Main enzymes used
in Molecular biology
(theory and practice)
by Jean-Pierre Herveg and a lot of friends at
the Brussels Branch of the Ludwig Institute for Cancer research (Licr) and the Christian de
Duve* Institute for cellular Patholgy (ICP).
April 2006
Université Catholique de Louvain
Avenue E. Mounier, 1200 Brussels (Belgium)
---------------------*Christian de Duve got the Noble Prize in 1974. He and his team discovered both lysosomes
and peroxysomes.
DNA pol is an EC 2 Transferase
DNA Polymerases
Enzymes classification
(in fact this classification is more a classification of reactions than a classification of enzymes.
There are 6 classes of reactions and thus six classes of enzymes.
The DNA pols are in the EC2 goup, the second group.
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
EC 2 Transferases: transfer a functional group (a nucleotide)
EC 3 Hydrolases: catalyze the hydrolysis of various bonds
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
EC 5 Isomerases: catalyze isomerization: changes within a single molecule
EC 6 Ligases: join two molecules creating covalent bonds
How does the EC classification work ?
Exemples:
EC 2.-.-.- transferases (any transfer).
EC 2.7.-.- transferring phosphorous-containing groups.
EC 2.7.7.- transferring nucleotides: nucleotidyltransferases.
EC 2.7.7.7
DNA-directed DNA pol.
EC 2.7.7.49
RNA-directed Dna pol.
A reverse transcriptase is an RNA- directed DNA pol
EC 2.7.7.49. This enzyme is in the EC2.7.7. Group
____________________
Question:
what is the difference between DNA pol and RT-DNA pol ?
DNA Polymerases
Enzymes that replicate DNA using a DNA or RNA templates*.
DNA-directed DNA polymerase EC 2.7.7.7
RNA-directed DNA polymerase (Reverse transcriptase) EC 2.7.7.49
Most organisms have more than one type of DNA polymerase
(for example, E. coli has five DNA polymerases), but all work using the same 4 basic rules.
Polymerization
1. requires a template (DNA or RNA) to copy the complementary strand.
2. requires a pre-existing primer (DNA or RNA) from which to extend 5’ to 3’.
3. occurs only in the 5' to 3’ direction
4. requires 4 dNTPs: dATP, dGTP, dCTP, dTTP
however, the reaction works also with ddNTPs even when they are bound to large
molecules like fluorochromes etc.
In this case, the size of catalytic core of the DNA pol had to be increased)
For replication, in most organism, the primer is a RNA molecule
(in some viruses the primer is a protein).
Bacteria have 5 known DNA polymerases:
* Pol I: Is implicated in DNA repair
has both 5'->3' and
3'->5' exonuclease activity.
* Pol II: Pol II is involved in replication of damaged DNA
has a 3'->5' exonuclease activity (proof reading).
* Pol III: is the main polymerase in bacteria (elongates in DNA replication)
as such it has 3'->5' exonuclease proofreading ability.
* Pol IV: is a Y-family DNA polymerase
* Pol V: is a Y-family DNA polymerase and participates in bypassing DNA damage
E. coli DNA Polymerase I
DNA Polymerase I from E. coli was the first DNA polymerase characterized.
The enzyme is a single large protein with a molecular weight of approximately 103 kDa
The enzyme requires a divalent cation (Mg++) for activity
It has 3 enzymatic activities:
1. 5'-to-3' DNA Pol activity
2. 3'-to-5' exonuclease (Proofreading activity)
3. 5'-to-3' exonuclease (Nick translation or Taqman activity)
The rate of DNA synthesis by pol is only 20 nucleotides/second (1200 nt/minutes)
much slower than the rate of 1,000 nucleotides/second measured for the replication of E. coli DNA.
DNA polI is not the main enzyme used to replicate DNA.
DNA polymerase I removes the RNA primer from the lagging strand and fills in the necessary
nucleotides.
-------------------------questions:
1. What is the lagging strand ?
It has 3 enzymatic activities:
1. 5'-to-3' DNA Pol activity
2. 3'-to-5' exonuclease (Proofreading activity)
3. 5'-to-3' exonuclease (Nick translation or Taqman activity)
------------------------------------Questions:
1. Cites the activities of E. Coli DNA polymerase I.
2. What is the rate of DNA synthesis of the E. Coli DNA polymerase I.
3. What does “proofreading activity means”?
4. What do “nick translation and Taqman activity mean”?
5. What is the rate of DNA synthesis by E. Coli DNA pol ?
It has 3 enzymatic activities:
1. 5’ to 3' DNA Pol activity
5‘ tatctggttgatcctgcc 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgatcctgccagtattatatgctgaattcag 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
2. 3’ to 5' exonuclease (Proofreading activity)
5‘ tatctggttgatcctgccagtagtatatgctaaaatcagagattaaggcatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
3. 5’ to 3' exonuclease (Nick translation and Taqman activity)
5‘ tatctggttgatcctgcc
tgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgatcctgcc
ttaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
DNA polymerase III in the replisome
is the primary enzyme complex involved in prokaryotic DNA replication.
The complex has high processivity (i.e. the number of nucleotides added per binding event) and,
specifically referring to the replication of the E.coli genome,
works in conjunction with four other DNA polymerases (Pol I, Pol II, Pol IV, and Pol V).
Being the primary holoenzyme involved in replication activity, the DNA Pol III holoenzyme
also has proofreading capabilities that correct replication mistakes by means of
exonuclease activity working 3'->5'.
DNA Pol III is a component of the replisome, which is located at the replication fork.
The replisome is composed of the following:
2 DNA Pol III enzymes, made up of α, ε, θ subunits.
the α subunit has polymerization activity.
the ε subunit has proofreading activity.
the θ subunit stimulates the ε subunit's proofreading.
2 β units which act as sliding DNA clamps, they keep the polymerase binded to the DNA.
It has two subunits.
2 τ units which connect the 2 DNA Pol III enzymes.
1 γ unit which acts as a clamp loader for the lagging strand Okazaki fragments,
helping the two β subunits to form a unit and bind to DNA.
The γ unit is made up of 5 γ subunits.
------------------------------Question:
1.
What is processivity ?
2.
What’s an Okazaki fragment ?
Thermoresistant DNA pol
Thermoresistant DNA pols are used in PCR. Thermoresistant DNA pols exist in extremophile
Bacteria and Archaea. Extremophile means living in extreme condtions. Some Bacteria and
Archaea are living in hot geyser or in the bottoms of ocean, near erupting volcanoes. Their
DNA pol resists then to high temperature.
They are used at 68 to 72° C. At this temperature, the primers are annealed to perfectly
Complementary sequences. In addition we don’t need to add DNA pols after each PCR cycle
As it was the case with non thermoresistant DNA pols.
The qualities of these DNA pols is expressed by the time they can be incubated at 94° C before
Showing a decrease of half or their activity.
Bacterial DNA pols
They don’t have a proof reading activity. This means that they don’t have a 3’to 5’ endonuclease
Activity. On example is the Taq DNA pol. The gene coding this enzyme is prepared from
Thermus aquaticus. Taq DNA pol incorporates 20 nt per second at their optimum pH and
temperature
Archaeal DNA pols
They have a proof reading activity, but they are 10 times less active. Pfu is an example of
this kind of DNA pol.
--------------------------------question:
Why is Pfu DNA pol 10 times less active than Taq DNA pol ?
Home made thermoresistant DNA pols.
Instead of giving their money to rich international companies, many laboratories prepare their
own thermoresistant DNA pol.
They have a strain of E. coli, transformed by a plamid coding such an enzymze.
The protein which is expressed is thermoresistant. Then, the broth in which this bacteria was grown
is heated for one hour at 70° C. The only protein which resist is the thermoresistant DNA pol.
This heated solution can be used directly as an enzyme.
The heated preparation could also be further purified by passing it through a resin.
This resin is expensive.
--------------------------Question
How can we prepare a “home made” thermoresistant DNA pol ?
Restriction enzymes
type II restriction enzyme: EC 3.1.21.4
These Enzymes are hydrolases that hydrolase ester bonds:
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
EC 2 Transferases: transfer a functional group
EC 3 Hydrolases: catalyze the hydrolysis of various bonds
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
EC 5 Isomerases: catalyze isomerization changes within a single molecule
EC 6 Ligases: join two molecules with covalent bonds
EC3.1.
E.C.3.1.-.- Acting on ester bonds. [ 1076 PDB entries ]
E.C.3.2.-.- Glycosylases. [ 1465 PDB entries ]
E.C.3.3.-.- Acting on ether bonds. [ 16 PDB entries ]
E.C.3.4.-.- Acting on peptide bonds (peptide hydrolases). [ 1582 PDB entries ]
E.C.3.5.-.- Acting on carbon-nitrogen bonds, other than peptide bonds. [ 306 PDB entries ]
E.C.3.6.-.- Acting on acid anhydrides. [ 153 PDB entries ]
E.C.3.7.-.- Acting on carbon-carbon bonds. [ 8 PDB entries ]
E.C.3.8.-.- Acting on halide bonds. [ 31 PDB entries ]
E.C.3.9.-.- Acting on phosphorus-nitrogen bonds. [-]
E.C.3.10.-.- Acting on sulfur-nitrogen bonds. [-]
E.C.3.11.-.- Acting on carbon-phosphorus bonds. [-]
E.C.3.12.-.- Acting on sulfur-sulfur bonds. [-]
E.C.3.13.-.- Acting on carbon-sulfur bonds. [-]
EC 3.1.21.4
Common name: type II site-specific deoxyribonuclease
Other name: type II restriction enzyme
Reaction: Endonucleolytic cleavage of DNA giving specific double-stranded fragments
with terminal 5'-phosphates
These enzymes recognize specific short DNA sequences (palindrome) and cleave
wthin the palindrome. Palindromic sequences are called restriction sites
Example:
gaattc
cttaag
g aattc
cttaa g
The fragments created by a restriction endonuclease possess either a restiction site
at both ends, or at one end only, depending of their position on the dsDNA molecule:
Restriction sites are palindromic element:
What is a palindrome ?
A palindrome is a word, phrase, verse, or sentence that reads the same backward or forward.
Exemple with words: a nut for a jar of tuna.
Exemple with a restriction site:
The site recognised by Eco RI is
gaattc
cttaag
The site recognised by Hae III is
ggcc
ccgg
Each time these enzymes recognize their specific sites, they bind to it and cleave
within it.
------------------------------Question:
1.
What’s a palindrome in molecular biology, give an example of a palindrome ?
Palindrome
5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
a SNP within a palindrome
5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgatcctgccagtattatatgctgtattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacataagtctctaattcggtacgtacacat 5‘
----------------------question:
1. What‘s a SNP within a palindrome.
2. What‘s a RFLP ?
Each time these enzymes recognize their specific sites, they bind to it and cleave
within it, giving restriction fragments
example with Eco RI (sticky ends):
5‘ tatctggttgaattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaacttaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttg 3‘
3‘ atagaccaacttaa 5‘
+
5‘ aattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘
gcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
example with Hae III (blunt ends):
5‘ tatctggttggccttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttgg 3‘
3‘ atagaccaacc 5‘
+
5‘ ccttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ ggaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
Restriction sites: cohesive and blunt ends
1. Cohesive ends (sticky ends)
extremos pegajosos o cohesivos
5’ protrusion (5’ saliente) (Eco RI):
5‘ tatctggttgaattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaacttaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttg 3‘
3‘ atagaccaacttaa 5‘
+
5‘ aattcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘
gcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
3’ protrusion (3’ saliente) (Pst I):
5‘ tatctggttctgcaggccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaagacgtccggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttctgca 3‘
3‘ atagaccaag 5‘
+
5‘
ggccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ acgtccggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
1. Blunt ends
extremos romos
example: Pvu II
5‘ tatctggttcagctgttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaagtcgacaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ tatctggttcag 3‘
3‘ atagaccaagtc 5‘
+
5‘ ctgttcgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ gacaagcggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
The Klenow fragment of DNA pol I:
Filling or suppressing the protruding ends
the Klenow fragment has two acitivities:
5’ --> 3’ DNA pol activity
5’ --> 3’ exonuclease activity
1. filling a 5’ protruding end using the DNA pol activity.
The molecule could be labeled if t is either radioactive or fluorescent
5' tatctggttg 3’
3' atagaccaacttaa 5’
------>
5' tatctggttgaatt 3’
3' atagaccaacttaa 5’
2. suppressing a 3’ protruding end using the exonuclease activity.
5‘ tatctggttctgca 3‘
3‘ atagaccaag 5‘
------>
5‘ tatctggttc 3‘
3‘ atagaccaag 5‘ + t, g, c and a
Frequency of restriction sites (sf):
1/4N
N = number of bases in the site
4 means that in DNA we have 4 nucleotides (A,G, T and C)
Examples:
1. Eco RI, N = 6, sf = 1/46 = 1/4096
This means that we could encounter one site every 4096 nucleotide (nt).
2. Hae III, N = 6, sf = 1/44 = 1/256
This means that we could encounter one site every 256 nucleotide (nt).
5‘ tatctggttggccttcgccagtattggatcctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataacctaggacttaagtctctaattcggtacgtacacat 5‘
Asp 718 (5’ protruding)
5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5‘
Kpn I (3’ protruding)
5‘ tatctggttggccttcgccagtattggatc ctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataac ctaggacttaagtctctaattcggtacgtacacat 5‘
Sau 3AI (5’ protruding)
5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5‘
Bam HI (5’ protruding)
5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5‘
but
Sau 3AI
5‘ tatctggttggccttcgccagtattg gatcctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataacctag gacttaagtctctaattcggtacgtacacat 5
Sau 3AI
5‘ tatctggttggccttcgccagtatta gatcgtgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaaccggaagcggtcataatctag cacttaagtctctaattcggtacgtacacat 5
example: a mammalian vector: why a mammalian ?
T 7 Promoter
Eco RI
Not I
T3 promter
5’ ttaatacgactcactataggctagcctcgagaattcacgcgtggtacctctagagtcgacccgggcggccgcttccctttagtgagggttaatg 3’.
---------------------------Question:
What’s a cloning sites
DNA Ligase (EC 6)
DNA ligases catalyze formation of a phosphodiester bond between the 5' phosphate
of one strand of dsDNA and the 3' hydroxyl of an other.
This enzyme is used to covalently link or ligate fragments of dsDNA together.
Most commonly, the reaction involves ligating a fragment of DNA into a plasmid vector, which is
a fundamental technique in recombinant DNA work.
A DNA ligase repair the cut made by a restriction endonuclease.
The most widely used DNA ligase is derived from the T4 bacteriophage.
T4 DNA ligase requires ATP as a cofactor.
EC 6.5.1.1 Common name: T4 DNA ligase (ATP)
A DNA ligase from E. coli is also available, but is not commonly used.
it uses NAD as a cofactor.
EC 6.5.1.2Common name: DNA ligase (NAD+)
----------------------------Questions:
1. In T4 DNA ligase, what does T4 means ?
2. What is the cofactor used by T4 DNA ligase ?
Among all the enzyme reactions, the ligase reaction is classified
as EC 6 in the EC number classification of enzyme reactions:
EC 1 Oxidoreductions. oxydoreductases
EC 2 Transferts: transferases transfer a functional group (a methyl or phosphate group)
EC 3 Hydrolysis: hydrolases catalyze the hydrolysis of various bonds
EC 4 Lyase reactions: Lyases cleave various bonds by means other than hydrolysis
and oxidation
EC 5 Isomerisations: isomerases catalyze isomerization changes within a single molecule
EC 6 Ligation: ligases join two molecules with covalent bonds
A ligase is an enzyme that catalyses the joining of two molecules
by forming a new chemical bond, with accompanying hydrolysis of
ATP or similar molecules.
A DNA ligase is an enzyme that catalyze the joining of two DNA molecules
by forming a phosphodiester bond between the 5' phosphate of one strand of DNA
and the 3' hydroxyl of the another.
with the accompanying hydrolysis of ATP (EC 6.5.1.1) or NAD (EC 6.5.1.2).
The common names of ligases sometimes include the word "ligase," such as
”DNA ligase”, an enzyme joining DNA fragments.
But, ligase could be absent of the name
”Synthetase" (when ligases are used to synthesize new molecules
”Carboxylase" (when ligases are used to add carbon dioxide to a molecule).
Classification.
Ligases are classified into six subclasses:
EC 6.1 includes ligases used to form carbon-oxygen bonds: acylates a tRNA with its aa
aa-tRNA ligases
EC 6.2 includes ligases used to form carbon-sulfur bonds: synthesizes acyl-CoA derivatives.
EC 6.3 includes ligases used to form carbon-nitrogen bonds: amide synthases
EC 6.4 includes ligases used to form carbon-carbon bonds: carboxylating enzymes,
mostly biotinyl-proteins
EC 6.5 includes ligases used to form phosphoric ester bonds,
DNA ligase = EC 6.5.1.1 and EC 6.5.1.2
EC 6.6 includes ligases used to form nitrogen-metal bonds
-----------------------------Question:
Explain the reaction catalyzed by T4 DNA ligase and ATP.
A ligation reaction requires 3 reagents :
At least 2 fragments of DNA
With either blunt or
compatible cohesive ("sticky") ends.
A buffer which contains ATP.
The buffer is usually provided or prepared as a 10X concentrate which,
after dilution, yields an ATP concentration of roughly 0.25 to 1 mM.
Most restriction enzyme buffers will work if supplemented with ATP.
T4 DNA ligase.
A typical reaction for inserting a fragment into a plasmid vector
would utilize about
0.01 (sticky ends) to
1.0 (blunt ends) units of ligase.
Intermolecular ligation:
(liagación intermolecular)
1. With cohesive ends (sticky ends, here Eco RI gaattc/cttaag):
5‘ tatctggttgatcctgccaattattatatgctgOH
3‘ atagaccaactaggacgttcataatatacgaccttaaPOH
+
OHPaattcagagattaagccatgcatgtgta 3‘
HOgtctctaattcggtacgtacacat 5‘
+ T4 DNA ligase + ATP
5‘ tatctggttgatcctgccaattattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacgttcataatatacgaccttaagtctctaattcggtacgtacacat 5‘
2. With bunt ends
5‘ tatctggttgatcctgccaattattatatgctOH
3‘ atagaccaactaggacgttcataatatacgacPOH
+
OHPagagattaagccatgcatgtgta 3‘
HOtctctaattcggtacgtacacat 5‘
+ T4 DNA ligase + ATP
5‘ tatctggttgatcctgccaattattatatgctagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacgttcataatatacgactctctaattcggtacgtacacat 5‘
Intramolecular ligation:
(liagación intramolecular)
Phage l (fagos l)
Cos R
Cos L
+ T4 DNA ligase + ATP
Phage l
cut and paste:
1. Cut the DNA to be inserted, using Eco RI and Not I:
5‘ tatctggttgagaattctcctgccagtagtatatgctaaaatcagaggcggccgcattaaggcatgcatgtgta 3‘
3‘ atagaccaactcttaagaggacggtcataatatacgacttaagtctccgccggcgtaattcggtacgtacacat 5‘
Eco RI
Not I
5‘ tatctggttgagaattctcctgccagtagtatatgctaaaatcagaggcggccgcattaaggcatgcatgtgta 3‘
3‘ atagaccaactcttaagaggacggtcataatatacgacttaagtctccgccggcgtaattcggtacgtacacat 5‘
+ Eco RI
5‘ tatctggttgag aattctcctgccagtagtatatgctaaaatcagaggcggccgcattaaggcatgcatgtgta 3‘
3‘ atagaccaactcttaa gaggacggtcataatatacgacttaagtctccgccggcgtaattcggtacgtacacat 5‘
+ Not I
5‘ tatctggttgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcattaaggcatgcatgtgta 3‘
3‘ atagaccaactcttaa gaggacggtcataatatacgacttaagtctccgccgg cgtaattcggtacgtacacat 5‘
fragment ready to be pasted:
5‘aattctcctgccagtagtatatgctaaaatcagaggc 3‘
3‘
gaggacggtcataatatacgacttaagtctccgccgg 5‘
5‘ tatctggttgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcattaaggcatgcatgtgta 3‘
3‘ atagaccaactcttaa gaggacggtcataatatacgacttaagtctccgccgg cgtaattcggtacgtacacat 5‘
-----------------------------------question:
explain The „cut and paste“ reaction in molecular biology.
example: our mammalian vector
2. Cut the vector, using Eco RI and Not I:
-------------------Question:
1.
Why are there T4 and SP6 promoter elements at both ends of the cloning site ?
2.
What’s a MCS ?
3.
Why is there an intron before the cloning site in this vector ?
T 7 Promoter
Eco RI
Not I
T3 promter
5’ ttaatacgactcactataggctagcctcgagaattcacgcgtggtacctctagagtcgacccgggcggccgcttccctttagtgagggttaatg 3’.
5’ …agcctcgagaattcacgcgtggtacctctagagtcgacccgggcggccgcttcccttta… 3’
3’ …tcggagctcttaagtgcgcaccatggagatctcagctgggcccgccggcgaagggaaat… 5’
5’ …agcctcgag aattcacgcgtggtacctctagagtcgacccgggc ggccgcttcccttta… 3’
3’ …tcggagctcttaa gtgcgcaccatggagatctcagctgggcccgccgg cgaagggaaat… 5’
5’ …agcctcgag aattcacgcgtggtacctctagagtcgacccgggc ggccgcttcccttta… 3’
3’ …tcggagctcttaa gtgcgcaccatggagatctcagctgggcccgccgg cgaagggaaat… 5’
5’ …agcctcgag aattcacgcgtggtacctctagagtcgacccgggc ggccgcttcccttta… 3’
3’ …tcggagctcttaa gtgcgcaccatggagatctcagctgggcccgccgg cgaagggaaat… 5’
2. Paste DNA into the vector using Ligase + ATP
fragment to be pasted
aaaaaaaaaaaa5‘ aattctcctgccagtagtatatgctaaaatcagaggc 3‘
aaaaaaaaaaaa3‘aaaaagaggacggtcataatatacgacttaagtctccgccgg 5‘
open vector
5’ …agcctcgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcttcccttta… 3’
3’ …tcggagctcttaag aggacggtcataatatacgacttaagtctccgccgg cgaagggaaat… 5’
5’ …agcctcgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcttcccttta… 3’
3’ …tcggagctcttaag aggacggtcataatatacgacttaagtctccgccgg cgaagggaaat… 5’
+ ligase and ATP
5’ …agcctcgag aattctcctgccagtagtatatgctaaaatcagaggc ggccgcttcccttta… 3’
3’ …tcggagctcttaag aggacggtcataatatacgacttaagtctccgccgg cgaagggaaat… 5’
Isoschizomers:
enzymes that recognize the same site
Asp 718
Kpn I
Bam HI
5’
3’
5’
3’
5’
3’
g gatcc
cctag g
ggatc c
c ctagg
g gatcc
cctag g
3’
5’
3’
5’
3’
5’
Enzyme’s family:
enzymes producing the same endings
gatc: Sau 3AI, Bgl I, Bam HI, Bcl II, Xho II
ctag: Mae I, Spe I, Nhe I, Avr I, Xba I
Bam HI
Sau 3AI
5’ g gatcc 3’
3’ cctag g 5’
5’ -gatc 3’
3’ ctag- 5’
DNA dependent RNA polymerases (RNA pol)
Enzymes:
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
EC 2 Transferases: transfer a functional group
EC 3 Hydrolases: catalyze the hydrolysis of various bonds
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
EC 5 Isomerases: catalyze isomerization changes within a single molecule
EC 6 Ligases: join two molecules with covalent bonds
Exemples:
EC 2.-.-.- Transferases.
EC 2.7.-.- Transferring phosphorous-containing groups.
EC 2.7.7.- Nucleotidyltransferases.
EC 2.7.7.6. DNA-dependent RNA polymerase II
EC 2.7.7.7 DNA-directed Dna polymerase.
-----------------------------Question:
1. What does “DNA dependant” means in DNA dependant RNA pol ?
2. Does the RNA pol need a primer ?
T7 RNA Polymerase
a DNA-dependent RNA polymerase from a bacteriophage
T7 RNA Polymerase is a DNA-dependent RNA polymerase that exhibits extremely high specificity
for its cognate promoter sequences: 5’ tgtaatacgactcactataggg 3’ (In red, the promotion start site)
Only DNA cloned downstream from a T7 promoter can serve as a template
for T7 RNA Polymerase-directed RNA synthesis.
It is generally provided (10-20 u /µl) with another tube containing the
concentrated (5x or 10 x) reaction buffer you should use.
It is used in molecular biology for the production of:
1.
2.
3.
4.
RNA templates for in vitro translation.
probes for nucleic acid hybridizations (microarrays).
RNA processing substrates.
antisense RNA.
Other phage RNA pol available
SP6 RNA pol, T3 RNA pol,
They are produced by recombinant technology.
--------------------------Questions
What are the uses of T7 RNA pol.
T7 promoter
Eco RI
Bam HI
5’ tgtaatacgactcactatagggcgaattcgagctcggtacccggggatcct
Hind III
SP6 promoter
ctagagtcgacctgcaggcatgcaagcttgagtattctatagtgtcacctaaat 3’
DNA primase ( in replication) is a DNA dependent RNA pol
DNA Primase and replication:
5‘ ugguugauccugcc 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ ugguugauccugccagtattatatgctgaattcagagattaagccatgcat---> 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
AMV reverse transcriptase
(AMV = Avian Myeloblastosis Virus)
Enzymes:
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
EC 2 Transferases: transfer a functional group
EC 3 Hydrolases: catalyze the hydrolysis of various bonds
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
EC 5 Isomerases: catalyze isomerization changes within a single molecule
EC 6 Ligases: join two molecules with covalent bonds
Exemples:
EC 2.-.-.- Transferases.
EC 2.7.-.- Transferring phosphorous-containing groups.
EC 2.7.7.- Nucleotidyltransferases.
EC 2.7.7.7 DNA-directed Dna polymerase.
EC 2.7.7.49 RNA-directed DNA polymerase
M-MLV RT DNA pol
AMV RT DNA pol (AMV RT DNA pol)
Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT)
catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrid
This enzyme is DNA/RNA dependent !
It requires
1. primer (DNA primers are more efficient than RNA primers)
2. Mg2+ or Mn2+ ions.
The enzyme possesses an intrinsic RNase H activity.
Both nonionic detergents and sulfhydryl compounds stabilize the enzyme activity in vitro.
AMV RT is the preferred reverse transcriptase for templates with high secondary structure
due to its stability at higher reaction temperatures (37–58°C).
It is generally provided (5-10 u /µl) with another tube containing the
concentrated (5x or 10 x) reaction buffer you should use.
Applications
First- and second-strand synthesis of cDNA.
Primer extensions.
RT-PCR. Up to 10 µl of an RT reaction containing AMV RT and the supplied AMV RT Reaction Buffer
can be added to a 50 µl PCR amplification reaction that uses Taq DNA Polymerase (20 %).
-----------------------Question:
What’s a RNA high secondary structure.
M-MLV RT DNA pol (reverse transcriptase)
(EC 2.7.7.49 RNA-directed DNA polymerase)
Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT)
is an RNA-dependent DNA polymerase that can be used in cDNA synthesis
with long messenger RNA templates (>5kb).
The enzyme is a product of the pol gene of M-MLV: it consists of a single subunit
with a molecular weight of 71kDa.
The RNase H activity of M-MLV RT is weaker than the commonly
used Avian Myeloblastosis Virus (AMV) reverse transcriptase.
Applications
First-strand synthesis of cDNA.
Primer extensions.
RT-PCR:
Up to 10 µl of an RT reaction containing AMV RT and the supplied
AMV RT Reaction Buffer can be added to a 50 µl PCR amplification reaction that uses
Taq DNA Polymerase.
T4 Polynucleotide Kinase
T4 Polynucleotide Kinase catalyzes the transfer of the g-phosphate from ATP to the
5´-terminus of polynucleotides or to mononucleotides bearing a 5´-OH group.
The enzyme is used to end phosphorylate RNA, DNA and synthetic oligonucleotides
prior to subsequent manipulations such as ligation.
The DNA 5´ End-Labeling System is a complete system for phosphorylating both
double- and single-stranded DNA and RNA with T4 Polynucleotide Kinase and [g-32P] ATP.
The system includes enzymes, buffers and control DNA standards to measure reaction efficiencies.
Calf or Chrimp Intestinal Alkaline Phosphatase is included for removal of the 5´-phosphate
prior to labeling with T4 Polynucleotide Kinase
applications
1.
2.
5´ end-labeling of single- or double-stranded DNA and RNA molecules for use as probes,
for sequencing or for DNA-protein footprinting.
2. Phosphorylation of DNA prior to cloning.
1.
An alcaline phosphatase cleave off the 5’ phosphates
5‘P tatctggttgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaacttaagtctctaattcggtacgtacacat P 5‘
+ alcaline phosphatase
5‘
3‘
1.
tatctggttgaattcagagattaagccatgcatgtgta 3‘
atagaccaacttaagtctctaattcggtacgtacacat 5‘
Polynucleotide kinase replaces this phosphate by the labeleed phosphate of
a radioactive phosphate from a radioactive ATP (a gamma phosphate!)
5‘P tatctggttgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaacttaagtctctaattcggtacgtacacat P 5‘
ATP
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookATP.html
Probe labelling
Among the various methods of labelling a probe we will describe
The following ones:
1. Random priming
2. Nick translation
3. Polynucleotid kinase
Random priming
Random primers:
5‘ ctggtt
5‘ gccagt
.........
One labelled nucleotide, t
5‘ctggtt3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘ctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘gccagt 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
5‘gccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
Nick translation
Using DNAase, we made a few number of nick (here in red)
5‘ tatctggttgatcctgccagtattatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
The 5‘ --> 3‘ exonucleasic activity of DNA pol I makes a gap fom a nick
5‘ tatctggttgatc----------tatatgctgaattcagagattaagccatgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
The DNA pol activity of DNA pol I uses the left sequence as a primer
5‘ tatctggttgatcctgccagtattatatgctgaattcag---------tgcatgtgta 3‘
3‘ atagaccaactaggacggtcataatatacgacttaagtctctaattcggtacgtacacat 5‘
Alkaline Phosphatase from the Calf Intestine (CIAP)
Alkaline Phosphatase from the Calf Intestine (CIAP),
catalyzes the hydrolysis of 5´-phosphate groups from DNA, RNA,
and ribo- and deoxyribonucleoside triphosphates.
This enzyme is used
1. to prevent recircularization and religation of linearized cloning vector DN
by removing phosphate groups from both 5´-termini
2. for the dephosphorylation of 5´ phosphorylated ends of DNA or RNA
for subsequent labeling with [32P]ATP and T4 Polynucleotide Kinase.
CIAP is active on 5´ overhangs, 5´ recessed and blunt ends.
Proteinase K
produced by the fungus Tritirachium album Limber, is a serine protease.
It exhibits a very broad cleavage specificity.
It cleaves peptide bonds adjacent to the carboxylic group
of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples .
It has been purified to be free of RNase and Dnase activities.
The stability of Proteinase K in urea and SDS and its ability to digest native proteins
make it useful for a variety of applications, including preparation of chromosomal DNA
for pulsed-field gel electrophoresis (2), protein fingerprinting (3,4) and removal of nucleases
topoisomerase
A class of enzymes that alter the supercoiling of double-stranded DNA.
(In supercoiling the DNA molecule coils up like a telephone cord, which shortens the molecule.)
The topoisomerases act by transiently cutting one or both strands of the DNA.
Topoisomerase type I (EC 5.99.1.2 ) cuts one strand
Topoisomerase type II (EC 5.99.1.3) cuts both strands of the DNA
to relax the coil and extend the DNA molecule..
Aside from topoisomerases I and II, there are more discovered topoisomerases.
Topoisomerase III may regulate recombination while topoisomerase IV regulates
the process of segregating newly replicated chromosomes from one another.
Drugs
Many drugs operate through interference with the topoisomerases.
The broad-spectrum fluoroquinolone antibiotics act by disrupting the function of
bacterial type II topoisomerases.
Some chemotherapy drugs work by interfering with topoisomerases
in cancer cells:
type 1 is inhibited by irinotecan and topotecan
type 2 is inhibited by etoposide and teniposide.
1. DNA Transciption and replication:
The regulation of DNA supercoiling is essential to DNA transcription and replication,
when the DNA helix must unwind to permit the proper function of the enzymatic machinery
involved in these processes.
Topoisomerases serve to maintain both the transcription and replication of DNA.
2. Negative supercoiling
All the naturally occuring double stranded DNAs are negatively supercoiled.
(1) Negative supercoiling is important for packing of long DNA molecules into highly restricted spaces
such as viral cells or chromosomal structures in nucleus because supercoils generate compact structures.
For example:Length of a human chromosome is of the order of centimeters,
the condensed chromosome that contain this DNA only a few nanometers long.
If DNA is constrained to be linear, it would not fit into a cell.
(2) Negative supercoiling facilitates the DNA-strand separation during replication,
transcription and recombination of DNA.
DNA supercoiling is regulated in every cell that influences many aspects of DNA metabolism.
The normal biological functioning of DNA occurs only if it is in the proper topological state.
SUPERHELIX TOPOLOGY
In circular double helical DNA (as in bacteria), both strands are covalently joined to form a circular
duplex molecule. A geometric property of such an assembly is that its number of coils
cannot be changed, without first cleaving at least one of its strands. Two strands are said to be
topologically bonded to each other because they cannot be separated without breaking covalent bonds.
The conformation of circular duplex DNA can be characterized by 3 parameters.
(1)Linking number:L
(2)Twist:T
(3)Writhing number: W
These parameters are related by the equation:
L= T+W (http://education.vsnl.com/kedar/)
(1) L
L is the number of times that one DNA strand winds about the other strand.
L is constant in an intact circular duplex DNA. L can be changed only if a covalent linkage
in DNA backbone is broken and reformed.
(2) T
T is the number of complete revolutions that one strand makes around the duplex axis.
In a particular conformation "T" is positive for right handed duplex turns.
For B-DNA the T equals the number of base pairs divided by 10.5 (10.5 base pairs /helical turn).
(3) W
is the number of turns that duplex axis makes about the superhelix axis in the conformation.
It is the measure of DNA superhelicity.
For a relaxed circular duplex DNA, W = 0 , Therefore L = T. Hence for relaxed duplex DNA L is
simply the number of base pairs /10.5 i. e. one linking number for 10.5 base pairs.
Two ways of introducing one supercoil in a DNA with 10 duplex turns. The two closed circular forms
are topologically equivalent i. e. they are interconvertible without breaking any covalent bonds.
The two DNA conformations have same linking number "L" but differ in their twists and writhing number.
topoisomerases and supercoil
In a "relaxed" double-helical segment of DNA,
the two strands twist around the helical axis once every 10.4 base pairs of sequence.
To add or subtract twists (vueltas), as some enzymes can do, is to impose a strain (tensión).
This strain is positive (adding twists) or negative (substrating twists).
If a DNA segment under twist strain (+or -) were to be closed into a circle by joining its two ends
and then allowed to move freely, the circular DNA would contort into the shape of an 8,
DNA topoisomerases type I (EC 5.99.1.2 )
Type I topoisomerases function by nicking one of the strands of the DNA double helix
twisting it around the other strand, and religating the nicked strand. Topoisomerases I change
the linking number in steps of 1. They pass a single DNA strand through a nick
This process doesn’t need energy.The torque (par de torsión)present in the DNA drives the uncoiling.
Type IA topoisomerases change the linking number of a circular DNA strand by units of strictly 1
Type IB topoisomerases change the linking number by multiples of 1.
-------------------------Question:
How does topoisomerase I and II work ?
DNA topoisomerases type II (EC 5.99.1.3 )
Type II topoisomerases cut both strands of the DNA helix simultaneously.
Topoisomerases II change the linking number (L + T + W) in steps of 2 by passing both strands of
double-stranded DNA through a break.
Once cut, the ends of the DNA are separated, and a second DNA duplex is passed through the break.
Following passage, the cut DNA is resealed.
This reaction allows type II topoisomerases to change the linking number of a DNA loop by +/-2,
and promotes chromosome disentanglement.
For example, DNA gyrase, a type II isomerase observed in E. coli and most other prokaryotes,
introduces negative supercoils and decreases the linking number by 2. Gyrase also is able to
remove knots from the bacterial chromosome.
Get the E. dispar rRNA, DNA sequence Z49256
ncbi: national center for biotechnology informations
nlm: national library of medicine
nih: national institutes od health
gov: US government
Learn how tu use PubMd: http://www.nlm.nih.gov/bsd/disted/pubmed.html
SSU: Small subunit
EDISSSURR (1212896): E. Dipar SSU rRNA
accession number: Z49256
-------------------------search for: buscar
accession: nf adquisición
record (nombre): (aquí) documento, archivo
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
SOURCE
ORGANISM
EDISSSURR
1949 bp
DNA
linear
E.dispar gene for small subunit ribosomal RNA.
INV 01-MAR-1996
Z49256
Z49256.1 GI:1212896
small subunit ribosomal RNA.
Entamoeba dispar
Entamoeba dispar
Eukaryota; Entamoebidae; Entamoeba.
REFERENCE
1 (bases 1 to 1949)
AUTHORS
Novati,S., Sironi,M., Granata,S., Scaglia,M. and Bandi,C.
TITLE
Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba
dispar and the desing of primers for rapid differentiation from
Entamoeba histolytica
JOURNAL
Unpublished
REFERENCE
2 (bases 1 to 1949)
AUTHORS
Bandi,C.
TITLE
Direct Submission
JOURNAL
Submitted (12-MAY-1995) Claudio Bandi, Ist. di Patologia Generale
Veterinaria, Universita', di Milano, Via Celoria 10, Milano, 20133,
Italy
FEATURES
Location/Qualifiers
source
1..1949
/organism="Entamoeba dispar"
/mol_type="genomic DNA"
/db_xref="taxon:46681"
rRNA
1..1949
/product="small subunit ribosomal RNA"
ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
tatctggttg
agtataaaga
tggttagtaa
atttgtatta
gagttaggat
ttctgatcta
gaattggggt
agcaggcgcg
agttgagtaa
ttggagggca
agttgctgtg
ctttanntaa
gtaattgagt
tgaatattcg
ttaaaaggaa
tataagatgc
gttaggggat
aggattggat
atcaatacta
gtatggtcac
tgcggcttaa
cagattaaga
gagtgatttg
tgcctataag
acatttcaat
gtgggaaaaa
tgggccgcac
tattaggcta
ggaaaactca
aggaattcct
acaccgcccg
agatagaaaa
ccgtaggtga
atcctgccag
ccaagtagga
agtacaagga
gtacaaagtg
gccacgacaa
tcaatcagtt
tcgacatcgg
taaattaccc
aatcaattct
agtctggtgc
attaaaacgc
gtgaagtttc
tgttattact
agcatgggac
caattggggt
acgagagcga
cgaagacgat
gaaattcaga
ccttgttcag
aaggctgaaa
tttgactcaa
gttctttcat
tcaggttaat
acagaaatgt
tgtcctattt
gaaaaaggaa
gcgcgctaca
tgtctaatag
aaagaacgta
tgtaatatcg
tcgctcctac
atggatttaa
acctgcggaa
tattatatgc
tgaaactgcg
tagctttgtg
gccaatttat
ttgtagaaca
ggtagtatcg
agagggagct
actttcgaat
tgaaggaatg
cagcagccgc
tcgtagttga
tagaaatgtt
ttgaataaaa
aatgctgagg
gattcagaaa
aagcatttca
cagataccgt
tgtacaaaga
aacttaaaga
cttaaaggaa
cacgggaaaa
gatttattgg
tccggtaacg
tcgcaagaac
taattgttag
gcattcagca
atggagttac
gtagggatag
catgacaggg
agtcattaac
cgattgaata
atctccttat
ggatcatta
tgatgttaga
gacggctcat
aatgataaag
gtaagtaaat
cacagtgttt
aggactacca
ttacagatgg
tgaagaggta
agtaggaggt
ggtaattcca
attaaaatgt
aaattaaaat
taaggtgttt
ggatgtcaat
ataacgggag
ctcaactggg
cgtagtccta
tgaagaaaca
gaaatcttga
ttgacggaag
cttaccaaga
gtagtggtgc
aacgagactg
aggtgcgtaa
ttatctaatt
ataacaggtc
tagagagcat
taagtggtgt
ataaatgatt
tcgagatgaa
aagaggtgaa
ttagaggaag
gattaagcca
tataacagta
ataatacttg
tgagaaatga
aacaagtaac
agattataac
ctaccacttc
gtgacgacac
aaattctcct
gctccaatag
gattttatac
caaagaagga
aaagcaaaac
tagacatttc
aggtgaaaat
tccattaatc
actataaacg
ttgtttctaa
gtttatggac
ggcacaccag
ccgaacagta
atggccgttc
aaacctatta
gtaccacttc
tcgattagaa
tgtgatgccc
tttatcattt
accgagattg
ggaattattt
tacgtccctg
attctaggat
gagaagtcgt
tgcatgtgta
atagtttctt
agacgatcca
cattctaagt
caatgagaat
ggataacgag
taaggaaggc
ataactctag
acgaaatcaa
tgtatattaa
attttgaaga
gacnnttcaa
attatgttaa
gagagaagga
ccatgatcgg
aagaacgaaa
atgtcaacca
atccaagtat
ttcaggggga
gagtggagcc
gaaggaatga
ttagttggtg
attagttttc
ttaaagggac
ctcttttaac
ttagacatct
acaccttatt
aaatagttaa
gttttgaacg
ccctttgtac
tctgtcttat
aacaaggttt
Get the E. histolytica rRNA, DNA sequence X56991
X56991. Reports
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
E.histolytica gen...[gi:9283]
Links
EH16SRRNA
1947 bp
DNA
linear
INV 29-OCT-1992
E.histolytica gene for small subunit ribosomal RNA (16S-like).
X56991
X56991.1 GI:9283
16S ribosomal RNA homologue; ribosomal RNA; small subunit ribosomal
RNA.
SOURCE
Entamoeba histolytica
ORGANISM Entamoeba histolytica
Eukaryota; Entamoebidae; Entamoeba.
REFERENCE
1
AUTHORS
Sogin,M.L., Edman,U., Elwood,H.E. and Agabian,N.
TITLE
Small subunit ribosomal RNA from Entamoeba histolytica
JOURNAL
Nucleic Acids Res.
REFERENCE
2 (bases 1 to 1947)
AUTHORS
Sogin,M.L.
TITLE
Direct Submission
JOURNAL
Submitted (14-DEC-1990) M.L. Sogin, MARINE BIOLOGICAL LAB, CENTER
FOR MOL EVOLUTION, WOODS HOLE MA 02543, U S A
FEATURES
Location/Qualifiers
source
1..1947
/organism="Entamoeba histolytica"
/mol_type="genomic DNA"
/db_xref="taxon:5759"
rRNA
1..1947
/product="small subunit ribosomal RNA (16S-like)
ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
//
tatctggttg
agtataaaga
tggttagtaa
gtttgtatta
agttaggatg
tctgatctat
aattggggtt
gcaggcgcgt
gttgagtaaa
tggagggcaa
gttgctgtga
tttatgtaag
taattgagtt
gaatattcaa
taaaaggaac
ataagatgca
ttaggggatc
ggattggatg
tcaatattac
tatggtcaca
gcggcttaat
agattaagag
agtgatttgt
gcctataaga
catttcaatt
gggaaaaaga
ggccgcacgc
ttaggctttg
aaaactcaaa
gaattccttg
accgcccgtc
atagaaaaat
gtaggtgaac
atcctgccag
ccaagtagga
aatacaagga
gtacaaaatg
ccacgacaat
caatcagttg
cgacatcgga
aaattaccca
atcaattctt
gtctggtgcc
ttaaaacgct
taaagtttct
gttattactt
gcatgggaca
aattggggtg
cgagagcgaa
gaagacgatc
aaattcagat
cttgttcaga
aggctgaaac
ttgactcaac
ttctttcatg
caggttaatt
cagaaatgtt
gtcctatttt
aaaaggaagc
gcgctacaat
tctaataatt
agaacgtaca
taatatcgag
gctcctaccg
ggatttaaat
ctgcggaagg
tattatatgc
tgaaactgcg
tagctttgtg
gccaattcat
tgtagaacac
gtagtatcga
gagggagctt
ctttcgaatt
gaaggaatga
agcagccgcg
cgtagttgaa
agaaatgtta
tgaataaaat
atgctgaggg
attcagaaaa
agcatttcac
agataccgtc
gtacaaagat
acttaaagag
ttaaaggaat
acgggaaaac
atttattggg
ccggtaacga
cgcaagaaca
aattgtagtt
attcagcaat
ggagttacta
aaggatagta
tgacagggat
tcattaactc
attgaataaa
ctccttattt
atcatta
tgatgttaaa
gacggctcat
aatgataaag
tcaatgaatt
acagtgttta
ggactaccaa
tacagatggc
gaagaggtag
gtaggaggta
gtaattccag
ttaaaatgtg
aattaaaatc
aaggtgttta
gatgtcaata
taacgggaga
tcaactgtgt
gtagtcctaa
agagaagcat
aaatcttgag
tgacggaagg
ttaccaagac
tagtggtgca
acgagactga
ggtgcgtaag
atctaatttc
aacaggtctg
gagagtattt
agtggtgtac
aaatgattgg
gagatgaata
gaggtgaaat
agaggaagga
gattaagcca
tataacagta
ataatacttg
gagaaatgac
acaagtaacc
gattataacg
taccacttct
tgacgacaca
aattctccta
ctccaatagt
gttttataca
aaagaaggaa
aagcaaaaca
agacatttcg
ggtgaaaatc
ccattaatca
ctataaacga
tgtttctaga
tttatggact
gcacaccagg
cgaacagtag
tggccgttct
aacctattaa
taccacttct
ggttagacct
tgatgccctt
tatcatttac
cgagattgaa
aattatttgt
cgtccctgcc
tctaggattc
gaagtcgtaa
tgcatgtgta
atagtttctt
agacgatcca
attctaagtg
aatgagaatt
gataacgagg
aaggaaggca
taactctaga
cgaaatcaat
gtatattaaa
ttttgaagac
acaattcaag
ttatgttaat
agagaaggat
catgatcgct
agaacgaaag
tgtcaaccaa
tctgagtata
tcagggggag
agtggagcct
aaggaatgac
tagttggtgg
ttagttttct
taaagggaca
cttttaacgt
agacatcttg
accttattta
atagttaagg
tttgaacgag
ctttgtacac
tgtcttatag
caaggtttcc
blast
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences.
The program compares nucleotide or protein sequences to sequence databases and calculates
the statistical significance of matches.
BLAST can be used to infer functional and evolutionary relationships between sequences
as well as help identify members of gene families.
Query: 1
Sbjct: 1
Query: 61
Sbjct: 61
Query: 121
Sbjct: 121
Query: 181
Sbjct: 181
Query: 241
Sbjct: 240
tatctggttgatcctgccagtattatatgctgatgttagagattaagccatgcatgtgta 60
|||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||
tatctggttgatcctgccagtattatatgctgatgttaaagattaagccatgcatgtgta 60
agtataaagaccaagtaggatgaaactgcggacggctcattataacagtaatagtttctt 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
agtataaagaccaagtaggatgaaactgcggacggctcattataacagtaatagtttctt 120
tggttagtaaagtacaaggatagctttgtgaatgataaagataatacttgagacgatcca 180
||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||
tggttagtaaaatacaaggatagctttgtgaatgataaagataatacttgagacgatcca 180
atttgtattagtacaaagtggccaatttatgtaagtaaattgagaaatgacattctaagt 240
|||||||||||||||| ||||||||| || || | |||||||||||||||||||||||
gtttgtattagtacaaaatggccaattcattcaa-tgaattgagaaatgacattctaagt 239
gagttaggatgccacgacaattgtagaacacacagtgtttaacaagtaaccaatgagaat 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
gagttaggatgccacgacaattgtagaacacacagtgtttaacaagtaaccaatgagaat 299
Query: 961
Sbjct: 960
gttaggggatcgaagacgatcagataccgtcgtagtcctaactataaacgatgtcaacca 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
gttaggggatcgaagacgatcagataccgtcgtagtcctaactataaacgatgtcaacca 1019
Query: 1021 aggattggatgaaattcagatgtacaaagatgaagaaacattgtttctaaatccaagtat 1080
||||||||||||||||||||||||||||||| |||| ||||||||||| ||| |||||
Sbjct: 1020 aggattggatgaaattcagatgtacaaagatagagaagcattgtttctagatctgagtat 1079
Query: 1081 atcaatactaccttgttcagaacttaaagagaaatcttgagtttatggacttcaggggga 1140
||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 1080 atcaatattaccttgttcagaacttaaagagaaatcttgagtttatggacttcaggggga 1139
Blg I site agatct (agatct is a palindrome) is only present in Entamoeba histolytica
DNA strider data.
questions
1. What are the differences between DNA pol and RT-DNA pol ?
2. Cites the activities of E. Coli DNA polymerase I
3. What is the rate of DNA synthesis of the E. Coli DNA polymerase I.
4. What does “proofreading activity” means”?
5. What do “nick translation and Taqman activity” mean”?
6. Why is Pfu DNA pol 10 times less active than Taq DNA pol ?
7. What’ s a palindromic element ?
8. What‘s a SNP within a palindrome.
9. Explain the reaction catalyzed by T4 DNA ligase and ATP.
10. Why is it better for a mammalian vector to have an intron before the insertion site ?
11.explain The „cut and paste“ reaction in molecular biology.
12. Why are there sometimes T4 and SP6 promoter elements at both ends of an insertion site ?
13. What does “DNA dependant” means in “DNA dependant RNA pol” ?
14. Does the RNA pol need a primer ?
15. How does topoisomerase I and II work ?