BSM-4023

Genomes and Molecular Genetics

1) Genome structure, organisation and methods of analysis
2) DNA replication: The process and regulation of.
3) The mechanisms of DNA damage and repair (Dr. Edgar Hartsuiker)
4) Transcription: The process, roles of RNA transcripts and epigenetic regulation
5) Translation: The process and regulation of.
6) The mitotic cell cycle
7) The meiotic cell cycle (Dr. Edgar Hartsuiker)
8) Cell signaling - 1
9) Cell signaling - 2
10) Stem cells, cell ageing and programmed cell death

11) Gene isolation and cDNA (Dr. Anil Shirsat)

12) Genomic libraries and reporter genes (Dr. Anil Shirsat)
These lectures will be examined by mini-review/essay (20%)
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Course Books
(Some versions available on the Web)

Introductory level only!

Campbell/Reece's Biology, Seventh Edition
Login name: dpryce
Password: password1
 
General Course books
The Cell a molecular approach
The NCBI Bookshelf
Human Molecular Genetics, 2nd edition
Genomes, 2nd edition
The Stem Cell Book

BlackBoard site
Lecture slides,
some relevant book chapters and research papers.
Some example MCQ type and short answer questions
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 Genomes and Molecular Genetics

Molecular genetics
“Primarily concerned with the inter-relationship between the information
macromolecules deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and
how these molecules are used to synthesize polypeptides, the basic
component of all proteins”.

Genome structure, organisation and

methods of analysis

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DNA and RNA molecules are made of a “string”
of covalently linked nucleotides.
Each nucleotide has 3 main components

• A five carbon sugar

• A nitrogenous base

• Phosphate group

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Ribose:
Sugars can exist in two optical isomers (enantiomers) called the D- and L-isoforms.
The D-isoforms are the most abundant in biological organisms

The Fisher Convention

The Fisher (D- L-) system is an historical

system in which an enantiomer is
designated by its similarity to one of the
two-optical isomers of Glyceraldehyde.

D- and L- ribose
BSM-4023 2010 Fisher projections 5
Ribose exists primarily in “cyclic” form
Cyclic formation occurs via an intra-molecular reaction between the anomeric carbon
of the carbonyl group and an alcohol group. Cyclic sugars exist as α (alpha) or β
(beta) “anomers”. Both have different physical and chemical properties

Anomeric “Haworth projections”

carbon atom
Anomeric
carbon atom
1

5
β-anomer
2 deoxyribose is the sugar moiety
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in DNA
Nucleotides of DNA and RNA
A SUGAR, nitrogenous BASE and PHOSPHATE group(s) form the nucleotide
building blocks of nucleic acid macromolecules

Nitrogen base moieties

Purines
Within RNA
molecules thymine
is replaced with
uracil (uridine)

Pyrimidines

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 Nitrogen bases of nucleotides can be chemically
modified via enzymes and/or environmental ‘damage’.
Deamination
of adenine
Purines

CH3 Deamination
Pyrimidines of cytosine

Methylation
and
Deamination
Important between
pyrimidines
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Important “modified” cellular nucleotides

Purines

Pyrimidines

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Nucleotide/nucleoside structure and naming conventions:

cyclic AMP (cAMP)

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 “Watson and Crick” complementary base pairing

HYDROGEN BONDS can form between COMPLEMTARY

purine and pyrimidine nitrogenous bases

ADENINE pairs with THYMINE

(uracil in RNA)
2 H bonds

GUANINE pairs with CYTOSINE

3 H bonds

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 Helical forms A, B, and Z-DNA.

B DNA Z DNA
A DNA
right turn Left turn
Right turn

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 ‘Simple’ in vivo DNA molecules
Plasmids: circular, double-stranded DNA (dsDNA) molecules.

A ‘typical’ eukaryotic plasmid used in gene cloning

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‘Simple’ in vivo DNA molecules

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 GENOMES
-The ‘primary’ DNA content of an organism

• Nanoarchaeum equitans 490,885 bp [490 kb]

(552 genes)
• Bacterium- E.coli – 4,700 kb
(4,377genes)
• Fission Yeast- S.pombe – 141,000 kb [14.1 Mb]
(5036 genes)
• Humans – 3.2 Gb
(20,000-25000 genes)
• “whisk fern” 250 Gb

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 Viral nucleic acids

Image from Molecular Biology of the Cell

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 Bacterial cellular DNA
The “Nucleoid”
Generally a single circular DNA molecule

Nucleoid is found free floating Nucleoid composed of highly condensed

in the cytoplasm “domains” of about 10 kb
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 Human cellular DNA
Human nucleus
Asynchronous
HeLa cells

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Human chromosomes
Humans:
Haploid Ploidy number
(n) = 23

23 chromosomes
-21 autosomes
-2 sex chromosome

Approximately
2 meters of DNA per
nucleus!

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 A typical eukaryotic chromosome

Main ‘domains’:
Centromere p-arm
Telomere
Heterchromatin (short)
Euchromatin

q-arm
(long)

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 Satellite DNA
highly repetitive “tandem repeat”
DNA sequences

Satellite arrays occur at a few specific

and/or many different chromosomal
locations.

Satellite DNA regions have a different

density from “bulk” genomic DNA and can
therefore form discrete bands when
genomic DNA is separated via density
gradient centrifugation.

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 Satellite DNA
highly repetitive “tandem repeat”
DNA sequences

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Human centromeric DNA largely consists of
various ‘families’ of satellite DNA.

3–5% of each chromosome

GATC Sau3A

tandem repetition of ATTCC sequence

sequence

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 Minisatellites
90% located at telomeric and sub-telomeric loci

~1% of minisatellites are hypervariable

6- bp Human telomere repeat:

TTAGGG
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 Microsatellites: tandem repeated and “short”

The use of microsatellite analysis in genetic profiling.

microsatellites located on the short arm of chromosome 6 have been amplified
by PCR and labeled with a blue or green fluorescent marker.
No two individuals have the same microsatellite genetic profile,
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 Transposons: Jumping genes!
Long terminal repeats (LTRs)
seqences of DNA that repeat hundreds
or thousands of times.
LTRs are present in some retroviral DNA
Human endogenous retroviruses
(HERVs)

DNA transposons: make up ∼3% of the human genome

At least 40 human DNA transposon families (∼33 Mb)
No evidence of “movement” for more than ∼37 Million years!
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 Transposons: Jumping genes!
A small proportion of active elements
(Alu, L1 and SVA and possibly HERV-K)

The Alu SINE element:

Karyotype from a female human
lymphocyte
Chromosomes were hybridized with a
probe for Alu sequences (green) and
counterstained with TOPRO-3 (red).
Alu sequences can serve as a marker
for chromosome bands rich in genes.

LINE elements
SINE elements
Retrotransposons

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“other” genome-wide repeat sequences

Processed pseudogene:
thought to arise by integration
into the genome of a copy of the
mRNA transcribed from a
functional gene.

Pseudogenes: genes that have lost their

coding ability or are no longer expressed.

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 The exons…
(1.5%) of the total DNA content!

Human Genome project

OMIM - Online Mendelian Inheritance in Man

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 A typical human ‘gene rich’ region 50kb region of chromosome 12

50 kb segment of chromosome 12
a) Four genes.
b) 88 genome-wide repeat sequences.
c) Seven microsatellites
d) 30% nongenic, nonrepetitive, single-copy DNA of no known function or significance.
Ultraconserved regions (UCRs)
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Ultraconserved regions (UCRs)
Some regions of the human genome are almost completely
conserved among various species!
UCRs
481 segments longer than
200 base pairs (bp) that are
absolutely conserved
(100% identity with no
insertions or deletions)
between orthologous
regions of the human, rat,
and mouse genomes.

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Studying Genomes
Medically relevant DNA “visualization” methods

in vivo methods:-
Microscopy-
non specific DNA ‘stains’
specific hybridization techniques using labeled ‘probes’

in vitro methods:-
DNA isolation –
specific hybridization techniques using labeled ‘probes’
various types of PCR based DNA sequencing

This will be discussed in the DNA replication lecture

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 visualizing DNA
DNA can be “stained” using chemical dyes that either bind within the double helix
structure or to the double helix-phosphate backbone

DAPI or 4',6-diamidino-2-phenylindole
Hoechst 33342.

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visualizing DNA
General DNA stains can be used to determine A ‘normal’ human male
KARYOTYPES G-banding pattern karyogram:

Banding patterns can be used for chromosome

identification

Table of DNA stains and names of subsequent

banding patterns

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visualizing DNA
Nucleic acid hybridization

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visualizing DNA
Nucleic acid hybridization
Melting temperature and hybridization stringency

TM: Denaturation of dsDNA

Probe optimization:
Strand length
(500-800bp)
base composition –
%GC / AT composition
Chemical environment –
the presence of monovalent cations
(e.g. Na+ ions) BSM-4023 2010 36
visualizing DNA
Types of Nucleic acid molecules used in hybridization based
research and diagnosis assays

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 Methods of nucleic acid labeling

1) 3)

2)

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 Incorporation of labeled nucleotides into “probes”

1) Random priming 2) End labeling

4) Nick-translation
3) Direct
Chemical Labeling

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In vitro methods of DNA visualization
Southern blot assays

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In vitro methods of DNA visualization
Southern blot assays

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 In vitro methods of DNA visualization
Southern blot assays

phosphorimager
Slot blot apparatus DIG labeled and other antibody
detected probes

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In vivo methods of DNA visualization
Fluorescence microscopy
Structure of common fluorophores
Fluorescein-dUTP. Rhodamine

Fluorescence microscopy
The excitation filter barrier “filters” light

Light reflected by the dichroic (beam-

splitting) mirror

Longer wavelength of the emitted light

passes through the dichroic mirror.

Light passes through a second color barrier

filter which blocks unwanted fluorescent
signals
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Fluorescence in situ hybridization (FISH)
Chromosome “painting”

Spectral karyotyping (SKY),

multiplexed FISH (M-FISH) and
Comparative genome hybridization
(CGH) database
BSM-4023 2010
Bolzer et al (2005) Three-Dimensional
Maps of All Chromosomes in Human Male
Fibroblast Nuclei and Prometaphase
44
Rosettes
 Using fluorescence in situ hybridization to define “chromosome territories”.

Chromsome regions are found

in “fixed” areas of the nucleus
termed “chromosomal territories”

These regions are highly

conserved

Human chromosome 19 and 18

territories compared to the apes.

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Using fluorescence in situ hybridization to define a translocation breakpoint.

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Summary – background/further reading
Specific chapters for relevant for this lecture:
Basic introduction-
Campbell Biology - Chapter 16: The Molecular Basis of Inheritance
Human Molecular Genetics –Chapter 2
Genomes – Chapter 5

Specific topics -
Human Molecular Genetics Chapter 8 Genome projects and model organisms

Website book searches

Fluorescence in situ hybridization

Specific Papers reviews-

Nuclear genome organization common themes and individual patterns
Human Genome Ultraconserved Elements Are Ultraselected

Next –
2) DNA replication: The process and regulation of.

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