Review of Genome Language and some Facts

1
Review of Genome Language and some Facts
Life is specified by genomes. Every organism,
including humans, has a genome that contains all
of the biological information needed to build and
maintain a living example of that organism. The
biological information contained in a genome is
encoded in its deoxyribonucleic acid (DNA) and
divided into discrete units called genes. Genes
code for proteins that attach to the genome at
the appropriate positions and switch on a series
of reactions called gene expression. In 1909,
Danish botanist Wilhelm Johanssen coined the word
gene for the hereditary unit found on a
chromosome. Nearly 50 years earlier, Gregor
Mendel had characterized hereditary units as
factors observable differences that were passed
from parent to offspring. Today we know that a
single gene consists of a unique sequence of DNA
that provides the complete instructions to make a
functional product, called a protein. Genes
instruct each cell type such as skin, brain, and
liverto make discrete sets of proteins at just
the right times, and it is through this
specificity that unique organisms arise.

2
The cell nucleus

http//www.nature.com/genomics/human/slide-show/1.
html
3
DNA fibres

http//www.nature.com/genomics/human/slide-show/2.
html
4
Nuclear DNA
A DNA chain, also called a strand, has a sense of
direction, in which one end is chemically
different than the other. The so-called 5' end
terminates in a 5' phosphate group (-PO4) the 3'
end terminates in a 3' hydroxyl group (-OH).
This is important because DNA strands are always
synthesized in the 5' to 3' direction. The DNA
that constitutes a gene is a double-stranded
molecule consisting of two chains running in
opposite directions. The chemical nature of the
bases in double-stranded DNA creates a slight
twisting force that gives DNA its characteristic
gently coiled structure, known as the double
helix. The two strands are connected to each
other by chemical pairing of each base on one
strand to a specific partner on the other strand.
Adenine (A) pairs with thymine (T), and guanine
(G) pairs with cytosine (C). Thus, A-T and G-C
base pairs are said to be complementary. This
complementary base pairing is what makes DNA a
suitable molecule for carrying our genetic
informationone strand of DNA can act as a
template to direct the synthesis of a
complementary strand. In this way, the
information in a DNA sequence is readily copied
and passed on to the next generation of cells.

5
Ribonucleic Acids

• Just like DNA, ribonucleic acid (RNA) is a chain
  of nucleotides with the same 5' to 3' direction
  of its strands. The ribose sugar component of RNA
  is slightly different than that of DNA RNA has a
  2' oxygen atom not present in DNA.
• Other fundamental structural differences
• uracil (U) takes the place of the thymine (T)
  nucleotide found in DNA
• RNA is, for the most part, a single-stranded
  molecule.
• DNA directs the synthesis of a variety of RNA
  molecules, each with a unique role in cellular
  function. E.g. all genes that code for proteins
  are first made into an RNA strand in the nucleus
  called a messenger RNA (mRNA). The mRNA carries
  the information encoded in DNA out of the nucleus
  to the protein assembly machinery, the ribosome,
  in the cytoplasm. The ribosome complex uses mRNA
  as a template to synthesize the exact protein
  coded for by the gene.
• In addition to mRNA, DNA codes for other forms of
  RNA, including ribosomal RNAs (rRNAs), transfer
  RNAs (tRNAs), and small nuclear RNAs (snRNAs).
  rRNAs and tRNAs participate in protein assembly
  whereas snRNAs aid in a process called splicing
  the process of editing of mRNA before it can be
  used as a template for protein synthesis.

6
Central Dogma of Molecular Genetics

• DNA---------gtRNA---------gtProtein
• This diagram depicts the flow of genetic
  information from DNA into protein, the molecule
  most often associated with a specific phenotype.
• The three molecular events that maintain the
  genetic integrity and convert DNA information
  into a protein molecule are replication,
  transcription and translation. For some viral
  species, reverse transcription is also important.
  Each of these events are enzymatically driven and
  some of the enzymes involved in these steps are
  important for molecular studies.
• In particular these enzymes are
• DNA polymerase - synthesizes DNA from a DNA
  template
• DNA ligase - forms a covalent bond between free
  single-stranded ends of DNA molecules during
  replication
• Reverse transcriptase - synthesizes DNA from a
  RNA template

http//www.cc.ndsu.nodak.edu/instruct/mcclean/plsc
431/431g.htm
7
cloning
8
Restriction-Modification System of Bacteria
The most widely recognizable enzymes used in
molecular genetics are restriction enzymes. They
are part of the restriction-modification system
that bacterial species use to prevent foreign
organisms from overtaking their cells.
Presumably, each species has one or more of these
systems consisting of a restriction enzyme that
cleaves DNA at a specific sequence and a
methylase that protects the host DNA from being
cleaved. E.g. for one E. coli system the
restriction enzyme site is m 5' - G A A T T C
- 3' 3' - C T T A A G - 5' The restriction
enzyme EcoRI cuts this site between G and A. This
site is protected in the bacteria by the action
of the enzyme EcoRI methylase which adds a methyl
group to the 3'-adenine. The DNA that is cut at
the EcoRI site will have the following "sticky"
ends. 5' - G - 3' 5' - A A T T C - 3' 3' -
C T T A A - 5' 3' - G - 5' Invading viral
DNA will not be methylated and can be cut by the
restriction enzyme. Foreign DNA proliferation is
therefore restricted in the cell by the
restriction enzyme, but bacterial DNA is modified
by the methylase to prevent cleavage by the
restriction enzyme.

9
Cloning Vectors

• The molecular analysis of DNA has been made
  possible by the cloning of DNA. The two molecules
  that are required for cloning are the DNA to be
  cloned and a cloning vector.
• Cloning vector - a DNA molecule that carries
  foreign DNA into a host cell, replicates inside a
  bacterial (or yeast) cell and produces many
  copies of itself and the foreign DNA
• Three features of all cloning vectors
• sequences that permit the propagation of
  itself in bacteria (or in yeast for YACs)
• a cloning site to insert foreign DNA the
  most versatile vectors contain a site that can
  be cut by many restriction enzymes
• a method of selecting for bacteria (or yeast
  for YACs) containing a vector with foreign DNA
  usually accomplished by selectable markers for
  drug resistance.

10
Types of Cloning Vectors

• Plasmid - an extrachromosomal circular DNA
  molecule that autonomously replicates inside the
  bacterial cell cloning limit 100 to 10,000 base
  pairs or 0.1-10 kilobases (kb)
• Phage - derivatives of bacteriophage lambda
  linear DNA molecules, whose region can be
  replaced with foreign DNA without disrupting its
  life cycle cloning limit 8-20 kb
• Cosmids - an extrachromosomal circular DNA
  molecule that combines features of plasmids and
  phage cloning limit - 35-50 kb
• Bacterial Artificial Chromosomes (BAC) - based on
  bacterial mini-F plasmids. cloning limit 75-300
  kb
• Yeast Artificial Chromosomes (YAC) - an
  artificial chromosome that contains telomeres,
  origin of replication, a yeast centromere, and a
  selectable marker for identification in yeast
  cells cloning limit 100-1000 kb

11
cDNA cloning
The cloning described sofar will work for any
random piece of DNA. But since the goal of many
cloning experiments is to obtain a sequence of
DNA that directs the production of a specific
protein, any procedure that optimizes cloning
will be beneficial. One such technique is cDNA
cloning. The principle behind this technique is
that an mRNA population isolated from a specific
developmental stage should contain mRNAs specific
for any protein expressed during that stage.
Thus, if the mRNA can be isolated, the gene can
be studied. mRNA cannot be cloned directly, but a
DNA copy of the mRNA can be cloned. (The term
cDNA is short for "copy DNA".) This conversion is
accomplished by the action of reverse
transcriptase and DNA polymerase. The reverse
transcriptase makes a single-stranded DNA copy of
the mRNA. The second DNA strand is generated by
DNA polymerase and the double- stranded product
is introduced into an appropriate plasmid or
lambda vector.

12
DNA Sequencing

• These cloning techniques have been widely used to
  isolate many genes from nearly all species. Once
  these genes have been isolated what can they be
  used for?
• The nucleic acid sequence of the gene can be
  derived. If a partial or complete sequence of the
  protein that it encodes is available the gene can
  be confirmed in this manner. If the protein
  product is not known then the sequence of the
  gene can be compared with those of known genes to
  try to derive a function for that gene.
• The clone can then be used to study the sequences
  of the regulatory region of the gene. This is
  possible only for genomic clones because cDNA
  clones just contain coding sequences.
• The clone can be used to isolate similar genes
  from other organisms. Thus it can serve as a
  heterologous probe.
• If the gene is of clinical importance, the clone
  can be used for diagnostic purposes. E.g. one
  type of hemophilia.

13
sequencing physical mapping
14
Goals of molecular genetics
A major goal is to correlate the sequence of a
gene with its function. Thus obtaining the
sequence is of primary importance. DNA sequencing
is nowadays performed by the the
dideoxy-chain-termination procedure that is a DNA
polymerase-based technique. This technique is
based on the ability of a specific nucleotide
(dideoxy nucleotide) to terminate the DNA
polymerase reaction. These nucleotides do not
have a free 3'-OH group, an absolute requirement
for DNA polymerase activity. Thus, any time this
nucleotide is inserted into the growing chain DNA
synthesis stops. Technically, four polymerase
reactions are performed, each containing the four
nucleotides dATP, dTTP, dCTP and dGTP. In
addition the reactions contain a limited amount
of one of the four dideoxybases so that all
possible terminations can occur. After the
reactions are finished, the products from the
four reactions are separated side-by-side on a
polyacrylamide gel. Each of the fragments within
a lane ends with the base corresponding to the
dideoxy nucleotide used in the reaction. Thus by
reading the four lanes from the bottom of the gel
to the top, the sequence of the DNA can be
obtained.

15
Sanger Sequencing Process sequence short DNA
pieces
In this much-automated method the single-stranded
DNA to be sequenced is "primed" for replication
with a short complementary strand at one end.
This preparation is then divided into four
batches, and each is treated with a different
replication-halting nucleotide, together with the
four "usual" nucleotides. Each replication
reaction then proceeds until a reaction-terminatin
g nucleotide is incorporated into the growing
strand, whereupon replication stops. Thus, the
"C" reaction produces new strands that terminate
at positions corresponding to the G's in the
strand being sequenced. Gel electrophoresis - one
lane per reaction mixture - is then used to
separate the replication products, from which the
sequence of the original single strand can be
inferred.

16
DNA Sequencing Process readout
Variation use fluorescently labelled
replication-halting nucleotides. The image shows
a portion of a fluorescence-based sequence gel.
Each column of colored bars represents labeled
DNA fragments which can be read as follows blue
C, green A, yellow G, red T.

17
Genome mapping

http//www.nature.com/genomics/human/slide-show/3.
html
18
Physical mapping the principle
Physical mapping of the genome recovers different
levels. Broad definition position nucleotidic
sequences with respect to longer nucleotidic
sequences (DNA matrix). For instance, placing a
gene responsible for a disease on the chromosome
in which it is contained. The importance of this
kind of information for genome projects is
evident. The biggest chunk of DNA which can
nowaday be sequenced is at most 1000 nucleotides
long (1 kb). As it is not possible to cut the
human genome in bits of neighboring pieces of 1
kb, it is necessary to first cut it in bigger
pieces, which will be themselves cut into smaller
pieces, etc. Cutting DNA is performed by
restriction enzymes. The resulting fragments are
usually inserted into bacterias or other
micro-organisms (or clones). This allows for
their conservation and mass production of DNA.
How are all these cloned fragments reorganized
in the corresponding order on the chromosomes
they come from ? That is the role of physical
mapping techniques.

http//www.pasteur.fr/recherche/unites/biophyadn/e
-mapping.html
19
Linear ordering of clones
None of today's techniques allows for a precise
positioning of the probes down to one nucleotidic
base. It is thus necessary to use overlapping
clones, that is, clones with a common part.
Covering of a region of the genome can then be
done by a set of partially overlaping clones,
also called a contig (for contiguous clones).
Building a contig of clones for a given region
is thus the first step of physical mapping.
Basically, one picks up clones out of a clone
library obtained by systematic cloning of pieces
resulting from the enzymatic digestion of the
whole genome. These clones are chosen when they
are positive for markers specific of the studied
region, and have to be organised by physical
mapping one thus obtains a minimal continuous
string of overlapping clones which can eventually
be sequenced.

20
Techniques using FISH
Different techniques have been developed in the
last few years to precisely measure the
respective position of clones onto a partially
linearized DNA fiber. All these techniques use
FISH (or fluorescent in situ hybridization). The
detection of nucleotidic sequences (probes) on a
DNA matrix is performed indirectly by hybridizing
the nucleotidic sequences with the matrix DNA.
If the probes are synthesized with incorporated
fluorescent molecules, the relative position of
the probes can be visualized directly.

A STS map indicating which cosmids were used in
the experiment. STSs are represented as vertical
ticks separated by an arbitrary distance. The
relative orientation of the contigs was unknown.
B Images of representative hybridizations of
pairs of cosmids. Bar indicates 10 microns, i.e.
20 kb. C Final map.
21
Fine Structure Mapping of Chromosomes
Molecular maps can be used to identify a marker
for a specific gene. These markers are quite
useful for a specific gene that is difficult to
score or is expressed late in the life cycle.
Maps can also be used as a starting point for
cloning a gene. A fine structure map of the
species is quite useful for this purpose. Yeast
artifical chromosome (YAC) clones and bacterial
artificial chromosome (BAC) clones are key tools
for developing a fine structure map. In
principle, a YAC or BAC clone library should
contain a series of clones that overlap each
other. The key is to order each of these clones.
The ordering of the clones often relies upon
sequence tagged sites (STS). STS are short
sequences of DNA that are sequenced. PCR primers
are developed, and if the same PCR product can be
amplified from any two YAC or BAC clones, the two
clones must overlap. In practise, a large number
of clones are scored for different STS sites, and
the data is analyzed to order the different
clones. The following table is an example of such
data. "" means that the STS is product is
obtained from that clone, and "-" means the
product is not amplified from the clone.

22
Contig map
This stretch of four clones is called a contig
map. The goal of fine structure mapping is to
develop complete contig maps for each chromosome
of the species. If these complete maps are
available, it is a simple matter to take the
molecular marker you have obtained and select a
clone to which it hybridized. Then you are
immediately working at the molecular level for
that species and are on your way to cloning that
species.

23
structural organisation of DNA
24
Eukaryotic Chromosome Structure
The length of DNA in the nucleus is far greater
than the size of the compartment in which it is
contained. Therefore, the DNA has to be condensed
in some manner expressed by its packing ratio -
the length of DNA divided by the length into
which it is packaged. E.g. the shortest human
chromosome contains 4.6 x 107 bp. This is
equivalent to 14,000 µm of extended DNA. In its
most condensed state during mitosis, the
chromosome is about 2 µm long. This gives a
packing ratio of 7000 (14,000/2). To achieve the
overall packing ratio, DNA is not packaged
directly into the final structure of chromatin
but contains several hierarchies of
organization (a) DNA is wound around a protein
core to produce a "bead-like" structure called a
nucleosome. This gives a packing ratio of about 6
(2pr). This structure is invariant in both the
euchromatin and heterochromatin of all
chromosomes. (b) The second level of packing is
the coiling of beads in a helical structure
called the 30 nm fiber that is found in both
interphase chromatin and mitotic chromosomes.
This structure increases the packing ratio to
about 40. (c) The final packaging occurs when
the fiber is organized in loops, scaffolds and
domains that give a final packing ratio of about
1000 in interphase chromosomes and about 10,000
in mitotic chromosomes.

25
Nucleosome
The nucleosome consists of about 200 bp wrapped
around a histone octamer that contains two copies
of histone proteins H2A, H2B, H3 and H4. These
are known as the core histones. Histones are
basic proteins that have an affinity for DNA and
are the most abundant proteins associated with
DNA. The amino acid sequence of these four
histones is conserved suggesting a similar
function for all. The length of DNA that is
associated with the nucleosome unit varies
between species. But regardless of the size, two
DNA components are involved. Core DNA is the DNA
that is actually associated with the histone
octamer. This value is invariant and is 146 base
pairs. The core DNA forms two loops around the
octamer, and this permits two regions that are 80
bp apart to be brought into close proximity.
Thus, two sequences that are far apart can
interact with the same regulatory protein to
control gene expression. The DNA that is between
each histone octamer is called the linker DNA and
can vary in length from 8 to 114 base pairs. This
variation is species specific, but variation in
linker DNA length has also been associated with
the developmental stage of the organism or
specific regions of the genome.

26
30 nm and 700 nm fiber
The next level of organization of the chromatin
is the 30 nm fiber. This is a structure with
about 6 nucleosomes per turn yielding a packing
ratio of 40 (ca. 66). The stability of this
structure requires the presence of the last
member of the histone gene family, histone H1.
The final level of packaging is characterized
by the 700 nm structure seen in the metaphase
chromosome. The condensed piece of chromatin has
a characteristic scaffolding structure that can
be detected in metaphase chromosomes. This
appears to be the result of extensive looping of
the DNA in the chromosome. When chromosomes are
stained with dyes, they appear to have
alternating lightly and darkly stained regions.
The lightly-stained regions are euchromatin and
contain single-copy, genetically-active DNA. The
darkly-stained regions are heterochromatin and
contain repetitive sequences that are genetically
inactive.

27
Centromeres and Telomeres
Centromeres and telomeres are two essential
features of all eukaryotic chromosomes. Each
provide a unique function that is absolutely
necessary for the stability of the chromosome.
Centromeres are required for the segregation of
the centromere during meiosis and mitosis, and
telomeres provide terminal stability to the
chromosome and ensure its survival. Centromeres
are those condensed regions within the chromosome
that are responsible for the accurate segregation
of the replicated chromosome during mitosis and
meiosis. When chromosomes are stained they
typically show a dark-stained region that is the
centromere. During mitosis, the centromere that
is shared by the sister chromatids must divide so
that the chromatids can migrate to opposite poles
of the cell. On the other hand, during the first
meiotic division the centromere of sister
chromatids must remain intact, whereas during
meiosis II they must act as they do during
mitosis. Therefore the centromere is an important
component of chromosome structure and segregation.

28
Centromeres
Within the centromere region, most species have
several locations where spindle fibers attach,
and these sites consist of DNA as well as
protein. The actual location where the attachment
occurs is called the kinetochore and is composed
of both DNA and protein. The DNA sequence within
these regions is called CEN DNA. Because CEN DNA
can be moved from one chromosome to another and
still provide the chromosome with the ability to
segregate, these sequences must not provide any
other function. Typically CEN DNA is about 120
base pairs long and consists of several
sub-domains, CDE-I, CDE-II and CDE-III .
Mutations in the first two sub-domains have no
effect upon segregation, but a point mutation in
the CDE-III sub-domain completely eliminates the
ability of the centromere to function during
chromosome segregation. Therefore CDE-III must be
actively involved in the binding of the spindle
fibers to the centromere.

29
Telomeres
Telomeres are the region of DNA at the end of the
linear eukaryotic chromo-some that are required
for the replication and stability of the
chromosome. McClintock recognized their special
features when she noticed, that if two
chromosomes were broken in a cell, the end of one
could attach to the other and vice versa. What
she never observed was the attachment of the
broken end to the end of an unbroken chromosome.
Thus the ends of broken chromosomes are sticky,
whereas the normal end is not sticky, suggesting
the ends of chromosomes have unique features.
Usually, but not always, the telomeric DNA is
heterochromatic and contains direct tandemly
repeated sequences. The following table shows the
repeat sequences of several species. These are
often of the form (T/A)xGy where x is between 1
and 4 and y is greater than 1.

30
Telomere Repeat Sequences

31
repetitive sequences
32
Cot curve
The technique for determining the sequence
complexity of any genome involves the
denaturation and renaturation of DNA. DNA is
denatured by heating which melts the H-bonds and
renders the DNA single-stranded. If the DNA is
rapidly cooled, the DNA remains single-stranded.
But if the DNA is allowed to cool slowly,
sequences that are complementary will find each
other and eventually base pair again. The rate at
which the DNA reanneals (another term for
renature) is a function of the species from which
the DNA was isolated. The so-called Cot curve
plots the percent of DNA that remains single
stranded (expressed as a ratio of the
concentration of single-stranded DNA to the total
concentration of the starting DNA) against the
log of the product of the initial concentration
of DNA multiplied by length of time the reaction
proceeded. The Cot curve is rather smooth which
indicates that reannealing occurs slowing but
gradually over a period of time. At Cot½ , half
of the DNA has reannealed.

33
DNA Denaturation and Renaturation Experiments
The shape of a "Cot" curve for a given species
is a function of two factors - the size or
complexity of the genome - the amount of
repetitive DNA within the genome The "Cot"
curves of the genome of bacteriophage lambda, E.
coli and yeast have the same shape, but Cot½ of
yeast is largest, E. coli next and lambda
smallest. Physically, the larger the genome size
the longer it will take for any one sequence to
encounter its complementary sequence in the
solution. This is because two complementary
sequences must encounter each other before they
can pair. The more complex the genome, the longer
it will take for any two complementary sequences
to encounter each other and pair.

34
Repeated DNA sequences, DNA sequences that are
found more than once in the genome of the
species, have distinctive effects on "Cot"
curves. If a specific sequence is represented
twice in the genome it will have two
complementary sequences to pair with and as such
will have a Cot value half as large as a sequence
represented only once in the genome.
35
Repetitive DNA
Eukaryotic genomes actually have a wide array of
sequences that are represented at different
levels of repetition. Single copy sequences are
found once or a few times in the genome. Many of
the sequences which encode functional genes fall
into this class. Middle repetitive DNA are found
from 10s - 1000 times in the genome. Examples of
these would include rRNA and tRNA genes and
storage proteins in plants such as corn. Middle
repetitive DNA can vary from 100-300 bp to 5000
bp and can be dispersed throughout the genome.
The most abundant sequences are found in the
highly repetitive DNA class. These sequences are
found from 100,000 to 1 million times in the
genome and can range in size from a few to
several hundred bases in length. These sequences
are found in regions of the chromosome such as
heterochromatin, centromeres and telomeres and
tend to be arranged as a tandem repeats. The
following is an example of a tandemly repeated
sequence ATTATA ATTATA ATTATA // ATTATA

36
Cot Plots reflect degree of repetitive sequences
Genomes that contain these different classes of
sequences reanneal in a different manner than
genomes with only single copy sequences. Instead
of having a single smooth "Cot" curve, three
distinct curves can be seen, each representing a
different repetition class. The first sequences
to reanneal are the highly repetitive sequences
because so many copies of them exist in the
genome, and because they have a low sequence
complexity. The second portion of the genome to
reanneal is the middle repetitive DNA, and the
final portion to reanneal is the single copy DNA.
The following diagram depicts the "Cot" curve for
a "typical" eukaryotic genome

37
Sequence distribution for selected species

38
Sequence Interspersion
Even though the genomes of higher organisms
contain single copy, middle repetitive and highly
repetitive DNA sequences, these sequences are not
arranged similarly in all species. The
prominent arrangement is called short period
interspersion. This arrangement is characterized
by repeated sequences 100-200 bp in length
interspersed among single copy sequences that are
1000-2000 bp in length. This arrangement is found
in animals, fungi and plants. The second type
of arrangement is long-period interspersion. This
is characterized by 5000 bp stretches of repeated
sequences interspersed within regions of 35,000
bp of single copy DNA. Drosophila is an example
of a species with this uncommon sequence
arrangement. In both cases, the repeated
sequences are usually from the middle repetitive
class.

39
C-value paradox
In addition to describing the genome of an
organism by its number of chromosomes, it is also
described by the amount of DNA in a haploid cell.
This is usually expressed as the amount of DNA
per haploid cell and is called the C value. One
immediate feature of eukaryotic organisms
highlights a specific anomaly that was detected
early in molecular research Even though
eukaryotic organisms appear to have only 2-10
times as many genes as prokaryotes, they have
many orders of magnitude more DNA in the cell.
Furthermore, the amount of DNA per genome is not
correlated with the presumed evolutionary
complexity of a species. This is stated as the C
value paradox the amount of DNA in the haploid
cell of an organism is not related to its
evolutionary complexity. Another important
point to keep in mind is that there is no
relationship between the number of chromosomes
and the presumed evolutionary complexity of an
organism.

40
C-Value paradox
A dramatic example of the range of C values can
be seen in the plant kingdom where Arabidopsis
represents the low end and lily (1.0 x 108
kb/haploid genome) the high end of complexity.
In weight terms this is 0.07 picograms per
haploid Arabidopsis genome and 100 picograms per
haploid lily genome.

41
The genetic code
42
The genetic code

• The genetic code consists of 64 triplets of
  nucleotides. These triplets are called codons.
  With three exceptions, each codon encodes for one
  of the 20 amino acids used in the synthesis of
  proteins. That produces some redundancy in the
  code. One codon, AUG, serves two related
  functions
• it signals the start of translation
• it codes for incorporating the amino acid Met
  into the growing polypeptide chain.
• The genetic code can be expressed as either RNA
  codons or DNA codons.
• RNA codons occur in messenger RNA (mRNA) and are
  the codons that are actually "read" during the
  synthesis of polypeptides (the process called
  translation). But each mRNA molecule acquires its
  sequence of nucleotides by transcription from the
  corresponding gene. Because DNA sequencing has
  become so rapid and because most genes are now
  being discovered at the level of DNA before they
  are discovered as mRNA or as a protein product,
  it is extremely useful to have a table of codons
  expressed as DNA.
• There are also exceptions to the genetic code but
  we will not mention these here.

43
The genetic code RNA
U C A G
U U UUU Phe UCU Ser UCU Ser UAU Tyr UGU Cys
UUC Phe UCC Ser UCC Ser UAC Tyr UGC Cys
UUA Leu UCA Ser UCA Ser UAA STOP UGA STOP
UUG Leu UCG Ser UCG Ser UAG STOP UGG Trp
C C CUU Leu CCU Pro CCU Pro CAU His CGU Arg
CUC Leu CCC Pro CCC Pro CAC His CGC Arg
CUA Leu CCA Pro CCA Pro CAA Gln CGA Arg
CUG Leu CCG Pro CCG Pro CAG Gln CGG Arg
A A AUU Ile ACU Thr ACU Thr AAU Asn AGU Ser
AUC Ile ACC Thr ACC Thr AAC Asn AGC Ser
AUA Ile ACA Thr ACA Thr AAA Lys AGA Arg
AUG Met or START ACG Thr ACG Thr AAG Lys AGG Arg
G G GUU Val GCU Ala GCU Ala GAU Asp GGU Gly
GUC Val GCC Ala GCC Ala GAC Asp GGC Gly
GUA Val GCA Ala GCA Ala GAA Glu GGA Gly
GUG Val GCG Ala GCG Ala GAG Glu GGG Gly

44
The genetic code DNA
The DNA Codons These are the codons as they are
read on the sense (5' to 3') strand of DNA.
Except that the nucleotide thymidine (T) is
found in place of uridine (U), they read the same
as RNA codons. However, mRNA is actually
synthesized using the antisense strand of DNA (3'
to 5') as the template. This table could well be
called the Rosetta Stone of life.
T C A G
T T TTT Phe TCT Ser TCT Ser TAT Tyr TGT Cys
TTC Phe TCC Ser TCC Ser TAC Tyr TGC Cys
TTA Leu TCA Ser TCA Ser TAA STOP TGA STOP
TTG Leu TCG Ser TCG Ser TAG STOP TGG Trp
C C CTT Leu CCT Pro CCT Pro CAT His CGT Arg
CTC Leu CCC Pro CCC Pro CAC His CGC Arg
CTA Leu CCA Pro CCA Pro CAA Gln CGA Arg
CTG Leu CCG Pro CCG Pro CAG Gln CGG Arg
A A ATT Ile ACT Thr ACT Thr AAT Asn AGT Ser
ATC Ile ACC Thr ACC Thr AAC Asn AGC Ser
ATA Ile ACA Thr ACA Thr AAA Lys AGA Arg
ATG Met or START ACG Thr ACG Thr AAG Lys AGG Arg
G G GTT Val GCT Ala GCT Ala GAT Asp GGT Gly
GTC Val GCC Ala GCC Ala GAC Asp GGC Gly
GTA Val GCA Ala GCA Ala GAA Glu GGA Gly
GTG Val GCG Ala GCG Ala GAG Glu GGG Gly

45
Codon usage Cytochrome P450
or how the genome affects protein composition
110 non-allelic cytochrome P450 genes from man
(n30), rat (n38), rabbit (n24), and mouse
(n18) for which complete cDNA or gene sequences
are available were analyzed.  Codon usage bias
(the tendency to use a limited subset of codons)
was estimated by summing the usage of the
preferred codon for each of the 18 amino acids
for which synonymous codons exist and expressing
it as a percentage of all the synonymous codons
in that gene.  Thus, genes with a high codon
usage bias tend to use a subset of all possible
codons (i.e., preferred codons) rather than the
full range of codons available.   Porter, T.D.,
"Correlation between codon usage, regional
genomic nucleotide composition, and amino acid
composition in the cytochrome P-450 gene
superfamily", Biochim. Biophys. Acta 1261,
394-400, 1995. borrowed from http//www.uky.edu/
Pharmacy/ps/porter/CodonUsage/p450_codon_usage.htm

46
Codon Usage Bias Not Correlated with
Evolutionary Age
Thus, genes that have arisen early in evolution
and have been maintained in an organism do not
necessarily "optimize" their codon usage pattern
(e.g., P450 families 19 and 7, shown on lower
right of graph.

Codon usage bias is plotted against the estimated
evolutionary distance of 18 P450 subfamilies. 
The points on each line represent one or more
P450 sequences in the respective family or
subfamily evolutionary distance represents the
branch point at which a given group diverges from
all other P450 groups.  Thus, the most recently
evolved P450s are closest to the X origin.
47
Codon Usage Bias Not Correlated with Evolutionary
Conservation
It has been suggested that highly conserved
proteins may exhibit greater codon usage bias
than less well conserved proteins.  However, a
comparison of 11 P450 orthologues between rat and
man demonstrates that highly conserved
orthologues exhibit no greater bias than less
well conserved proteins.  This graph also
demonstrates that codon usage bias is not
conserved across species for orthologous P450
genes.

Codon usage bias is plotted against amino acid
identity for 11 rat-human orthologues (each pair
is connected by a line).  Highly conserved
orthologues exhibit high amino acid identity, and
are at the right of the graph, while less
conserved orthologues are at the left.
48
Codon Usage Bias is not Tissue-Specific
Some evidence has indicated that codon usage
might differ for genes expressed only in specific
tissues, such as muscle or liver.  But an
analysis of P450 genes expressed predominantly in
a single tissue does not support this hypothesis.

The average bias in P450 codon usage is shown for
each tissue or organ.  Each group includes all
P450s that are expressed predominantly or
exclusively in that tissue or organ.  No
statistically significant differences were noted. 
49
Codon Usage Bias Correlates with 3rd Position
CG Content

The increasing CG content at the codon 3rd
position is the 'silent position' in many codons
because it often does not influence amino acid
specificity.  This graph demonstrates that
preferred P450 codons in these four mammals
usually end in C or G.
50
Codon Positional CG Content Correlates with
Regional Genomic CG Content
For reasons that are not yet understood (1995),
the composition of mammalian genomes is not
homogeneous some segments (isochores) are high
in CG content, while some regions are AT rich. 
As shown here, genes located in CG-rich segments
exhibit high CG content at the third codon
position (i.e., codon usage bias, closed
circles), and to a lesser extent at the first and
second codon positions (open circles). 

The CG content at the codon third position
(closed circles) and the first and second codon
positions (open circles) for 31 P450 genes
available at the time of this analysis are
plotted against the non-exonic CG content of
these genes.  Flank intron CG content is taken
as an indicator of the CG composition of the
corresponding region (isochore) of the genome.
51
Amino Acid Composition Correlates with Isochore
Composition
The correspondence of CG content in the first
and second codon positions with isochore
composition suggests that genes located in
regions of high CG content should have a
relative abundance of amino acids encoded by
C/G-rich codons, and a relative deficit of amino
acids encoded by C/G-poor codons.  As shown here,
this holds true for the 31 P450 genes analyzed
above.  As flankintron CG content increases so
does the abundance of amino acids encoded by
CG-rich codons (Pro, Ala, Arg, Gly) a
corresponding decrease in amino acids encoded by
CG-poor codons is also seen (Phe, Ile, Met, Tyr,
Asn, Lys).

52
Codon usage Cytochrome P450
Amino Acid Composition Correlates with Codon
Usage Bias As noted earlier, codon 3rd position
CG content (or codon usage bias) correlates with
regional genomic nucleotide composition.  Thus
codon usage bias can be taken as a proxy for
isochore composition.  This is illustrated by the
figures to the right, where amino acid content
correlates with codon 3rd position CG
content.   Thus, the regional genomic nucleotide
composition influences the composition of genes
and, surprisingly, their encoded proteins.  

53
Codon usage Conclusions

• Codon usage bias in mammals appears to reflect
  the composition of the genome in which the gene
  lies genes in GC-rich regions of the genome will
  exhibit biased codon usage, in which a majority
  of the codons end in C or G.
• This genomic influence extends to the first and
  second codon positions, where increased CG
  content will increase those amino acids encoded
  by CG-rich codons (Pro, Ala, Arg, Gly) and
  decrease those amino acids encoded by CG-poor
  codons (Phe, Ile, Met, Tyr, Asn, Lys).
• The total variation in amino acid composition
  between genes with high and low codon usage bias
  is approximately 20, and the content of any one
  amino acid changes from 2-6.  This is sufficient
  to alter the characteristics of the encoded
  protein, and reveals an important and previously
  unrecognized force that affects protein evolution.

54
Codon usage in different species

http//www.uky.edu/Pharmacy/ps/porter/CodonUsage/p
referred_codons.htm
55
organelle DNA
56
Organelle DNA
Not all genetic information is found in nuclear
DNA. Both plants and animals have an organellea
"little organ" within the cell called the
mitochondrion. Each mitochondrion has its own set
of genes. (Plants also have a second organelle,
the chloroplast, which also has its own DNA.)
Cells often have multiple mitochondria,
particularly cells requiring lots of energy, such
as active muscle cells. This is because
mitochondria are responsible for converting the
energy stored in macromolecules into a form
usable by the cell, namely, the adenosine
triphosphate (ATP) molecule. Thus, they are often
referred to as the power generators of the
cell. Unlike nuclear DNA (the DNA found within
the nucleus of a cell), half of which comes from
our mother and half from our father,
mitochondrial DNA is only inherited from our
mother. This is because mitochondria are only
found in the female gametes or "eggs" of sexually
reproducing animals, not in the male gamete, or
sperm. Mitochondrial DNA also does not recombine
there is no shuffling of genes from one
generation to the other, as there is with nuclear
genes.

57
Why is there a separate mitochondrial genome?
The energy-conversion process that takes place in
the mitochondria takes place aerobically, in the
presence of oxygen. Other energy conversion
processes in the cell take place anaerobically,
or without oxygen. The independent aerobic
function of these organelles is thought to have
evolved from bacteria that lived inside of other
simple organisms in a mutually beneficial, or
symbiotic, relationship, providing them with
aerobic capacity. Through the process of
evolution, these tiny organisms became
incorporated into the cell, and their genetic
systems and cellular functions became integrated
to form a single functioning cellular unit.
Because mitochondria have their own DNA, RNA, and
ribosomes, this scenario is quite possible. This
theory is also supported by the existence of a
eukaryotic organism, called the amoeba, which
lacks mitochondria. Therefore, amoeba must always
have a symbiotic relationship with an aerobic
bacterium.

58
Why study mitochondria
There are many diseases caused by mutations in
mitochondrial DNA (mtDNA). Because the
mitochondria produce energy in cells, symptoms of
mitochondrial diseases often involve degeneration
or functional failure of tissue. For example,
mtDNA mutations have been identified in some
forms of diabetes, deafness, and certain
inherited heart diseases. In addition, mutations
in mtDNA are able accumulate throughout an
individual's lifetime. This is different from
mutations in nuclear DNA, which has sophisticated
repair mechanisms to limit the accumulation of
mutations. Mitochondrial DNA mutations can also
concentrate in the mitochondria of specific
tissues. A variety of deadly diseases are
attributable to a large number of accumulated
mutations in mitochondria. There is even a
theory, the Mitochondrial Theory of Aging, that
suggests that accumulation of mutations in
mitochondria contributes to, or drives, the aging
process.

59
exons introns, splicing
60
Introns and Exons
Genes make up only about 1 percent of the total
DNA in our genome. In the human genome, the
coding portions of a gene, called exons, are
interrupted by intervening sequences, called
introns. In addition, a eukaryotic gene does not
code for a protein in one continuous stretch of
DNA. Both exons and introns are "transcribed"
into mRNA, but before it is transported to the
ribosome, the primary mRNA transcript is edited.
This editing process removes the introns, joins
the exons together, and adds unique features to
each end of the transcript to make a "mature"
mRNA. One might then ask what the purpose of an
intron is if it is spliced out after it is
transcribed? It is still unclear what all the
functions of introns are, but scientists believe
that some serve as the site for recombination,
the process by which progeny derive a combination
of genes different from that of either parent,
resulting in novel genes with new combinations of
exons, the key to evolution.

61
Recombination
Recombination involves pairing between
complementary strands of two parental duplex DNAs
(top and middle panel). This process creates a
stretch of hybrid DNA (bottom panel) in which the
single strand of one duplex is paired with its
complement from the other duplex

62
Alternative Splicing
Since each exon in a eukaryotic gene encodes a
portion of a protein it is possible, by altering
how the pre-mRNA is spliced, to produce different
versions of the mRNA and ultimately, different
proteins. This has been demonstrated in a number
of cases and two such cases will be described
here. The first involves processing of mRNAs
that will be translated into parts of antibody
molecules (immunoglobulins). On the next slide
two possibilities are shown for one such gene,
the gene for the m heavy chain of the mouse IgM
immunoglobulin.

63
Alternative Splicing
The top shows the DNA structure of this gene
region. The exons are shown as colored boxes, the
introns as lines. A pre-mRNA is transcribed from
this DNA. It can be spliced in two different ways.

On the left, the RNA is spliced to include the
exons S, V, Cm1, Cm2, Cm3, Cm4, and C (the
terminus of the secreted form of the protein).
This form is translated and sent out of the cell
as part of a secreted antibody. On the right is
shown a splicing pattern that includes S, V, Cm1,
Cm2, Cm3, Cm4 and then the M exons. This form of
the mRNA is translated into a protein with a
transmembrane anchor region (M) and therefore
winds up in the plasma membrane of the cell that
produces it. In this way the immune system can
produce two different forms of the protein one
that is sent out of the cell as a soluble
antibody, and the other that remains on the
surface of the cell to help identify it to other
cells of the immune system.
64
Alternative Splicing
Another example is the sex determination pattern
of Drosophila. There are three genes involved
(the names are derived from the phenotype of
mutations) Sxl (sex lethal) tra
(transformer) dsx (double sex). Each of these
genes produces a pre-mRNA that has two possible
splicing patterns, depending upon whether the fly
is male (XY) or female (XX).

65
Alternative Splicing
Middle row pre-mRNAs for each gene, splicing
pattern for female splicing pattern for male.

The product mRNAs are shown on left and right.
The inclusion of two exons (3 in Sxl and 2 in
tra) produces, in the male mRNAs, messengers that
have termination (stop) codons that yield
inactive proteins. The only active male product
is the protein translated from dsx, which in turn
inactivates all female -specific genes. The
female produces mRNAs without stop
codon-containing exons. The protein products of
Sxl and tra have a positive effect on the
splicing patterns observed, controlling the
choice of introns removed in the spliceosome
reaction. Thus we the spliceosome cycle is
modulated to produce a variety of products in the
eukaryotic nucleus. (Some RNA splicing events do
not require the action of spliceosome complexes).
66
knowledge about whole genomesgenome content and
annotation
67
Genome sequences Archaea

http//www.ebi.ac.uk
68
Protein length

http//www.ebi.ac.uk
69
Amino acid composition

http//www.ebi.ac.uk
70
Secondary and tertiary structure information
2nd structure information S. pombe 827 of 5040
proteins (16.41) Human 4601 of 28937 proteins
(15.90) S. cerevisae 785 of 6213 proteins
(12.63) 3rd structure information S. pombe 17
of 5040 proteins (0.34) human 1149 of 28937
proteins (3.97) S. cerevisae 266 of 6213
proteins (4.28)
http//www.ebi.ac.uk
71
Most common protein families

http//www.ebi.ac.uk
72
What comes after human genome sequence is
completed?
The working draft DNA sequence and the more
polished 2003 version represent an enormous
achievement. However, much work remains to
realize the full potential of the accomplishment.
Early explorations into the human genome, now
joined by projects on the genomes of a number of
other organisms, are generating data whose volume
and complex analyses are unprecedented in
biology. Genomic-scale technologies will be
needed to study and compare entire genomes, sets
of expressed RNAs or proteins, gene families from
a large number of species, variation among
individuals, and the classes of gene regulatory
elements.

73
Research challenges for the future

• Gene number, exact locations, and functions
• Gene regulation
• DNA sequence organization
• Chromosomal structure and organization
• Noncoding DNA types, amount, distribution,
  information content, and functions
• Coordination of gene expression, protein
  synthesis, and post-translational events
• Interaction of proteins in complex molecular
  machines
• Predicted vs experimentally determined gene
  function
• Evolutionary conservation among organisms
• Protein conservation (structure and function)
• Proteomes (total protein content and function)
  in organisms
• Correlation of SNPs with health and disease
• Disease-susceptibility prediction based on gene
  sequence variation
• Genes involved in complex traits and multigene
  diseases
• Complex systems biology including microbial
  consortia useful for environmental restoration
• Developmental genetics, genomics