Sorex_G2 MODULARIZACE VÝUKY EVOLUČNÍ A EKOLOGICKÉ BIOLOGIE CZ.1.07/2.2.00/15.0204 PF_72_100_grey_tr ubz_cz_black_transparent CYTOGENETIC METHODS http://image.tutorvista.com/content/heredity-and-evolution/chromosome-types.jpeg http://www.intl-pag.org/icons/telomere.gif http://www.vet.cam.ac.uk/cytogenetics/harlequin.jpg FISHchip http://media.wiley.com/mrw_images/els/articles/a0005002/image_n/nfgz003.gif analysis of chromosome microscopic structure the term „chromosome“ – 1888 Wilhelm Waldeyer chromosomal theory of heredity: 1st half of 20th century – Theodore Boveri, Walter S. Sutton, Thomas H. Morgan study of chromosomes: karyology, cytogenetics karyotype = arranged set of chromosomes in a cell chromosom Structure of metaphase chromosome telomere short arm centromere long arm secondary constriction satellite Classification of chromosomes according to position of centromere: metacentric submetacentric (subtelocentric) acrocentric telocentric http://image.tutorvista.com/content/heredity-and-evolution/chromosome-types.jpeg Mus1 History of cytogenetics Role of key technological innovations – 4-5 breakthroughs in the modern era: 1. Discovery of hypotonic treatment  spread of metaphase chromosomes 2. Cultivation of peripheral blood (leucocytes) and fibroblasts 3. Chromozome banding techniques 4. In situ hybridization (ISH) 5. Immunochemical methods used along with ISH  non-radioisotopic detection of hybridized probes (NISH) using different fluorochromes („chromosome painting“) Mitotic preparations 1. Choice of tissue with high mitotic activity root cap, embryos, larvae, regenerating tissues adult vertebrates: bone marrow, kidney, spleen, gonads, interstitial epithelium, corneal epithelium sometimes subcutaneous stimulation, or intraperitoneal injection of phytohemagglutinin, pokeweed (Phytolacca americana), or active yeast suspension Mitotic preparations 2. Discontinuation of mitotic divisions in vivo or in vitro cytostatic: colchicine, colcemid, vinblastine in vivo: advantage: cheaper, simpler disadvantage: necessary to sacrifice in vitro: peripheral blood cultivation (short-term) and fibroblasts (long-term) advantage: possibility to synchronize cell divisions  reduction of variation in chromosome condensation, increased quality, reduced consumption of cytostatic disadvantage: more laborious, expensive, time-consuming, fewer chromosomes Mitotic preparations 3. Hypotonization of cells 0,075 M KCl solution, distilled water also possible 4. Fixation Carnoy mixture = methanol : acetic acid (glacial) 3:1 multiple changes (in squash preparations: ethanol instead of methanol) Mitotic preparations 5. Slide preparation 2 basic techniques: „squash“ (rozmačkání): maceration or gentle grinding of tissue pieces on a slide and squashing with silicone cover slip „splash“ (nakapání): cell suspension is poured in drops onto a cover slip using Pasteur pipette  chromosome spread due to surface tension; after dripping either „air-drying“, or „flame-drying“ kultivace Blood cultivation Meiotic preparations testes, pollen mother cells hypotonization with sodium citrate, procedure similar to mitotic preparation meiotic progress and importance of particular stages; synaptonemal complexes (SC), lampbrush chromosomes Differential staining - chromosome banding mechowi_Q http://media.wiley.com/mrw_images/els/articles/a0005777/image_n/nfgz001.gif Q-banding (quinacrine): differential fluorochrome excitation and extinction depending on presence of AT bases quinacrine staining, UV  short period of visibility mechowi_G rypoš obří (Fukomys mechowi) Differential staining - chromosome banding G-banding (Giemsa, GTG-banding): effect of denaturation agents on stability of protein and nucleic chromatin constituents positive (dark) bands  areas rich of AT bases (isochores) effect of trypsin (chymotrypsin, NaOH) Giemsa staining Sorex_G2 rejsek obecný (Sorex araneus) Anicka_G Homo sapiens Lemur catta_R Lemur catta Differential staining - chromosome banding R-banding (reverse banding): denaturation by alcaline treatment at high temperature (80–90C) followed by DNA renaturation dark bands  isochores rich of GC bases Giemsa or acridine orange staining myvalovec_C psík mývalovitý (Nyctereutes procyonides) Differential staining - chromosome banding C-banding (constitutive heterochromatin): treatment first with strong acid (1M HCl), followed by alcaline (Ba(OH)2) and heterochromatin renaturation in saline buffer (2SSC) at high temperature (60C) euchromatin dissolving Giemsa staining (visualization of satellite DNA) kolčava a hranostaj mechowi_C mechowi_G rypoš obří (Fukomys mechowi) lasice_C kolčava rypoš obří (Fukomys mechowi) kolcava_NOR mechowi_NOR Differential staining - chromosome banding Ag-NOR: gelatine + formic acid, AgNO3 staining nucleolus organizer visualization (only active NORs) 2_11 Differential staining - chromosome banding BrdU: replication with artificial precursor (5-bromo-2´-deoxyuridine)  visualization of sister chromatid interchanges in situ hybridization of chromosomes with flurescently labelled probe possibility of simultaneous application of several probes visualization: antibodies specific for biotin (avidin, streptavidin) are conjugated either with fluorochrome (e.g. fluoresceine isothiocyanate, FITC), or enzymes (e.g. alcaline phosphatase, peroxidase), reaction with specific substrate Fluorescent in situ hybridization (FISH) CISS, chromosome in situ supression hybridization PRINS, primed in situ labelling GISH, whole genome in situ hybridization FACS, fluorescence activated cell sorting „chromosome painting“ Fluorescent in situ hybridization (FISH) 799px-FISH_(Fluorescent_In_Situ_Hybridization) FISH_(technique) Urovysion_on_Duet Bcrablmet FISHchip Microdissection Electrophoresis Unit MINI | The Classic Horizontal Line | Horizontal Systems | Electrophoresis Units | Electrophoresis | Life Science | Carl Roth - France ELECTROPHORESIS of enzymes and other proteins http://web.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg Agarose gel Electrophoresis – Site Title AAT Phenotype Identification by Isoelectric Focusing. | Semantic Scholar electrophoresis: from Greek, "to bear electrons" = motion of particles under influence of electric field until end of 1950’s, studies of genetic variation in natural populations only based on Mendelian morphological traits or polytene chromosomes  To what extent these traits represent real genetic variability in nature? amino acid substitutions can be detected by sequencing – if this is impossible, we can use protein electrophoresis *) isoelectric point of 20 AA, 3 bear positive charge (Arg, Lys, His), 2 negative charge (Asp, Glu) besides charge, also macromolecule size and conformation (-S-S- bridges, van der Waals forces, hydrogen bonds, electrostatic forces); buffer pH electric charge stabilization → specific buffer of high ionic strength and pH as different from given protein’s pI*) as possible: pH 3–10, most often pH 6,5–9,5 charge of most proteins at pH 8–9 negative  migration to anode •Principle of electrophoresis known since end of the 19th century •1937 – Thisselius: “moving boundary” method •1949 – Linus Pauling: filter paper – abnormal Hb (sickle cell anemia) •1955 – Oliver Smithies: starch •1957 – Hunter & Moeller: employment of catalytical abilities of enzymes (histochemical staining) •1966 – application on natural populations: Harry Harris (humans), Richard Lewontin & John Hubby (fruit fly) Media (gels): starch (SGE): molecule size + charge cellulose acetate (CAGE): charge agar, agarose (AGE): charge polyacrylamide (PAGE): molecule size + charge Electrophoretic methods Agarose Gel Electrophoresis - an overview | ScienceDirect Topics Plant Life: Electrophoresis Capillary Electrophoresis: PA 800 plus horizontal vertical capillary 3_1 3_2 Prince Technologies :: Introduction to Capillary Electrophoresis http://web.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg Electrophoretic methods 1. ELFO in continuous buffer 2. ELFO in discontinuous buffer (multiphasic ELFO): 2 gels of different concentrations - concentrating and separating gels protein „sandwiching“ on boundary between „leading“ a „dragged“ ions; on its own = isotachophoresis •3. Isoelectric focusing, IEF: •= separation of molecules by differences in their isoelectric points •solution of ampholytes (syntetic polyamino polycarbonates) with a range of pI put in gel; in electric field  stable pH gradient; ampholytes kept in gel by strong acid at anode and strong alcali at cathode •molecules stop where zero charge (pI point) • http://www.fda.gov/ucm/groups/fdagov-public/documents/image/ucm059972.jpg http://www.channelwolf.com/lvv/sem6/index_files/image1749.jpg http://www.channelwolf.com/lvv/sem6/index_files/image1749.jpg 4. urea and SDS ELFO: SDS = sodium dodecyl sulphate (= anion detergent): can dissolve some proteins and cleave some polymers SDS brings about strong charge of proteins, migration only based on molecular weight urea: similar to SDS, but protein charge normal – migration based on total charge (likewise, proteins can be denatured by increased teperature  ELFO) 5. Two-dimensional (2-D) ELFO: electric field applied first in one direction and then perpendicularly e.g. 1. stage = IEF, 2. stage = SDS ELFO – combination of pI and molecular weight http://www.prlog.org/10827617-2d-gel-electrophoresis.jpg Electrophoretic methods Ability to separate blood plasma proteins: CAGE: 5 bands SGE: 15 bands PAGE: 19 bands IEF > 30 bands 2-D ELFO ~300 spots 75-100 polypeptides Protein detection non-specific: amido black, Coomassie Brilliant Blue R specific: dyes for glycoproteins, lipoproteins histochemical staining of enzymes: catalysis of specific substrate processing connected with staining reaction - nitro tetrazolic salts (MTT, NBT) + PMS (phenazine methosulphate); Fast Blue RR; Fast Garnett GBC, Fast Black K - reduction of NAD+, NADP+ - sometimes necessary to add other enzymes 3_6 3_6 stained gel = in general electrophoretogram, if enzymes specifically stained = zymogram (enzymogram) bands = „electromorphs“, „alelles“, „alellomorphs“ isozymes, allozymes 3_8 3_9