2. Lecture WS 2003/04Bioinformatics III1 Review of Genome Language and some Facts Life is specified by genomes. Every organism, including humans, has a

2. Lecture WS 2003/04

Bioinformatics III 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 liver—to make discrete sets of proteins at just the right times, and it is

through this specificity that unique organisms arise.



The cell nucleus

http://www.nature.com/genomics/human/slide-show/1.html



DNA fibres




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 information—one 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.



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.



Central Dogma of Molecular Genetics

DNA--------->RNA--------->Protein

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/plsc431/431g.htm



cloning



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.



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

1. sequences that permit the propagation of itself in bacteria (or in yeast

for YACs)

2. a cloning site to insert foreign DNA; the most versatile vectors contain a

site that can be cut by many restriction enzymes

3. a method of selecting for bacteria (or yeast for YACs) containing a

vector with foreign DNA; usually accomplished by selectable markers for

drug resistance.



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



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.



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?

1. 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.

2. 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.

3. The clone can be used to isolate similar genes from other organisms.

Thus it can serve as a heterologous probe.

4. If the gene is of clinical importance, the clone can be used for diagnostic

purposes. E.g. one type of hemophilia.



sequencing + physical mapping



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.



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-terminating 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.



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.



Genome mapping




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



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.



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.



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.



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.



structural organisation of DNA



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 (2*πr). 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.



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.



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.

6*6). 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.



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.



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.



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.



Telomere Repeat Sequences



repetitive sequences



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.



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.



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.



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



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



Sequence distribution for selected species



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.



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.



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.



The genetic code



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.



The genetic code: RNA

U C A G

U UUU Phe UCU Ser UAU Tyr UGU Cys

UUC Phe UCC Ser UAC Tyr UGC Cys

UUA Leu UCA Ser UAA STOP UGA STOP

UUG Leu UCG Ser UAG STOP UGG Trp

C CUU Leu CCU Pro CAU His CGU Arg

CUC Leu CCC Pro CAC His CGC Arg

CUA Leu CCA Pro CAA Gln CGA Arg

CUG Leu CCG Pro CAG Gln CGG Arg

A AUU Ile ACU Thr AAU Asn AGU Ser

AUC Ile ACC Thr AAC Asn AGC Ser

AUA Ile ACA Thr AAA Lys AGA Arg

AUG Met or START ACG Thr AAG Lys AGG Arg

G GUU Val GCU Ala GAU Asp GGU Gly

GUC Val GCC Ala GAC Asp GGC Gly

GUA Val GCA Ala GAA Glu GGA Gly

GUG Val GCG Ala GAG Glu GGG Gly



The genetic code: DNA

T C A G

T TTT Phe TCT Ser TAT Tyr TGT Cys

TTC Phe TCC Ser TAC Tyr TGC Cys

TTA Leu TCA Ser TAA STOP TGA STOP

TTG Leu TCG Ser TAG STOP TGG Trp

C CTT Leu CCT Pro CAT His CGT Arg

CTC Leu CCC Pro CAC His CGC Arg

CTA Leu CCA Pro CAA Gln CGA Arg

CTG Leu CCG Pro CAG Gln CGG Arg

A ATT Ile ACT Thr AAT Asn AGT Ser

ATC Ile ACC Thr AAC Asn AGC Ser

ATA Ile ACA Thr AAA Lys AGA Arg

ATG Met or START

ACG Thr AAG Lys AGG Arg

G GTT Val GCT Ala GAT Asp GGT Gly

GTC Val GCC Ala GAC Asp GGC Gly

GTA Val GCA Ala GAA Glu GGA Gly

GTG Val GCG Ala GAG Glu GGG Gly

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.



Codon usage: Cytochrome P450

or how the genome affects protein composition

110 non-allelic cytochrome P450 genes from man (n=30), rat (n=38), rabbit

(n=24), and mouse (n=18) 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



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.



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.



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.



Codon Usage Bias Correlates with 3rd Position C+G Content

The increasing C+G 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.



Codon Positional C+G Content Correlates with Regional Genomic C+G Content

For reasons that are not yet understood (1995), the composition of mammalian genomes is not homogeneous; some segments (isochores) are high in C+G content, while some regions are A+T rich. As shown here, genes located in CG-rich segments exhibit high C+G 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 C+G 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 C+G content of these genes. Flank + intron C+G content is taken as an indicator of the C+G composition of the corresponding region (isochore) of the genome.



Amino Acid Composition Correlates with Isochore Composition

The correspondence of C+G content in the

first and second codon positions with

isochore composition suggests that genes

located in regions of high C+G 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 flank+intron C+G 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).



Codon usage: Cytochrome P450

Amino Acid Composition Correlates with

Codon Usage Bias

As noted earlier, codon 3rd position C+G

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 C+G

content.

Thus, the regional genomic nucleotide

composition influences the composition of

genes and, surprisingly, their encoded

proteins.



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 C+G 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.



Codon usage in different species

http://www.uky.edu/Pharmacy/ps/porter/CodonUsage/preferred_codons.htm



organelle DNA



Organelle DNA

Not all genetic information is found in nuclear DNA. Both plants and animals have

an organelle—a "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.



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.



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.



exons + introns, splicing



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.



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



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.




On the left, the RNA is spliced to include the exons S, V, C1, C2, C3, C4, 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, C1, C2, C3, C4 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.

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.




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).




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).

Middle row:

pre-mRNAs for each gene,

splicing pattern for female

splicing pattern for male.



knowledge about whole genomes:genome content and annotation



Genome sequences: Archaea

http://www.ebi.ac.uk



Protein length




Amino acid composition




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%)




Most common protein families




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.



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

Documents

2. Lecture WS 2003/04Bioinformatics III1 Review of Genome Language and some Facts Life is specified by genomes. Every organism, including humans, has a