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Chapter 8

Chapter 8. Chromosomal Structure and Chromosomal Mutations. Objectives. Define mutations and polymorphisms. Distinguish the three types of DNA mutations: genome, chromosomal, and gene. Diagram a human chromosome and label the centromere, q arm, p arm, and telomere.

MikeCarlo
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Chapter 8

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  1. Chapter 8 Chromosomal Structure and Chromosomal Mutations

  2. Objectives • Define mutations and polymorphisms. • Distinguish the three types of DNA mutations: genome, chromosomal, and gene. • Diagram a human chromosome and label the centromere, q arm, p arm, and telomere. • Illustrate the different types of structural mutations that occur in chromosomes. • Show how karyotypes reveal chromosomal abnormalities. • Describe interphase and metaphase FISH analyses.

  3. Mutations and Polymorphisms • Mutation: a permanent transmissable change in the genetic material, usually in a single gene • Polymorphism:two or more genetically determined, proportionally represented phenotypes in the same population

  4. Types of Mutations • Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy) • Chromosomal: abnormal chromosome structure • Gene: DNA sequence changes in specific genes

  5. Chromosome Morphology • Telomere: chromosome ends • Centromere: site of spindle attachment • Constriction of the metaphase chromosome at the centromere defines two arms • Nucleosome: DNA double helix wrapped around histone proteins

  6. Chromosome Morphology Telomere Short arm (p) Arm Centromere Long arm (q) Telomere Metacentric Submetacentric Acrocentric

  7. Arm Region Band Subband 3 2 2 1 2 2 1 p 1 5 1 4 1 3 2 1 1 17 q 1 1 . 2 2 1 1 3 1 2 2 q 3 1 3 2, 3 4 1 2 2 4 Chromosome 17 3 Defining Chromosomal Location

  8. Chromosome Morphology Changes During the Cell Division Cycle. • DNA double helix: 2nm diameter Interphase (G1, S, G2) • Chromatin “beads on a string:” 11nm • Chromatin in nucleosomes: 30nmMetaphase (Mitosis) • Extended metaphase chromosomes: 300 nm • Condensed metaphase chromosomes: 700 nm

  9. Cell Division Cycle Metaphase (300–700 nm fibers) Interphase (11–30 nm fibers) G1 S G2 M Mitosis: Prophase Anaphase Metaphase Telophase

  10. Visualizing Metaphase Chromosomes • Patient cells are incubated and divide in tissue culture. • Phytohemagglutinin (PHA): stimulates cell division • Colcemid: arrests cells in metaphase • 3:1 Methanol:Acetic Acid: fixes metaphase chromosomes for staining

  11. Giemsa-, reverse- or centromere-stained metaphase chromosomes Visualizing Metaphase Chromosomes (Banding) G-Bands R-Bands C-Bands

  12. Karyotype • International System for Human Cytogenetic Nomenclature (ISCN) • 46, XX – normal female • 46, XY – normal male • G-banded chromosomes are identified by band pattern.

  13. Normal Female Karyotype (46, XX)(G Banding)

  14. Normal Female Karyotype(High-Resolution G Banding)

  15. Chromosome Number AbnormalityAneuploidy (48, XXXX)

  16. Chromosome Number AbnormalityTrisomy 21 (47, XX, +21)

  17. Deletion Translocation Inversion Isochromosome Insertion Ring chromosome Derivative chromosome Chromosome Structure Abnormalities

  18. Chromosome Structure Abnormality:Balanced Translocation 45, XY, t(14q21q)

  19. Fluorescent in situ Hybridization (FISH) • Hybridization of complementary gene- or region-specific fluorescent probes to chromosomes. Interphase or metaphase cells on slide (in situ) Probe Microscopic signal (interphase)

  20. Fluorescent in situ Hybridization (FISH) • Metaphase FISH • Chromosome painting • Spectral karyotyping • Interphase FISH

  21. Uses of Fluorescent in situ Hybridization (FISH) • Identification and characterization of numerical and structural chromosome abnormalities. • Detection of microscopically invisible deletions. • Detection of sub-telomeric aberrations. • Prenatal diagnosis of the common aneuploidies (interphase FISH).

  22. FISH Probes • Chromosome-specific centromere probes (CEP) • Hybridize to centromere region • Detect aneuploidy in interphase and metaphase • Chromosome painting probes (WCP) • Hybridize to whole chromosomes or regions • Characterize chromosomal structural changes in metaphase cells • Unique DNA sequence probes (LSI) • Hybridize to unique DNA sequences • Detect gene rearrangements, deletions, and amplifications

  23. FISH Probes • Telomere-specific probes (TEL) • Hybridize to subtelomeric regions • Detect subtelomeric deletions and rearrangements Probe binding site Telomere 100–200 kb 3–20 kb Unique sequences Telomere associated repeats (TTAGGG)n

  24. Genetic Abnormalities by Interphase FISH LSI Probe • Greater or less than two signals per nucleus is considered abnormal. Cell nucleus Normal diploid signal Trisomy or insertion Monosomy or deletion

  25. Structural Abnormality by Interphase FISH LSI Probe (Fusion Probe)

  26. Structural Abnormality by Interphase FISH LSI Probe (Break Apart Probe)

  27. Translocation by Metaphase FISHWCP Probe (Whole-Chromosome Painting)

  28. Summary • Mutations are heritable changes in DNA. • Mutations include changes in chromosome number, structure, and gene mutations. • Chromosomes are analyzed by Giemsa staining and karyotyping. • Karyotyping detects changes in chromosome number and large structural changes. • Structural changes include translocation, duplication, and deletion of chromosomal regions. • More subtle chromosomal changes can be detected by metaphase or interphase FISH.

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