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BPS 594
Pharmacogenomics and Molecular Pharmacology
Genes and Genetics
Debra A. Tonetti, Ph.D.
COP 453
[email protected]
Human Molecular Genetics, Strachan & Read, 3rd Edition
Chapter 1
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.
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.
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
Common bases found in nucleic acids with
corresponding nucleosides and nucleotides
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
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.
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 righthanded 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
Intramolecular hydrogen bonding in DNA and RNA
(A) Double-stranded hairpin loop with a single DNA strand.
(B) Transfer RNA (tRNA) has extensive secondary structure.
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.
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
The chromosome of complex organisms have multiple replication origins
Table 1.2. The five classes of mammalian DNA polymerase
High Fidelity Class
a
b
g
d
e
Location
Nuclear
Nuclear
Mitochondrial
Nuclear
Nuclear
Function
Synthesis and
priming of lagging
strand
DNA
repair
Replicates
mitochondrial
DNA
Synthesis of
leading
strand
--
3’→ 5’
exonuclease
DNA repair
No
No
Yes
Yes
Yes
-
Base
excision
mtDNA repair
Nucleotide
and base
excision
Nucleotide
and base
excision
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 3’OH and 5’-phosphate groups.
• Single-stranded binding proteins: Maintains the stability of the
replication fork, prevents single-stranded DNA degradation.
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.)
2. Translation: mRNA translated at ribosomes
(cytoplasm and mito) into protein.
NOT QUITE TRUE ANYMORE!!!
Gene Expression in an Animal Cell
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.
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.
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.
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
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
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
RNA splicing involves endonucleolytic cleavage and removal of intronic RNA
segments and splicing of exonic RNA segments
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
Mechanism of RNA splicing (GU-AG introns)
The 5’ end of eukaryotic mRNA molecules is protected
by a specialized nucleotide (capping)
A methylated nucleoside, 7methylguanosine is
linked by a 5’-5’phosphodiester bond.
Several possible functions of
the cap:
1. To protect the transcripts
from 5’-3’ exonuclease
attack.
2. To facilitate transport from
the nucleus to the
cytoplasm.
3. Aid the attachment of the
40S subunit of the
cytoplasmic ribosomes to
the mRNA.
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
Histone mRNAs are not polyadenylated: 3’ cleavage
occurs by secondary structure of the transcript
The Poly(A) tail has several
possible functions:
1. Transport of the mRNA
from cytoplasm to the
nucleus
2. mRNA stabilization
3. Enhanced recognigion of
the mRNA by the
ribosomal machinery.
Expression of the human b-Globin Gene
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.
tRNA Molecule
Each tRNA has a specific trinucleotide sequence called the anticodon and provides
the specificity to interpret the genetic code.
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)3=64 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.
Table 1.5. Codon-anticodon pairing admits relaxed base-pairing (wobbles) at
the third base position of codons
Base at 5’ end
of tRNA anticodon
A
C
Base recognized at 3 ‘
end of mRNA codon
U
only
G
only
G
C or
U
U
A or
G
Structure of the Amino Acids
Polypeptides are synthesized by peptide bond formation
between successive amino acids
Table 1.6. Major types of modification of polypeptides
Type of modification (group added)
Target amino acids
Comments
Phosphorylation (PO4-)
Tyrosine, serine,
threonine
Achieved by specific kinases. May be
reversed by phosphatases
Methylation (CH3)
Lysine
Achieved by methylases and undone by
demethylases
Hydroxylation (OH)
Proline, lysine, aspartic
acid
Hydroxyproline and hydroxylysine are
particularly common in collagens
Acetylation (CH3CO)
Lysine
Achieved by an acetylase and undone by
deacetylase
Carboxylation (COOH)
Glutamate
Achieved by g-carboxylase
N-glycosylation (complex
carbohydrate)
Asparagine, usually in the
sequence: Asn-X-Ser/Thr
Takes place initially in the endoplasmic
reticulum; X is any amino acid other than
proline
O-glycosylation (complex
carbohydrate)
Serine, threonine,
hydroxylysine
Takes place in the Golgi apparatus; less
common than N-glycosylation
GPI (glycolipid)
Aspartate at C terminus
Serves to anchor protein to outer layer of
plasma membrane
Myristoylation (C14 fatty acyl
group)
Glycine at N terminus
(see text)
Serves as membrane anchor
Palmitoylation (C16 fatty acyl
group)
Cysteine to form Spalmitoyl link.
Serves as membrane anchor
Farnesylation (C15 prenyl group)
Cysteine at C terminus
(see text)
Serves as membrane anchor
Geranylgeranylation (C20 prenyl
group)
Cysteine at C terminus
(see text)
Serves as membrane anchor
Insulin Synthesis Involves Multiple Post-Translational
Cleavages of Polypeptide Precursors
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.
Regions of secondary structure in polypeptides
are often dominated by intrachain hydrogen bonding
Intrachain and interchain disulfide bridges in human insulin
Chromosome structure and Function
Molecular Biology of the Cell
Chapter 2
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.
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).
The haploid sperm and egg originate by meiosis from diploid precursors
Packaging DNA into Chromosomes Requires Multiple
Hierarchies of DNA folding
From DNA Duplex to Metaphase Chromosome
Compaction ratios: 1:6 for nucleosomes; 1:36 for 30 nm fiber; 1:10,000 for metaphase
chromosome
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.
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”.
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.
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
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.
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.
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
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 320 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.
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
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.
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.
Heterochromatin is responsible for the proper functioning of telomeres and
centromeres.
Heterochromatin is dynamic, it can spread and retract and it is tends to be
inherited from a cell to its progeny.
An Outline of Cell Division by Mitosis
In the human lifetime, there are ~1017 mitotic divisions.
During mitosis, each chromosome in the diploid set act independently,
paternal and maternal homologs do not associate at all.
Development of the Germ-line
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)
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
Meiosis I - Recombination
At zygotene, a
synaptonemal
complex is formed.
Chiasma (Chiasmata)
marks a chrossover
point
Meiosis I
Meiosis II
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-2phenylindole) 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.
G-Banded Chromosome 1 at Different Banding Resolutions
G-banded prometaphase karyogram (karyotype) of mitotic
chromosomes from lymphocytes of a normal female
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
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
[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.
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.
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.
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).
Table 2.4. Consequences of numerical chromosomal
abnormalities
Polyploidy
Triploidy
(69,XXX, XXY or XYY)
1 3% of all conceptions; almost never liveborn; do not
survive
nullisomy (missing a pair
of homologs)
Preimplantation lethal
monosomy (one
chromosome missing)
Embryonic lethal
trisomy (one extra
chromosome)
Usually embryonic or fetal lethal
Aneuploidy
Autosomes
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 (47,XXX, 47,XXY, 47,XYY Relatively minor problems, normal lifespan
chromosomes
Lacking a sex
chromosome
45,X =Turner syndrome - 99% abort spontaneously; survivors are of
normal intelligence but infertile and show minor physical
signs
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.
Possible stable results of 2 breaks on a single chromosome
Origins of Translocations