The School of Biomedical Sciences Wiki - User contributions [en]

Sanger “dideoxy” method

2012-10-26T13:08:54Z

110025641:

The Sanger dideoxy method of DNA sequencing allows large volumes of DNA to be sequenced in vitro. This method uses a single stranded, DNA template strand, a specific primer of 18-20 bases, DNA polymerase , deoxyribonucleoside triphosphates(dNTP’s) and their respective derivatives dideoxyribonucleoside triphosphates (ddNTP’s) which differ from the dNTP’s in that instead of a 3’ OH group they have a 3’ H which is intergral to their function as chain terminators. This is because with the 3’ H prevents chain elongation as phosphodiester bonds cannot be created with the next ribonucleoside (Hames and Hooper, 2000:260)<ref>Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd</ref>

 A DNA primer binds to the 5' end of the single-stranded DNA molecule to be sequenced (the template strand) to allow the DNA polymeras eenzyme to attach and synthesize a new DNA chain against the template strand through Watson-Crick complementary base pairing. There are 4 different dideoxyribonucleoside triphosphate chain-terminating molecules (ddCTP, ddGTP, ddATP, ddTTP) each containing one of the DNA bases, so ddCTP contains the base ‘C’ (Cytosine) etc complemtary to a G on the template strand. These 4 different molecules are used in 4 separate DNA synthesis reactions on copies of the same single-stranded DNA template strand to be sequenced. When a ddNTP is incorporated into the growing newly synthesised chain, it terminates synthesis at that point so each reaction produces fragments of different lengths complementary to the template strand, which have terminated at different points. (Alberts, et al, 2008:549)<ref>Alberts, Bruce, et al, 2008, Molecular Biology of the Cell, 5th edition, New York, Garland Science</ref>

 These DNA fragments are then separated by gel electrophoresis which allows us to determine how long each fragment is in relation to how far through the gel it has travelled by performing autoradiography. The sequence is read bottom to top so the band which travelled furthest through the gel must have been the sequence which terminated at the first base. The band which was second furthest through the gel must have been terminated at the second base in the sequence.ddNTPs can be colour coded for ease of identification. (Hames and Hooper, 2000:261)<ref>Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd</ref> By looking at which ddNTP bound to each of these DNA molecules you can determine the base on the template strand at the position it terminated because of complementary base pairing. 

 

References

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Watson-Crick base pairing

2011-12-02T17:00:59Z

110025641:

= Watson- Crick base pairing  =

=== Background ===

In 1953, James D Watson and Francis Crick discovered the structure of DNA using X ray Crystallography. They worked out that [[DNA|DNA]] was a double helix using Rosalind Franklin's X ray diffraction pattern.<ref>BBC NEWS. Science/Nature Available: http://news.bbc.co.uk/1/hi/sci/tech/2804545.stm
[accessed 2 December 2011]</ref> At first, it was thought that [[DNA|DNA]] was made up of many chemicals, which proved too difficult to analyse, but the researchers persisitence led to the discovery of complementary base pairing.

=== Pairing ===

It has been found that DNA contains four bases namly adenine (A), thymine(T), guanine(G) and cytosine(C). A and T occur in same amounts and C and G occur in same amounts, thus the two possible base pair combinations.<ref>DNA tutorial Available :http://www.dnatutorial.com/BasePairing.shtml [accessed 2 December 2011]</ref>

Watson-Crick base pairing

2011-12-02T16:56:28Z

110025641: Created page with "= Watson- Crick base pairing  = === Background === In 1953, James D Watson and Francis Crick discovered the structure of DNA using X ray Crystallography. They worked out..."

Phosphatase

2011-12-02T16:47:37Z

110025641: Created page with "=  Phospahatase = Phosphatase is a eznyme that removes a phosphate group from an organic molecule. They are important in the metabolism and absorption of carbohydrates, nuc..."

=  Phospahatase =

Phosphatase is a eznyme that removes a phosphate group from an organic molecule. They are important in the metabolism and absorption of carbohydrates, nucleotides and phospholipids.<ref>The Free Dictionary, Available : http://medical-dictionary.thefreedictionary.com/phosphatase [accessed 2 December 2011</ref>

NAD+

2011-12-02T13:20:35Z

110025641: Created page with " NAD stands for nicotinamide adenine dinucluotide. NAD+ is the oxidised form, and NADH is the reduced form which exist in an equilibrium, with NAD+ bei..."

NAD stands for nicotinamide adenine dinucluotide. NAD+ is the oxidised form, and NADH is the reduced form which exist in an equilibrium, with NAD+ being favoured. Oxidation of fuel molecules produces electrons which are used to reduce NAD+ in the reaction of ethanol + [[dehydrogenase|dehydrogenase]] to form ethanal + NADH + H+. Also NADH+ is oxidised to NAD in [[Oxidative_phosphorylation|oxidative phosphoryation]] which is directly linked to [[ATP|ATP]] synthesis. <ref>Alberts et al., 2008, Molecular Biology of the Cell,5th ed.,New York, Garland Science, Taylor and Francis Group p.86</ref>

Adenosine

2011-12-02T13:09:40Z

110025641: Created page with " Adenosine is a nucleoside with three phospahte groups. It is the main constituent of important molecules likeATP, and ADP."

 Adenosine is a [[Nucleoside|nucleoside]] with three phospahte groups. It is the main constituent of important molecules like[[ATP|ATP]], and [[ADP|ADP]].

Condensation reaction

2011-12-02T13:07:37Z

110025641: Created page with " A condensation reaction is a reaction in which a water molecule is formed , usually in bond making, for example, carbohydrates in making glycosidic bonds."

 A condensation reaction is a reaction in which a water molecule is formed , usually in bond making, for example, carbohydrates in making glycosidic bonds.

Gene expression

2011-12-02T13:04:41Z

110025641:

= Gene expression =

[[DNA|DNA]] is transcribed into [[MRNA|mRNA]] which translates into protein. Gene expression uses the information from a [[Gene|gene]] to produce a protein. It is believed that eukaryotes, prokaryotes and archae undergo gene expression, with speculation still going on about viruses.

== Basics about Gene expression ==

Gene activity is mainly controlled at the [[Transcription|transcription]] level. Genes that are always being expressed are siad to undergo constituve expression and those only expressed conditionally undergo regulated expression.<ref>Berg,J.M, Tymoczko,J.L, Stryer,L,2007, Biochemistry, 6th ed.,W.H Freeman</ref>&nbsp;Gene expression control involves the interactions between [[DNA|DNA]], DNA binding proteins and regulatory proteins. Both eukaryotes and prokaryotes use [[RNA polymerase|RNA polymerase]]&nbsp;to initiate [[Transcription|transcription]],&nbsp;which binds to specific binding sites on [[DNA|DNA]]&nbsp;called&nbsp;&nbsp;[[Promoter|promoter sequences&nbsp;]]. Regulatory proteins either repress or activate[[Transcription|transcription]].

 

== Gene expression in Prokaryotes ==

More information is available on how prokaryotes carry out cell reactions, with the model organism being[[Escherichia coli|''E.coli'']]&nbsp;which has its whole genome sequenced,is &nbsp;easy to grow and can be genetically modified. Bacteria use lactose as an energy source when glucose is in limited amounts. Researchers used X-gal, a coloured compund, to observe that in the presence of glucose, a few&nbsp;β-galactosidase molecules were present, whereas with lactose, 1000's of&nbsp;β-galactosidase were presnt. This suggested that levels of enzyme expression changed with changes in environment and his was co-ordinated by an expression unit called an&nbsp;[[Operon|operon]].<ref>Berg,J.M, Tymoczko,J.L, Stryer, L, 2007, Biochemistry, 6th ed., W.H. Freeman p 897</ref>&nbsp;More specifically, the[[The Lac Operon|lac operon]] is made up of a regulator gene which codes for the repressor, an operator site and structural genes z, y and a.

[[Image:Lac operon - basic.JPG]]

'''Transcription'''

[[Helicase|Helicase]] is used to unwind double stranded [[DNA|DNA]] into single stranded DNA, to which [[RNA polymerase|RNA polymerase]] and [[RNTP|rNTPs]] are added.

==== Initiation ====

The RNA polymerase binds onto the promoter which consitutes of a -35 sequence TTGACA, a -10 sequence TATAAT , both upstream of the start site ( TG/ AT). The sequences are known as [[Consensus sequence|consensus sequences]] as they produce maximum efficiency transcription. [[RNA polymerase|RNA polymerase]] is Mg2+ dependent and has a core made of 2&nbsp;α, 1β, 1&nbsp;β'&nbsp;and 1 ω subunits.<ref>Andersen-Lykke,J, Christiansen,J, 1998,Nucleic Acid Research,Oxford Journals, [online] 26 (24) Available at: naroxfordjournals.org/content/26/24/5630.full [accesses 2 December 2011]</ref>&nbsp;The core combines with a&nbsp;σ70&nbsp; to form a [[Holoenzyme|holoenzyme]], with the sigma factor&nbsp;makeing sure that [[RNA polymerase|RNA polymerase]] binds to correct [[DNA|DNA]]&nbsp;sequence.

Gene expression

2011-12-02T13:04:06Z

110025641:

= Gene expression =

[[DNA|DNA]] is transcribed into [[MRNA|mRNA]] which translates into protein. Gene expression uses the information from a [[Gene|gene]] to produce a protein. It is believed that eukaryotes, prokaryotes and archae undergo gene expression, with speculation still going on about viruses.

== Basics about Gene expression ==

Gene activity is mainly controlled at the [[Transcription|transcription]] level. Genes that are always being expressed are siad to undergo constituve expression and those only expressed conditionally undergo regulated expression.<ref>Berg,J.M, Tymoczko,J.L, Stryer,L,2007, Biochemistry, 6th ed.,W.H Freeman</ref>&nbsp;Gene expression control involves the interactions between [[DNA|DNA]], DNA binding proteins and regulatory proteins. Both eukaryotes and prokaryotes use [[RNA polymerase|RNA polymerase]]&nbsp;to initiate [[Transcription|transcription]],&nbsp;which binds to specific binding sites on [[DNA|DNA]]&nbsp;called&nbsp;&nbsp;[[Promoter|promoter sequences&nbsp;]]. Regulatory proteins either repress or activate[[Transcription|transcription]].

 

== Gene expression in Prokaryotes ==

More information is available on how prokaryotes carry out cell reactions, with the model organism being[[Escherichia coli|''E.coli'']]&nbsp;which has its whole genome sequenced,is &nbsp;easy to grow and can be genetically modified. Bacteria use lactose as an energy source when glucose is in limited amounts. Researchers used X-gal, a coloured compund, to observe that in the presence of glucose, a few&nbsp;β-galactosidase molecules were present, whereas with lactose, 1000's of&nbsp;β-galactosidase were presnt. This suggested that levels of enzyme expression changed with changes in environment and his was co-ordinated by an expression unit called an&nbsp;[[Operon|operon]].<ref>Berg,J.M, Tymoczko,J.L, Stryer, L, 2007, Biochemistry, 6th ed., W.H. Freeman p 897</ref>&nbsp;More specifically, the[[The Lac Operon|lac operon]] is made up of a regulator gene which codes for the repressor, an operator site and structural genes z, y and a.

[[Image:Lac operon - basic.JPG]]

'''Transcription'''

[[Helicase|Helicase]] is used to unwind double stranded [[DNA|DNA]] into single stranded DNA, to which [[RNA polymerase|RNA polymerase]] and [[RNTP|rNTPs]] are added.

====== Initiation ======

The RNA polymerase binds onto the promoter which consitutes of a -35 sequence TTGACA, a -10 sequence TATAAT , both upstream of the start site ( TG/ AT). The sequences are known as [[Consensus sequence|consensus sequences]] as they produce maximum efficiency transcription. [[RNA polymerase|RNA polymerase]] is Mg2+ dependent and has a core made of 2&nbsp;α, 1β, 1&nbsp;β'&nbsp;and 1 ω subunits.<ref>Andersen-Lykke,J, Christiansen,J, 1998,Nucleic Acid Research,Oxford Journals, [online] 26 (24) Available at: naroxfordjournals.org/content/26/24/5630.full [accesses 2 December 2011]</ref>&nbsp;The core combines with a&nbsp;σ70&nbsp; to form a [[Holoenzyme|holoenzyme]], with the sigma factor&nbsp;makeing sure that [[RNA polymerase|RNA polymerase]] binds to correct [[DNA|DNA]]&nbsp;sequence.

Enzyme

2011-12-01T20:47:18Z

110025641:

Enzymes <ref>Molecular Biology of the Cell 5th ed (2007) Alberts et.al. 159-169</ref> act as specific [[Catalysts|catalysts]]. That is to say each enzyme accelerates one or more specific chemical reactions without affecting the final [[Equilibrium|equilibrium]] concentrations of reactants and products. In [[Thermodynamics|thermodynamic]] language, enzymes reduce the [[Activation energy|activation energy]] of a reaction but do not affect the [[Free energy|free energy]] change of the overall reaction. Many enzymes are so effective that they will [[Catalyse|catalyse]] intracellular reactions which are too slow to be observed at all under comparable conditions in the absence of enzyme catalysis. Enzymes are often highly specific, both for the [[Molecule|molecules]] they will accept as [[Substrate|substrates]] and for the precise chemical changes that they will catalyse, and the presence of active enzymes is essential to form most of the [[Molecule|molecules]] in the [[Cell|cell]].

Enzyme reactions can be either [[Anabolic|anabolic]] or [[Catabolic|catabolic]] in nature <ref>Nigel P. O. Green (1989). Biological Science. 2nd ed. Cambridge: Cambridge University Press. p.167.</ref>.  

Enzymes and substrates must first interact to form an enzyme-substrate complex before any reaction can occur. This happens through molecular motions where all of the molecules in a cell are constantly moving and colliding; however only a few collisions will result in a reaction. The rate of encounter between the enzyme and the substrate is primarily dependant on the concentration on the substrate; meaning that, to increase the enzyme activity you must increase the substrate concentration. 

=== The mechanism of Enzyme Action: ===

Enzymes increase the rate of reaction between 2 reactants in various possible ways:

*They improve the Proximity of the substrate being that they increase the local concentration of the substrate
*They affect the orientation and hold the [[Atom|atoms]] in positions that favour the reaction
*They produce strain distortion; they put strains on the bonds that are associated with the reaction
*In acid-base catalysis they aid in the exchange of H+ or generation of –OH <ref>Molecular Biology of the Cell 5th ed (2007) Alberts et.al. 159-169</ref> 

=== Specificity ===

Enzymes are specific to the point of being able to distinguish between optical isomers.

The [[Amino acids|amino acids]] forming the active site mainly determine the specificity of the enzyme; a change in only a few [[Amino acids|amino acids]] in this region can result in a large change in the shape of the active site and this could then vastly change to affinity for the substrate or even change the substrate the enzyme is specific for. 

The specificity of enzymes is exhibited in the ‘Lock and Key’ mechanism: 

 

[[Image:Lock and key.jpg]] 

 

Taken from [http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A1031 http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A1031] <ref>http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;part=A1031</ref>

This illustrates the Lock and Key mechanisms and how the shape of the substrate is exactly complememtary to the shape of the active site. However the lock and key model doesn't fully explain enzymatic activity. The model indicates that the enzyme and substrate are unable to change shape.

A modification to the Lock and Key Model of enzymes is the Induced Fit Hypothesis, also known as the "Hand-shake Model" <ref>http://www.portlandpress.com/pp/books/online/glick/searchresdet.cfm?Term=induced-fit%20theory%20%28rack%20model%29</ref>. This hypothesis states that the structure of both the enzyme and substrate can change on binding. In essence the enzyme can wrap itself around the substrate molecule, untill the substrate is completley bound. This produces an enzyme-substrate complex, which places strain on a particular bond, therefore weakening said bond; to a point where it can interact with the enzyme amino acid groups, further non-organic groups or further bound substrates.

The change in the shape of the enzyme is known as a conformational change; the purpose of which is two fold:

#As mentioned above, the conformational change places strain on the desired bond, allowing for a more efficient reaction to take place,
#The new conformation brings amino acid groups essential to the enzyme reaction, which in the unbound conformation may distant from the active site, into the active site. These groups ensure the catalytic reaction will be optimal <ref>The World of the Cell, 3rd Edition, (1996) Becker et.al. p146, p147</ref>. The most common groups to be brought into the Active Site of the Enzyme are those relating to Acid/Base chemistry - therefore promoting the reaction and ensuring optimal conditions <ref>The World of the Cell, 3rd Edition, (1996) Becker et.al. p146</ref>.

=== Substrates and the Active Site ===

Whether an enzymatic reaction will occur is dependant on the substrate colliding and binding to the active site. Once a substrate binds onto the active site, it is held there by a variety of interactions. These interactions take place between charged residual groups of the amino acids in the conformed active site. Hydrogen Bonds and Ionic bonds generally occur - however they are very weak. These weak interactions are of the order of '''3 - 12 kcal mol-1 (12.5 - 50.2 kJ mol''''''-1'''''') '''<ref>Royal Society of Chemistry [RSC]fckLRfckLRhttp://www.rsc.org/ebooks/archive/free/SP9780851869209/SP9780851869209-FP015.pdf</ref> this is of the order of 1/10th the strength of an, on average, single covalent bond <ref>The World of the Cell, 3rd Edition, (1996) Becker et.al. p146</ref>. 

This ensures that enzyme-substrate formation is a reversible process.

=== Allostery ===

An allosteric enzyme couples the effector levels to enzyme activity; it couples signal to functionality. Allosteric enzymes have multiple binding sites (allosteric sites) and show cooperative binding <ref>J.Mol.Biol. (2004) 336, 263-273</ref>.

Allosteric control of enzymes can be positive or negative and can have effects such as up regulate or down regulate activity. 

=== Enzyme Types ===

There are many enzymes used in labs, one of which is the [[Restriction enzyme|restriction enzyme]].

Restriction [[Endonucleases|endonucleases]] are used naturally in a wide range of [[Prokaryotes|prokaryotes]] as a self-defence mechanism against foreign [[DNA|DNA]] [[Molecule|molecules]].  The prokaryotes own [[DNA|DNA]] is methylated so it will not be cut by the enzyme.They recognise a specific 4-8 base pair [[Palindromic sequence|palindromic sequence]] and by carrying out a hydrolysis reaction cut at that specific point. They may cut to form a blunt end or a sticky end. A blunt end is when the enzyme cut the [[DNA|DNA]] symmetrically. Asymmetrical cleavage leaves sticky end, these are unpaired bases. These sticky end can anneal to complementary bases on another strand <ref>Berg J., Tymoczko J and Stryer L. (2007) Biochemistry, 6th edition, New York: WH Freeman</ref>. 

=== Kinetics ===

Kinetic parameters:

Two important enzyme parameters in a simple enzyme catalysed reaction are the [[Michaelis-Menten constant|Michaelis-Menten constant]] ([[Michaelis-Menten constant|K]][[Michaelis-Menten constant|m]]) and the [[Maximum reaction|maximum reaction]] velocity ([[Maximum reaction|V]][[Maximum reaction|max]])

*Km is the approximate measure of the enzyme affinity for the substrate. This can be calculated from the graph as ½ Vmax. Generally a lower Km value signifies a higher affinity for the substrate.
*Kd is the dissociation constant for substrate binding to enzyme
*Kcat is the turnover number for the enzyme
*Vmax is the maximal activity of the enzyme when all of the active sites are saturated.

The michaelis-menten equation:

 V = Vmax [S]/Km [S]

 To obtain Vmax and Km the enzyme activity must be recorded and then plotted on a double reciprocal plot, a [[Lineweaver-Burk plot|Lineweaver-Burk plot]], and the Michaelis Menten equation is then rearranged to look like this: 1/V = (Km/Vmax)(1/S)+1/Vmax <ref>Molecular Biology of the Cell 5th ed (2007) Alberts et.al. 159-169</ref>.

[[Image:350px-Lineweaver-Burke plot svg.png]] 

Taken from [http://www.search.com/reference/Lineweaver-Burk_plot www.search.com/reference/Lineweaver-Burk_plot] <ref>http://www.search.com/reference/Lineweaver-Burk_plot</ref> A Lineweaver-Burk plot showing all the necessary parameters. 

=== Inhibition ===

Enzymes can be inhibited by denaturing which is when a protein is changed in structure to form a randomly coiled peptide which exhibits none of its usual functions. Denaturing can result from extreme temperatures and [[PH|pHs]] as these alter the bonding in the molecule.

Inhibition can also be initiated by the binding of specific molecules called inhibitors. These can be split into categories:

#Irreversible Inhibitors are molecules that permanently bind to the enzyme's active site or specific side chain, commonly to the serine (CH2OH) or cysteine (CH2SH) by [[Covalent|covalent]] bonds. Thsi inactivates the enzyme so the substrate cannt bind.
#Competitive Inhibitors are competing molecules that will have a very similar structure to that of the natural substrate and thus will be complementary to the enzyme active site. Vmax stays the same, but Km increases This type of inhibition can be overcome by an increase in substrate concentration.
#Non-competitive inhibitors bind to a region on the enzyme other than the active site, causing changes to enzyme shape resulting in disruption of the active site. This decreases the turnover number of the enzyme rather than preventing substrate binding- Vmax decreases but Km stays the same. This cannot be overcome with an increase in substrate concentration.
#Uncompetitive inhibitors only bind to an enzyme-substrate complex; so both Km and Vmax decreaae as it takes longer for the substrate to leave the active site. This also cannot be overcome by an increase in substrate concentration. We are able to distinguish the types of inhibition occurring by looking at the graph of enzyme activity <ref>Biochemistry 6th (2006) Stryer et.al. pg51</ref>.

[[Image:Inhibitor graph.jpg]] Taken from [http://ibhow.files.wordpress.com/2010/06/7-6-4.jpg?w=399&h=275 http://ibhow.files.wordpress.com/2010/06/7-6-4.jpg?w=399&h=275] <ref>http://ibhow.files.wordpress.com/2010/06/7-6-4.jpg?w=399&amp;amp;amp;amp;amp;amp;amp;amp;amp;h=275</ref>

Competative Inhibitors show the same Vmax value however we see an increased Km value and non-competitive inhibitors show a decreased Vmax but the same Km. 

=== Targets for drug action ===

We are able to exploit these mechanisms of inhibition to engineer drugs with therapeutic effects. By targeting an enzyme in drug therapy we have the ability to change whole metabolic reactions that are catalysed by that particular enzyme. We can investigate possible new drugs by exploring drug-reaction interactions and drug-pathway interactions <ref>BMC Bioinformatics 2010, 11:501</ref>.  Some examples of Drug Inhibitors:

*Irreversible drug inhibitors: [[Penicillin|Penicillin]] the antibiotic which inhibits transpeptidate in bacteria rendering them unable to synthesise cell walls. Aspirin which decreases the inflammatory response by inhibiting Cyclooxegenase.
*[[Competitive inhibitors|Competitive inhibitors]]: Methotrexate which inhibits dihydrofolate reductase which is involved in the synthesis of [[Purine|purines]] and [[Pyrimidine|pyrimidines]].
*[[Non-competative inhibitor|Non-competitive inhibitor]]: NNRT1 in the treatment of HIV <ref>Biochemistry 6th (2006) Stryer et al.</ref>. 

=== References ===

<references />