PREFI LEC: CHROMOSOMAL STRUCTURE & CHROMOSOMAL MUTATIONS

Question 1

All of the genes found in a single individual

Answer 1

HUMAN GENOME

Question 2

_______ nucleotide base pairs of DNA organized in _____

Answer 2

2.9 billion; 23 chromosomes

Question 3

Diploid organisms

Answer 3

46 chromosomes

Question 4

Each chromosome is a double helix of
______

Answer 4

DNA

Question 5

largest chromosome

Answer 5

Chromosome 1

Question 6

smallest chromosome

Answer 6

Chromosome 21

Question 7

A difference in DNA sequence found in 1%-2% or more of a given population

Answer 7

POLYMORPHISM

Question 8

Examples: blood types in humans, color of hair & eyes, separation of most higher organisms into male & female sexes, polymorphisms used for human ID & paternity testing

Answer 8

POLYMORPHISM

Question 9

A change in the order/sequence of nucleotide in DNA found in <1%2% of a given population (due to errors in DNA replication, exposure to mutagens, or other factors)

Answer 9

MUTATION

Question 10

Maintained a population through a balance of positive & negative phenotype

Answer 10

BALANCED POLYMORPHISM

Question 11

Abnormal RBCs resulting into resistance to infection by Plasmodium species (malaria)

Answer 11

Sickle cell anemia

Question 12

Does not have any detectable negative effects on an organism’s health/fitness

Answer 12

BENIGN POLYMORPHISM

Question 13

Typically common in populations, do not lead to disease/other adverse phenotypic outcomes

Answer 13

BENIGN POLYMORPHISM

Question 14

Examples: blood type (ABO blood group), eye color, facial features, skin pigmentation

Answer 14

BENIGN POLYMORPHISM

Question 15

Affect single genes & are often, but
not always, small changes in the DNA sequence

Answer 15

Gene Mutations

Question 16

Affect the structures of entire chromosomes

Answer 16

Chromosome Mutations

Question 17

Changes in the number of
chromosomes (aneuploidy)

Answer 17

Genome Mutations

Question 18

Process by which the long, linear DNA molecules of eukaryotic chromosomes are organized & packaged into highly condensed structures

Answer 18

CHROMOSOMAL COMPACTION

Question 19

IMPORTANT:
1. Chromosome behavior is dependent on chromosome structure & DNA sequences (position effect)
2. Chromosome topology affects gene activity (highly compacted DNA = less available for RNA transcription)

Answer 19

CHROMOSOMAL COMPACTION

Question 20

Group A

Answer 20

1, 2 (large metacentric)
3 (Large submetacentric)

Question 21

Group B

Answer 21

4, 5 (Large submetacentric)

Question 22

Group C

Answer 22

6-12, X (Medium-sized submetacentric)

Question 23

Group D

Answer 23

13-15 (Medium-sized acrocentric with satellite)

Question 24

Group E

Answer 24

16 (Short Metacentric)
17, 18 (Short Submetacentric)

Question 25

Goup F

Answer 25

19, 20 (Short Metacentric)

Question 26

Group G

Answer 26

21, 22 (Short acrocentric with satellite) Y (Short acrocentric)

Question 27

TASM

Answer 27

TELOCENTRIC TO ACROCENTRIC TO SUBMETACENTRIC TO METACENTRIC

Question 28

7 TYPES OF VISUALIZING CHROMOSOMES

Answer 28

Conventional cytological stains Fluorescent dyes Chemical dye Harsher treatment of chromosomes Alkali treatment Nucleolar organizing region (NOR) staining 4’6-diamidino-2phenylindole (DAPI)

Question 29

Feulgen, Wright, & hematoxylin

Answer 29

Conventional cytological stains

Question 30

▪ Quinacrine & quinacrine mustard ▪ Pattern: Q bands – 1st demonstrated by Caspersson, Zech, & Johansson (1970)

Answer 30

Fluorescent dyes

Question 31

▪ Giemsa stain ▪ Pattern: G bands

Answer 31

Chemical dye

Question 32

▪ Pattern: R bands (can be visualized after staining w/ acridine orange)

Answer 32

Harsher treatment of chromosomes (87ºC for 10 min, then cooling to 70ºC) before Giemsa staining)

Question 33

▪ Centromere staining ▪ Pattern: C bands

Answer 33

Alkali treatment

Question 34

Silver nitrate: stain specifically at the constricted regions, or stalks, on the acrocentric chromosomes

Answer 34

Nucleolar organizing region (NOR) staining

Question 35

▪ Binds to the surface grooves of dsDNA Fluoresces blue under UV light (353-nm wavelength) ▪ Visualization of chromosomes & whole nuclei

Answer 35

4’6-diamidino-2phenylindole (DAPI)

Question 36

Chromosome banding facilitates the:

Answer 36

▪ Detection of deletions, insertions, inversions, & other abnormalities ▪ ID of distinct chromosomal locations

Question 37

▪ Indirect method of detecting genome mutations, or aneuploidy ▪ Measuring DNA content of individual cells ▪ Aneuploidy is reflected by a change in the amount of DNA

Answer 37

FLOW CYTOMETRY

Question 38

Direct method of detecting genome mutations, or aneuploidy 🡪 observation of metaphase chromosome structure by arranging them according to size

Answer 38

KARYOTYPING

Question 39

chart that shows the complete set of chromosome in a cell

Answer 39

Karyotype

Question 40

Performed in light microscope

Answer 40

KARYOTYPING

Question 41

Exchange of genetic material between chromosomes

Answer 41

Translocation

Question 42

2 TYPES OF Translocation

Answer 42

Reciprocal Robertsonian

Question 43

Parts of 2 chromosomes exchange

Answer 43

Reciprocal

Question 44

Movement of long arm of an acrocentric chromosome to the centromere of another acrocentric chromosome

Answer 44

Robertsonian

Question 45

Loss of chromosomal material

Answer 45

Deletion

Question 46

Gain of chromosomal material

Answer 46

Insertion

Question 47

Result from excision, flipping, & reconnecting chromosomal material w/in the same chromosome

Answer 47

Inversions

Question 48

include centromere in the inverted region

Answer 48

Pericentric

Question 49

involve sequences within 1 arm of the chromosome

Answer 49

Paracentric

Question 50

Metacentric chromosome resulting from transverse splitting of centromere during cell division

Answer 50

Isochromosome

Question 51

Results from deletion of genetic regions from ends of the chromosome and a joining of the ends to form a ring

Answer 51

Ring chromosome

Question 52

Translocated / otherwise rearranged parts from 2 or more unidentified chromosomes joined to a normal chromosome

Answer 52

Derivative chromosome

Question 53

that the 1st chromosome mutations associated with human disease were visualized in the _____ in leukemia cells

Answer 53

1960s

Question 54

_______ observed an abnormally small chromosome 22 in leukemia cells (Philadelphia chromosome)

Answer 54

Peter Nowell and David Hungerford

Question 55

1st described the translocation between chromosomes 8 & 21 in patients with acute myeloblastic leukemia

Answer 55

1972: Janet Rowley

Question 56

▪ Method widely used to detect proteins & nucleic acids ▪ Targets specific sequences of chromosomes w/ fluorescent probes 🡪 designed to hybridize to critical areas that are amplified/deleted/translocated/ otherwise rearranged in disease states

Answer 56

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Question 57

Requires fluorescence microscope & special filters

Answer 57

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Question 58

2 TYPES OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Answer 58

interphase FISH & metaphase FISH

Question 59

▪ Used to study the genetic content & organization of non-dividing cells, particularly cells in the interphase stage of the cell cycle ▪ Bound probe is visualized under a fluorescent microscope as a point of fluorescent light in the nucleus of the cell

Answer 59

Interphase FISH

Question 60

______= 1 signal

Answer 60

Deletion

Question 61

_____= more than 2 signals

Answer 61

Duplication

Question 62

Commonly used to study prenatal samples, tumors, & hematological malignancies

Answer 62

Interphase FISH

Question 63

bind to regions spanning the breakpoint of translocations

Answer 63

Dual-color probes/dual-fusion probes

Question 64

bind to the intact chromosome flanking the translocation breakpoint

Answer 64

Break-apart probes

Question 65

▪ Hybridize to highly repetitive alpha satellite sequences surrounding centromeres ▪ Detect aneusomy of any chromosome ▪ + region-specific probes = to confirm deletions / amplifications in specific chromosomes ▪ + dual-color probes = tricolor probe serves as a control for amplification/loss of 1 of the chromosomes involved in the translocation ▪ Example: IGH/MYC/CEP 8 Tri-color probes

Answer 65

Centromeric (CEN) probe

Question 66

▪ Designed to specifically target & visualize the telomeric regions of chromosomes ▪ Useful for detection of chromosome structural abnormalities (cryptic translocations/sub-telomeric deletions) that are not easily visualized by standard karyotyping

Answer 66

Telomeric probes

Question 67

Preparation of sample

Answer 67

▪ Permeabilize the cells for optimal-target interaction & to maintain cell morphology ▪ Fresh interphase cells are incubated overnight (aging) after deposition on slides ▪ Cells are treated with protease and fixed with 1% formaldehyde ▪ Cells are dehydrated in graded concentration of ethanol ▪ Denaturation

Question 68

Under a fluorescent microscope w/ appropriate color-distinction filters: signal should be bright, specific to the target in the cell nuclei, & free of high background noise

Answer 68

Quality of the probe

Question 69

▪ 1-10 µg of probe may be used in a hybridization volume of 3-10µL ▪ Hybridization: 37ºC-42ºC in a humidified chamber ▪ Slides are cover-slipped & sealed

Answer 69

Both probe & target must be denatured prior to hybridization

Question 70

Rinsing off of the unbound probe

Answer 70

Washing

Question 71

▪ Sample is observed under fluorescent microscope ▪ Probe signals should be visible from entire intact nuclei ▪ Adequate number of cells must be visible

Answer 71

Microscopic analysis

Question 72

▪ Allows analysis of small regions not visible by regular chromosome banding ▪ Probes that cover the entire chromosome/whole chromosome paints ▪ Combination of 5 fluors & using a special imaging software 🡪 spectral karyotyping can distinguish all 23 chromosomes by chromosome-specific colors (detect abnormalities that affect multiple chromosomes) ▪ Telomeric & centromeric probes are also used 🡪 detection of aneuploidy & other genomic mutations

Answer 72

Metaphase FISH

Question 73

PROCEDURE OF Metaphase FISH

Answer 73

▪ Culture of cells for 72 hours ▪ 45 mins before harvesting, colcemid is added ▪ Cells are suspended in a hypotonic medium (0.075 M KCl) & fixed with methanol/acetic acid (3:1) ▪ Fixed cell suspension is applied to an inclined slide & allowed to dry ▪ 2nd treatment w/ 70% acetic acid ▪ Under a phase contrast microscope 🡪 chromosome should appear well separated w/ sharp borders & cytoplasm should not be visible ▪ Dry the slide & proceed to hybridization

Question 74

▪ Simultaneous use of combinations of different locus-specific probes & chromosome paints ▪ Uses multiple fluorescently labeled probes that target different chromosomal regions/genes ▪ Identifies specific chromosomal regions based on the presence/absence of the probe color visualized with specific filters ▪ May show cryptic translocations & insertions

Answer 74

MULTICOLOR FISH (QMFISH or MFISH)

Question 75

▪ Detection of intrachromosomal amplifications/deletions ▪ Test DNA is isolated & labeled along with a reference DNA

Answer 75

COMPARATIVE GENOME HYBRIDIZATION (CGH)

Question 76

fluoresces @ 550 nm “green” (reference DNA)

Answer 76

Cy3

Question 77

fluoresces @ 650-667 nm “red” (test DNA)

Answer 77

Cy5

Question 78

fluoresces in the red-orange region

Answer 78

Cy3.5

Question 79

can result in cells w/ too few or too many copies of a chromosome

Answer 79

Error in cell division

Question 80

chromosomal errors can appear in eggs as women age

Answer 80

Maternal age

Question 81

conclusive evidence is currently lacking

Answer 81

Environmental conditions