Genetics - DNA/Chromosome Structure, Gene Expression, Mutation, and Repair Flashcards
When is DNA at its most loose?
When is it most compacted?
Interphase
Metaphase
How does
- phosphorylation
- methylation
of histones
affect DNA packaging?
Phosphorylation - loosens DNA
Methylation - condenses DNA
What are the functions of the centromere?
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)
What are telomeres? What are their functions?
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)
Samples from which chromosomes can be analyzed
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
Describe steps involved in chromosome analysis from a blood sample
- 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
Describe acrocentric chromosomes
vs meta-centric (mid) and sub-centric (1/3 from the top)
Centromere is located towards the very top of the chromosome, with virtually no p (short) arm present
13-15, 21-22
What is heterochromatin?
The darker bands of G-banded chromatin
A-T rich
Gene-poor regions
What is euchromatin?
The lighter bands of G-banded chromatin
G-C rich
Gene-rich regions
What is the minimum size chromosome aberrations must be to be detected by G-banding under light microscope?
At least 5 mega-bases in size (5 million base pairs)
Stages of mitosis
DNA replication = 2 copies of each chromosome
- Prophase - chromosomes condense, spindle apparatus assembles
- Pro-metaphase - nuclear membrane disassociates, tubulin fibers enter nucleus and attach at kinetochore around centromere
- Metaphase - chromosomes at most condensed, spindle fiber tension > chromosomes line up at metaphase plate
- Anaphase - sister chromatids separate and drawn to opposite sites of cell at spindle poles
- Telophase - nuclear membrane starts to assemble around each pair of sister chromatids at spindle poles
- Cytokinesis - cleave of cell membrane to create 2 genetically identical daughter cells
Stages of meiosis
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
Define DNA mutation
Permanent change in DNA sequence (can be a single base pair, large segment of chromosome, or whole chromosome)
Describe various methods by which DNA mutations could arise:
- Inherited/hereditary
- De novo
- Somatic
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)
Define DNA polymorphism
Occurs within at least 1% of the population
Considered a normal variant of the DNA sequence
Different types of DNA mutations (and examples of diseases caused by them)
- Missense
- Nonsense
- Insertion
- Duplication
- Frameshift
- Repeat expansion
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
How does chromosomal mosaicism occur?
Early errors in chromosome segregation during mitosis
The earlier in embryogenesis the error occurs, the greater proportion of cells will be affected
Types of aneuploidies:
- Trisomy
- Monosomy
- Triploidy
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
What type of DNA mutation is the leading cause of pregnancy loss and developmental disability in humans?
Chromosomal aneuploidy
Why is sex chromosome aneuploidy less devastating in general than other aneuploidies?
X chromosome inactivation
Few genes on the Y chromosome
Structural chromosome aberrations
- Balanced
- Unbalanced
“Stable”
- 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
What is a para vs peri-centric inversion?
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
Chromosome translocations
- Balanced
- Unbalanced
- Reciprocal
- Robertsonian
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
Different DNA repair pathways:
1. Direct repair
2. Base-excision repair
3. Nucleotide-excision repair
4. Mismatch repair
5. Postreplication repair
- Direct repair: utilizes a specific enzyme to reverse the DNA damage
- Base-excision repair: removal of the damaged base and several surrounding bases by cutting sugar-phosphate backbone > then uses template strand to repair
- Nucleotide-excision repair: removes thymidine dimers and chemically modified bases
- Mismatch repair: corrects mismatched base pairs (if defective, NHPCC, breast cancer)
- Postreplication repair: repairs damage that has resulted in double-stranded breaks, uses other chromosome as a template to copy material that has been deleted
