Genetics - DNA/Chromosome Structure, Gene Expression, Mutation, and Repair

Question 1

When is DNA at its most loose?

When is it most compacted?

Answer 1

Interphase

Metaphase

Question 2

How does
- phosphorylation
- methylation
of histones
affect DNA packaging?

Answer 2

Phosphorylation - loosens DNA

Methylation - condenses DNA

Question 3

What are the functions of the centromere?

Answer 3

Centromere - specialized region of DNA (highly repetitive sequences with large degree of homology) localized within the constriction between sister chromatids on DNA

Key Functions:
- Hold together the 2 chromatids formed after DNA replication until cell division is complete
- Attachment site for mitotic spindle prior to separation of the sister chromatids (therefore essential to proper segregation)

Question 4

What are telomeres? What are their functions?

Answer 4

Caps at terminal ends of telomeres.

TTAGGG (highly relative sequence) to allow the minimal amount of DNA lost with each replication.

Functions:
- Seal ends of chromosomes to prevent fusion with other chromosomes
- Enabling ends of chromosomes to be replicated (with every division, ends of telomeres are shortened, as very ends of chromosomes cannot be replicated; eventually, telomeres become critically short and chromosome can no longer be replicated)

Question 5

Samples from which chromosomes can be analyzed

Answer 5

Chromosomes can be analyzed from any dividing cell that contains DNA
- CVS
- Amniotic fluid
- Spontaneous abortus (*abortus cells cannot be cultured as they are not capable of continuing to replicate)
- Bone marrow
- Blood

Question 6

Describe steps involved in chromosome analysis from a blood sample

Answer 6

• Remember, only WBCs contain DNA, not RBCs. Therefore, RBCs are removed first.
  1. WBCs are cultured with PHA (phytohemagglutinin) to induce mitosis x 71 hours
  2. Treat with colchicine/colcemid (breaks down the mitotic spindle to arrest in metaphase)
  3. Hypotonic solution to induce cell swelling
  4. Centrifuge > fix > Giemsa stain (G-banding) to visualize on microscope

*The earlier in the metaphase, the longer the chromosomes are, and the better the G-bands are visible

Question 7

Describe acrocentric chromosomes

vs meta-centric (mid) and sub-centric (1/3 from the top)

Answer 7

Centromere is located towards the very top of the chromosome, with virtually no p (short) arm present

13-15, 21-22

Question 8

What is heterochromatin?

Answer 8

The darker bands of G-banded chromatin

A-T rich

Gene-poor regions

Question 9

What is euchromatin?

Answer 9

The lighter bands of G-banded chromatin

G-C rich

Gene-rich regions

Question 10

What is the minimum size chromosome aberrations must be to be detected by G-banding under light microscope?

Answer 10

At least 5 mega-bases in size (5 million base pairs)

Question 11

Stages of mitosis

Answer 11

DNA replication = 2 copies of each chromosome

1. Prophase - chromosomes condense, spindle apparatus assembles
2. Pro-metaphase - nuclear membrane disassociates, tubulin fibers enter nucleus and attach at kinetochore around centromere
3. Metaphase - chromosomes at most condensed, spindle fiber tension > chromosomes line up at metaphase plate
4. Anaphase - sister chromatids separate and drawn to opposite sites of cell at spindle poles
5. Telophase - nuclear membrane starts to assemble around each pair of sister chromatids at spindle poles
6. Cytokinesis - cleave of cell membrane to create 2 genetically identical daughter cells

Question 12

Stages of meiosis

Answer 12

Meiosis I - reductional division
1. Prophase I - 5 stages
- 1. Leptotene
- 2. Zygotene
- 3. Pachytene: crossing over
*the longer the chromosome, the more sites of crossing over
- 4. Diplotene
in females, oocyte arrested in this stage at 20 weeks gestation until ovulation
- 5. Diakinesis: separation into 2 daughter cells (HAPLOID)
2. Metaphase I
3. Anaphase I
4. Telophase I
5. Cytokinesis I
* no DNA replication occurs again
End result of meiosis I: 2 genetically different daughter cells with 23 chromosomes (haploid), but 2 chromatids

Meiosis II - equational division
1. Prophase II
2. Metaphase II
3. Anaphase II
4. Telophase II
5. Cytokinesis II
End result of meiosis II: 4 genetically different daughter cells with 23 chromosomes (haploid)

• In males, meiosis is initiated in puberty and never arrests
• In females, meiosis is initiated in fetal life (12 weeks gestation) > arrested in meiosis I, prophase I, diplotene I at 20 weeks gestation until ovulation > arrested in meiosis II, metaphase II until fertilization (if fertilization does not occur, oocyte shed without completing meiosis II)
• Primary oocyte > Meiosis I complete > Secondary oocyte > Meiosis II

Question 13

Define DNA mutation

Answer 13

Permanent change in DNA sequence (can be a single base pair, large segment of chromosome, or whole chromosome)

Question 14

Describe various methods by which DNA mutations could arise:
- Inherited/hereditary
- De novo
- Somatic

Answer 14

Inherited/hereditary: passed from parent > offspring, present in all cells

De novo: new mutations in egg or sperm or shortly after fertilization, present in all or most cells

Somatic: arises sporadically during a lifetime, mosaic (present only in the cells derived from the affected cells)

Question 15

Define DNA polymorphism

Answer 15

Occurs within at least 1% of the population

Considered a normal variant of the DNA sequence

Question 16

Different types of DNA mutations (and examples of diseases caused by them)
- Missense
- Nonsense
- Insertion
- Duplication
- Frameshift
- Repeat expansion

Answer 16

Missense mutation: single nucleotide altered (e.g. C instead of A) > changes assembled amino acid in polypeptide chain
- Achondroplasia
- Sickle cell disease

Nonsense mutation: single nucleotide altered which early produces a stop codon, causing protein shortening
- Duchenne muscular dystrophy
- Cystic fibrosis

Insertion mutation: 1+ nucleotides inserted > alters gene length, alters some/all of the AAs
- Hemophilia
- CF

Deletion mutation: similar to insertion, but with 1+ nucleotide deletion
- CF
- Hemophilia
- Duchenne muscular dystrophy
- Di George syndrome
- Cri du chat syndrome
- Prader willi syndrome
- Thalassemia

Duplication mutation: segment of DNA copied, producing longer protein/chromosome
- Beckwidth-Widemann syndrome
- Charcot-Marie-Tooth disease

Frameshift mutation: caused by duplications, insertions, deletions; reading frame of DNA altered > typically alters all the AAs following mutation and cause non-functional protein
- Tay-Sachs
- CF

Repeat expansion mutation:
- e.g. CAG repeats (Fragile X), Huntington, myotonic dystrophy
- Can be expanded (how many times is it repeated?)
- Usually threshold for how many repeats can be tolerated for protein to function normally
*Copy number variation (CNV): number of repeats varies across individuals, surprisingly common (occur in >10% of human DNA), does not cause disease

Question 17

How does chromosomal mosaicism occur?

Answer 17

Early errors in chromosome segregation during mitosis

The earlier in embryogenesis the error occurs, the greater proportion of cells will be affected

Question 18

Types of aneuploidies:
- Trisomy
- Monosomy
- Triploidy

Answer 18

Aneuploidy = numerical chromosome aberration
*Vast majority of aneuploidies will be spontaneously aborted

Trisomy = presence of additional chromosome
- Viable trisomies: T21 (Down), T18 (Edward), T13 (Patau), Sex chromosome : XXY (Klinefelter), XXX (Triple X syndrome)
*Vast majority of these still result in SAB

Monosomy = loss of a chromosome
- Viable monosomy: Monosomy X (Turner)

Triploidy = extra copy of every chrosome, NON-viable

Question 19

What type of DNA mutation is the leading cause of pregnancy loss and developmental disability in humans?

Answer 19

Chromosomal aneuploidy

Question 20

Why is sex chromosome aneuploidy less devastating in general than other aneuploidies?

Answer 20

X chromosome inactivation

Few genes on the Y chromosome

Question 21

Structural chromosome aberrations
- Balanced
- Unbalanced
“Stable”

Answer 21

• Occur spontaneously
• Can be passed on/inherited
• Can be induced (chemicals, viral infections, rare inherited conditions)

Can be identified by karyotyping if > 5 Mb)

*Many structural rearrangements are specific to families and difficult to counsel regarding
*Majority of structural rearrangements result in SAB

Balanced (all chromosome material present)
- In general, no effect, unless:
- Break in functional gene
- If aberration is too small to be detected by karyotyping

Unbalanced (extra/missing material)
- Can be deleterious
- Partial monosomy or trisomy or both duplication and deletion
- Risk of passing to offspring depends on size of unbalanced segment, whether imbalance is monosomic/trisomic, which genes are involved, whether break points occur within a gene

Stable - passed through cell divisions if functional centromere + telomeres on p and q arms

Question 22

What is a para vs peri-centric inversion?

Answer 22

A type of structural chromosomal rearrangement in which there is an inversion (flip) of a DNA segment.

Paracentric: DNA segment does not involve centromere

Pericentric: DNA segment includes centromere

*Generally balanced, unless there is loss of material around break points

Can affect future offspring if genetic recombination occurs at inversion sites

Question 23

Chromosome translocations
- Balanced
- Unbalanced
- Reciprocal
- Robertsonian

Answer 23

Chromosome translocations are a special type of structural rearrangement

Requires at least 2 breaks, 1 in each chromosome

Balanced - if all genetic material remains present (however, during meiosis, chromosomes can segregate resulting in unbalanced SR in offspring)

Reciprocal - translocation involving 2 autosomes

Robertsonian - translocation involving 2 acrocentric chromosomes

Question 24

Different DNA repair pathways:
1. Direct repair
2. Base-excision repair
3. Nucleotide-excision repair
4. Mismatch repair
5. Postreplication repair

Answer 24

1. Direct repair: utilizes a specific enzyme to reverse the DNA damage
2. Base-excision repair: removal of the damaged base and several surrounding bases by cutting sugar-phosphate backbone > then uses template strand to repair
3. Nucleotide-excision repair: removes thymidine dimers and chemically modified bases
4. Mismatch repair: corrects mismatched base pairs (if defective, NHPCC, breast cancer)
5. Postreplication repair: repairs damage that has resulted in double-stranded breaks, uses other chromosome as a template to copy material that has been deleted