You are on page 1of 56

Seminarski rad 1: Opisati postupak kloniranja gena organizma

iji genom jo uvek nije sekvenciran


GRUPA 1: studenti sa parnim rednim brojevima na listi
Veliina nepoznatog gena oko 40 Kb
Pojedini delovi gena koji kodiraju odreene proteinske domene pokazuju visok
stepen homologije na nukleotidnom nivou sa odgovarajuim regionima ortolognih
gena ije su sekvence poznate i deponovane
Navesti ta je potrebno za postupak kloniranja
Dati detaljan prikaz eksperimenta kloniranja


GRUPA 2 : studenti sa neparnim rednim brojevimana listi
Veliina nepoznatog gena oko 5 kb
Gen pokazuje izuzetno visok stepen homologije na nukleotidnom nivou sa
ortolognim genima ije su sekvence poznate i deponovane
Navesti ta je potrebno za postupak kloniranja
Dati detaljan prikaz eksperimenta kloniranja

Seminarski rad otprintovan na najvie 2 strane formata A4 sa imenom
studenta, brojem indeksa i potpisom dostaviti na poetku predavanja koje e
biti odrano 11.04.2013.g.
Organism
Genome
Size (Mb)
Gene Number
Hepatitis D virus 0.0017 1
Hepatitis B virus 0.0032 4
HIV-1 0.0092 9
Bacteriophage l 0.0485 80
Escherichia coli 4.6392 4400
S. cerevisiae (yeast) 12.155 6300
C. elegans (nematode) 97 19000
D. melanogaster (fruit fly) 137 13600
Mus musculus (mouse) 3000 20000-30000
Homo sapiens (human) 3000 20000-30000
1 Mb = 1 million base pairs (for double-stranded DNA or RNA)
The genomes of prominent organisms
E.coli genome
CIRCULAR MAP
Genetic map: linkage
map of known genes or
functional sites
Often given in minutes
(the time which it takes
to each male
chromosome to move
into female cell)
Physical: restriction
sites of mapped genes


Organisation of E.coli genome
A prokaryotic genome has to squise into
relatively tiny space with the help of DNA
binding proteins that package the genome in
an organised fashion

The circular genome is supercoiled: in a
typical prokaryote the genome is a single
circular DNA molecule is organized in
NUCLEOID

The protein component of nucleoid include
DNA gyrase and DNA topoisomerase I- the
two enzymes that are primarly responsible for
maintaining the supercoiled state

A set of at least four additional proteins are
beleived to have a more specific roles in
packaging of bacterial DNA

The circular E. coli chromosome has a
circumference of 1.6 mm (E.coli cell has
dimension 1.0 x 2.0 mm)
Remove of a few turns
of the double
helix (underwinding)
results
in negative supercoiling
NUCLEOID
Bacteria genome is a nucleoid
Although bacteria do not display structure with the distinct
morphological features of the eucaryotic chromosomes, bacteria
genomes are organized into definite bodies-NUCLEOID.
Protein core
Between 50 and
100 supercoiled
loops of DNA
radiate from the
central protein
core
Structures of new Rhs elements
Unique DNA sequences from the E. coli K-12 chromosomal framework are
designated ORFs f256, o205, and o77 (2). The Rhs elements are aligned so that the
start codon of the core ORF is base 1. RhsE and RhsH of ECOR-45 are linked in
tandem (indicated by the diagonal dashed line). The core of ECOR-45 RhsG
contains a 587-bp deletion, indicated by .
Wang etal., JBC
Journal of Bacteriology,
1998, 180, 4102-4110.
Transposable Elements in Prokaryotes
Insertion Sequences (IS)

Noncomposite transposons

Composite Transposons
Insertion Sequences (IS)
Simplest transposable elements: Segments of DNA in bacteria
that can move from one position to another.

Normal constituents of bacterial chromosome and plasmids.

Cause increase in the size of the genome

IS elements only contain genes required to mobilize the
element and insert the element at a new location.

When IS elements transpose, promoters within IS elements
themselves may alter expression of nearby genes.
Insertion Sequences: IS
Insertion elements are mobile genetic elements that occasionally insert into
chromosomal sequences, often disrupting gene.

IS are a special class of transposable elements found in prokaryotes
(studied extensively in Escherichia coli K12)

They usually range in size from 1 to 2 kilobasepairs (kb) and contain
perfect or nearly perfect inverted terminal repeat sequences. These terminal
repeats likely are recognition sites for an enzyme responsible for the
insertion.

Mobility of the element depends only on the element itself; it is an
autonomous element. Thus, it must carry the coding ability for the
transposase recognizing the inverted terminal repeats.

Insertion Sequences (IS)

The terminal sequences flank a unique central sequence with at least one long open
reading frame coding, presumably, for the transposase protein: All IS elements are
made up of transposae gene flanked by inverted repeats (IRs).

IS1 first identified in E. colis glactose operon is 768 bp long and is present with 4-
19 copies in the E. coli chromosome

Ends of all known IS elements show inverted terminal repeats (ITRs)

Mechanizm of IS insertion
Staggered cut made at target site
IS element inserted, joined to single
stranded ends: DNA Pol and DNA
ligase fills in gaps.
Produced target site duplication
flanking the IS element.
The direct repeats externally
flanking the inverted repeats are not
part of the insertion sequence.
Instead, they are chromosomal
sequences that become duplicated
upon insertion, with one copy at
each end; this is called target-site
duplication.
Orientation of IS Elements: IS
elements can insert in any
orientation.


Composite Transposons
Created when two IS elements insert near each other. The region between
them can then be transposed by joint action of flanking IS elements (i.e.
two IS elements "capture" a DNA sequence and endow it with the ability to
transpose).
In many cases, these "captured" sequences are antibiotic resistance genes.
These transposons can jump onto conjugation plasmids and be transferred
from one strain or even one species to another. This is of great medical
significance.
R-factors are multidrug-resistance plasmids containing multiple
transposons bearing different antibiotic resistance genes.
Composite
transposons:
Have two ISs at
their ends, the
DNA between the
two ISs can
encode resistance
genes or virulence
factors. In many
cases only one of
the two
transposases is
functional.

Noncomposite transposons
Noncomposite transposons combine the qualities of both IS elements and
composite transposons.
Like IS elements, they have single IR sequences at each end.
Like composite transposons, they carry selectable marker genes.

Consists of a transposase gene, plus other genes, flanked by inverted
repeat (IR) sequences

IR sequences can form "stem-loop" structures when DNA containing transposon is
denatured and ssDNA allowed to reanneal.
Evolutionary role of insertion sequences

Selfish or parasitic DNA sequences which are maintained in the population, even in
the face of adverse natural selection, as a result of their ability to transpose and
become horizontally transmitted among strains by means of hitchhiking in
plasmids.

Insertion sequences also play an important role in the evolution of transposons and
plasmids:
One evolutionary implication of insertion sequences derives from their mutagenic
activity in causing insertion mutations.

A pair of insertion sequences flanking a central sequence can transpose as a unit,
and such composite transposons containing antibiotic-resistance genes, for
example,Tn5, Tn9 and TnlO, are well documented

Insertion sequences and transposons can also transpose into plasmids and remold
their structure, and in general change the genetic capabilities of plasmids

The distribution of numbers of insertion sequences in the genome is also of some
interest in understanding the population dynamics of the elements.


Streptomycetes
Comprises a large group of filamentous soil gram-
positive bacteria
The most important group of industrial
microorganisms: producing approximately two-thirds
of all known varieties of antibiotics
Produces many other useful secondary metabolites
and extra cellular enzymes
Phenomenon of genetic instability
Instability of certain genetic traits: mutations frequently exhibit
pleiotropy- several traits being altered at the same time
Molecular studies have shown that trait losses are due to large
deletions (ten to thousands of KB long) in the chromosomal DNA
These large chromosomal DNA (in average 8 MB in size) have
terminal inverted repeats
Deletions are frequently accompanied by tandem amplifications of
up to several hundred copies of specific DNA sequences termed
Amplifiable Units of DNA (AUD)
This phenomenon is explained by fact that chromosomes of many
Streptomycetes species are linear DNA molecules
Proteins are covalently attached to the 5-terminal ends of
chromosomal DNAs which presumably act as the primers of
replication
Unstable regions are at the both termini of the chromosome
Essentially all deletions involve one, or both, telomeres
Complete genome sequence of the model actinomycete
Streptomyces coelicolor A3(2), 2002, Nature 417, 141 -147
S coelicolor is a soil bacterium that has
many different metabolic processes and
biotransformations that allow it to live
under a wide range of conditions in the
soil and use a wide range of metabolic
pathways to, for example, degrade
insoluble remains of other organisms.
S coelicolor is unusual, in that it is a
multicellular bacterium which forms into
different sorts of 'tissues'. The complexity
of its metabolic pathways give rise to two
thirds of modern antibiotics, as well as
anti-tumour and immuno-suppressant
agents.
All this complexity arises from the longest
known eubacterial genome (8.7 million
base pairs and 7,825 genes; about one
quarter the number of human genes).

Yeast Genome
Yeast Genome: Saccharomyces cerevisiae
The entire genome sequence was released in April, 1996 and was the first
eukaryotic genome to be sequenced.
The haploid genome contains 16 chromosomes ranging in size from 220 to 1500 kb
Analysis of the genome reveals 6,183 potential ORFs
Compared to the genomes of other organisms, the yeast genes are very efficiently
spaced on the 16 chromosomes with a density of 1 gene/2 kb of DNA.
Most yeast genes do not contain introns
There is little 'junk' DNA in the intergenic regions commonly seen in other eukaryotes.
A total genome size of approximately 13,000kb:
Chromosomes 85% of the total yeast DNA
The 2mm-plasmid accounts for about 5%,
Mitochondrial DNA 10%
The 6.3 kb 2mm-plasmid plasmid has no known function in yeast other than to
replicate, but it is not deleterious to the cell. Each cell contains 60-100 copies of
this plasmid. Researchers have taken advantage of this naturally occurring high
copy number plasmid to create yeast plasmid vectors containing the 2mm-plasmid
origin of replication.
Life with 6000 genes
Science,1996, Vol. 274, p546
Reports on the complete sequencing of the genome of the
yeast Saccharomyces cerevisiae.
The sequence of 12,068 kilobases defining:
6183 potential ORFs of at least 100 amino acids in length. However,
there are likely additional ORFs smaller than 100 amino acids and not
all of the 6,183 potential ORFs are protein encoding genes.
5885 potential protein-encoding genes
140 genes specifying ribosomal RNA;
40 genes for small nuclear RNA molecules;
275 transfer RNA genes
Providing of information about the higher order organization
of yeast's 16 chromosomes and allowance of some insight into
their evolutionary history
The total number of real protein-coding genes
It has been proposed a revised yeast gene catalog consisting of 5538 ORFs 100 amino acids.
This reflects the proposed elimination of 503 ORFs.

Only two-thirds of these have been experimentally validated (known), and the remaining
~2000 ORFs are currently annotated as hypothetical. The total number of real protein-coding
genes has been a subject of considerable debate, with estimates ranging from 4,800 to 6,400
genes.
Saccharomyces cerevisiae complete
genome
The size of DNA in each chromosome of the yeast S. cerevisiae.
The elements are some 6 kb flanked by long terminal repeats (LTRs; some 300 bp in length),
which are named delta (for Ty1/2), sigma (for Ty3) and tau (for Ty4).
Ty elements resemble retroviral genomes in yet other aspects: there are two overlapping (in a
+1 mode) open reading frames. ORF
TyA encodes the gag (envelope) protein of the virus-like particles;
TyB provides a polycistronic message the product of which is processed by the endogenous Asp-type
protease to yield a Ty integrase and a retro transcriptase.
Though the elements share a relatively high degree of homology, Ty1/2/4 belong to the 'copia'
class of retroelements, while Ty3 is a member of the 'gipsy' family.
The majority of the Ty elements were found to transpose upstream of tRNA genes, in a
sequence non-specific manner. These sites appear to represent less harmful targets than genes
or other intergenic regions.
Retrotransposons
in yeast

-Ty1- Ty5.
-Ty1 through Ty4 have
been identified to
retrotranspose via an
RNA intermediate
-Ty5 does appear to be
no longer
capable of transposition

The genome sequence of Schizosaccharomyces pombe
Nature. 2002 Feb 21;415(6874):871-80.
The genome of fission yeast (Schizosaccharomyces pombe) contains the
smallest number of protein-coding genes yet recorded for a eukaryote:
4,824
The centromeres are between 35 and 110 kilobases (kb) and contain related
repeats including a highly conserved 1.8-kb element.
Regions upstream of genes are longer than in budding yeast
(Saccharomyces cerevisiae), possibly reflecting more-extended control
regions.
Some 43% of the genes contain introns.
50 genes have significant similarity with human disease genes; half of these
are cancer related.
Highly conserved genes important for eukaryotic cell organization
including those required for the cytoskeleton, compartmentation, cell-cycle
control, proteolysis, protein phosphorylation and RNA splicing are
identified. These genes may have originated with the appearance of
eukaryotic life.
Few conserved genes that are important for multicellular organization were
identified, suggesting that the transition from prokaryotes to eukaryotes
required more new genes than did the transition from unicellular to
multicellular organization.


Genetic mapping
A pair of homologous chromosomes can exchange parts by
crossing-over

Recombination produces genotypes with new combinations of
parental alleles

Two genes close together on the same chromosome pair do not
assort independently at meiosis

Gene loci on chromosome can be mapped by measuring the
frequencies of recombinants produced by crossing-over
Meiotic recombination
Process that generates a haploid product with
genotype that differs from both haploid genotypes
that constituted the meiotic diploid cell

The product of meiosis generated by recombination is
called RECOMBINANT
Haplotype
Recombination will rarely separate loci which lie close
together on a chromosome

A set of alleles on the small chromosomal segment tend to be
transmitted as a block through a pedigree

Such block of alleles is known as HAPLOTYPE

Haplotypes mark recognizable chromosomal segments which
can be traced through pedigrees and through population
Genetic distance
The recombination fraction is a measure of the
distance between two loci

The recombination fraction define genetic distance
Two loci which show 1% recombination are defined
as being 1 CENTIMORGAN apart on a genetic map
a)
b)
a
b
A B
A B
a b
a B
A b
a b
A B
a b
A B
a b
a B
A b
Heterozigotni roditelj
Produkti mejotike
rekombinacije
Dobijena
frekvenca
45%
45%
5%
5%
90% roditeljski
10% rekombinant
Heterozigotni roditelj
Produkti mejotike
rekombinacije Oekivana
frekvenca
25%
25%
25%
25%
50% roditeljski
50% ne-roditeljski
Nezavisna segregacija
Genetic markers
Mendelian characters which are sufficiently polymorphic to
give a reasonable chance that randomly selected person will be
heterozygous
Highly polymorphic and informative
Randomly distributed
Easy to score
High PIC values
PIC (polymorphism information content) a measure
of the informativness of genetic marker
STS (sequence tagged site): any piece of DNA
whose sequence is known and for which a
specific PCR assay has been designed


EST (expressed tagged site): a short sequence
of cDNA for which a PCR assay is available
RFLP

Restriction Fragment Length Polymorphism
A polymorphism due to difference in size of
allelic restriction fragments as a result of
restriction site polymorphism
Effects at the protein level
Effects at the DNA level
Normal red blood cells (top)
and sickle cells (bottom)
There are effects at the cellular level.
When red blood cells carrying mutant hemoglobin are
deprived of oxygen, they become sickle-shaped instead of the
usual round shape (see picture). This shape can sometimes
interrupt blood flow.
There are negative effects at the whole organism level.
Under conditions such as high elevation and intense exercise, a carrier of
the sickle cell allele may occasionally show symptoms such as pain and
fatigue.
There are positive effects at the whole organism level.
Carriers of the sickle cell allele are resistant to malaria, because the
parasites that cause this disease are killed inside sickle-shaped blood cells.
Sickle Cell Anemia
RFLP and sickle
cell mutation
CCTTAGG
GGAATCC
CCTGAGG
GGACTCC
CCTTAGG
GGAATCC
CCTTAGG
GGAATCC
CCTGTGG
GGAAACC
CCTTAGG
GGAATCC
0.2 KB 1.1 KB
1.3 KB
Mst I I Mst I I Mst I I
Mst I I Mst I I
b
a
b
s
RFLP and sickle cell mutation
Vezanost markera
Kada se ispituje odreeno svojstvo (osobina, oboljenje) prvi
korak je odreivanje na kom hromozomu se nalazi traeni
genski lokus

To se radi indirektno traenjem neke druge genetike
karakteristike (odnosno genetikog markera) koji se nasleuje
u korelaciji sa bolesnim alelom

Vezanost je korelacija u nasleivanju nekog RFLP alela (ili
nekog drugog genetikog markera) i bolesnog alela
Linkage Analyis: analiza vezanosti
markera


Tendencija da se markeri na specifinom lokusu
nasleuju zajedno kao posledica njihove fizike
vezanosti na pojedinanim hromozomima
A probe P detects two DNA
morphs when DNA is cut
by a certain restriction
enzyme (RE).

The pedigree of analysis of
dominant disease phenotype
D shows:
-Linkage of the D locus to
the RFLP locus in childs 2,
3, 6 and 7
-Absence of linkage in child
8 ?????
The detection and inheritance of RFLP
The detection
and
inheritance of
RFLP

The pedigree of
dominant disease
phenotype D shows
linkage of the D locus to
the RFLP locus.

Only child 8
is recombinant
Mapping using microsatellite repeats as
molecular markers
Disease allele P is probably linked to M
Mapping using DNA Fingerprint bands as molecular marker
DNA fingerprint
The specific pattern
of DNA fragments
formed when DNA
is cut with
restriction enzymes,
separate and
hybridize

Alele P possible
linked to C or I
A
B
C
D
E
F
G
I
H
F and H: Always inherited together- linked?
A and B: in progeny always either A or B- allelic?
A and D: Four combinations; A and D, A, D or neither- unlinked?
F, H and E: Always either F or H or E- closely linked in trans?
DNA profiling
In violent crimes such a murder or rape and
in paternity (or grand-paternity) cases.
Fortunately DNA is quite stable and resistant
to degradation.
With the latest procedures available
sufficient DNA may be obtained from just a
single hair follicle.


Autoradiograph from an actual rape case
showing the DNA profiles for one VNTR
locus.
The lanes marked "M" show a "ladder" of
DNA fragments of known sizes. These are
loaded onto the gel to provide an internal
ruler--allowing the sizes of the VNTR alleles
to be estimated more accurately.
As can be seen there is a match of the DNA
profile of defendant 1 and the forensic
sample.

LOD SCORE
Mera verovatnoe o genetikoj vezanosti
izmeu lokusa

Lod score > +3 dokaz vezanost
Lod score < -2 dokaz odsustva vezanosti

You might also like