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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 visokstepen 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 saortolognim 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 straneformata A4 sa imenomstudenta, brojem indeksa i potpisom dostaviti na poetku predavanjakoje e
biti odrano 11.04.2013.g.
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Organism GenomeSize(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 13600Mus 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
http://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10565http://www.web-books.com/MoBio/Free/Ch3H1.htmhttp://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10119http://www.web-books.com/MoBio/Free/Ch3H3.htmhttp://www.web-books.com/MoBio/Free/Ch3H5.htmhttp://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=celegans.infhttp://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=7227http://www.ncbi.nlm.nih.gov/genome/guide/mouse/http://www.ncbi.nlm.nih.gov/genome/guide/humanhttp://www.ncbi.nlm.nih.gov/genome/guide/humanhttp://www.ncbi.nlm.nih.gov/genome/guide/mouse/http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=7227http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=celegans.infhttp://www.web-books.com/MoBio/Free/Ch3H5.htmhttp://www.web-books.com/MoBio/Free/Ch3H3.htmhttp://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10119http://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.web-books.com/MoBio/Free/Ch3H2.htmhttp://www.web-books.com/MoBio/Free/Ch3H1.htmhttp://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=105658/11/2019 2014 Predavanje Broj 3 Manipulisanje genima
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E.coligenome
CIRCULAR MAP
Genetic map: linkagemap of known genes or
functional sites Often given in minutes(the time which it takesto each malechromosome to move
into female cell) Physical: restriction
sites of mapped genes
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Organisation ofE.coligenome A prokaryotic genome has to squise into
relatively tiny space with the help of DNAbinding proteins that package the genome in
an organised fashion
The circular genome is supercoiled: in atypical prokaryote the genome is a singlecircular DNA molecule is organized in
NUCLEOID
The protein component of nucleoid includeDNA gyraseand DNA topoisomerase I- the
two enzymes that are primarly responsible formaintaining the supercoiled state
A set of at least four additional proteinsarebeleived to have a more specific roles in
packaging of bacterial DNA
The circularE. colichromosome has acircumference of 1.6 mm(E.colicell has
dimension 1.0 x 2.0 mm)
Remove of a few turns
of the doublehelix (underwinding)
results
in negative supercoiling
NUCLEOID
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Bacteria genome is a nucleoid
Although bacteria do not display structure with the distinctmorphological 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
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Structures of newRhselements
Unique DNA sequences from theE. coliK-12 chromosomal framework aredesignated ORFs f256, o205, and o77 (2). TheRhselements are aligned so that thestart codon of the core ORF is base 1.RhsEandRhsHof ECOR-45 are linked intandem (indicated by the diagonal dashed line). The core of ECOR-45RhsGcontains a 587-bp deletion, indicated by .
Wang etal., JBC
Journal of Bacteriology,
1998, 180, 4102-4110.
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Transposable Elements in Prokaryotes
Insertion Sequences (IS)
Noncomposite transposons
Composite Transposons
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Insertion Sequences (IS)
Simplest transposable elements: Segments of DNA in bacteriathat 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 theelement and insert the element at a new location.
When IS elements transpose, promoters within IS elementsthemselves may alter expression of nearby genes.
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Insertion Sequences: IS
Insertion elements are mobile genetic elementsthat occasionally insert intochromosomal sequences, often disrupting gene.
IS are a special class of transposable elements found in prokaryotes(studied extensively inEscherichia coli K12)
They usually range in size from 1 to 2 kilobasepairs(kb) and containperfect or nearly perfect inverted terminal repeatsequences. These terminalrepeats 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 thetransposase recognizing the inverted terminal repeats.
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Insertion Sequences (IS)
The terminal sequences flank a unique central sequence with at least one long openreading framecoding, presumably, for the transposase protein: All IS elements are
made up of transposae gene flanked by inverted repeats(IRs).
IS1first identified inE. colis glactose operon is 768 bp long and is present with 4-19 copies in theE. colichromosome
Ends of all known IS elements show inverted terminal repeats (ITRs)
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Mechanizm of IS insertion Staggered cut made at target site
IS element inserted, joined to singlestranded ends: DNA Pol and DNAligase fills in gaps.
Produced target site duplicationflanking the IS element.
The direct repeats externallyflanking the inverted repeats are notpart of the insertion sequence.Instead, they are chromosomalsequences that become duplicatedupon insertion, with one copy ateach end; this is called target-site
duplication. Orientation of IS Elements: IS
elements can insert in anyorientation.
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Composite Transposons
Created when two IS elements insert near each other. The region betweenthem 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 totranspose).
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 medicalsignificance.
R-factorsare multidrug-resistance plasmidscontaining multipletransposons bearing different antibiotic resistance genes.
Compositetransposons:
Have two ISs at
their ends, theDNA between thetwo ISs canencode resistancegenes or virulencefactors. In manycases only one of
the twotransposases isfunctional.
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Noncomposite transposons
Noncomposite transposons combine the qualities of both IS elements andcomposite 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 invertedrepeat (IR) sequences
IR sequences can form "stem-loop" structures when DNA containing transposon is
denatured and ssDNA allowed to reanneal.
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Evolutionary role of insertion sequences
Selfish or parasitic DNAsequences which are maintained in the population, even inthe face of adverse natural selection, as a result of their ability to transpose and
become horizontally transmitted among strains by means of hitchhiking inplasmids.
Insertion sequences also play an important role in the evolutionof transposons and
plasmids: One evolutionary implication of insertion sequences derives from their mutagenicactivity 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 remoldtheir structure, and in general change the genetic capabilities of plasmids
The distribution of numbers of insertion sequences in the genome is also of someinterest in understanding the population dynamics of the elements.
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Streptomycetes
Comprises a large group of filamentous soil gram-
positive bacteria
The most important group of industrial
microorganisms: producing approximately two-thirdsof all known varieties of antibiotics
Produces many other useful secondary metabolites
and extra cellular enzymes
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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 largedeletions (ten to thousands of KB long) in the chromosomal DNA
These large chromosomal DNA (in average 8 MB in size) haveterminal inverted repeats
Deletions are frequently accompanied by tandem amplifications ofup to several hundred copies of specific DNA sequences termedAmplifiable Units of DNA (AUD)
This phenomenon is explained by fact that chromosomesof manyStreptomycetes species are linear DNA molecules
Proteins are covalently attached to the 5-terminal ends of
chromosomal DNAs which presumably act as the primers ofreplication
Unstable regions are at the both termini of the chromosome
Essentially all deletions involve one, or both, telomeres
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Complete genome sequence of the model actinomycete
Streptomyces coelicolorA3(2), 2002, Nature 417, 141 -147 S coelicoloris a soil bacterium that has
many different metabolic processes andbiotransformations that allow it to liveunder a wide range of conditions in thesoil and use a wide range of metabolic
pathways to, for example, degrade
insoluble remains of other organisms. S coelicoloris unusual, in that it is a
multicellular bacterium which forms intodifferent sorts of 'tissues'. The complexityof its metabolic pathways give rise to twothirds of modern antibiotics, as well as
anti-tumour and immuno-suppressantagents.
All this complexity arises from the longestknown eubacterial genome (8.7million
base pairs and 7,825 genes; about onequarter the number of human genes).
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Yeast Genome
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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 chromosomesranging 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 efficientlyspacedon the 16 chromosomes with a density of 1 gene/2 kb of DNA.
Most yeast genes do not contain introns
There is little 'junk' DNAin 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-plasmidplasmid has no known function in yeast other than toreplicate, but it is not deleterious to the cell. Each cell contains 60-100 copiesof
this plasmid. Researchers have taken advantage of this naturally occurring highcopy number plasmid to create yeast plasmid vectorscontaining the 2mm-plasmid
origin of replication.
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Life with 6000 genesScience,1996, Vol. 274, p546
Reports on the complete sequencing of the genome of theyeast Saccharomyces cerevisiae.
The sequence of 12,068 kilobases defining:
6183 potential ORFsof at least 100 amino acids in length. However,
there are likely additional ORFs smaller than 100 amino acids and notall of the 6,183 potential ORFs are protein encoding genes.
5885potential protein-encoding genes
140genes specifying ribosomal RNA;
40genes for small nuclear RNA molecules;
275transfer RNA genes Providing of information about the higher order organization
of yeast's 16 chromosomes and allowance of some insight intotheir evolutionary history
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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-codinggenes has been a subject of considerable debate, with estimates ranging from 4,800 to 6,400
genes.
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Saccharomyces cerevisiaecomplete
genome
The size of DNA in each chromosome of the yeast S. cerevisiae.
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R t t
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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 TyAencodes the gag(envelope) protein of the virus-like particles; TyBprovides a polycistronic message the product of which is processed by the endogenous Asp-type
protease to yield a Ty integraseand 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 tRNAgenes, in asequence non-specific manner. These sites appear to represent less harmful targetsthan genes
or other intergenic regions.
Retrotransposons
in yeast
-Ty1- Ty5.-Ty1 through Ty4 havebeen identified to
retrotranspose via an
RNA intermediate
-Ty5 does appear to be
no longercapable of transposition
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The genome sequence of Schizosaccharomyces pombe
Nature. 2002 Feb 21;415(6874):871-80.
The genome of fission yeast (Schizosaccharomyces pombe) contains thesmallest number of protein-coding genes yet recorded for a eukaryote:4,824
The centromeres are between 35 and 110 kilobases (kb) and contain relatedrepeats including a highly conserved 1.8-kb element.
Regions upstream of genes arelongerthan in budding yeast
(Saccharomyces cerevisiae), possibly reflecting more-extended controlregions.
Some 43% of the genes contain introns.
50 genes have significant similarity with human disease genes; halfof theseare cancer related.
Highly conserved genes important for eukaryotic cell organization
including those required for the cytoskeleton, compartmentation, cell-cyclecontrol, proteolysis, protein phosphorylation and RNA splicing areidentified. These genes may have originated with the appearance of
eukaryotic life.
Few conserved genes that are important for multicellular organizationwereidentified, suggesting that the transition from prokaryotes to eukaryotes
required more new genes than did the transition from unicellular tomulticellular organization.
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Genetic mapping
A pair of homologous chromosomes can exchange parts bycrossing-over
Recombination produces genotypes with new combinationsof
parental alleles
Two genes close together on the same chromosome pair do notassort independently at meiosis
Gene loci on chromosome can be mapped by measuring thefrequencies of recombinantsproduced by crossing-over
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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
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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
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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% recombinationare defined
as being 1 CENTIMORGANapart on a genetic map
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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
rekombinacijeDobijena
frekvenca
45%
45%
5%
5%
90% roditeljski
10% rekombinant
Heterozigotni roditelj
Produkti mejotike
rekombinacije Oekivana
frekvenca
25%
25%
25%
25%
50% roditeljski
50% ne-roditeljskiNezavisna segregacija
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Genetic markers
Mendelian characters which are sufficiently polymorphic to
give a reasonable chance that randomly selected person will be
heterozygous
Highly polymorphic and informativeRandomly distributed
Easy to score
High PIC valuesPIC (polymorphism information content) a measure
of the informativness of genetic marker
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STS(sequence tagged site): any piece of DNA
whose sequence is known and for which aspecific PCR assay has been designed
EST(expressed tagged site): a short sequence
of cDNA for which a PCR assay is available
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RFLP
Restriction Fragment Length Polymorphism
A polymorphism due to difference in size ofallelic restriction fragments as a result of
restriction site polymorphism
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Effects at the protein levelEffects 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 theparasites that cause this disease are killed inside sickle-shaped blood cells.
Sickle Cell Anemia
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RFLP and sicklecell 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 IMst I I
ba
bs
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RFLP and sickle cell mutation
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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
Li k A l i li i
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Linkage Analyis: analiza vezanosti
markera
Tendencija da se markeri na specifinom lokusu
nasleuju zajedno kao posledica njihove fizikevezanosti na pojedinanim hromozomima
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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 tothe RFLP locus in childs 2,
3, 6 and 7
-Absence of linkage in child
8 ?????
The detection and inheritance of RFLP
i
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The detection
and
inheritance of
RFLP
The pedigree of
dominant diseasephenotype D shows
linkage of the D locus to
the RFLP locus.
Only child 8is recombinant
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Mapping using microsatellite repeats as
molecular markers
Disease allele P is probably linked to M
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Mapping using DNA Fingerprint bands as molecular marker
DNA fingerprint
The specific patternof DNA fragments
formed when DNA
is cut with
restriction enzymes,
separate and
hybridize
Alele P possible
linked to C or I
A
BCDE
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?
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DNA profiling
In violent crimes such a murder or rape andin paternity (or grand-paternity) cases.
Fortunately DNA is quite stable and resistantto degradation.
With the latest procedures availablesufficient DNA may be obtained from just asingle hair follicle.
Autoradiograph from an actual rape caseshowing the DNA profiles for one VNTRlocus.
The lanes marked "M" show a "ladder" ofDNA fragments of known sizes. These areloaded onto the gel to provide an internal
ruler--allowing the sizes of the VNTR allelesto be estimated more accurately.
As can be seen there is a match of the DNAprofile of defendant 1 and the forensicsample.
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LOD SCORE
Mera verovatnoe o genetikoj vezanosti
izmeu lokusa
Lod score > +3 dokaz vezanost
Lod score < -2 dokaz odsustva vezanosti
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