BPS 594

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BPS 594 Pharmacogenomics and Molecular
Pharmacology Genes and Genetics Debra A.
Tonetti, Ph.D. COP 453 dtonetti_at_uic.edu
Human Molecular Genetics, Strachan Read, 3rd
Edition Chapter 1
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Lecture Objectives

• Understand the composition and chemical bonds
  found in DNA, RNA and polypeptides.
• Know the structure of DNA
• Understand the processes of DNA replication, RNA
  transcription and gene expression.
• List the steps involved in RNA processing
• Know the basic steps involved in translation and
  post-translational processing.
• Understand the different levels of protein
  structure.

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Molecular Genetics

• Primarily concerned with the interaction between
  the information molecules (DNA and RNA) and how
  this information is translated into proteins.
• In eukaryotes, DNA molecules are found in the
  chromosomes of the nucleus, mitochondria and also
  chloroplasts of plant cells.

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Structure of Bases, Nucleosides and Nucleotides
Purines (A and G) 2 interlocked heterocyclic
rings of carbon and nitrogen Pyrimidines (C and
T) only one heterocyclic ring
DNA is consists of a linear backbone of
alternating sugar (deoxyribose) and phosphate
residues
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Common bases found in nucleic acids with
corresponding nucleosides and nucleotides
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A 3-5 Phosphodiester Bond
A phosphate group links the carbon atom 3 of a
sugar to the carbon atom 5 of the neighboring
sugar. Whereas RNA molecules normally exist as
single molecules, DNA exists as a double
helix. The DNA strands are held together by weak
hydrogen bonds to form a DNA duplex
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A-T base pairs have two connecting hydrogen
bonds G-C base pairs have three
Watson-Crick rules A specifically binds to T
and C specifically binds to G therefore A T
and G C.
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The Structure of DNA is a Double-Stranded,
Antiparallel Helix
B-DNA 10 bp/turn
DNA can adopt different helical structures
A-DNA and B-DNA are both right-handed helices
(helix spirals in a clockwise direction). Under
physiological conditions, most DNA is in the
B-DNA form. Z-DNA is a left handed helix
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Intramolecular hydrogen bonding in DNA and RNA

• Double-stranded hairpin loop with a single DNA
  strand.
• Transfer RNA (tRNA) has extensive secondary
  structure.

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DNA Replication is Semi-conservative
During DNA replication the 2 DNA strands are
unwound by a helicase, and each strand directs
the synthesis of a complementary DNA strand. 2
daughter DNA duplexes are formed that are
identical to the parent molecule. Chain growth
must be in the 5?3 direction.
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Asymmetry of Strand Synthesis during DNA
Replication
Synthesis of the leading strand (by DNA
Polymerase d) is continuous in the 5?3
direction, however the lagging strand must be
synthesized in the opposite direction of the
replication fork. 5?3 synthesis occurs is steps
by 100-1000 nucleotide fragments called Okazaki
fragments. RNA primers are first generated
(primase) to provide the free 3-OH group needed
by DNA polymerase a to start DNA synthesis.
These fragments are then joined by DNA ligase
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The chromosome of complex organisms have multiple
replication origins
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Table 1.2. The five classes of mammalian DNA
polymerase
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Major Classes of Proteins used in the DNA
Replication Machinery

• Topoisomerases unwind DNA by breaking a single
  DNA strand. Tension from the supercoil is
  released.
• Helicases Unwind the double strand.
• DNA polymerases
• DNA-directed DNA polymerases (some with DNA
  repair function)
• RNA-directed DNA polymerases (reverse
  transcriptases)
• Telomerase ends of linear chromosome
• Primases attach small RNA primer to provide
  3-OH group for DNA polymerase. Is degraded by
  ribonuclease.
• Ligase catalyzes the formation of a
  phosphodiester bond between adjacent 3OH and
  5-phosphate groups.
• Single-stranded binding proteins Maintains the
  stability of the replication fork, prevents
  single-stranded DNA degradation.

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RNA Transcription and Gene Expression

• The Central Dogma of Molecular Biology
• DNA ? RNA ? protein
• 1 2
• Involves
• 1. Transcription DNA-directed RNA polymerase
  (nucleus, mito.)
• Translation mRNA translated at ribosomes
  (cytoplasm and mito) into protein.
• NOT QUITE TRUE ANYMORE!!!

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Gene Expression in an Animal Cell
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RNA is transcribed as a single strand which is
complementary in base sequence to one strand
(template) of the gene
Only a small fraction of all DNA is
transcribed -different cells require different
genes to be transcribed -highly repetitive
non-coding DNA, pseudogenes Only a small portion
of RNA made by transcription is translated into
protein -noncoding RNA includes tRNA, rRNA,
microRNA (see 9.2.3) -primary transcript is
processed, much of it being discarded -only the
central part of the mature RNA is translated
sections on each end remain untranslated.
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Three Classes of Eukaryotic RNA Polymerases

• Class Genes transcribed
• I 28S rRNA 18S rRNA 5.8S rRNA
• II All genes encoding polypeptides
• III 5S rRNA tRNA genes,snRNAs.

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Trans-acting Transcription Factors and Cis-acting
regulating elements are required for Gene
Expression

• Short sequence elements in the vicinity of the
  gene (cis) are recognized by transcription
  factors (trans) to guide and recruit RNA
  polymerase.
• These sequences are often clustered upstream of
  the coding sequence of the gene and collectively
  define the promoter region.

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Eukaryotic Promoters
Some common cis-acting promoter elements the
TATA box TATAA usually -25 bp upstream the
GC box GGGCGG consensus, is found sometimes in
the absence of the TATA box, function in either
orientation the CAAT box CCAAT often at -80
position, functions in either orientation
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Additional Specific Recognition Elements (often
tissue specific)

• Enhancers located at a variable distances from
  the transcriptional start site
  orientation-independent enhance transcriptional
  activation
• TRE (TPA response element) GTGAGT(A/C)A
• transcription factor AP-1 family (Jun/Fos)
• Silencers similar to enhancers but inhibit
  transcriptional activity of specific genes

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Tissue-Specific Gene Expression

• The DNA content of every cell is identical
• What makes the different cell types unique??
• Only a portion of genes are expressed in any one
  cell type.
• How is this achieved??
• Transcriptionally inactive or active chromatin
• -determined by chromatin conformation
  condensed or open

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RNA splicing involves endonucleolytic cleavage
and removal of intronic RNA segments and
splicing of exonic RNA segments
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Consensus sequences at the DNA level for the
splice donar, splice acceptor and branch sites
in introns of complex eukaryotes
Splicesome large RNA-protein complex that
mediates the splicing reactions consists of 5
types of small nuclear RNA (snRNA) attached to
more that 50 specific proteins the reaction is
initiated by RNA-RNA base pairing between the
transcript and the snRNA
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Mechanism of RNA splicing (GU-AG introns)
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The 5 end of eukaryotic mRNA molecules is
protected by a specialized nucleotide (capping)

• A methylated nucleoside, 7-methylguanosine is
  linked by a 5-5-phosphodiester bond.
• Several possible functions of the cap
• To protect the transcripts from 5-3 exonuclease
  attack.
• To facilitate transport from the nucleus to the
  cytoplasm.
• Aid the attachment of the 40S subunit of the
  cytoplasmic ribosomes to the mRNA.

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The 3 end of most eukaryotic mRNA molecules is
polyadenylated

• RNA polymerase II Polyadenylation signal
  sequence AAUAAA
• Cleavage occurs 15-20 NT downstream followed by
  the addition of about 200 adenylate residues
  (AMP) by the enzyme Poly (A) polymerase
• The Poly(A) tail has several possible functions
• Transport of the mRNA from cytoplasm to the
  nucleus
• mRNA stabilization
• Enhanced recognigion of the mRNA by the ribosomal
  machinery.

Histone mRNAs are not polyadenylated 3 cleavage
occurs by secondary structure of the transcript
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Expression of the human b-Globin Gene
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The genetic code is deciphered by codon-anticodon
recognition
Ribosomes are large RNA-protein complexes that
form a structural framework for polypeptide
synthesis. In eukaryotes 60S and 40S
subunits 60S is comprised of 28S, 5.8 and 5S
rRNA and about 50 proteins 40S is comprised of
18S RNA and about 30 ribosomal proteins. It is
the RNA components that are primarily responsible
for the catalytic function of the ribosome. A
triplet genetic code directs the assembly of
amino acids. Groups of 3 nucleotides (codons)
specify individual amino acids.
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tRNA Molecule
Each tRNA has a specific trinucleotide sequence
called the anticodon and provides the specificity
to interpret the genetic code.
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The nuclear and mitochondrial genetic codes are
similar but not identical
AUG is recognized efficiently as an initiation
codon only when it is embedded in an initiation
codon recognition sequence GCCPu CCAUGG Codons
in blue are interpreted differently in the
nucleus and mitochondria.
The genetic code is a 3-letter code. There are 4
possible bases to choose from at each of 3 base
positions (4)364 possible codons. Since there
are only 20 major types of amino acids, each
amino acid is specified by at least 3 different
codons. Wobble Hypothesis Pairing of codon and
anticodon follow the normal A-U and G-C rules for
the 1st 2 base positions in the codon, the wobble
occurs at the 3rd position and G-U base pairs can
also be used.
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U only
A
C
G only
G
C or U
U
A or G
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Structure of the Amino Acids
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Polypeptides are synthesized by peptide bond
formation between successive amino acids
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Insulin Synthesis Involves Multiple
Post-Translational Cleavages of Polypeptide
Precursors
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Table 1.8. Levels of protein structure
Level
Definition
Comment
Primary
The linear sequence of amino acids in a
polypeptide
Can vary enormously in length from a small
peptide to thousands of amino acids long
Secondary
The path that a polypeptide backbone follows in
space
May vary locally, e.g. as a-helix or b-pleated
sheet, etc.
Tertiary
The overall three-dimensional structure of a
polypeptide
Can vary enormously, e.g. globular, rod-like,
tube, coil, sheet, etc.
Quaternary
The overall structure of a multimeric protein,
i.e. of a combination of protein subunits
Often stabilized by disulfide bridges and by
binding to ligands, etc.
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Regions of secondary structure in polypeptides
are often dominated by intrachain hydrogen bonding
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Intrachain and interchain disulfide bridges in
human insulin
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Chromosome structure and Function

• Molecular Biology of the Cell
• Chapter 2

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Lecture Objectives

• Understand the structure and function of
  chromosomes.
• Know the two types of cell division, mitosis and
  meiosis and be able to identify similarities and
  differences of these processes.
• Learn the nomenclature of chromosomal
  abnormalities and understand the functional
  consequences.

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Human Chromosomal DNA Content During the Cell
Cycle
N the number of different chromosomes in a
nucleated cell.. C the DNA content For humans
N 23 C 3.5 pg Ploidy refers to the
number of copies of chromosomes Most human cells
are diploid 2n and 2C (somatic cells) Sperm and
egg cells are haploid (n and C) (gametes).
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The haploid sperm and egg originate by meiosis
from diploid precursors
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Packaging DNA into Chromosomes Requires Multiple
Hierarchies of DNA folding From DNA Duplex to
Metaphase Chromosome
Compaction ratios 16 for nucleosomes 136 for
30 nm fiber 110,000 for metaphase chromosome
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DNA Molecules are Highly Condensed in Chromosomes
Stretched end-to-end, Chromosome 22 would extend
about 1.5 cm ( 48 million nucleotide pairs). In
a mitotic chromosome, 22 measures only 2 mm in
length. This is a compaction ratio of nearly
10,000-fold! The DNA of interphase chromosomes
have a compaction ratio of 1000-fold. This is
accomplished by proteins that successively coil
and fold the DNA into higher and higher levels of
organization.
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Nucleosomes Basic Unit of Eucaryotic Chromosome
Structure Comprised of both a Histone Protein
Core and DNA
A Electron Micrograph of chromatin isolated
from interphase B Chromatin that has been
experimentally decondensed to visualize the
nucleosomes or beads on a string.
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• 1st Level of DNA Packing
• Reduces the length of a chromatin thread to about
  1/3 its initial length.
• Core particle consists of 2 molecules each of 4
  different histones H2A, H2B, H3, H4.
• Sperm DNA is packaged using protamines (small
  basic proteins) instead of histones.

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The overall structural organization of the core
histones

• The N-terminal tail is subject to several forms
  of covalent modification
• The histone fold region, 3 a-helices connected by
  2 loops, participates in the handshake dimer
  interaction

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The assembly of a histone octamer H2A-H2B dimer
and H3-H4 dimers are formed by the handshake
interaction The H3-H4 tetramer forms the
scaffold for the octomer on to which the H2A-H2B
dimers are added. All 8 N-terminal tails of the
histones protrude from the disc-shaped core.
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Mechanisms to Form the 30 nm Fiber From Linear
Nucleosomes
Zigzag model of compaction involves several
mechanisms acting together. A larger histone,
H1, acts to pull nucleosomes together and the
histone tails may help to pull the nucleosomes
together.
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Model for the Formation of 30 nm Fiber Through
Histone Tails

• Evidence for the model
• X-ray crystal structure show tails of one
  nucleosome contact the histone core of the
  adjacent nucleosome.
• Histone tails interact with DNA

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Functional Elements of a Yeast Chromosome
Centromere Region where sister chromatids are
attached and is essential for segregaton during
cell division. Telomeres specialized structures
comprised of DNA and protein which cap the ends
of eukaryotic chromosomes. Repeated G rich
sequence on one strand in humans (TTAGGG)n,
typically spans 3-20 Kb. Autonomous Replicating
Sequence (ARS) In yeast, the ARS is about 50 bp
in length and consists of an AT-rich region with
a core consensus and some imperfect copies of the
consensus sequence.
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The Structure of a Human Centromere
There is no centromere-specific DNA sequence.
The centromere consists of short repeated DNA
sequences that are A-T-rich, known as a satellite
DNA. The centromere is defined mainly by the
assembly of proteins rather than by a specific
DNA sequence
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• Likely functions of telomeres
• Maintain structural integrity-loss of a telomere
  can result in fusion with another broken
  chromosome or can be degraded.
• Establish chromosome positioning
• Ensure complete replication. The end replication
  problem is solved by telomerase, an RNA-protein
  enzyme.
• Telomerase is a reverse transcriptase
• - RNA-dependent DNA polymerase
• - carries internal RNA component needed to prime
  the leading strand and provide the template for
  the lagging strand.

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Heterochromatin is Highly Organized and Usually
Resistant to Gene Expression
Two types of chromatin exist in interphase nuclei
of many higher eucaryotic cells Euchromatin is
less condensed and associated with genes that are
expressed. Heterochromatin is highly condensed
and usually does not contain genes. However
genes that are packaged into heterochromatin are
resistant to expression. Approximately 10 of
the genome is packaged into heterochromatin. Hete
rochromatin is responsible for the proper
functioning of telomeres and centromeres. Heteroc
hromatin is dynamic, it can spread and retract
and it is tends to be inherited from a cell to
its progeny.
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An Outline of Cell Division by Mitosis In the
human lifetime, there are 1017 mitotic divisions.
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During mitosis, each chromosome in the diploid
set act independently, paternal and maternal
homologs do not associate at all.
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Development of the Germ-line
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Table 2.2. Mitosis and meiosis compared
Mitosis
Meiosis
Location
All tissues
Only in testis and ovary
Products
Diploid somatic cells
Haploid sperm and egg cells
DNA replication and cell division
Normally one round of replication per cell
division
Only one round of replication but two cell
divisions
Extent of prophase
Short (30 min in human cells)
Meiosis I is long and complex can take years to
complete
Pairing of homologs
None
Yes (in meiosis I)
Recombination
Rare and abnormal
Normally at least once in each chromosome arm
Relationship between daughter cells
Genetically identical
Different (recombination and independent
assortment of homologs)
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Independent Assortment of Maternal and Paternal
Homologs at Meiosis I There are 223 or 8.4
million ways of picking one chromosome from each
of the 23 pairs in a diploid cell
This diagram ignores recombination
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Meiosis I - Recombination
At zygotene, a synaptonemal complex is
formed. Chiasma (Chiasmata) marks a chrossover
point
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Meiosis I
Meiosis II
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Chromosome Banding Techniques
G-banding - the chromosomes are subjected to
controlled digestion with trypsin before staining
with Giemsa, a DNA-binding chemical dye. Dark
bands are known as G bands. Pale bands are G
negative. Q-banding - the chromosomes are stained
with a fluorescent dye which binds preferentially
to AT-rich DNA, such as Quinacrine, DAPI (4
,6-diamidino-2-phenylindole) or Hoechst 33258,
and viewed by UV fluorescence. Fluorescing bands
are called Q bands and mark the same chromosomal
segments as G bands. R-banding - is essentially
the reverse of the G-banding pattern. The
chromosomes are heat-denatured in saline before
being stained with Giemsa. The heat treatment
denatures AT-rich DNA, and R bands are Q
negative. The same pattern can be produced by
binding GC-specific dyes such as chromomycin A3,
olivomycin or mithramycin. T-banding - identifies
a subset of the R bands which are especially
concentrated at the telomeres. The T bands are
the most intensely staining of the R bands and
are visualized by employing either a particularly
severe heat treatment of the chromosomes prior to
staining with Giemsa, or a combination of dyes
and fluorochromes. C-banding - is thought to
demonstrate constitutive heterochromatin, mainly
at the centromeres. The chromosomes are typically
exposed to denaturation with a saturated solution
of barium hydroxide, prior to Giemsa staining.
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G-Banded Chromosome 1 at Different Banding
Resolutions
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G-banded prometaphase karyogram (karyotype) of
mitotic chromosomes from lymphocytes of a normal
female
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Male Human Chromosomes Imaged by DNA
Hybridization During Mitosis Chromosome Painting
Each chromosome is painted a different color by
hybridization with chromosome-specific DNA probes
labeled with a fluorescent dye. The display of
the 46 chromosomes at mitosis is called the human
karyotype
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The banding patterns of human chromosomes are
unique as visualized by Giemsa staining.
Cytogeneticists can determine if parts of the
chromosome are lost or switched based on changes
in the banding pattern. These changes are
associated with inherited defects or cancer
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A Giemsa staining of chromosomes 4 and 12 from
a patient with ataxia, a progressive disease
affecting motor skills. B The same
chromosome pair stained by chromosome painting.
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Chromosome Abnormalites

• Changes resulting in a visible alteration of the
  chromosomes.
• FISH allows much smaller changes to be seen.
• Most chromosomal aberrations are produced by
  misrepair of broken chromosomes, improper
  recombination or by malsegregation of chromosomes
  during mitosis or meiosis.

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Types of Chromosomal Abnormality

• Constitutional abnormality present in all cells
  of the body.
• Somatic abnormality present in only certain
  cells or tissues of an individual.
• -this individual is a mosaic
• Most abnormalities are either numerical or
  structural.

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Numerical Abnormalities
Polyploidy 1-2 of human pregnancies are
triploid. Usually caused by 2 sperm fertilizing
the same egg. Constitutional polyploidy is rare
and lethal, all normal people have some polyploid
cells. Aneuploidy one or more individual
chromosomes is present in an extra copy or is
missing from a euploid set. Trisomy three
copies of a chromosome (trisomy 21, Down
syndrome).
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Table 2.4. Consequences of numerical chromosomal
abnormalities
Polyploidy
1  3 of all conceptions almost never liveborn
do not survive
(69,XXX, XXY or XYY)
  Triploidy
Aneuploidy
Preimplantation lethal
nullisomy (missing a pair of homologs)
Autosomes
Embryonic lethal
monosomy (one chromosome missing)
Usually embryonic or fetal lethal
trisomy (one extra chromosome)
Trisomy 13 (Patau syndrome) and trisomy 18
(Edwards syndrome) may survive to term
Trisomy 21 (Down syndrome) may survive to age 40
or longer
Aneuploidy (sex chromosomes) Additional Sex
chromosomes Lacking a sex chromosome
Relatively minor problems, normal lifespan
(47,XXX, 47,XXY, 47,XYY
Turner syndrome - 99 abort spontaneously
survivors are of normal intelligence but
infertile and show minor physical signs
45,X
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Structural Chromosomal Abnormalities

• Chromosome breaks occur as a result of DNA damage
  (radiation or chemicals) or as part of
  recombination.
• Arise when breaks are repaired incorrectly.
• A break that occurs in G2 results in a chromatid
  break affecting only one fof the 1 sister
  chromatids.
• Breaks occurring in G1, if not repaired before S
  phase, appear later as a chromosome break.

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Possible stable results of 2 breaks on a single
chromosome
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Origins of Translocations
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