1 1. Proteins polypeptide chains consist of amino acids (20) structure of amino acids peptide bond N-terminus, C-terminus structural levels: primary (sequence) secondary (α-helix, β-sheet) tertiary quaternery (subunits) functional categories: enzymes (catalysts, lowering of activation energy) structural regulatory receptor transport proteins

2. Lipids definition (low water-solubility: hydrophobic /non-polar/ nature) classification triglycerides, phospholipids, glycolipids, steroids, carotenoids structure, function, examples glycerol, fatty acids, ester bond amphipathic lipids

3. Carbohydrates definition (polyhydroxi aldehydes or ketones) (CH2O)n monosaccharides pentoses (ribose, deoxyribose) hexoses (glucose, fructose etc.) linear form, ring form disaccharides sucrose (glucose +fructose) lactose (glucose +galactose) oligosaccharides polysaccharides cellulose, starch, glycogene structure, function glycosaminoglycans structure, function, examples proteoglycans

2 4. Nucleosides, nucleotides nucleosides = base + sugar nucleotides = base + sugar + phosphate bases purines: A, G pyrimidines: T, U, C bind at 1’ position sugar (pentose) ribose, deoxyribose phosphate binds at 5’ or 3’ position role of nucleotides e.g. energy storage (ATP) regulatory function (cAMP, GTP) components of coenzymes (NAD, FAD) components of nucleic acids

5. Comparison of pro- and eukaryotic cells Prokaryotes

Eukaryotes

definition organisms

no nucleus bacteria

genetic apparatus

circular chromosomal DNA coupled transcriptiontranslation

contain nucleus unicellular, multicellular organisms linear DNA molecules

extrachromosomal DNA cellular organelles

plasmids cell membrane + cell wall no membranebounded organelles

cytoskeleton

ribosomes ∅

cell division

fission

endosymbiosis theory

transcription: nucleus translation: cytoplasm mitochondrial DNA cell membrane (cell wall) membrane-bounded organelles (nucleus, mitochondria, endoplasmic reticulum, lysosomes ribosomes microfilaments, microtubules, intermediate filaments mitosis meiosis

3 6. Methods of immunocytochemistry aim: microscopic detection of specific antigens (proteins) using labeled antibodies immunofluorescence microscopy light microscopical technique microscopic section → treatment with the specific antibody labeled with a fluorescent dye→fluorescence microscopy immunogold technique electron microscopical technique specific antibody is bound to gold particles → treatment of specimen →transmission electron microscopy examples: visualization of cytoskeleton elements visualization of nuclear lamins etc.

7. Restriction endonucleases bacterial enzymes cut double-stranded DNA at specific sequences palindromic sequences (=read the same in both strands from 5’ to 3’ end direct cleavage → blunt ends staggered cleavage → sticky ends protect the host cell from foreign DNA modification methylases methylate the same palindromic sequences→protect DNA from restriction enzyme cleavage application of restriction enzymes in molecular biology - restriction mapping - Southern blotting - cloning of DNA = ligation of a DNA fragment into a vector - construction of genomic and cDNA libraries - etc.

8. Southern blotting aim: to identify specific DNA fragments procedure: DNA sample → restriction endonuclease digestion → agarose gel electrophoresis (fractionation by size) → denaturation of DNA fragments (alkaline treatment) → blotting to membrane → hybridization with radioactive probe → autoradiography (with X-ray film)

4 9. DNA sequencing Sanger method (= chain termination method) in vitro DNA synthesis system (DNA template, synthetic primer, 4 dNTPs, DNA polymerase) dideoxyribonucleoside triphosphates → chain termination usually primer is labeled (radioactive or fluorescent labeling) size determination of products: denaturing PAGE

10. DNA chips sequencing by hybridization microchip with oligonucleotide arrays → hybridization with fluorescent DNA or RNA → microchip studied with a fluorescence microscope → computer determines the sequence applications: to identify point mutations to study gene expression

11. Genomic libraries genomic library = DNA fragments representing the total genome of a cell ligated into vector molecules insertion vectors (λ phage, plasmid etc.) are used construction of the library genomic DNA → cut with a restriction enzyme → ligation with λ arms → packaging with phage proteins → infection of E.coli cells → plaques screening of the library plaque transfer to nitrocellulose filter → DNA denaturation → hybridization with a radioactive probe → autoradiography

12. Polymerase chain reaction aim: in vitro amplification of a DNA region in vitro DNA synthesizing mixture (2 primers, 4 dNTPs, template DNA, Taq polymerase) one cycle: denaturation → primer-binding → DNA synthesis 20-40 cycles are used the region between the 2 primers is amplified

5 13. Transgenic organisms transgene = a foreign gene introduced into the germ line of an organism generation of transgenic organisms - microinjection of the transgene into the fertilized egg - gene transfer into embryonic stem cells → injection into blastocysts → chimera embryos →breeding → real transgenic animals

14. Targeted inhibition of endogenous gene function K.O. mutation by homologous recombination between the targeted gene and its inactivated form K.O. vector contains the inactivated gene region selection against random integration antisense oligonucleotide = complementary to a region of target RNA ribozyme = binds the target RNA and cuts it si RNA = small interfering RNA short dsRNA → binds to complementary RNA region → gene silencing complex → degradation of target RNA inhibition of protein function e.g. by antibodies by small inhibitors

15. cDNA libraries cDNA = DNA complementary to an RNA molecule cDNA library = a collection of cDNA clones corresponding to all mRNAs in a cell construction of a cDNA library total cellular mRNA →cDNA synthesis (4 dNTPs, oligo (dT) primer, mRNA, reverse transcriptase)→ adding sticky ends → ligation into vector (plasmid, phage) → transfer into host cells screening of the library - molecular hybridization - immunocreening (with antibody) expression vector

6 16. Northern blotting aim: to identify specific RNA molecules procedure: RNA sample → formaldehyde/agarose gel electrophoresis → blotting to membrane → hybridization with radioactive probe →autoradiography

17. Immunoprecipitation and Western-blotting immunoprecipitation aim: separation of a specific protein from a mixture of proteins procedure: Cell extract → mixed with antibody-covered agarose beads → centrifugation → pellet dissolved in a sample buffer → SDS–PAGE → visualization of protein Western-blotting aim: identification of a specific protein in a mixture of proteins procedure: cell extract → SDS-PAGE → fractionation by size → blotting to nitrocellulose filter → treatment with specific antibody → visualization of antibody

18. The structure of cell nucleus nuclear membrane outer membrane, inner membrane nuclear lamina – lamin proteins nuclear pore complex structure (nucleoporins) protein import nuclear localization signal importin Ran (monomeric G-protein) RNA export by proteins with nuclear export signal chromatin heterochromatin (peripheral, perinucleolar) highly condensed inactive (no RNA synthesis) euchromatin decondensed active (transcribed) nuclear matrix

7 19. The organization of chromatin levels of organization I. DNA double helix II. beads-on-a-string nucleosome = histone octamer + DNA linker DNA H1 histone III. solenoid 6 nucleosomes/turn IV. looped domains V. metaphase chromosome highly condensed

20. The structure of DNA DNA (=deoxyribonucleic acid) linear polynucleotide chains building blocks: nucleotides (see exam question # 4!) 3’ 5’- phosphodiester bonds primary structure = sequence of nucleotides secondary structure = double helix (Watson-Crick model) complementary base-pairing (A=T, G≡C) hydrogen bonds antiparallel orientation (= direction of phosphodiester bonds is opposite in the two strands) DNA denaturation, renaturation linear, circular DNA tertiary structure superhelix

21. DNA, the genetic material Pneumococcus experiment (bacterium transformation) S variant encapsulated → causes, pneumonia → death R variant (mutant form) noncapsulated → not virulent → survival heat-treated S variant → no pneumonia → survival R variant + heat-treated S variant → pneumonia → death → live S variant in the lungs conclusion: the genetic material is heat-resistant

8 (not proteins!) use of purified DNA → DNA is the transforming principle bacteriophage experiment phage with 32 P-labelled DNA and 35 S-labelled proteins → only 32 P is detected in infected E. coli cells → new phage particles are produced conclusion: DNA (not proteins) is the genetic material of bacterial viruses

22. Unique and repetitive sequences unique sequences = sequences present in 1 copy/haploid genome e.g. single-copy genes repetitive sequences more than one copy/haploid genome satellite DNA = short tandem repeats centromeric, telomeric DNA minisatellites highly polymorphic (variable number of tandem repeats, VNTR) DNA fingerprinting microsatellites e.g. trinucleotide repeats genetic instability → trinucleotide repeat expansion e.g. fragile X-syndrome Huntington disease polyglutamine disease interspersed repeats copies spread over the genome e.g. Alu family tandemly repeated genes e.g. rRNA genes gene amplification = increase in copy number gene families e.g. globin genes transposons = genetic elements that move from one site to another in the genome retrotransposons move via an RNA intermediate

23. The chemical composition of chromatin main components of chromatin DNA + proteins (histones, non-histone protein) histones basic proteins (lysine, arginine) nucleosomal histones → octamer (2x4 histones) linker histone (H1)

9 regulated by modifications (phosphorylation, acetylation) functions: structural elements regulate transcription non-histone proteins heterogenous group of proteins regulated by modifications (e.g. phosphorylation) functions: structural proteins enzymes transcription factors transport proteins etc.

24. The phases of cell cycle interphase G1 phase (=gap) diploid (2n) DNA content Go phase S phase (=synthesis) DNA replication centriole duplication G2 phase tetraploid (4n) DNA content M phase = cell division synchronisation of cell cultures serum starvation→ Go phase colchicine → metaphase FACS (= fluorescence-activated cell sorter) duration of phases generation time 3 H-thymidine labeling mitotic index

25. The regulation of cell cycle cyclin/Cdk complexes cyclin – regulatory subunit Cdk (=cyclin-dependent kinase) - catalytic subunit (Ser/Thr protein kinase) G1 /S transition regulated by G1 cyclin/Cdk complexes (SPF= S phase promoting factor) restriction point (=the point of no return) G2/M transition regulated by MPF (=M-phase promoting factor) mitotic cyclin/cdk complex completion of M phase

10 degradation of cyclin by APC (= anaphase promoting complex) regulation of Cdk activity cyclins Cdk-phosphorylation Cdk-inhibitors

26. Mitosis mitosis→ somatic cells meiosis→ germ cells mitotic apparatus = centrosome (microtubule organizing center) + mitotic spindle 2 centrioles – duplicate in S phase phases of mitosis prophase triggered by MPF chromatin condensation, breakdown of nuclear envelope, formation of mitotic spindle metaphase chromosomes in equatorial plane anaphase mitotic spindle - kinetochor microtubules→ chromatid segregation (anaphase A) - polar microtubules → elongation of cell (anaphase B) - astral microtubules telophase MPF degradation chromatin decondensation, nuclear envelope reformation cytokinesis = division of cytoplasm contractile ring (microfilaments)

27. General features of replication methods to study DNA replication in vitro (template DNA +DNA polymerase + dNTPs +primer) in vivo - density labeling (e.g. 15NH4 Cl) - radioactive labeling (e.g.[3H]thymidine) replication is semiconservative = the template strand is “old”, the other is newly synthesized Meselson-Stahl experiment 15 NH4 Cl labeling→ replicated DNA: one strand is heavy (15 N), the other is light (14 N) density measured by (CsCl gradient centrifugation template and primer-dependent template – strand determines the sequence of the new strand by complementary base-pairing primer

11 base-pairs with template→ provides a 3’-OH for DNA polymerase replication is bidirectional starts at the origin → proceeds in both directions→ replication bubble replication is semidiscontinuous leading strand → continuous synthesis lagging strand → discontinuous synthesis (Okazaki fragments)

28. The mechanism of replication in prokaryotes replication is performend by replisome initiator proteins → recognize the origin topoisomerase I→ superhelix relaxation DNA helicase→ separation of template strands ssb proteins→ prevent renaturation of template primase → synthesizes RNA primers DNA polymerase III 5’→3’ elongation activity → leading strand, Okazaki fragments 3’→5’ exonuclease activity → proofreading (= removal of non-complementary nucleotides) DNA polymerase I 5’→3’ exonuclease activity → removes primers 5’→3’ elongation activity →fills the gap DNA ligase → joins Okazaki fragments topoisomerase II→separation of daughter molecules; superspiralisation

29. Eukaryotic replication multiple origins (= replication starts at many sites) DNA polymerases α,δ,ε→ replication β→ repair γ→ mtDNA replication replication in chromatin → nucleosomes form on both daughter DNA molecules telomere replication telomeres = tandem repeats at the end of chromatids telomerase =RNA template + reverse transcriptase without it: telomere shortening telomerase→immortalisation of cell (cancer)

30. DNA repair base excision repair damage: depurination, abnormal base (e.g. methylated) repair: DNA glycosylase (removal of damaged base) → AP endonuclease (cleavage) → DNA polymerase (fills gap) →DNA ligase (seals nick) nucleotide excision repair

12 damage: bulky lesion (UV-induced thymine dimer, adducts) repair: recognition proteins → excinuclease (cleavage at both sides) → helicase (removal of cleaved strand) → DNA polymerase → ligase xeroderma pigmentosum = inherited defect in repair increased risk of skin cancer mismatch repair damage: non-complementary “base-pair” repair: recognition protein → endonuclease (cleavage) → helicase→ exonuclease (removal of abnormal nucleotide) → DNA polymerase→ ligase hereditary non-polyposis colon cancer (HNPCC) defective mismatch repair

31. The mechanism of prokaryotic transcription chromosome-polysome complex (→ coupled transcription-translation) transcription unit promoter – initiation site terminator RNA polymerase sigma factor – initiation core polymerase – elongation steps of transcription initiation promoter: - 35, - 10 site (Pribnow box) closed and open initiation complex elongation 5’→ 3’ direction formation of phosphodiester bonds triphosphate end termination rho – dependent, rho – independent

32. General features of eukaryotic transcription in chromatin heterochromatin - inactive euchromatin – active RNA polymerases RNA polymerase I nucleolar → pre-rRNA synthesis α - amanitin resistant RNA polymerase II extranucleolar → pre-mRNA synthesis highly α-amanitin–sensitive RNA polymerase III extranucleolar→ small RNAs

13 moderately α-amanitin-sensitive

33. Synthesis and processing of eukaryotic rRNA nucleolus perinucleolar chromatin fibrillar centers nucleolar organizing regions fibrillar component = pre–rRNP granular component = preribosomal particles chromatin spreading → “Christmas-trees” spacers (transcribed, non-transcribed) rRNA transcription units tandem repeats → 45s pre-rRNA → processing → 18S, 5.8S, 28S rRNA 5S rRNA - exranucleolar synthesis assembly of ribosomal subunits (rRNA+proteins)

34. Synthesis of pre-mRNA in eukaryotes, cap formation and polyadenylation pre-mRNA synthesis initiation promoter TATA box, initiator region → bind general transcription factors enhancer elements→ bind regulatory transcription factors RNA polymerase II elongation formation of phosphodiester bonds 5’→3’ direction formation of 5’-cap = addition of guanine nucleotide to the 5’-end through 5’-5’ triphosphate bond cap is methylated promotes ribosome-binding termination and polyadenylation poly(A) signal→ cleavage of transcript→ addition of poly(A) tail (poly(A) polymerase) poly(A) tail protects from degradation

14 35. Pre-mRNA splicing pre-mRNA coding genes exons = represented in mature mRNAs introns =sequences absent from mature mRNAs splicing =pre-mRNA→removal of introns + joining of exons splicing of adenovirus late mRNAs late region in DNA: 1 promoter, 5 poly(A) signals exons: leader sequences, body sequence introns alternative splicing mechanism of splicing spliceosome = pre-mRNA + snRNAs + proteins binding of U1 snRNP to the 5’-splice site→ binding of U2 snRNP to intron →cleavage at 5’-splice site + lariat formation → binding of U4/U6, U5 snRNP at 3’-splice site→ cleavage at 3’-splice site + exon ligation

36. The structure and classes of RNA RNA = ribonucleic acid usually single-stranded (with double-stranded regions) consists of nucleotides (see question #4) ribose + base (A, G, U, C) + phosphate 3’5’ phosphodiester bonds 5’-end, 3’-end types of RNA mRNA (=messenger RNA) template of translation rRNA (=ribosomal RNA) prokaryotes: 5S, 16S, 23S eukaryotes: 5S, 5.8S,18S, 28S tRNA (= transfer RNA) carry amino acids RNA precursors e.g. pre-mRNA, pre-rRNA snRNAs (=small nuclear RNAs) telomerase RNA ribozymes = catalytic RNAs e.g. snRNAs – splicing rRNA – peptide-bond formation

15 37. Synthesis of aminoacyl-tRNA tRNA = transfer RNA carries an amino acid adaptor function = binds specific amino acid (3’-end) and codon (anticodon) aminoacyl–tRNA synthesis 1. activation of amino acid amino acid + ATP → aminoacyl-AMP + pyrophosphate 2. formation of aminoacyl-tRNA aminoacyl-AMP + tRNA→aminoacyl-tRNA+AMP catalyzed by aminoacyl-tRNA synthases (bind to the D loop of tRNA)

38. The structure and function of ribosomes prokaryotic ribosomes 70S monomer 30S small subunit =16S rRNA + proteins 50S large subunit = 23S, 5S rRNA + proteins eukaryotic ribosomes 80S monomer 40S small subunit =18S rRNA + proteins 60 large subunit =28S, 5.8S, 5S rRNA + proteins function of ribosomes provide surface for translation binding of: mRNA (small subunit) aminoacyl-tRNA (A site) peptidyl-tRNA (P site) peptide bond formation peptidyl transferase (ribozyme in large subunit)

39. The genetic code = the way the sequence of nucleotides in mRNA determines the sequence of amino acids in the protein methods in vitro translation of synthetic polynucleotides aminoacyl-tRNA binding assay features of the genetic code triplet code = 3 nucleotides →1 amino acid 61 sense codons, 3 stop codons redundant (degenerate)

16 = one amino acid coded by several codons wobble = degenerate codons differ at the third position unambiguous = a codon codes for one amino acid only continuous no punctuations, no overlapping open reading frame (ORF)= coding region mRNAs – monocystronic (=one ORF) polycystronic (=several ORFs) universal =conserved in the living world

40. Initiation of translation translation = protein synthesis initiation in prokaryotes formation of 30S initiation complex 30S subunit → binds initiation factors→ binds formyl-methionyl-tRNA and mRNA (through Shine-Dalgarno sequence) formation of 70S initiation complex 30S initiation complex → initiation factors released → binding of 50S subunit initiation in eukaryotes ribosome binding to mRNA via 5’-cap

41. Elongation and termination of translation elongation 70 initiation complex→binding of second aminoacyl-tRNA to A site (EF-Tu/GTP required) → peptide bond formation (peptidyl transferase =ribozyme) → first tRNA released→ translocation of ribosome (EF-G/GTP required) →A site becomes vacant→ elongation cycle repeated termination stop codon → releasing factors bind to ribosome→ polypeptide, tRNA, ribosomal subunits released from mRNA 42. General features of translation direction 5’ end→ 3’ end on the mRNA N terminus → C terminus on the protein polysomes = mRNA + ribosomes are formed during translation only one polypeptide chain synthesized on one ribosome energy requirement Aa-tRNA synthesis: ATP→AMP

17 Aa-tRNA binding: GTP→GDP translocation: GTP→GDP translation inhibiting antibiotics e.g. streptomycin, tetracycline, erythromycin etc.

43. The lactose operon operon = promoter + operator + structural gene(s) promoter = initiation site for RNA polymerase operator = repressor binding site structural genes = code for enzymes lactose operon inducible operon→ codes for catabolic enzymes regulation by lactose no lactose→ lac repressor binds to operator→ structural genes are not transcribed lactose→binds to lac repressor →released from operator→operon can be transcribed regulation by glucose no glucose → cAMP↑→ CAP activated→ binds to lac promoter→RNA polymerase binding→operon can be transcribed

44. The tryptophan operon operon (see #43) tryptophan operon repressible operon→codes for biosynthetic enzymes no tryptophan → trp repressor does not bind to operator→ structural genes transcribed tryptophan→binds to trp repressor (as corepressor) → repressor binds to operator→transcription of structural genes is blocked tryptophan is synthesized by the cell only if needed

45. Levels of regulation of eukaryotic gene expression transcription structure of chromatin histone phosphorylation, acetylation DNA methylation→inhibits transcription transcription factors (see #46) pre-mRNA processing e.g. alternative splicing (=1 pre-mRNA→several mRNAs) RNA export mRNA degradation role of 5’-cap, poly(A) tail translation protein degradation

18 lysosomal proteolysis ubiquitin-proteasome pathway protein-ubiquitination→proteasomal degradation protein function allosteric regulation (e.g. cAMP, GTP binding) modification (e.g. phosphorylation) protein-protein interactions (e.g. cyclin/Cdk complex)

46. Eukaryotic transcription factors transcription factors =bind to DNA, regulate transcription basal TFs→ promoter regulatory TFs→ enhancer elements domains – DNA binding - activation TF families helix-turn-helix proteins e.g. homeotic proteins amhipathic helix proteins (=dimerized by leucine zipper) leucine zipper proteins e.g. AP-1 (=FosJun) helix-loop-helix proteins e.g. MyoD Zinc finger proteins e.g. steroid receptors, WT-1 diseases tumors oncogenic TFs combined pituitary hormone deficiency mutation in Pit-1→ pituitary dwarfism

47. The mechanism of action of steroid hormones steroid receptors = ligand-regulated transcription factors intracellular receptors ligands: steroid hormones, vitamin D, retinoic acid etc. glucocorticoid action glucocorticoid enters the cell→ binds to cytosolic receptor→ inhibitory protein released→steroid-receptor complex translocates to nucleus→ binds to enhancer→ gene activation diseases testicular feminization no testosterone-receptor→ female phenotype

19

48. Rough endoplasmic reticulum rough ER = membranes + ribosomes synthesizes: secretory proteins, membrane proteins, lysosomal proteins cotranslational protein transport synthesis starts on free ribosome → N terminal signal sequence→ binds signal recognition particle (SRP) → binds SRP-receptor in rough ER membrane→ protein translocates to→ cleaved by signal peptidase posttranslational modification (in lumen) - glycosylation (N-linked) - formation of disulfide bonds - chaperones stabilize conformation

49. Golgi complex. Protein glycosylation Golgi complex cis-Golgi reticulum, cis-Golgi, median Golgi, trans-Golgi, trans-Golgi reticulum functions: secretion, protein processing, protein glycosylation, protein sorting protein glycosylation = addition of oligosaccharides N-linked dolichol in rough ER membrane→ oligosaccharide is synthesized →transferred to an asparagine in protein → oligosaccharide processing O-linked in Golgi sugars are added to protein one at a time by glycosyl transferases role of glycosylation cell-cell, cell-matrix interactions, receptors

50. The mechanisms of secretion protein secretion (see also #48) synthesis on rough ER → cis-Golgi→ transport through Golgi → trans-Golgi→ protein sorting types of secretion regulated e.g. insulin secretion secretory granules → exocytosis constitutive e.g. extracellular matrix proteins, antibodies

20

51. Endocytosis types of endocytosis phagocytosis = uptake of large particles pinocytosis = uptake of soluble molecules receptor-mediated endocytosis e.g. uptake of LDL particles LDL = low density lipoprotein cholesterol-ester + phospholipid monolayer + ApoB Protein LDL-receptors in clathrin-coated pits→ LDL-binding→ endocytosis →coated vesicle→ early endosome (no clathrin) → receptor recycling →LDL transported to late endosomes →+primary lysosomes →secondary lysosomes→digestion familial hypercholesterolemia inherited defect in LDL receptor→ serum LDL↑→atherosclerosis

52. The mechanism of vesicular transport components of transport donor compartment→ budding of coated transport vesicles→ shedding of coat → fusion with acceptor compartment (targeting proteins, Rab proteins, fusion proteins) coated vesicles clathrin-coated vesicles e.g. receptor-mediated endocytosis, formation of lysosomes COP-coated vesicles COP =coat protein between endoplasmic reticulum and Golgi

53. Lysosomes. Smooth endoplasmic reticulum lysosomes primary lysosomes contain acid hydrolases proton pump→pH 5 secondary lysosomes = fusion of primary lysosomes with vesicles function of lysosomes heterolysis e.g. phagocytosis→phagosome→+primary lysosome→ phagolysosome autolysis autophagosome→+primary lysosome→secondary lysosome

21

formation of lysosomes rough ER→synthesis of lysosomal enzymes→N-linked glycosylation→ Golgi→ synthesis of mannose-6-phosphate (M6P) → binding to M6P-receptor in Golgi membrane→clathrin-coated vesicles →primary lysosomes lysosomal storage diseases e.g. Tay-Sachs disease (gangliosidosis) I-cell disease (absence of M6P or M6P-receptor) smooth endoplasmic reticulum lipid synthesis e.g. phospholipids, steroids biotransformation of xenobiotics xenobiotics = chemicals of non-biological origin cytochrome P450 enzymes oxidative enzymes in ER biotransformation phase I: hydroxylation→ water-solubility↑ phase II: conjugation with acids→ excretion procarcinogens e.g. polycyclic aromatic hydrocarbons →oxydation→ bind to DNA →mutations→ cancer 54. Oxygen free radicals. Membrane damage. Lipid peroxidation Oxygen free radicals = have unpaired electrons e.g. superoxide, hydroxyl radical phagocytosis leukocyte membrane: NADPH oxidase-Rac complex → superoxide radical in phagosome →kills microbe DNA damage → mutations, cancer lipid peroxidation → membrane damage antioxidants enzymes (e.g. superoxyde dismutase) small molecules (e.g. vitamin C, E etc.)

55. The structure and function of mitochondria breakdown of glucose C6 H12 O6 +6O2 →6CO2 +6H2 O + 32ATP steps: glycolysis (cytosol) glucose→2 pyruvate molecules citric acid cycle (mitochondrial matrix) oxidative phosphorylation (inner mitochondrial membrane) structure of mitochondria outer membrane highly permeable (porin)

22 intermembrane space inner membrane cristae mitochondriales impermeable proteins: transport proteins (e.g. H+ /pyruvate symporter) electron transport chain ATP synthase mitochondrial matrix citric acid cycle genetic apparatus function of mitochondria pyruvate→acetyl-coenzymeA citric acid cycle Ac-CoA+ oxalacetate= citric acid production of GTP, reduced coenzymes (NADH, FADH2 ) oxidative phosphorylation (chemiosmosis mechanism) reduced coenzymes → electrons to electron transport chain → electrochemical proton gradient→ drives ATP synthase (Fo F1 complex) Fo = H+ channel F1 = enzyme

56. The genetic apparatus of mitochondria mtDNA circular, double-stranded several copies/mitochondrion mostly coding sequences codes for: 2 rRNAs 22 tRNAs 13 mRNAs (→ subunits of electron transport system, ATP synthase) symmetric transcription, no splicing high mutation rate mitochondrial protein import posttranslational free polysomes→ binding of chaperones→ translocation channel→ import to matrix → matrix protease

57. Mitochondrial diseases mtDNA mutations→ATP↓ hereditary diseases maternal inheritance homoplasmy (only normal mtDNA) heteroplasmy (=normal + mutant mtDNA) e.g. Leber’s hereditary optic neuropathy acquired diseases somatic mtDNA mutations→somatic heteroplasmy

23 e.g. aging Parkinson disease etc.

58. Microtubules

αβ tubulin dimers → protofilaments →microtubule plus end, minus end dynamic instability centrosome (=MTOC, microtubule organizing center) 2 centrioles + pericentriolar material nucleation →binding of dimer/GTP complexes at plus end; release of dimer/GDP complexes at minus end inhibitors of polymerization e.g. colchicin, vincristine etc. motor proteins (use ATP) kinesins (→ move to plus end ) dyneins (→ move to minus end)

59. Microfilaments structure and assembly actin monomer: G actin, filament: F actin dynamic instability actin/ATP complexes bind at plus end→growth→ ATP hydrolysis→actin/ADP complexes are released at minus end inhibitors: e.g. phalloidin myosins (=actin-activated ATP-ases) motor proteins ATP hydrolysis → myosin moves toward plus end of microfilament functions: muscle contraction, vesicle transport etc. organization of microfilaments actin bundles (e.g. stress fibers) actin networks cell membrane/microfilament connections (e.g. focal adhesion) Duchenne muscular dystrophy dystrophin – binds microfilaments to membrane X-linked, recessive mutation→muscle degeneration

60. Intermediate filaments organisation monomer (rodlike) → dimer→tetramer→ protofilament→intermediate filament function – mechanical scaffold types (are tissue-specific – diagnosis of tumors) keratins (epithelial cells) vimentin (connective tissue etc.)

24 desmin (muscle) peripherin (peripheral nervous) neurofilaments (neurons of central nervous system) lamins (ubiquitous) mutations can cause diseases (e.g. epidermolysis bullosa simplex)

61. The cell membrane fluid mosaic model =continuous lipid bilayer + embedded proteins lipid bilayer amphipathic lipids (=hydrophobic + hydrophilic part) (see # 2) phospholipids glycolipids cholesterol asymmetric bilayer (e.g. glycolipids - extracellular surface inositol lipids - cytosolic surface) liposomes (=artificial lipid vesicles) membrane proteins amphipathic proteins integral (transmembrane) and peripheral proteins caveolae specialized membrane domains contain caveolin multiple functions (endocytosis, signaling etc.)

62. Cell – cell junctions transient junctions e.g. leukocyte-endothel stable junctions occludin junctions tight junction occludin - transmembrane protein anchoring junctions – linked to cytoskeleton actin-linked belt desmosome focal-adhesion (cell→matrix) intermediate filament - linked spot desmosome cadherins, catenins pemphigus (anti-cadherin auto-antibodies) hemidesmosomes (cell→basal membrane) communicating junctions gap junctions (= connexin channels) e.g. between heart muscle cells

25 63. Passive transport passive transport driven by concentration gradient → no ATP required simple diffusion gases, small uncharged molecules osmosis membrane impermeable to solutes→transport of water e.g. plasmolysis, osmotic hemolysis facilitated diffusion performed by membrane proteins channel proteins e.g. K+, Na+ channels in action potential CFTR protein chloride ion channel mutation→cystic fibrosis aquaporins (=water channels) e.g. kidney tubules (water reabsorption) (mutation→nephrogenic diabetes insipidus) carrier proteins uniporters (e.g. glucose transporter) cotransporters symporters (e.g. Na+/glucose symporter) antiporters (e.g. Na+/Ca++ antiporter)

64. Active transport active transport generates concentration gradients→requires ATP ATP–dependent transporters (hydrolyze ATP) P-type ATPases (are phosphorylated) Na+K+ATPase pumps Na+ outward, K+ inward ++ Ca ATPase pumps Ca++ outward V-type ATPases (in vesicle membranes) proton pumps (e.g. lysosomes) F-type ATPases (Fo F1 complex) e.g. in mitochondria (ATP synthase) ABC–transporters e.g. multidrug transporter ion-dependent transporters e.g. Na+/glucose symporter in epithelial cells → transepithelial transport of glucose

26 65. The extracellular matrix functions: scaffold for cells morphogenesis regulates cell shape, signaling, gene expression extracellular matrix proteins collagen triple helical structure synthesis on rough ER → lumen → hydroxylation, glycosylation→ triple helix → Golgi → exocytosis → crosslinking of tropocollagen → collagen fiber collagen present in bones, ligaments, basal membrane etc. diseases scurvy (no vitamin C→ no hydroxylation→ collagen secretion ↓) osteogenesis imperfecta (mutation → collagen secretion ↓ → fragile bones) glycosaminoglycans, proteoglycans (see #3) matrix proteoglycans e.g. aggrecan cell-surface proteoglycans e.g. fibroglycan multiadhesive proteins (bind to other proteins) e.g. laminin (in basal lamina) integrins (=matrix receptors) (see # 66) αβ heterodimers in focal adhesions, hemidesmosomes

66. Cell-matrix connections cell-matrix connections (see # 62, 65) focal adhesions hemidesmosomes integrin signaling integrins (see # 65) mechanical stimulus→ αβ integrin dimer→ focal adhesion kinase/Src→tyrosine phosphorylation→binding of SH2 proteins signaling pathways (see also # 70, 71) →Ras/Erk→proliferation, differentiation →JNK→stress response →PI3K→survival →actin cytoskeleton→movement, cell shape

67. Types of chemical signaling intercellular and intracellular signaling signal generating cell – produces the ligand ligand – binds to receptor target cell – expresses receptor (intracellular or cell surface) types of chemical signaling endocrine (ligand: hormone)

27 secretory and target cell for apart→blood stream involved paracrine (ligand: local chemical mediator) secretory and target cell close to each other juxtacrine (ligand: cell surface protein) direct cell-cell contact autocrine secretory and target cell the same intracrine intracellular ligand and receptor types of receptors intracellular receptors (see # 47) for membrane permeable ligands (e.g. steroids) cell surface receptors for membrane impermeable ligands (e.g. adrenalin, growth factors)

68. cAMP – mediated signal transduction G-protein-linked receptors = heptahelical receptor family heterotrimeric G proteins αβγ subunits G-protein cycle ligand-binding to receptor→G-nucleotide exchange (activation) →α/GTP complex and βγ dissociate → stimulation of effector proteins→ GTP hydrolysis (by GTPase of α)→α/GDP complex and βγ reassociate (inactivation) cAMP pathway e.g. adrenalin signaling in liver binding of adrenalin to receptor→Gs protein→ adenylate cyclase →cyclic AMP from ATP (inactivation by cAMP phosphodiesterase) →activation of protein kinase A →Ser/Thr phosphorylation of target proteins CREB (=cAMP response element binding protein) diseases e.g. cholera cholera toxin → Gs activation→ loss of water and salts in small intestine

69. Phospholipid-derived second messengers G-protein linked receptors (see # 68) inositol phospholipid pathway e.g. acetylcholine 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 → phosphorylation of target proteins (e.g. AP-1) IP3 → Ca++ channel in ER membrane →release of Ca++ into cytosol→calmodulin → calmodulin-dependent kinase →phosphorylation of target proteins (e.g. CREB)

28 clinical aspects phorbol esters (see # 88) DAG analogs → act as tumor promoters

70. Growth factor signaling growth factors polypeptides (e.g. PDGF, EGF, FGF, NGF, insulin, VEGF etc.) functions: regulation of proliferation, differentiation, survival, metabolism GF receptors (=tyrosine protein kinase receptors) domains: ligand binding domain transmembrane domain kinase domain receptor activation ligand binding →dimerization→autophosphorylation→binding of SH2domain proteins→ Tyr - phosphorylation→ activation SH2 domain (=Src homology 2) → pTyr binding SH3 domain →effector protein Ras/ERK pathway Ras proteins monomer G proteins Ras cycle GF→receptor→adapter protein→G nucleotide exchange factor (GEF) → Ras/GTP (active) → effector proteins→GTPase (GAP helps) →Ras/GDP (inactive) MAPK cascades (=mitogen-activated protein kinase) MAPKKK→MAPKK→MAPK ERK pathway (=extracellular signal-regulated kinase) Ras→Raf→MEK (=MAPK/ERK kinase) →ERK→phosphorylation of target proteins SRF (=serum response factor) binds to SRE (=serum response element) PLC pathway (see # 69) PI3K pathway (see # 69) PIP2→PI3K: PIP3 (phosphatidylinositol-trisphosphate) → PKB → target proteins function: survival PTEN (=phosphatase and tensin homolog) hydrolyzes PIP3 →counteracts PI3K tumor suppressor protein

71. The role of protein kinases protein kinases phosphorylate proteins using ATP→negative charges introduced→conformational change→functional change types of protein kinases

29 Ser/Thr specific kinases e.g. PKA, PKB, PKC, ERK etc. Tyr specific kinases Tyr-phosphorylation→creates protein binding sites (SH2-proteins) e.g. GF-receptors, Src, Abl etc. diseases not enough kinase activity e.g. achondroplasia (FGF receptor mutation) too much kinase activity e.g. chronic myeloid leukemia (Bcr/Abl protein)

72. Cytokine signaling. Stress signaling cytokines polypeptides (e.g. interferons, interleukins, etc.) cytokine signaling ligand →receptor dimerization→binding of cytosolic tyrosine kinase (e.g. JAK) →JAK-phosphorylation→binding of STAT (=signal transducer and activator of transcription) →phosphorylation of STAT→translocation to nucleus→gene activation stress signaling cellular stress (e.g. radiation, heat, DNA damage, oxidative stress etc.) stress response: survival (ERK, P13K pathway) or apoptosis (JNK, p38 pathway) JNK-pathway (=c-Jun N-terminal kinase) p38-patway MAPK cascades NFκB-pathway stress→IκB phosphorylation→IκB degradation→NFκB released from complex with IκB→translocates to nucleus→gene activation often activated in chronic inflammation (e.g. rheumatoid arthritis)

73. General features of signal transduction molecular mechanisms second messengers (=small, diffusible molecules→target proteins) e.g. cAMP, IP3, Ca++ , DAG, PIP3 protein phosphorylation (see # 71) macromolecular interactions protein-protein interactions e.g. SH2, SH3 domains (see #70) lipid-protein interactions e.g. DAG→PKC DNA- protein interactions e.g. CREB→CRE general features redundant signaling (=different ligands→same effect)

30 pleiotropic effects (=same ligand→different effect) signal amplification (=one ligand→many signaling events) signal termination (role to maintain normal regulation) signaling networks (= diverging and converging pathways)

74. Molecular basis of development genomic equivalence = all diploid cells in an organism have the same genome early mammalian development fertilized egg→divisions→morula→blastocyst (=throphoblast + inner cell mass) → organogenesis cellular processes of development proliferation (=mitotic divisions) determination (=engagement toward a developmental direction) cell memory (cytoplasmic, autocrine, nuclear) differentiation (=phenotypic changes toward a developmental direction) cell migration apoptosis (=elimination of unwanted cells) pattern formation signal transduction (cell-matrix, cell-cell communication, growth factors etc.) homeotic genes → code for master regulatory proteins (transcription factors) congenital abnormalities e.g. achondroplasia (inherited dwarfism) FGF receptor mutation

75. The physiological and pathological role of apoptosis apoptosis = programmed cell death shrinking of cell no membrane damage → no inflammation chromatin condensation → internucleosomal DNA fragmentation dead cell phagocytosed by neighboring cells necrosis caused by severe damage swelling of cells → membrane damage →inflammation chromatin condensation → random DNA fragmentation physiological role of apoptosis - development - tissue homeostasis - quality control - aging diseases of apoptosis too much ⎯AIDS, Alzheimer-d. etc. too little ⎯ tumors, autoimmune diseases etc.

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76. The mechanism of apoptosis phases of apoptosis 1. initiation death ligands/receptors growth factor withdrawal DNA damage 2. signaling p53 tumor suppressor protein Bcl-2 proteins (pro- and antiapoptotic) cytochrome C (released from mitochondria) + caspase + caspase activating protein = apoptosome 3. effector phase caspases ( proteolytic enzymes) caspase targets (lamins, cytoskeletal proteins, nuclear proteins etc.) 4. degradation = phagocytosis

77. General features of the tumor cell classification benign tumor malignant tumor (carcinoma, sarcoma, leukemia, lymphoma) morphology of tumor cells nucleus increased euchromatin content nucleolus hypertrophy segmentation of nucleus cytoplasm cytoskeleton changes many desmosomes mitochondrium degeneration functional features malignant transformation loss of contact inhibition growth factor independence anchorage independence → metastasis immortality increased angiogenesis

78. Oncogenic DNA viruses SV40/polyoma permissive cells → lytic infection nonpermissive cells → permanent transformation adenoviruses

32 lytic infection or transformation hepatitis B virus increased frequency of liver cancer papillomaviruses human cervical cancer herpesviruses Epstein-Barr virus mononucleosis infectiosa Burkitt lymphoma human herpesvirus-8 causes Kaposi-sarcoma (in AIDS) codes for proteins that stimulate cell growth or inhibit apoptosis

79. Retroviruses retroviruses = oncogenic RNA viruses with reverse transcriptase infectious cycle binding on cell surface → fusion with cell membrane →genome reverse transcribed into host genome (provirus) → transcription (from promoter in LTR) → translation → assembly of new virus particles → budding strongly oncogenic retroviruses e.g. Rous sarcoma virus (RSV) rapid oncogenesis polyclonal tumor transform cells in culture contain an oncogene weakly oncogenic viruses e.g. avian leukemia virus (ALV) slow oncogenesis monoclonal tumor do not transform cells in culture no oncogene

80. Retroviral oncogenes isolation of src RSV RNA →reverse transcribed into cDNA fragments → hybridised to RNA of transformation-defective RSV→ unhybridised cDNA: corresponds to v-src v-src = retroviral oncogene c-src = protooncogene ALV provirus = 5’-LTR- gag-pol-env–LTR-3’ RSV provirus = 5- LTR- gag-pol-env–src-LTR-3’ origin of retroviral oncogenes by transduction (=acquiring cellular proto-oncogenic sequences by a weakly oncogenic retrovirus)

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81. Identification of cellular oncogenes by gene transfer transfection = introduction of purified DNA into eukaryotic cells human tumor DNA → transfected into mouse fibroblast cell line → transformed foci → DNA → 2nd transfection into mouse fibroblasts → transformed foci → DNA → construction of a genomic library→ screening with a human Alu probe → phage containing human oncogenes (rasH gene)

82. Oncogenesis by weakly transforming retroviruses insertional mutagenesis = integration of provirus activates a proto-oncogene e.g. ALV→ provirus integration into the c-myc gene→ deletion → expression from the downstream LTR of the provirus→ increased, constitutive expression of the c-Myc protein → malignant transformation

83. Mechanisms of cellular oncogene activation proto-oncogenes are converted into cellular oncogenes by activating mutations 1. point mutation (see question #81) 2. insertional mutagenesis (see question #82) 3. reciprocal translocation e.g. chronic myeloid leukemia (CML) translocation between chromosome 9 and 22 → Philadelphia chromosome → bcr/abl fusion gene→ Bcr/Abl fusion protein → leukemogenesis Glivec e.g. Burkitt lymphoma B, T cell malignancies 4. gene amplification homologously staining region – double minute chromosomes e.g. her2 amplification in breast cancer Herceptin

84. General features of tumor suppressor genes tumor suppressor genes identified by cell fusion experiments: hybrids of normal and tumor cells→ often non-tumorigenic→tumor phenotype suppressed by genes in the normal cell loss-of function mutations of tumor suppressor genes→ carcinogenesis function of tumor suppressor proteins: - inhibition of proliferation (arrest in Go) - induction of differentiation - induction of apoptosis

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85. Rb and p53 retinoblastoma childhood eye tumor hereditary form: early onset, bilateral, multiple tumors sporadic form: late onset, unilateral, single tumor Knudson’s two-hit hypothesis hereditary form: 1 germ line + 1 somatic mutation sporadic form: 2 somatic mutations Rb protein sequesters a key transcription factor→ inhibits transcription of S phase genes phosphorylation by cyclin/cdk complex → transcription of S phase genes p53 codes for a transcription factor DNA damage→ p53↑→ induction of Cdk inhibitor, DNA repair enzymes, proapoptotic proteins “guardian of the genome” mutated in many tumors loss of p53→ chemotherapy and radiotherapy resistant tumor

86. The role of tumor suppressor genes in Wilms tumor, neurofibromatosis, colon and breast cancer tumor suppressor genes (see #84) Kundson hypothesis WT1 mutated in Wilms tumor (childhood kidney cancer) hereditary, sporadic form codes for a zinc finger transcription factor required for kidney development NF1 (→ neurofibromin) mutated in neurofibromatosis codes for a Ras - GAP→ loss-of–function → Ras activation APC (=adenomatous polyposis coli) mutation→ polyposis → colon cancer BRCA1 and BRCA2 mutation→ familiar breast cancer

87. The role of oncogenes in cell cycle regulation cell cycle regulation – via growth factor signaling (see # 70) proto-oncogenes/cellular oncogenes in growth factor signaling (see # 81-83) sis (= subunit of PDGF) HER 2 (= member of EGF receptor family) Src, Abl (=non-receptor tyrosine protein kinases) RasH, RasK, RasN (=monomeric G proteins)

35 Raf (=MAPKKK) Fos, Jun, Myc (=transcription factors) tumor suppressors in growth factor signaling (see # 84-86) NF1 (neurofibromin) (=Ras GAP) Rb (retinoblastoma protein) (=regulator of S-phase genes) p53 (=transcription factor) p15, p16 (=Cdk inhibitors) oncogenic DNA viruses in growth factor signaling (see # 78) SV40, polyoma, adenovirus, papillomavirus – oncoproteins inhibit Rb, p53

88. The multistage mechanism of carcinogenesis multistep carcinogenesis = tumors are caused by several mutations most carcinogens are mutagens direct carcinogens (=cause cancer as they are) indirect carcinogens (=cause cancer after metabolic activation) steps of experimental carcinogenesis 1. initiation – caused by mutation 2. tumor promotion – stimulation of cell division tumor promoters (= not carcinogenic, but increase the effect of carcinogens) e.g. phorbol esters (stimulate PKC) 3. tumor progression karyotypic instability →chromosomal abnormality clinical stages of carcinogenesis e.g. skin cancer initiated cell →epithelial dysplasia (=abnormal cell growth) →in situ (=local) carcinoma→invasion to neighboring tissues →metastasis oncogene cooperation in colon carcinoma loss of APC → familial adenomatous polyposis →rasK activation→adenoma→deletion in chromosome 18, loss of p53 →malignant colon cancer mutator genes = genes involved in DNA repair their mutation→genomic instability →cancer risk↑ e.g. XP genes, mismatch repair genes (see # 30) BRCA 1, 2 (see # 86)

89. Meiosis meiosis = cell division that produces haploid gametes from diploid cells I. meiotic division preceded by DNA replication → the cell becomes tetraploid (4n) prophase - crossing over between homologous chromosomes metaphase anaphase – separation of homologous chromosomes

36 telophase II. meiotic division preceded by a short interphase (no DNA replication!) prophase metaphase anaphase – separation of sister chromatids telophase → four haploid (n) cells

90. Autosomal chromosomal abnormalities normal kariotype 23 pairs: 22 pairs of autosomes 1 pair of sex chromosomes (XX or XY) diagnosis of chromosomal abnormalities (= methods of cytogenetics) karyotype analysis lymphocytes→cultured with phytohaemagglutinin →+ colchicine →metaphase chromosomes→staining banding techniques e.g. Giemsa banding→heterochromatin structural chromosome abnormalities e.g. deletion, inversion, translocation numerical chromosome abnormalities aneuploidy (monosomy, trisomy) Down syndrome (=trisomy 21) growth and mental retardation congenital heart and kidney disorders caused by – meiotic non-dysjunction (age of mother!) significance of translocation (e.g. 15/21) mitotic non-dysjunction (→mosaicism)

91. Sex chromosome abnormalities normal karyotype (see # 90) diagnosis of chromosomal abnormalities (see # 90) sex chromatin (=Barr body) X chromosome inactivation (= one X chromosome becomes heterochromatin) sex chromosome abnormalities caused by non-dysjunction Klinefelter syndrome (44 + XXY) non-functional testes azoospermia (=lack of sperm production) → infertility reduced testosterone production→feminine features Turner syndrome (=44 + X0) non-functional ovary infertility reduced estrogen production→primary amenorrhea (=absence of menstrual period)

37 short stature, infantile female features multiple X syndrome (=44 + 3-5X) problems in menstruation double Y syndrome (=44 + XYY) agressivity, mental retardation

92. Autosomal inheritance of diseases 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 autosomes and sex chromosomes homozygote=carries identical alleles in a locus of homologous chromosomes heterozygote=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 that the normal incompletely dominant disorder=the normal and abnormal genes have similar strength autosomal dominant diseases caused by gain-of function mutations vertical type of inheritance (=appear in successive generations) e.g. achondroplasia (=mutation in FGF receptor gene; see # 74) Huntington’s disease (=triplet repeat expansion in huntingtin gene; see # 22) osteogenesis imperfecta (=mutation in collagen gene; see # 65) autosomal recessive diseases caused by loss-of-function mutations horizontal type of inheritance (= affected persons in the same generation) consanguinity increases the risk e.g. cystic fibrosis (=mutation in CFTR gene; see # 63) lysosomal storage diseases (see # 53) phenylketonuria (=mutation in the gene of phenylalanine hydroxylase) albinism (=mutation in the gene of tyrosine hydroxylase→decreased melanin production)

93. Sex-linked inheritance of diseases basic genetic terms (see # 92) X-linked recessive diseases common in males (hemizygotes), transmitted by healthy females (carriers) no male-to-male inheritance e.g. hemophilia A (=absence of clotting fator VIII) Duchenne’s muscular dystrophy (see # 59)

38 colour blindness

94. Molecular diagnosis of inherited diseases point mutations (= alteration of a single basepair) e.g. sickle cell anemia (point mutation in β-globin gene) RFLP analysis (= restriction fragment length polymorphism) Southern blotting (see # 8) PCR (see # 12) dot-blot hybridization with allele-specific oligonucleotides sequence analysis larger lesions (e.g. deletions, translocations) Southern blotting PCR FISH =fluorescence in situ hybridization) applications of molecular diagnosis - heterozygote screening - prenatal diagnosis - confirmation of diagnosis 95. Molecular diagnosis of tumors and infectious diseases tumors mutations in proto-oncogenes and tumor suppressor genes point mutations (e.g. ras genes) deletions (e.g. Rb gene) translocations (e.g. bcr/abl fusion gene) Southern blotting, PCR (see # 94) gene amplification (e.g. HER-2) Southern blotting infectious diseases fast, sensitive techniques: Southern blotting, PCR (→ to detect DNA of infectious agent) Northern blotting, RT-PCR (→to detect RNA of virus) (see # 12, 16) e.g. tuberculosis bacterium, papillomavirus, HIV etc.

96. Methods of gene transfer nonviral vectors naked DNA liposomes (see # 61) viral vectors retroviruses (see # 79) advantage: efficient transfer into dividing cells integration of provirus→sustained effect danger: insertional mutagenesis (see # 82) adenoviruses (see # 78)

39 advantage: transfer into non-dividing cells larger cDNAs can be used disadvantage: no integration→transient effect

97. Human gene therapy oligonucleotide therapy (see # 14) → inhibition of gene expression antisense oligonucleotides e.g. CML (see # 83), AIDS ribozymes siRNAs real gene therapy = manipulation of endogenous genes gene substitution (= introduction of missing gene) gene inactivation (= K.O. mutation, see # 14) gene correction (= targeted gene replacement) types: ex vivo (=cells taken from the body→gene therapy in cell culture →returned to the body) in situ, in vivo, in utero target cells: somatic cells germ-line cells (not permitted!)