Lecture 2 Flashcards
What is the cell cycle?
G1 - Hrs to years depending on cell type
G0 - Cells permanently in G1
S - Synthesis of DNA occurs
G2 - Prepping the cell for mitosis/meiosis
M - Nuclear and Cytoplasmic division
Interphase
G1 + S + G2
- Chromosomes resemble fine fibers.
- Extremely decondensed
- Individual chromosomes are unrecognizable
- Has gene transcription
During M phase what happens to the chromosomes?
Chromosomes become more condensed before M phase, and continue to condense during.
Metaphase
- Replicated chromosomes line up on the spindle apparatus.
- Analysis requires disruption of spindle apparatus to get a spread.
How are G-bands produced?
By trypsin pretreatment, then staining with Giemsa.
Which autosome pair have unique banding?
X & Y
What are the Pseudoautosmal regions on X & Y?
Region 1 = 2.5 mb DNA with no less than 24 genes
Region 2 = 230 kb DNA with 4 genes
Autosomes
What chromosomes are collectively called in a karyotype.
A Group
(1;2;3)
Metacentric
B Group
(4;5)
Submetacentric
C Group
(6;7;8;9;10;11;12)
D Group
(13;14;15)
Acrocentric
E Group
(16;17;18)
F Group
(19;20)
G Group
(21;22)
Acrocentric
What groups are acrocentric with stalks and satellites?
Groups D & G
Homologues
2 pairs of a chromosome
Telomere
- Protects the integrity of a chromosome structure
- Without it, the chromosome would fuse and produce unstable structures
- Has 6 base pair repeat
- Shortens with each mitotic division
What has telomerase that can activate/elongate telomeres?
Stem cells
Centromeres
- Site at which chromosomes attach to the spindle apparatus during cell division.
- Composed of satellite DNA
- Repetitive non-coding highly compacted DNA
- Size varies between chromosomes and person to person.
- Alpha-satellite DNA is predominate type
Kinetochore
Specialized protein that participates in the function of chromosome segregation
Chromatin
- Composes centromere of one linear molecule of DNA and 2 classes of proteins.
- Undergoes levels of folding and compaction to fit nucleus
- Has 3.3. billion base pairs of DNA
- Is 2 meters long
What are the 2 classes of proteins in chromosomes?
Histone - 5 major types (H1, H2, H2B, H3, H4)
Nonhistone - Have various functions
What are Nonhistone functions?
- Structural
- DNA replication / DNA polymerase
- Chromosome segregation
- Gene expression
Of the Nonhistone functions, which is the largest group?
Gene expression
What do the stalks of the acrocentric chromosomes do?
Code for rRNA at the Nuclear Organizing Regions (NOR)
What are the largest and smallest chromosomes?
Largest - Chromosome 1 - 250 million bp
Smallest - Chromosome 21 - 50 million bp
Nucleosome
- Is the 1st level of chromatin compaction
- Has a Histone core with 2 copies of H2A, H2B, H3, H4
- Has a segment of DNA wrapped around the core twice
- Has 160 base pairs per core
- Is not evenly spaced but placement is crucial
- This placement is transmitted from parent to daughter cell with high fidelity.
What is a chromatin compaction made of?
- Nucleosome
- 40 base pairs of Linker DNA between Nucleosomes
- H1 that binds DNA where it enters or leaves the nucleosome
What does it mean that a chromatin structure is dynamic?
- Changes allow protein access to DNA during Interphase
- Permits processes like gene transcription and replication
What are the 2 types of chromatin?
Euchromatin and Heterochromatin
Euchromatin
- Contains protein coding DNA
- Genes actively transcribed
Heterochromatin
Has 2 types:
Constitutive - Repetitive DNA sequences and does not contain genes
Facultative - Euchromatin that is inactivated and genes are not transcribed
What are the 2 regulations of Chromatin?
Upper panel:
- Methylation promotes tightly packed nucleosomes
- Transcription factors cannot bind DNA
- Genes not expressed
Lower panel:
- Acetylation of histones results in loosely packed nucleosomes
- Transcription factors access DNA
- Genes expressed
Constitutive Heterochromatin
- Always inactive
- Vary in length and size
- Vary in staining intensity of satellites
- Occasional variation in # of satellites
- Located in:
- proximal long arms of chromosomes 1;9;16
- distal long arm of Y- short arms and satellites of acrocentric
- chromosomes
- short arms and satellites of acrocentric
Facultative Heterochromatin
- Can go back and forth between active and inactive
- Inactive X is best example
- Transcriptional inactivation of all X chromosomes in excess of one copy
- Provides dosage compensation between males and females.
- Appears in interphase cell as condensed darkly stained body (aka Barr body)
Process of Inactive X
- Both X’s active at conception
- During blastocyst stage one X becomes inactive
- Inactive X turned back on during oogenesis
- Becomes euchromatin again
X Chromosome Inactivation
- Inactivation occurs through the action of the Xist gene.
- Usually random
- All descendants of a cell inactivate the same X
What is Xist stand for and how is it regulated?
X (inactive) Specific Transcript
- Located on proximal Xq
- Untranslated RNA coats X to help silence it
-It is regulated by methylation of CpG islands of promoters
Non-random or Skewed Inactivation
- Lethal gene mutation on one X
- Serious X-linked recessive diseases
- Structural abnormality of X
Examples of X-linked recessive diseases
Duchenne Muscular Dystrophy
Hemophilia
Skewed inactivation in manifesting females
What percentage escape inactivation?
10% to 15%
Numerical Abnormalities
Aneuploidy - gain trisomy or loss monosomy of whole chromosome
Polyploidy - gain of haploid or diploid set of chromosomes
Structural Abnormalities
Balanced - rearrangement without gain or loss of chromatin
Unbalanced - rearrangement with gain, loss or both of varying amounts of chromatin
Uniparental Disomy (UPD)
Gain of DNA from one parent AND loss of same DNA from other parent
Constitutional Abnormalities
Present at birth
G-Banded Chromosome Analysis
- Requires live cells arrested in metaphase
- Used to detect:
- Numerical abnormalities
- Balanced and unbalanced structural abnormalities affection more than 5-10 MB of DNA
FISH
- Can detect numerical and structural abnormalities
- Applicable to interphase or metaphase cells
- Detects / sees abnormalities too small to be seen by microscope
FISH Process
- Fluorescently labeled molecule attached to piece of DNA from a specific chromosome region to create a probe
- Probe solution applied to patient cells on a slide
- Probe attaches to complementary sequences on specific chromosome
- Counterstained and viewed with fluorescent microscope
Example of microdeletion syndromes (22q11.2)?
DiGeorge Syndrome
Velocardiofacial Syndrome
Interphase FISH
- Detection of gene rearrangements in oncology studies
- Dual color / dual fusion probe sets
- Break apart probe sets
Advantages and disadvantages of FISH?
Advantages:
- Increased resolution over G-banded analysis
- Faster TAT for interphase FISH
- Genetic sex determination in newborn with ambiguous genitalia
- Rule out lethal trisomy in critically ill newborn
- Detection of mosaicism missed in dividing cells
- Archival tissue (formalin fixed paraffin embedded) may be used in some cases
Disadvantages:
- Only targets specific chromosome region
- Probes not readily available for all known abnormalities
Chromosomal Microarray Analysis (CMA)
Detects:
- Numerical abnormalities
- Unbalanced structural abnormalities including ones too small to be seen by conventional karyotyping or FISH
- Some cases of uniparental disomy if array contains single nucleotide polymorphism SNP probes
- Will not detect balanced structural rearrangements
- Cannot determine exact nature of rearrangement
- Uses extracted DNA to detect copy number abnormalities
- Patient and control DNA labeled with different color fluorescent dyes
- Laser scanner measures dye intensities of each probe
- Computer software compares patient and control data
Aneuploidy
- Affects 4% to 5% pregnancies.
- Affects 0.3% Newborns
- Up to 4% Stillbirths
- Most end in miscarriage.
- Trisomies are most common groub seen (16;22;21;15) (Trisomy 16 most common)
Full Trisomy
- Autosomal
- All cells abnormal
- The following can result in live births (13;18;21)
- Others are incompatible with life
Mosaic Trisomy
- Autosomal
- Trisomic cells and normal cells (normal cells lessen severity)
- (8;9) Well known syndromes
Trisomy 21
- Down Syndrome
- Most common autosomal trisomy
- Common cause of pregnancy loss.
- Phenotypic result of 3 copies of 21
Example: 47,XY,+21
Down Syndrome Phenotypes
- Upslanting palpebral fissures
- Flat nasal bridge
- Small mouth
- Protruding tongue
- Single palmar crease
- Increased risk of heart defect
- Short stature
- Hypotonia in infancy
- Variable degrees of intellectual disability
- Life expectancy 60 yrs
Trisomy 18
- Edwards Syndrome
- More common at conception
- 95% of conceptions end in miscarriage
- Severe growth impairment
- Failure to thrive after birth
- Profound neurological impairment
Example: 47,XY,+18
Trisomy 18 Phenotype
- Severe neurological impairment
- Very hypertonic – note contractures
- Cardiac anomalies
- Growth retardation pre and postnatally
- Rocker bottom feet
- Clenched hands
Trisomy 13
- Patau Syndrome
- Majority of trisomy 13 conceptions lost prenatally
- Median survival 2.5 days
- Only 5% survive 6 months
- Recurrence risk < 1%
Example: 47,XX,+13
Trisomy 13 Phenotype
- IUGR/Failure to Thrive
- Severe Neurological Impairment
- Craniofacial anomalies
- Cleft lip and palate
- Micropthalmia (very small eyes)
- Malformed and low set ears
- Scalp Defects
- Microcephaly
- Holoprosencephaly
- Cardiac anomalies (~80%)
- Polycystic kidneys
What causes Aneuploidy?
- Recombination failure
- Nondisjunction
- Premature homologue separation
- Premature sister chromatid separation
- Anaphase lag
DNA Replication Process
- Double helix unwinds (both strands serve as a template)
- DNA Polymerase adds new nucleotides
- Synthesis occurs from 5’ to 3’
- Leading strand synthesized continuously/lagging strand in short segments joined by ligase
- Multiple origins of replication
- Each DNA segment has own characteristic timing of replication
- DNA replication only one part of process
True or False, Heterochromatin is late replicating
True
What is required to complete replication of telomeres at 5’ end?
Telomerase
What are the effects of Low and Hight telomerase activity?
- Low telomerase activity leads to progressive shortening of telomeres with each cell division
- High telomerase activity seen in many tumor cells
M (Mitosis) Phase of Somatic Cells
- At end of S phase, each chromosome consists of two double helix strands (sister chromatids) held together at the centromere
- During metaphase, chromosomes align on spindle apparatus
- At anaphase, cohesin degrades allowing separation to opposite poles
- Result is two daughter nuclei with exact same chromosome complement
Cohesin
Protein that wraps around chromosome to prevent premature separation of sister chromatids during Mitosis.
Cytokinesis
Division of cytoplasm
Meiosis
1 round of DNA synthesis
2 rounds of nuclear/cytoplasmic division
Synapsis
A another term for pairing during Meiosis
What initiates chromosome pairing during Meiosis?
Synaptonemal complex forms between two homologous chromosomes
Meiosis Basic Process
- Diploid Germ Cell
- Normal Miosis I
- Haploid Gametocyte
- Normal Miosis II
- Normal Gametes
Prophase of Meiosis
- Chiasmata (physical links) form between homologues
- Mediated by programmed double strand breaks
- Allows exchange of chromatin between parental chromosomes
- Helps homologous chromosomes remain paired until end of MI
Pairing of X and Y in Male Meiosis
- X and Y pair only in PAR 1 and PAR 2
- Unpaired sequences transcriptionally silenced during pachytene
Meiotic sex chromosome inactivation (MSCI)
Unpaired sequences transcriptionally silenced during pachytene
What is essential for male fertility?
Sequences becoming heterochromatin for duration of spermatogenesis during paring in male meiosis.
What happens when there is a lack of Chiasmata?
- Numerical abnormalities of X and Y are common
- Important for each chromosome arm to have at least one chiasma otherwise there will be Recombination failure
Meiosis I (Part 1)
-Synaptonemal complex breaks down at end of prophase I
-Bivalents begin to separate
(Still held together at the points of the chiasmata)
-Metaphase I
(Bivalents align on spindle still connected by chiasmata)
Anaphase I
- Disjunction of homologous chromosomes
- Chiasmata migrate toward telomeres and are removed
- Cohesin between chromosome arms is cleaved
- Cohesin around each centromere protected by protein called Shugoshin (guardian spirit) and thus not degraded
- Homologous centromeres drawn toward opposite poles
Meiosis I (Part 2)
-Independent assorting
-Telophase I
(One haploid set of chromosomes now grouped at each pole)
-Cytokinesis:
Cytoplasm equally divided between two haploid daughter cells in spermatogenesis
Cytoplasm shunted mostly to one daughter cell in oogenesis
Daughter cell with little cytoplasm becomes first polar body
Meiosis II
- No S phase in meiosis II
- Similar to mitosis, sister chromatids separate
- Shugoshin and cohesion around the centromeres break down to allow separation
The end result of Meiosis II is?
- Spermatogenesis is four sperm
- Oogenesis is one oocyte and two polar bodies
When does Oogenesis begin and complete?
-Oogenesis begins during prenatal period but not complete until after fertilization of egg
-Oocytes arrested in prophase I before birth
MI resumes after puberty at ovulation
-MII not completed unless ooctye is fertilized
Breakdown of Meiosis
meiosis I (prophase I, metaphase I, anaphase I, telophase I) and meiosis II (prophase II, metaphase II, anaphase II, telophase II).
Breakdown of Mitosis
prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis
Maternal Age Effect
- Advancing maternal age increases risk of nondisjunction.
- Most pronounced in Trisomy 21
Meiosis I errors that lead to Aneuploidy.
- Asynapsis of homologous chromosomes
- Recombination failure
- Premature homologue separation
- True nondisjunction
- Premature sister chromatid separation
Asynapsis of homologous chromosomes
- Homologues do not pair
- Segregate randomly and independently
Recombination failure
- Chiasmata do not form between paired homologues
- When synaptonemal complex breaks down, two homologues segregate randomly and independently
Premature homologue separation
- Premature resolution of chiasmata
- Reduced number of chiasmata
- Abnormal placement of chiasmata
-Segregate randomly and independently
True nondisjunction
Chiasma not resolved
Both homologues pulled to same pole
Premature sister chromatid separation
Premature loss of shugoshin/cohesin between sister centromeres
Sister chromatids separate in MI instead of M2
Meiosis II errors that may lead to aneuploidy
- Nondisjunction
- Premature sister chromatid separation
Nondisjunction
Failure to resolve the connection between the sister centromeres
Anaphase Lag
- Delayed movement of homologue or chromatid to one of the poles
- May be due to failure to attach to spindle
- Results in bivalent or chromatid not being included in nuclear membrane
- Micronucleus may form in cytoplasm that contains the lone chromosome
- Eventually lost during a subsequent cell division
- May occur in meiosis or mitosis
Most Aneuploidy Autosomal errors occur where?
Most autosomal aneuploidies result from errors in maternal meiosis
Chromosomal differences in maternal meiosis
Trisomy 16 – almost all are M1 errors
Trisomy 21 – majority are M1 errors
Trisomy 18 – majority are M2 errors
Mitotic Errors After Normal Conception
- Nondisjunction or premature sister chromatid separation occurs in cell of developing embryo or fetus
- Result is one trisomic cell and one monosomic cell
- Monosomy 21 cell not viable
- Trisomy 21 cell continues to divide producing a trisomy 21 cell line
- Result: Mosaicism for abnormal cell line and normal cell line
Timing of Mitotic Error
Timing and location of mitotic error determines type and location of mosaicism
- Somatic Mosaicism
- Confined placental Mosaicism
- Gonadal Mosaicism
Somatic Mosaicism
Prior to formation of inner and outer cell masses likely leads to mosaicism in placenta and baby
Confined placental Mosaicism
Nondisjunction only in outer cell mass likely leads to it
Gonadal Mosaicism
Nondisjunction in primordial germ cell may lead to it
-Individual does not show phenotype but can transmit abnormality to next generation
47,XX,+13[9]/46,XX[11]
Example of Mosaicism
Sex Chromosome Aneuploidy
- Phenotypic manifestations of sex chromosome trisomies less severe than autosomal trisomies
- Mitotic nondisjunction leads to mosaicism
- Can see multiple different abnormal cell lines
45,X
- Turner Syndrome
- Only viable monosomy
-99% miscarry by 28 weeks
Remaining 1% survive to term
Phenotypic Features of Turner Syndrome
- Phenotypic females
- Short stature (under 5 feet)
-Sexual immaturity
Lack of 2° sex characteristics
Streak gonads
Primary amenorrhea
-Common presentation in utero Cystic hygroma (top panel)
- Lymphedema of hands and feet may be seen in newborns
- Webbed neck
- Broad shield shaped chest
- Cubitus valgus
- Cardiac defects in ~ 50%
- Renal anomalies ~ 1/3
- Cognitive function/possible learning disabilities
47,XXX
-Triple X Syndrome
-Phenotype unremarkable
May be taller than normal siblings
May have mild dysmorphism
-IQ usually in normal range
Learning disabilities common
-Fertility:
Usually normal
May have earlier menopause
May be some risk for chromosomally abnormal children
47,XXY
- Klinefelter Syndrome
- 1 in 1,000 newborn males
- Taller than chromosomally normal brothers
-Hypogonadism:
May enter puberty normally or may experience delayed puberty
Secondary sexual characteristics underdeveloped
Often requires testosterone supplementation
-IQ is usually within the normal range
(10 or so points lower than normal siblings)
Increased risk of learning disabilities and speech delay
Klinefelter syndrome patient comes to attention through:
- Prenatal diagnosis for advanced maternal age or parental concern
- Work up for speech delay as a child
-As an adolescent:
Development of a somewhat female body shape
Gynecomastia seen in up to 50%
-Work up for infertility
Spermatogenesis not complete
47,XYY
-Incidence of ~ 1 in 1,000 males
-Phenotype subtle
Height tends toward tall beginning in childhood
Tendency toward truncal weight gain
Hypertelorism
-IQ usually within normal range
-Normal fertility
No empirically increased risk for chromosomally abnormal children
-May see behavior problems Attention deficits Hyperactivity Impulsiveness Temper tantrums and low tolerance for frustration
Paternal meiotic errors common in sex chromosome aneuploidy
47,XXX – 10% due to paternal errors
47,XXY – 50%
47,XYY – 100%
What are the 2 Polyploidy?
Tetraploidy - gain of one diploid set of chromosomes
Triploidy - gain of one haploid set of chromosomes
Tetraploidy
- 92 chromosomes
- Common mosaicism with diploid cell line in long term cell cultures
- Usually considered culture artifact or confined placental mosaicism
- Mosaicism detected in the placenta but not in the fetus
- Diploid/tetraploidy mosaicism documented in liveborn individuals
Triploidy
- Occurs in 1-3% of all conceptions
- One of the most common chromosome abnormalities observed in first trimester miscarriages
- Small percentage survive to term
- Usually survive only a few days
- Longer survival possible if normal cell line present
- Rare
Endoreduplication
DNA synthesis without subsequent cell division
Possible karyograms:
69,XXX
69,XXY
69,XYY
Examples of Triploidy
Parental origin of Triploidy
-Diandry (paternal) Moderate growth retardation of fetus Enlarged abnormal placentas Partial hydatidiform molar changes Focal cell proliferation with mixture of normal and cystic type cells
-Digyny (maternal)
Severe growth retardation of trunk and limbs
Macrocephaly
Placenta small and non-cystic
Etiologies (causes) of Triploidy
- Dispermy - Simultaneous fertilization of one egg by two sperm
- Fertilization of diploid egg by haploid sperm
- Post-zygotic event leading to mosaicism with normal cell line
- NOT considered hereditary
