Chromosomal Structure and Chromosomal Mutations
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1 Chromosomal Structure and Chromosomal Mutations Chapter 8 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.
2 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 Types of Mutations Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy) Chromosomal: abnormal chromosome structure Gene: DNA sequence changes in specific genes
3 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 Telomere Centromere Chromosome Morphology Arm Short arm (p) Telomere Long arm (q) Metacentric Submetacentric Acrocentric 3
4 Defining Chromosomal Location Arm Region Band Subband p q Chromosome , q. Chromosome Morphology Changes During the Cell Division Cycle DNA double helix: nm diameter Interphase (G, S, G) Chromatin beads on a string : nm Chromatin in nucleosomes: 30 nm Metaphase (mitosis) Extended metaphase chromosomes: 300 nm Condensed metaphase chromosomes: 700 nm 4
5 Cell Division Cycle G S Interphase ( 30 nm fibers) M Mitosis: Prophase Anaphase Metaphase Telophase G Metaphase ( nm fibers) Visualizing Metaphase Chromosomes Patient cells are incubated and divide in tissue culture. Phytohemagglutinin (PHA): stimulates cell division Colcemid: arrests cells in metaphase 3: methanol:acetic acid: fixes metaphase chromosomes for staining 5
6 Visualizing Metaphase Chromosomes (Banding) Giemsa, reverse, or centromere stained metaphase chromosomes G-bands R-bands C-bands Karyotype International System for Human Cytogenetic Nomenclature (ISCN) 46, XX: normal female 46, XY: normal male G banded chromosomes are identified by band pattern. 6
7 Normal Female Karyotype (46, XX) (G banding) Normal Female Karyotype (High Resolution G banding) 7
8 Chromosome Number Abnormality Aneuploidy (48, XXXX) Chromosome Number Abnormality Trisomy (47, XX, +) 8
9 Chromosome Structure Abnormalities Translocation Deletion Inversion Isochromosome Derivative chromosome Insertion Ring chromosome Chromosome Structure Abnormality: Balanced Translocation 45, XY, t(4qq) 9
10 Fluorescent in situ Hybridization (FISH) Hybridization of complementary gene or regionspecific fluorescent probes to chromosomes Interphase or metaphase cells on slide (in situ) Probe Microscopic signal (interphase) Fluorescent in situ Hybridization (FISH) Metaphase FISH Chromosome painting Spectral karyotyping Interphase FISH 0
11 Uses of Fluorescent in situ Hybridization (FISH) Identification and characterization of numerical and structural chromosome abnormalities Detection of microscopically invisible deletions Detection of subtelomeric aberrations Prenatal diagnosis of the common aneuploidies (interphase FISH) 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
12 FISH Probes Telomere specific probes (TEL) Hybridize to subtelomeric regions Detect subtelomeric deletions and rearrangements Probe binding site Telomere Unique sequences kb 3 0 kb Telomere associated repeats (TTAGGG)n Genetic Abnormalities by Interphase FISH LSI Probe Greater or fewer than two signals per nucleus is considered abnormal. Cell nucleus Normal diploid signal Trisomy or insertion Monosomy or deletion
13 Structural Abnormality by Interphase FISH LSI Probe (Fusion Probe) Structural Abnormality by Interphase FISH LSI Probe (Break Apart Probe) 3
14 Translocation by Metaphase FISH WCP Probe (Whole Chromosome Painting) 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. 4
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