transport of a molecule is coupled to passive transport of an ion e.g. Na+/glucose transporter transepithelial transport of glucose

41. CELL MEMBRANE AND THE EXRACELLULAR MATRIX (Cooper: pages 504-509) Extracellular matrix functions: - forms a scaffold between cells - morphogenesis - determines the shape of cells - is involved in signal transduction - can influence gene expression

Collagens fibrillar proteins tripla helical structure contain hydroxylated amino acids synthesized on the endoplasmic reticulum → translocation to the lumen (soluble procollagen) → hydroxylation, glycosylation → triple helix → Golgi complex → exocytosis → tropocollagen → crosslinking → collagen fiber types of collagens - fiber-forming collagens (e.g. bones, ligaments etc.) osteogenesis imperfecta - fiber-associated collagens - network-forming collagens e.g. basal membrane

Glycoseaminoglycans, proteoglycans glycoseaminoglycans highly hydrophilic polysaccharides → form hydrated gels e.g. hyaluronan in cartilage proteoglycans = proteins + glycoseaminoglycans matrix proteoglycans e.g. aggrecan = corre protein + chondroitin sulfate + dermatan sulfate + linking protein cell-surface proteoglycans e.g. syndecan fibroglycan

Multiadhesive proteins bind to cell surface receptors, collagen, proteoglycans e.g. laminin in lamina basalis fibronectin in connective tissues

Integrins transmembrane proteins receptors αβ heterodimers are highly tissue specific bind to actin filaments (focal adhesion) bind to intermediate filaments (hemidesmosomes) abnormal integrins in: tumors leukocyte adhesion deficiency

42. SIGNAL TRANSDUCTION I: SIGNALLING MOLECULES AND THEIR RECEPTORS (Cooper: pages 521-529) Phases of signal transduction intercellular and intracellular signalling signal generating cell → ligand → target cell → intracellular signalling → biological response

Types of chemical signalling endocrine signalling ligand: hormone bloodstream is involved paracrine signalling ligand: local chemical mediator juxtacrine signalling ligand: cell surface protein direct cell-cell contact autocrine signalling secretory and target cell are the same e.g. some tumor cells intracrine signalling ligand and receptor are intracellular (orphan receptor)

Types of receptors intracellular receptors for small, hydrophobic ligands (e.g. steroids, thyroid hormone, retinoic acid) cell surface receptors for charged or large ligands (e.g. adrenalin, insulin, growth factors)

Identification of cell-surface receptors binding of labelled ligand radioactive or fluorescent ligand specific and nonspecific binding affinity labelling labelled ligand → crosslinked to receptor → membrane fraction → SDS-PAGE affinity chromatography ligand is linked to the affinity matrix expression cloning of receptor cDNA cDNA library from a receptor-positive cell → transfection into receptor-negative cells → selection of cells expressing the receptor → isolation of recombinant vector from these cells

43. SIGNAL TRANSDUCTION II: HETEROTRIMER G PROTEIN-MEDIATED SIGNALLING (Cooper: pages 529-531, 537-544) Types of cell surface receptors - ion channel receptors = ligand-gated channels - G protein-linked receptors - catalytic receptors e.g. tyrosine protein kinase receptors - tyrosine kinase-linked receptors

G proteins GDP-bound (inactive) and GTP-bound (acitve) state monomer G proteins heterotrimer G proteins αβγ subunits signalling by heterotrimer G proteins ligand binds to receptor → GDP/GTP exchange on α subunit → α•GTP dissociates from βγ → stimulates effector proteins → hydrolysis of GTP by GTPase of α subunit → α•GDP binds to βγ dimer G protein-linked receptors heptahelical proteins

cAMP-pathway

e.g. β-adrenerg receptor-mediated signalling binding of adrenalin → G protein → adenylate cyclase → cAMP from ATP → (inactivated to AMP by cAMP phosphodiesterase) → activation of protein kinaseA by cAMP → serine/threonine phosphprylation of target proteins (e.g. enzymes, membrane proteins, transcription factors) CREB = cAMP response element binding protein G s and Gi proteins

Inositol phospholipid pathway e.g. acetyl-choline in exocrine pancreas binding to receptor → Gq protein → phospholipase C → hydrolysis of phosphatidylinositol-bisphosphate (PIP2) to diacylglycerol (DAG) and inositoltrisphosphate (IP3) DAG → protein kinase C → target proteins (e.g. AP-1 transcription factor) IP3 → Ca++ channel in endoplasmic reticulum → increased cytosolic Ca++ → calmodulin → Ca++/calmodulin-dependent protein kinase (CaM kinase) → phosphorylation of target proteins (e.g. CREB)

44. SIGNAL TRANSDUCTION III: SIGNALLING THROUGH TYROSINE PROTEIN KINASE RECEPTORS (Cooper: pages 525-527, 531-534, 544-549) Tyrosine protein kinase receptors catalytic receptors ligands: growth factors e.g. PDGF (= platelet-derived growth factor) EGF (= epidermal growth factor) FGF (= fibroblast growth factor) NGF (= nerve growth factor) insulin IGF (= insulin-like growth factor) domain structure ligand binding domain transmembrane domain kinase domain receptor activation ligand binding → receptor dimerization → autophosphorylation → binding of signaling proteins (e.g. adapter proteins, PLC etc.) → Tyr phosphorylation → activation SH2 domain → pTyr-binding SH3 domain → target binding

Ras/MAPK pathway Ras proteins monomer G proteins mechanism of activation activated receptor → adapter protein → G nucleotide exchange factor (GEF) → GDP/GTP exchange → Ras•GTP (active) → effector proteins → GTP hydrolyzed to GDP (GAP helps = GTPase activating protein) MAPK cascades = mitogen-acivated protein kinase MAPKKK → MAPKK → MAPK ERK pathway = extracellular signal-regulated kinase activated Ras → Raf activation → MEK (= MAPK/ERK kinase) → ERK → phosphorylation of target proteins (e.g. transcription factors) SRE = serum response element enhancer SRF = serum response factor

Other signalling pathways phospholipase C (PLC) pathway → generation of phospholipid-derived second messengers (DAG, IP3) phosphatidylinositol-3-kinase (PI3K) pathway PIP 2 PI3KÆ PIP 3 (phosphatidylinositol trisphosphate) → target proteins involved in cell survival, proliferation

45. SIGNAL TRANSDUCTION IV. STRESS RESPONSE, CYTOKINES, INTEGRIN SIGNALLING (Cooper: pages 535-536, 549-553) Stress signalling stress response can be evoked by: radiations, heat shock, DNA damage, oxidative stress, extracellular ligands, osmotic shock, toxic compounds etc. survival signalling (ERK-pathway, PI3K-pathway) apoptotic signalling (JNK-pathway, p38-pathway) JNK-pathway = c-Jun N-terminal kinase JNK phosphorylates c-Jun in AP-1 complex

Cytokine signalling cytokine family polypeptide ligands e.g. interferons, interleukins, erythropoietin, growth hormone, prolactin mechanism of signalling ligand binding → receptor dimerisation → binding of cytosolic non-receptor tyrosine kinase (e.g. JAK = Janus kinase) → JAK phosphorylation → binding of SH2containing STAT proteins (= signal transducer and activator of transcription) → STAT dimer → translocation to the nucleus → induction of target genes

Integrin signalling integrins transmembrane proteins link the extracellular matrix to the cytoskeleton are involved in: cell adhesion determination of cell shape cell movements signal transduction mechanism of signalling binding of extracellular matrix, mechanical stress → αβ integrin dimer → focal adhesion kinase/Src → tyrosine phosphorylation → binding of SH2-containing signalling proteins → - actin binding proteins → stress fibers → cytoskeleton rearrangements → changes in cell shape, movements - Ras/ERK-pathway → proliferation, differentiation, migration, and/or survival - JNK-pathway → stress response - PI3K-pathway → survival

46. SIGNAL TRANSDUCTION V. GENERAL CONCLUSIONS, PRACTICAL ASPECTS (Cooper: pages 548-549) General features of signal transduction specificity of signalling redundant signalling = different ligands → similar effects overlapping pathways pleiotropic effects = same ligand → different response in different cells molecular mechanisms in signalling second messengers = small, diffusible molecules have allosteric effects on target proteins water-soluble agents (e.g. cAMP, IP3, Ca++) or lipids (e.g. DAG, PIP3) protein phosphorylation by protein kinases - Ser/Thr specific kinases - Tyr specific kinases - dual specificity kinases macromolecular interactions protein-protein interactions e.g. SH2 domains SH3 domains compartmentalization of signalling proteins lipid-protein interactions e.g. DAG-protein kinase C DNA-protein interactions e.g. transcription factor-enhancer signal amplification signal termination signalling networks diverging and converging pathways combinatorial signalling

Clinical aspects non-insulin dependent diabetes mellitus (NIDDM) = type II diabetes mellitus insulin signalling insulin → receptor → insulin receptor substrate (IRS) proteins → → Ras/Erk-pathway → mitogenic response → PI3K-pathway → exocytosis of glucose transporter containing vesicles → increased glucose uptake NIDDM mutations in receptor, IRS etc. genes nephrogenic diabetes insipidus mutations in vasopressin receptor gene in aquaporin gene cholera cholera toxin → activation of a G s protein → loss of water and salts tumor mutations in genes coding for proteins of mitogenic signalling

47. CELLULAR AND MOLECULAR MECHANISMS OF DEVELOPMENT (Cooper: pages 589-592) Genomic equivalence = same genome - different phenotype based on differential gene expression

Early development of mammals

zygote (= fertilized egy) → successive cell divisions → totipotent cells → morula → differentiation → blastocyst (= trophoblasts + inner cell mass) → organogenesis

Cellular processes of development cell proliferation series of mitotic divisions short cycles determination = engagement toward a defined developmental direction is inhereted to the daughter cells based on cell memory - cytoplasmyc memory = production of transcription factors products of selector genes - autocrine memory - nuclear memory is based on heritable chromatin modifications e.g. X chromosome inactivation genomic imprinting differentiation = phenotypic changes toward a defined developmental direction terminal differentiation cell movements cell migration is important for morphogenesis programmed cell death (apoptosis) → elimination of unwanted cells pattern formation is based on position dependent determination of cells morphogen gradients are important

Signal transduction and embryonal development various forms of signalling - direct cell-cell communication (e.g. gap junctions) - cell-matrix communication (e.g. integrins) - growth factor signalling EGF → epithelial cells PDGF → smooth muscle, glia FGF → mesoderm induction activin → mesoderm induction IGF → skeletal elements

Homeotic selector genes

→ code for master regulatory proteins (helix-turn-helix transcription factors) contain homeoboxes → code for DNA binding homeodomains

Congenital abnormalities of development = abnormal morphogenesis achondroplasia → inherited dwarfism craniosynostosis syndromes FGF receptor mutations = fusion of skull sutures Albright syndrome = hereditary osteodystrophy mutation in GSα gene combined pituitary hormone deficiency → dwarfism caused by mutation in a homeotic gene (Pit 1)

48. APOPTOSIS (Cooper: pages 592-593) Apoptosis

= programmed cell death can be physiological and pathological

Necrosis and apoptosis necrosis caused by severe chemical or physical damage swelling of cells chromatin condensation membrane damage inflammation apoptosis shrinking of cells chromatin condensation, internucleosomal DNA fragmentation no inflammation apoptotic bodies → phagocytosed

The physiological role of apoptosis - development (elimination of unwanted structures) - tissue homeostasis - tissue involution - physiological aging

Phases of apoptosis 1. initiation external stimuli: e.g. tumor necrosis factor α (TNFα) → death receptor growth factor withdrawal 2. signal transduction signaling proteins are involved: e.g. adapter proteins Bcl-2 - antiapoptotic p53 - induced by DNA damage triggers apoptosis 3. effector phase executioners: caspases (proteolytic enzymes) target proteins e.g. lamins → chromatin condensation actin-binding proteins, intermediate filaments → cytoskeletal changes nuclear proteins → altered gene expression DNase → DNA fragmentation 4. degradation phase ends with phagocytosis

Diseases of apoptosis

too much apoptosis → AIDS Alzheimer disease Parkinson disease alcoholic liver damage etc. not enough apoptosis → tumors autoimmune diseases viral diseases etc.

49. TUMOR BIOLOGY I: THE TUMOR CELL (Cooper: pages 599-601, 604-608) Classification of tumors benign tumors malignant tumors invasion, metastasis carcinoma = epithelial tumor sarcoma = mesodermal solid tumor leukaemia = blood cell tumor lymphoma = immune cell tumor

Morphology of tumor cells differentiated tumor dedifferentiated (anaplastic) tumor alterations of the nucleus high mitotic index euchromatinisation nucleolar abnormalities (hypertrophy etc.) hypersegmentation of the nucleus pseudoinclusions karyopycnosis, karyolysis, apoptotic changes cytoplasmic and membrane alterations mitochondrium degeneration endoplasmic reticulum ↓ → free ribosomes ↑ desmososmes ↑ cytoskeleton rearrangement

Functional features of tumor cells transformation of cultured cells malignant trensformation foci behavior of tumor cells disappearance of contact inhibition decreased growth factor dependence autocrine stimulation anchorage independence anoikis immortality telomerase expression changes in gene expression e.g. matrix proteases ↑ → invasion

50. TUMOR BIOLOGY II: ONCOGENIC DNA VIRUSES (Cooper: pages 608-611) Oncogenic viruses in culture: malignant transformation in vivo: tumor formation their genetic information integrates into the cellular genome DNA or RNA viruses

Oncogenic DNA viruses SV40 and polyoma virus virus genome small, circular, double-stranded DNA early region → early proteins (large T, small t) late region → virion proteins lytic infection in permissive cells virus replicates permanent transformation in nonpermissive cells no virus replication stable integration of virus DNA adenoviruses linear, double-stranded DNA early and late genes lytic infection or transformation hepatitis B virus acute hepatitis → chronic hepatitis → cirrhosis → hepatocellular carcinoma papillomaviruses some cause benign warts certain types cause cervix carcinoma, anogenital cancers spread through sexual contact early and late genes transforming proteins inactivate tumor suppressor proteins (p53, Rb) herpesviruses large genome Epstein-Barr virus (EBV) causes mononucleosis infectiosa may cause Burkitt lymphoma herpesvirus associated with Kaposi-sarcoma Kaposi sarcoma: frequently associated with AIDS virus codes for: - angiogenic growth factor - cyclin - antiapoptotic protein

51. TUMOR BIOLOGY III: ONCOGENIC RNA VIRUSES (Cooper: pages 611-613) Retroviruses contain reverse transcriptase envelope: phospholipid bilayer + Env protein nucleocapsid: 2 RNA molecules + Gag protein + reverse transcriptase

Infections cycle of retroviruses

virus adsorbs to cell surface → fusion with cell membrane → genome is reverse transcribed → double-stranded cDNA (LTR sequences at both ends) → transport to nucleus → integration into host genome → provirus →transcription → translation → assembly of new virus particles → released by budding infection is usually non-lytic cell may be transformed endogenic retroviruses

Strongly and weakly oncogenic retroviruses strongly oncogenic viruses e.g. Rous sarcoma virus (RSV) rapid oncogenesis polyclonal tumor transform cells in culture contain oncogene weakly oncogenic viruses e.g. avian leukaemia virus (ALV) slow oncogenesis monoclonal tumor do not transform cells in culture do not contain oncogene

Retroviral oncogenes transformation-defective RSV (td RSV) mutant Bishop-Varmus experiment retroviral oncogenes (e.g. v-src) protooncogenes (e.g. c-src) ALV genome: 5'-LTR-gag-pol-env-LTR-3' RSV genome: 5'-LTR-gag-pol-env-src-LTR-3' other retroviral oncogenes: myc, erbA, erbB, jun, fos, abl, raf, ras, sis

Origin of retroviral oncogenes

infection by weakly oncogenic retrovirus → integration → transcription → virus genome aquires a cellular protooncogene (transduction)

Human retroviruses HTLV1 and 2 (= human T cell leukaemia virus) cause leukaemia HIV (= human immunodeficiency virus) causes AIDS endogenous human retroviruses may cause autoimmune diseases (e.g. SLE, Sjögren syndrome)

52. TUMOR BIOLOGY IV: CELLULAR ONCOGENES (Cooper: pages 613-622) Transfection experiments with the DNA of: - RSV-transformed cells - nonviral tumors - naturally occuring human tumors the first human oncogene from bladder tumor cells constitutively active rasH gene → GTPase activity of RasH is lost → constitutively active RasH

Mechanisms of oncogene activation point mutation e.g. rasH - bladder, skin etc. cancer rasK - colon, pancreas etc cancer rasN - neuroblastoma, leukaemia neu - neuroblastoma insertional mutagenesis caused by integration of the provirus of a weakly oncogenic retrovirus e.g. ALV → c-myc overexpression translocation chronic myeloid leukaemia Philadelphia chromosome reciprocal translocation between chromosome 9 and 22 → bcr/abl fusion gene → constitutively active Brc/Abl fusion protein bcr (= breakpoint cluster region) Burkitt lymphoma Ig promoter/c-myc fusion gene → c-Myc is overexpressed many other B or T cell malignancies e.g. follicular B cell lymphoma bcl-2 → codes for an antiapoptotic preotein gene amplification → homogenously staining region or double minute chromosomes e.g. N-myc amplification in neuroblastoma

53. TUMOR BIOLOGY V: TUMOR SUPPRESSOR GENES (Cooper: pages 623-629) Identification of tumor suppressor genes by cell fusion experiments

hybrids of normal and tumor cells → are often non-tumorigenic → recessive oncogenes exist tumor suppressor genes inhibit malignant proliferation of cells by - arresting them in G0 phase - inducing terminal differentiation - causing apoptosis lead to carcinogenesis by loss-of-function mutations

Identification of the retinoblastoma (rb) gene retinoblastoma childhood eye tumor hereditary form autosomal dominant inheritance early onset bilateral, multiple tumors sporadic form no family clustering later onset unilateral, single tumor Knudson hypothesis = two-hit hypothesis hereditary form: one germ line mutation + one somatic mutation sporadic form: two somatic mutations cloning of the rb gene

The role of tumor suppressor genes in human cancer rb p53

mutation involved in retinoblastoma, osteosarcoma, other tumors inhibits transcription of S phase genes

mutation involved in many tumors codes for a transcription factor inhibits cell cycle progression or causes apoptosis "quardian of the genome" Cdk inhibitors e.g. p16 mutation in familial melanoma APC mutation causes adenomatous polyposis coli → colorectal cancer protein involved in cell adhesion WT1 mutation causes Wilms tumor (childhood kidney cancer) codes for a zinc finger transcription factor NF1 mutation causes von Recklinghausen neurofibromatosis codes for a Ras-GAP (neurofibronin) BRCA1 and BRCA2 their mutations cause familial breast cancer

Mutator genes

their products are involved in DNA repair → muttaions increase genetic instability → increase the frequency of protooncogene or tumor suppressor gene mutations e.g. BRCA1 and 2, XP genes, mismatch repair genes

54. TUMOR BIOLOGY VI: MULTISTEP CARCINOGENESIS (Cooper: pages 599-603) Multistep carcinogenesis = tumors are caused by several mutations long latency risk of most cancers increases with age

Experimental carcinogenesis: clonal evolution of tumors carcinogenic agents are mutagenic (genotoxic) direct and indirect carcinogens stages 1. tumor initiation caused by mutation irreversible 2. tumor promotion caused by tumor promoters (cocarcinogens) e.g. phorbol ester (stimulates PKC → mitogenic effect) reversible 3. tumor progression initiated cell becomes malignant karyotypic instability → chromosomal abnormalities irreversible

Clinical stages of carcinogenesis example: skin cancer initiated cell → epithelial dysplasia → in situ carcinoma → invasive carcinoma (secretion of matrix proteases) → metastasis

Oncogene cooperation in carcinogenesis = cooperation between mutations in cellular oncogenes and tumor suppressor genes e.g. ras + myc synergize for - malignant transformation in cell culture - tumorigenesis in transgenic mice

A modell: colon carcinoma most common mutations: - loss of APC gene → familial adenomatous polyposis - activation of rasK gene → adenoma - deletion in chromosome 18 - inactivation of the p53 gene → malignant colon carcinoma

55. AN INTRODUCTION TO CLINICAL GENETICS I: HEREDITARY DISEASES (Gelehrter: pages 27-47) Meiosis

diploid cells → haploid gametes I. meiotic division separation of homologous chromosomes crossing over between homologous chromosomes interphase no DNA replication II. meiotic division separation of sister chromatids

Basic genetic terms gene = region of DNA that codes for a protein or stable RNA molecule alleles = variant forms of a gene locus = the site of a gene in a chromosome homologous chromosomes = members of a chromosome pair somatic chromosomes and sex chromosomes homozygote = carries identical alleles in a locus of homologous chromosomes heretozygote = carries different alleles in a locus of homologous chromosomes genotype = genetic constitution phenotype = features that appear monogenic inheritance = the trait is inherited by a single pair of genes polygenic inheritance = the trait is inherited by multiple pairs of genes dominant disorder = the abnormal allele is stronger than the normal recessive disorder = the abnormal allele is weaker than the normal incompletely dominant disorder = the normal and abnormal genes have similar strength

Monogenic diseases autosomal dominant diseases are caused by gain-of-function mutations vertical type of inheritance e.g. achondroplasia Huntington's disease osteogenesis imperfecta autosomal recessive diseases are caused by loss-of-function mutations horizontal type of inheritance role of consanguinity e.g. cystic fibrosis lysosomal storage diseases phenylketonuria = phenylalanine hydroxylase deficiency albinism = tyrosine hydroxylase deficiency sickle cell anaemia X-linked recessive diseases much more common in males (hemizygotes) no male-to-male inheritance e.g. haemophilia A Duchenne's muscular dystrophy colour blindness

Polygenic diseases polygenic traits: Gaussian distribution multifactorial abnormalities - inheritance - environmental factors

twin studies e.g. diabetes mellitus hypertension schizophrenia

56. AN INTRODUCTION TO CLINICAL GENETICS II. CHROMOSOMAL ABNORMALITIES (see Gelehrter: pages 159-191) Methods of cytogenetics karyotype analysis blood lymphocytes → cultured in the presence of a mitogen (phytohaemagglutinin) → colchicine → metaphase chromosomes → staining → photography → karyotype banding techniques e.g. Giemsa banding → stains heterochromatin fluorescence in situ hybridisation (FISH) to visualize: - centromores - telomeres - whole chromosomes - single genes interphase cytogenetics comparative genome hybridisation to identify deletions or duplications (amplifications) flow cytometry for fast karyotype analysis, fractionation of chromosomes

The normal karyotype Denver system 23 pairs: 22 pairs of autosomes 1 pair of sex chromosomes (XX or XY) metacentric, submetacentric, acrocentric chromosomes

Structural chromosome abnormalities are caused by breaks types: deletion inversion translocation - reciprocal translocation - Robertsonian translocation are frequent in cancers in congenital disorders e.g. Lejeunne syndrome (cri du chat)

Numerical chromosome abnormalities polyploidy (e.g. triploidy) aneuploidy - monosomy - trisomy autosomal chromosome abnormalities Down syndrome (trisomy 21) growth and mental retardation congenital heart and kidney disorders caused by - meiotic non-dysjunction (age of the mother!) - mitotic non-dysjunction Patau syndrome (trisomy 13) Edwards syndrome (trisomy 18)

Sex chromosome abnormalities sex determination zfy gene on the Y chromosome → codes for a zinc finger transcription factor (testis determination factor) X chromosome inactivation one X chromosome becomes heterochromatin (sex chromatin, Barr body) Klinefelter syndrome 44 + XXY non-functional testes azoospermia → infertility insufficient testosterone production → feminine features (e.g. gynecomastia) Turner syndrome 44 + X0 non-functional ovary → infertility → reduced estrogen production → primary amenorrhea short stature, infantile female features multiple X syndrome 44 + 3-5X problems in menstruation double Y syndrome 44 + XYY agressivity, antisocial behaviour, mental retardation

57. MOLECULAR MEDICINE I. MOLECULAR DIAGNOSIS (see Gelehrter: pages 275-283) Molecular medicine - molecular diagnosis - gene therapy

Molecular diagnosis of hereditary diseases identification of point mutations detection of the appearance or elimination of a restriction site by Southern blotting or PCR e.g. sickle cell anaemia hybridisation with allele specific oligonucleotides dot-blot hybridisation PCR using wild-type and mutant primers single-strand conformational polymorphism (SSCP) heteroduplex analysis sequence analysis e.g. DNA chips

identification of larger lesions e.g. deletions, translocations, inversions, insertions Southern blotting PCR (e.g. multiple PCR) FISH

use of genetic linkage analysis linkage to a polymorphic genetic marker (e.g. minisatellite)

applications -

confirmation of diagnosis heterozygote screening prenatal diagnosis preimplantation diagnosis

Molecular diagnosis of tumors somatic (somatimes germ line) mutations of protooncogenes and tumor suppressor genes e.g. bcr/abl fusion gene in CML N-myc amplification in neuroblastoma

Molecular diagnosis of infectious diseases fast, sensitive Southern, Northern blotting, PCR diagnostic kits are available (e.g. Mycobacterium tuberculosis, Neisseria gonorrheae, herpes simplex virus, hepatitis B virus, papillomavirus etc.)

58. MOLECULAR MEDICINE II. GENE THERAPY (see Gelehrter: pages 294-296) Oligonucleotide therapy = inhibition of the expression of specific genes using complementary oligonucleotides antisense oligonucleotides bind to specific mRNAs → inhibit their function e.g. chronic myleoid leuhemia (CML) anti-bcr/abl oligonucleotide various tumors viral infections (AIDS, hepatitis etc.) bacterial infections anti-Shine-Dalgarno oligo ribozymes catalytic RNAs → bind to the target mRNA and cleave it e.g. AIDS, CML etc.

Gene therapy by gene substitution, inactivation or correction types of gene therapy ex vivo in situ in vivo in utero vectors for gene therapy viral vectors retroviruses adenoviruses adeno-associated viruses nonviral vectors naked DNA liposomes clinical trials 1990 - first human gene therapy adenosine deaminase deficiency → ex vivo introduction of ADA-cDNA into T lymphocytes somatic cell gene therapy = target cells are somatic cells → will not be inherited gene substitution for loss-of-function mutations e.g. ADA deficiency cystic fibrosis familial hypercholesteroloemia haemophilia suicide genes → tumors DNA vaccines gene inactivation (K.O.) gene correction for gain-of-function mutations germ cell gene therapy germ-line cells are involved → would be inherited not permitted!