from Wikipedia, the free encyclopedia

The cytogenetics (also cytogenetics , cell genetics ) is the branch of genetics that the chromosomes primarily with the light microscope analyzed. The number, shape, structure and function of the chromosomes are examined, because the DNA of a chromosome set in the cell nucleus contains most of the genetic information (the genome ) of a living being. Attempts are made to link chromosome abnormalities with their phenotypic effects.

Normal male karyotype: technically banded metaphase chromosomes (46, XY)

Capture chromosomes

Both during meiosis in prophase 1 (zygotene, pachytene, diplotene, diakinesis), metaphase 1 and metaphase 2, as well as during mitotic (pro) metaphase, chromosomes can be individually identified and the karyotype can be created. After endoreplication , enlarged chromosomes are visible in endometaphases and during the entire cell cycle as polytene chromosomes . The DNA content of a chromosome set reveals the genome size of an organism, shows the extent of possible endoreplication and also allows selective endoreplication to be measured .

The length of a stably hereditary chromosome arm is limited by the extent of the spindle axis in the anaphase. That is why the elements of a karyotype are to be understood as compartments of a large genome. Early attempts to determine their number in humans revealed 46 to 48 chromosomes; there was only agreement on the XX / XY system of gender determination. Reproducible analyzes in the 1950s gained widespread acceptance of the normal human karyotype formula 2n = 46.

Research history

Theodor Boveri and Walter Sutton independently developed the chromosome theory of inheritance . In short, it says: Individual chromosomes contain different hereditary factors ( genes ). The polar separation of the homologous chromosomes of a bivalent occurs in meiosis randomly and independently of that of other bivalent. The genes of the bivalent are distributed correspondingly randomly. This corresponds to the rules of Gregor Mendel . However, the genes are “linked” within the individual chromosomes.


The success of cytogenetics depended on the insight that tissue should not be cut up with a microtome , but rather that its cells should be separated from one another in hypotonic solution with fine needles. Gentle squeezing pushes the chromosomes into one plane. Dyes such as carmine , orcein or the Giemsa mixture turn chromosomes into color bodies in order to separate them from the cytoplasm . This is particularly successful with the Feulgen reaction ( Feulgen procedure), because it specifically stains the chromosomal DNA ( nuclear DNA ).

Slices of human tissue are examined as part of the pathological routine. In order to obtain a fraction of intact mitotic cell nuclei for cytogenetic problems, the microtome must be set to a section thickness of 15 μm.

Induced banding

However, the classic methods did not allow the human (metaphase) chromosomes to be unequivocally differentiated. This was only possible through special banding techniques on non- interphase nuclei . The fluorescent dye ( fluorochrome ) quinacrine creates a band pattern that identifies the individual chromosome pairs. Characteristic band patterns also arise when the DNA in the chromosomes is somewhat denatured. A standardized system of abbreviations describes each section of the technically banded chromosomes. The p stands for petit (French for small) and denotes the short arm of a chromosome. The following letter q was chosen for the long arm . Not only can abnormal chromosome numbers (such as trisomy 21) be determined, but structural chromosome changes ( deletions , duplications , translocations ) can also be recorded.

Gene activity and replication

In order to study the interphase in the cell cycle, living cells were offered radioactively labeled molecules. Incorporation of 3 H- uridine , specific for RNA , demonstrated the gene expression of active chromosome locations. Incorporation of 3 H- thymidine , specific for DNA, showed chromosomal DNA synthesis in the S phase. As beta radiation , electrons each left their traces as autoradiography in a film placed on the cells.

  • In later replication analyzes , bromodeoxyuridine replaced the radioactively labeled thymidine.
  • Advanced genetic engineering also made radioactive substances superfluous when it came to questions about gene activity. The detection shifts from the transcription level (RNA synthesis) to the completed translation : the time and place of activation of a gene-specific promoter are indicated by the protein produced by the reporter gene . As a prerequisite, the DNA sequence of the reporter gene must be attached after the promoter sequence of the gene to be examined. An organism is transformed with this DNA construct . The reporter gene, which codes for the green fluorescent protein (GFP), is popular. This system can be used to track gene expression in cells, tissues and organs.

Restriction enzymes

The isolation of numerous DNA restrictases represented a revolutionary and lasting breakthrough in cytogenetics and genetics in general. These enzymes, mostly obtained from bacteria, are molecular scissors : They cut up DNA using short, defined base sequences. The restrictases and the polymerase chain reaction (PCR) are the two pillars of modern molecular genetics and genetic engineering . They make it possible to sequence entire genomes or to carry out different types of fluorescence in situ hybridization ( FISH ). For the latter, DNA or RNA samples are marked with fluorochromes and hybridized to complementary DNA sequences of the targeted chromosomes . A fluorescence microscope is required for diagnosis .

Interphase -Zytogenetik or Molekularzytogenetik: by fluorescence in situ hybridization visualized chromosomal translocation t (9; 22)

Chromosomes painted with FISH

For the "paint" multiple probes simultaneously ( English probes ), with different fluorochromes labeled, hybridized to metaphase. Every chromosome pair in the karyotype is given its own color tone through chromosome painting . There are two ways of doing this.

  • Multicolor (multiplex) fluorescence in situ hybridization ( M-FISH ). A correspondingly large number of digital images are recorded of a metaphase decorated with different fluorochromes. A computer program analyzes the original recordings and combines them in a single image that shows the chromosome pairs in easily distinguishable false colors . The method is also ideally suited to display chromosome mutations in interphase cell nuclei (image: interphase cytogenetics).
  • Spectral karyotyping ( SKY ). This type of chromosome painting requires powerful computers. Because two partial beams are produced from each pixel of the digital image, which are brought to interference by means of an interferometer with a path difference. A Fourier transformation provides precise data on the original fluorescent spectrum from the interferogram . A downstream computer program uses this to generate easily distinguishable false colors for the individual chromosome pairs in the karyotype. As a Fourier spectroscopy, SKY has the advantage over M-FISH of greater distances between chromosomal signals and noise .

Dual hybridization

The chromogenic in situ hybridization ( CISH , including: Dual ISH) is a low-cost alternative for FISH. It is used to search for a specific mutation on a specific chromosome. CISH has several advantages over FISH: Use of two stable, absorbent colors that do not bleach; Diagnosis on the brightfield microscope, faster and cheaper. Question in breast cancer and other solid tumors: Is the HER2 gene on chromosome 17q12 for receptor 2 of human epidermal growth factor amplified? To do this, the HER2 DNA sample is hybridized with the first color on a cell nucleus in interphase. With the second color, CEN-17 is simultaneously hybridized as reference DNA for the centromere in chromosome 17. The ratio 2 or greater means HER2 amplification , combined with a poor medical prognosis .

Genomes in comparison

Comparative Genomic Hybridization ( CGH ) detects quantitative sequence changes in a genome more sensitively than FISH. For CGH, the DNA from cells to be examined is marked with a first fluorochrome. DNA from normal cells is also marked with a second fluorochrome of different emission wavelengths . Sample DNA and reference DNA are simultaneously hybridized to spread, normal metaphases in equal amounts. “Normal” means, as far as is known, a healthy organism as a donor. In CGH, the two DNAs "fight" (compete) for the same binding sites in the target chromosomes. The complex fluorescence is measured along the metaphase chromosomes. If both DNA samples match, their ratio is 1.0. The first fluorochrome (bound to the sample DNA) gives a value of 1.5 in the case of duplications in a chromosome. If both homologous chromosomes are doubled in the same place, the value is 2.0. In the case of deletions in a chromosome ( loss of heterozygosity ) the value is 0.5. If both homologous chromosomes are deleted, the value is 0.0 (no signal from the first fluorochrome). Molecular cytogenetics, also known as CGH, can analyze a single cell nucleus or reveal the DNA differences between the two sex-linked chromosomes.

Cell nuclei and numbers

Simple counting

The number of mitotic or meiotic chromosomes is constant for a species and its individuals: "The sex cells (eggs or spermatozoa) of an organism contain half as many chromosomes as the first embryonic cell from which this organism arose." This constant number is a species characteristic. The simple (haploid) number n is usually derived from 2n chromosomes of a mitotic metaphase. Because in the actually haploid nuclei of the sex cells, the chromosomes cannot be counted directly. However, when the bivalents pair through chiasmata, meiosis I shows 1n chromosomes.

Measure lengths

“The chromosomes are basically bilateral symmetrical or asymmetrical structures.” This sentence inspired many measurements to determine the length of chromosomes and the ratio of their short to their long arms. The position of the kinetochore classifies a symmetrical chromosome as metacentric and an asymmetrical one as submetacentric , subtelocentric or acrocentric . The position of a secondary constriction as the nucleolus organizer region must also be defined numerically. Such length measurements allow the identity of a chromosome in a karyotype with relatively few elements to be diagnosed.

Weigh cell nuclei

  • Microphotometry (also photomicrometry , microscope photometry or cytometry ). This technique is mainly used to determine the DNA content of whole cell nuclei and also their chromosomes. Work in the visible spectrum proved to be the most practical. After the nuclear DNA has been stained with the Feulgen procedure, measurements are taken at the absorption maximum (560 nm). In order to convert the resulting absorption units into picograms (pg) or megabase pairs (Mbp), reference nuclei with known DNA content must also be registered. The process is time consuming. It requires careful preparation on glass slides ( slide based microphotometry ), but offers the advantage of being able to repeat measurements and assess coherent tissue parts and cell types. A pioneering work defined the value 1 C as the DNA content of the (haploid) genome of a species.
  • Flow cytometry (also pulse cytophotometry ). For this procedure, the cell structure of a tissue is completely dissolved and the cells or their nuclei are usually colored with fluorochromes. It has the advantage of analyzing large samples in a short time.



Web links

Individual evidence

  1. ^ Ingo Schubert, JL Oud: There is an upper limit of chromosome size for normal development of an organism. In: Cell . Volume 88, 1997, pp. 515-520.
  2. Hans von Winiwarter: Études sur la spermatogenese humaine. In: Archives de Biologie (Liege) Volume 27, No. 93, 1912, pp. 147-149.
  3. ^ Theophilus S. Painter : Studies in mammalian spermatogenesis, II. The spermatogenesis of man. In: Journal of Experimental Zoology Volume 37, 1923, pp. 291-336, 1923.
  4. TC Hsu: Mammalian chromosomes in vitro, I. The karyotype in man. (PDF) In: Journal of Heredity Volume 43, 1952, pp. 167-172.
  5. Joe Hin Tjio, Albert Levan: The chromosome number of man. In: Hereditas Volume 42, 1956, pp. 1-6.
  6. Theodor Boveri : About multipolar mitoses as a means for analyzing the cell nucleus. In: Verh Phys-Med Ges Würzburg Volume NF 35, 1902, pp. 67-90, p. 81.
  7. Walter Sutton Stanborough: The chromosomes in heredity. In: Biol Bull Marine Biol Labor Woods Hole (Mass.) Volume 4, 1902 (or 1903), pp. 231-248.
  8. Rüdiger G. Steinbeck, Gert U. Auer, Anders D. Zetterberg: Reliability and significance of DNA measurements in interphase nuclei and division figures in histological sections. In: European Journal of Cancer Volume 35, No. 5, 1999, pp. 87-795.
  9. ^ Torbjörn Caspersson , Lore Zech, C. Johansson: Differential binding of alkylating fluorochromses in human chromosomes. In: Experimental Cell Research Volume 60, 1970, pp. 315-319.
  10. ^ Wolfgang Schnedl: Analysis of the human karyotype using a reassociation technique. In: Chromosoma Volume 34, 19971, pp. 448-454.
  11. JJ Yunis: High resolution of human chromosomes. In: Science . Volume 191, Number 4233, March 1976, pp. 1268-1270, PMID 1257746 . doi : 10.2307 / 1741162 (currently unavailable) .
  12. ^ LG Shaffer, J. McGowan-Jordan, M. Schmid (eds.): ISCN 2013. An international system for human cytogenetic nomenclature (2013): Recommendations of the international standing committee on human cytogenetic nomenclature, published in collaboration with "Cytogenetic and Genome Research "plus fold-out:" The Normal Human Karyotype G- and R-bands ". Karger, Basel 2012. ISBN 978-3-318-02253-7 .
  13. Claus Pelling: Chromosomal synthesis of ribonucleic acid as shown by incorporation of Uridine labeled with tritium. (PDF) In: Nature Volume 184, 4686, 1959, pp. 655-656, 1959.
  14. James Herbert Taylor , Philip S. Woods, Walter L. Hughes: The organization and duplication of No. chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. In: PNAS Volume 43, 1957, pp. 122-128. PMC 528395 (free full text)
  15. SR Kain, M. Adams, A. Kondepudi, TT Yang, WW Ward, P. Kitts: Green fluorescent protein as a reporter of gene expression and protein localization. In: BioTechniques Volume 19, No. 4, 1995, pp. 650-655.
  16. Werner Arber , S. Linn: DNA modification and restriction. In: Annual Review of Biochemistry Volume 38, 1969, pp. 467-500. ISSN  0066-4154 (print), ISSN  1545-4509 (electronic).
  17. Hamilton O. Smith, KW Wilcox: A restriction enzyme from Hemophilus influenzae, I. Purification and general properties. In: Journal of Molecular Biology Vol. 51, No. 2, 1970, pp. 379-391. ISSN  0022-2836 (Print), ISSN  1089-8638 (Electronic).
  18. K. Danna, Daniel Nathans: Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. In: PNAS Volume 68, No. 12, 1971, pp. 2913-2917. PMC 389558 (free full text)
  19. ^ Werner Arber: Restriction endonucleases. In: Angewandte Chemie (International Edition in English) Volume 17, No. 2, 1978, pp. 73-79. ISSN  1433-7851 (print), ISSN  1521-3773 (electronic).
  20. ^ Richard J. Roberts: How restriction enzymes became the workhorses of molecular biology. In: PNAS Volume 102, No. 17, 2005, pp. 5905-5908. PMC 1087929 (free full text)
  21. Thomas Ried, Evelin Schröck, Yi Ning, Johannes Wienberg: Chromosome painting: a useful art. In: Human Molecular Genetics Volume 7, No. 10, 1998, pp. 1619-1626.
  22. Michael R. Speicher, David C. Ward: The coloring of cytogenetics. In: Nature Medicine Vol. 2, No. 9, 1996, pp. 1046-1048, 1996. doi : 10.1038 / nm0996-1046 .
  23. ^ Evelin Schröck, S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, MA Ferguson-Smith, Y. Ning, DH Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, Thomas Ried: Multicolor spectral karyotyping of human chromosomes. In: Science Volume 273, No. 5274, 1996, pp. 494-497. doi : 10.1126 / science.273.5274.494 .
  24. H. Parsche, K. Luchner: Modern methods of spectral analysis. In: Physics in our time, Volume 6, 1975, pp. 151-161. Online ISSN  1521-3943 .
  25. Lim Sung-Jig, Alegria Cantillep, Philip M. Carpenter: Validation and workflow optimization of human epidermal growth factor receptor 2 testing using INFORM HER2 dual-color in situ hybridization. In: Human Pathology Vol. 44, No. 11, 2013, pp. 2590-2596.
  26. J. Mollerup, U. Henriksen, S. Müller, A. Schønau: Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: A large comparative study to current state of the art fluorescence in situ hybridization. In: BioMed Central Clinical Pathology Volume 12, 2012, p. 3.
  27. Anne Kallioniemi, Olli-P. Kallioniemi, Damir Sudar, Denis Rutovitz, Joe W. Gray, Fred Waldman, Dan Pinkel: Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. In: Science Volume 258, No. 5083, 1992, pp. 818-821.
  28. ^ Olli-P. Kallioniemi, Anne Kallioniemi, Damir Sudar, Denis Rutovitz, Joe W. Gray, Fred Waldman, Dan Pinkel: Comparative genomic hybridization: A rapid new method for detecting and mapping DNA amplification in tumors. In: Seminars in Cancer Biology Volume 4, 1993, pp. 41-46.
  29. Evelin Schröck et al. : Comparative genomic hybridization of human malignant gliomas reveals multiple amplification sites and nonrandom chromosomal gains and losses. In: The American Journal of Pathology Volume 144, No. 6, 1994, pp. 1203-1218. PMC 1887475 (free full text)
  30. Christoph A. Klein, O. Schmidt-Kittler, JA Schardt, K. Pantel, MR Speicher, Gert Riethmüller: Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. In: Proceedings of the National Academy of Sciences USA Vol. 96, No. 8, 1999, pp. 4494-4499. PMC 16360 (free full text)
  31. Walther Traut, Ulrike Eickhoff, Jan-C. Schorch: Identification and analysis of sex chromosomes by comparative genomic hybridization (CGH). In: Methods in Cell Science Vol. 23, 2001, pp. 155-161.
  32. Theodor Boveri: Cell Studies (3): About the behavior of the chromatic core substance during the formation of the directional bodies and during fertilization. In: Jenaische Zeitschrift für Naturwissenschaft Volume 24, 1890, pp. 314–401. There p. 372f.
  33. Georg Tischler: Chromosome number - form and individuality in the plant kingdom. In: Progress of Botany Volume 5, 1915, pp. 172-284.
  34. ^ Emil Heitz: The bilateral construction of the sex chromosomes and autosomes in Pellia fabbroniana, P. epiphylla and some other Jungermanniaceen. In: Planta Volume 5, 1928, pp. 725-768. There p. 764.
  35. ^ Helmut Zacharias, Boris Anokhin, Konstantin Khalturin, Thomas CG Bosch: Genome sizes and chromosomes in the basal metazoan Hydra. In: Zoology Volume 107, 2004, pp. 219-227.
  36. Hewson Swift: The constancy of desoxyribose nucleic acid in plant nuclei. In: PNAS Volume 36, No. 11, 1950, pp. 643-654. PMC 1063260 (free full text)
  37. J. Kusenda, M. Fajtova, A. Kovarikova: Monitoring of minimal residual disease in acute leukemia by multiparametric flow cytometry. In: Neoplasma Volume 61, No. 2, 2014, pp. 119–127. doi : 10.4149 / neo_2014_017 . See menu: 2014 .