Sunday, 16 th Jun 2012 Molecular Biology Lecture (5)
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- Myron Lewis
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1 ** These notes include extra information the doctor had mentioned in the lecture.. Related slides are slides # from the first set of slides,, and #1-40 from the next set.. Please make sure to refer to the slides -Slide #148 : The cell cycle is a process through which the cell undergoes a full turn of growth metabolism, replication and finally division into two daughter cells,, this cycle is divided into 4 phases : 1- G1 phase: For rapid growth and of cells for DNA replication. 2- S phase: For DNA synthesis. 3- G2 phase: More growth of the cell preparing it for cell division. 4- M (Mitosis) phase: Cell is divided into two identical daughter cells. *** Stem cells keep cycling between these four phases over and over, whereas differentiated cells (and sometimes Stem cells) enter another phase through their cell cycle which is the G0 phase, where they set there doing nothing. Cells don t grow anymore in the G0 phase, instead they differentiate into another type of cells. -Slide #149 : Cell cycle is a highly regulated process; many checkpoints are there. Each checkpoint allows the cell to stop and check for specific things to make sure it is ready to go through the next step. 1- G1 checkpoint: is the checkpoint where the cell sets, before going through the S phase, checking for: Cell size, nutrients, growth factors and DNA damage. 2- S checkpoint: Before going into the G2 phase, cell SCANS the DNA it synthesized to make sure it is clear of any mistakes. 3- G2 checkpoint: cell checks for its size and for DNA being replicated or not. 4- M checkpoint: the cell checks for the efficiency of the available machinery for cell division. -Slide #150 : - Those checkpoints are regulated by what so-called CYCLINS, cyclins also regulate the activation of other proteins (Kinases) known as Cyclin-dependent kinases (CDKs). Cyclins bind to these kinases and activate them.
2 - These regulatory protein are called cyclins because they CYCLE in there levels! Meaning they are expressed in high amounts, and are then degraded - Notice that Cyclin D is continuously expressed in ALL phases, but it eventually degraded! - Cyclin B is important for M phase, Cyclin A regulates G2 phase and Cyclin E is necessary for the regulation of the transitional phase between G1 phase and S phase.. Sometimes it is said that cyclin E regulates the S phase only. ** Whereas Cyclin D shares in the regulation of the FOUR phases,, each one of the other 3 cyclins ( B,A & E ) is responsible for the regulation of only a SINGLE phase,, reason why? because each one of them is synthesized and degraded within the same phase. -Slide #151 : This figure illustrates the exact allocation of the cyclins and CDKs to the different phases of cell cycle,, Where : - Cyclin D activates CDK 4 & 6. - Cyclin E activates CDK 2, remember it regulates the G1-S transitional phase. - Cyclin A also activates ADK 2. It regulates the G2 phase. - Cyclin B activates CDK1, and regulates the M phase. - P16 & P27 are inhibitory proteins ( P stands for Protein, and the number indicates the molecular weight of that protein). P16 inhibits CDK 4 &6, whereas P27 inhibits CDK2. -Slide #152 : - In the figure,, arrows = activation,, blocked lines = inhibition. -E2F: Elongation Factor 2, is a protein responsible for translation of genes during protein synthesis. If E2F is not active, then the cell can NOT synthesize proteins, and the cell cycle stops. - E2F is regulated by Retinoblastoma protein; RB binds and inhibits the E2F, thus inhibiting the whole cell cycle, i.e. E2F Rb complex is essential for the activation of the cell cycle.
3 - PRb = Phosphorylated Retinoblastoma protein. It is phosphorylated by CDK2. - Once Rb is phosphorylated, it detaches from E2F leaving it FREE and continuously ACTIVE. - Some viruses can cause cancers by the OVER-activation of E2F caused after the continuous inhibition of the Rb. Rb Is inhibited by a viral protein called E7. E7 binds Rb, leaving a continuously active E2F protein (and so the cell cycle will not stop!). - For any CDK to be active, it must form a complex with its activator-cyclin, for example: Cyclin D forms a complex with CDK4 or 6. This complex (cyclind-cdk4&6) has two regulators: 1- It is inhibited by P16, a protein that is activated by signals of senescence (cell death) and differentiation. 2- It is activated by mitogens, growth factors, hormones or any other signal that causes cells division. ** Another VERY important tumor suppressor gene, P53, is a molecule that senses DNA damage, activates P21 (a protein that inhibits the formation of CyclinE-CDK2 complex, thus inhibits phosphorylation of the Rb protein keeping the E2F inactive and ultimately the cell cycle stops! - This P53 also helps the cell to decide whether to fix the DNA damage and continue the cell cycle, OR to induce cell death. Active P53 active P21 inactive CyclinE-CDK2 complex NO Rb phosphorylation ( E2F is inactive) cell cycle STOPS! Inactive P53 inactive P21 active CyclinE-CDK2 complex Rb is phosphorylated (E2F is active) cell cycle GOES ON! So this is the story of cell cycle! DNA mutations Moving on to the second set of slides, we will start talking about DNA mutations. There is nothing mentioned about this topic in the book, so you can go back to the reference mentioned in Slide #1.
4 -Slide #2 : - Micromutaions: occur at the level of a DNA molecule, and can NOT be seen under the microscope. - Macromutations: at the level of the chromosome,, this part will be explained by Pro. Mohammad AlKhatib insha a Allah. -Slide #3-4 : ** The first line (in black) amino acids sequence, notice that it begins with Phenylalanine amino acid. The second line (in red) mrna sequence. The third line (in blue) DNA sequence (codones). Now,, in a missense mutation, ONE nucleotide of ONE codon is changed, leading to a difference in the amino acids sequence in ONA a.a only (changing of T into A yeilds Leucine instead of Phenylalanine). - A nonsense mutation yields an EARLY stop codon, so as when the DNA is translated, it will give a truncated (shorter-than-normal) protein. This codon originates due to a micromutation in one nucleotide in the DNA sequence. This mutation has a significant effect over the to-be-synthesized protein only if it originates early in the DNA sequences! But if this is not the case, meaning the stop codon originates at the end of the gene, few residues before the original stop codon, then no significant effect will be demonstrated, and so the mutation is negligible!. - Frameshift mutation: is of two types: addition or deletion of nucleotides. Each DNA sequence has its own frame of codons, so any addition or deletion of one or two nucleotides will lead to a shift in this frame changing the amino acid sequence starting from the point of mutation and on (downstream). *What if you add THREE nucleotides to the DNA frame?! This is actually an addition of a full new amino acid. Sometimes it is harmful, sometimes it is not.
5 -Slide #5 : -Translocation a sequence of DNA goes from one site to another. - Insertions and duplications when you add new nucleotides to the sequence, increasing the original number of nucleotides. - Inversion Flipping of a DNA sequence. * These mutations usually occur at the level of the chromosome. -Slide #6-10: Errors in DNA replication arise when the DNA polymerase mispairs two nucleotides through its polymerization journey. Without its own proof reading mechanism, DNA polymerase can result in an inaccurate pair of nucleotides after every 10,000 nucleotides it pairs. BUT with the help of its proof reading machinery, it causes only one mispairing mistake for each 1000,000,000 base pairs it polymerizes! This inaccurate pairing is a result of an abnormal (not optimal) hydrogen bonding between nitrogenous bases of these nucleotides. ** Abnormal base pairs are called Tautomers. -Slide #11 : - In this example,, After the first replication of the DNA, the G is mistakenly read as an A in one daughter cell, and so, a T will be added (A-T). The other daughter cell has a normal DNA sequence identical to the parental one. - In the second replication, the normal daughter will give two normal cells, while the one with the A-T tautomer will give one normal cell that results from the normal pairing of G with C, and a MUTANT cell resulting from the pairing of an A to the T that was mistakenly added in the first replication. This mutation is called a TRANSITIN mutation as we changed a purine (G) with a purine(a). ** A Transversion mutation results from the mispairing of a purine with a pyrimidine, or vice versa.
6 Remember that the normal pair contains a purine & pyrimidine, so if this mutation hits this normal pair, substituting a purine for a pyrimidine, for example, then this pair is now abnormally composed of two purines!! -Slide #13-15 : These DNA errors happen when the DNA polymerase comes across a repeated sequence of DNA, where: - It may skip some of these repeats. If the skipped repeats are few in number, then it is a frameshift DNA error. But if MORE than few base pairs are skipped, then it is a deletion error! - OR it may duplicate the number of the repeats, or may roughly increase it (not necessarily by a double!),, and then it is a Duplication error. -Slide #16-19 : 1- Depurination: the DNA polymerase recognizes the depurinated nucleotide as a nucleotide missing its base. Here the polymerase either randomly gives that nucleotide any nitrogenous base to fill in the gape, OR it skips that nucleotide yielding a frameshift addition or deletion!!! sorry I couldn t get this point, so I just wrote it literally as the doctor said. 2- Deamination: results when Cytosine is deaminated to its precursor Uracil, meaning that the cytosine s amino group is converted into a ketone group. As Uracil is not normally present in DNA, the DNA polymerase will read it as a Thymine! So, the final result will be the conversion of a C into a T, and a G into an A on the opposite strand Is this a transition or a transversion mutation??! Of course it is a TRANSITION mutation, as you substituted a purine for a purie (G A), and a pyrimidine for a pyrimidine (C T). -Slide #20 : - methylation is a regulation process of genes. A gene that has a methylated Cytosine is suppressed inactivated. - If a mythelated Cytosine undergoes deamination, then it will become a Thymine (NOT Uracil). And again, it is a transition mutation! C T,, G A.
7 -Slide #21-22: - ROS can cause DNA damage by donating their extra electron to the DNA molecule. - 8-oxodG is a product of oxidative damage to a G! It is not normally recognized by the DNA polymerase.. so the polymerase reads it as a T and then mispairs it with an A resulting in a G T transversion mutation. (Purine Pyrimidine) Induced mutations : -Slide #23-28 : - Induced mutations are caused by the effect of environmental factors, food, Radiations..etc - Mutagen: is a chemical compound that can cause a mutation in the DNA! - Only SOME mutagens have carcinogenic potential, meaning that EVERY carcinogen is a mutagen, But NOT the opposite! - Base analogs look like nucleotides,, and so can be incorporated in the DNA instead of the normal nucleotides. - NORMAL 5-Bromouracil (5-BU) is recognized by the DNA polymerase as a T, so it is mispaired with an A. If 5-BU is IONIZED, then it is read as a C, and so it will be mispaired with a G! And this is a transition mutations. - Ethyl Guanine an example of a transition mutation because T mispairs with G instead of A..! -Slide #29 : Intercalating agents are flat and cyclic,, e.g. Acridine & Ethidium bromide (a DNA stain). As it is flat, an intercalating agent can intercalate between nucleotides increasing the original space between them. So, when the DNA polymerase reaches this space it either: 1- Skips it and continue reading the following nucleotides. 2- Skips it and skips the following nucleotides. 3- Tries to add new nucleotides to fill that space. This mutation is considered a Frameshift mutation, either deletion OR addition.
8 -Slide #30-32 : - AP sites A-Purenic OR A-Pyrimidinic nucleotides (A = Non). So an AP site is a nucleotide that lacks its nitrogenous base. -Slide #33-36: *What is different about Ames mutant strain of salmonella?! 1- Normal salmonella grows in the absence of Histidine, but his salmonella CANNOT! Ames induced a mutation in the enzyme that is responsible for synthesis of histidine in salmonella,, so the mutant strain cannot synthesize histidine by itself. 2- It can NOT repair any DNA mutation, and as a result, DNA mutations will accumulate!, and so this salmonella has a very high mutation rate! 3- Lacks the normal protective LPS layer, so any chemical you add to this bacteria will pass easily into the cell. * Ames used the reversion mutation in order to enhance the growth of his salmonella in Petri dishes... Without this mutation, Salmonella won t grow, as it lacks the enzyme essential for histidine synthesis. -Slide #37-38 : ** We can decide if a certain chemical is a mutagen only if it increases number of Salmonella colonies by double compared to the number of colonies before adding that chemical..! - Numbers in boxes refer to number of Salmonella colonies after the addition of the corresponding chemical (chemical-to-be-tested) - 10 colonies in water this is the normal number of colonies in the absence of any chemical,, so to consider a chemical is mutagenic, it must DOUBLE that number,, meaning it must induce the growth of 20 colonies or more, so let s see 1- Motor oil 50 colonies > 20,, then it is a mutagen! 2- Alcohol 43 > 20,, it is a mutagen too. 3- Drug X 9 < 29,, it is NOT a mutagen.!! mutagenic 200 >>> 20,, it is HIGHLY شيبس أبو 5 قروش 4-
9 -Slide #38- : - Liver enzymes are normally responsible for the DETOXIFICATION of harmful chemicals. In some cases, this action is inversed!,, meaning that these enzymes can create a toxic or mutagenic compound from an originally harmful substance! - Ames incorporated liver enzymes in his experiment, and the results (numbers of grown colonies) are illustrated in the table in slide #39 before(-) and after(+) the addition of liver enzymes where : 1- Water number of colonies increased from 10 to 12 after the addition of liver enzymes, it is not that much increase,, but notice that you have now to compare all your new results (+ liver enzymes) to the new number (12), meaning that a mutagen is the chemical that causes the growth of (24) colonies and more! 2- Motor oil 22 <24,, it was mutagenic before the addition of liver enzymes, but now it is NOT! What does that mean?!! It means that the mutagenicity of motor oil had diminished after the addition of liver enzymes, because these enzymes had detoxified this initially-mutagenic compound to a harmless one. 3- Alcohol 50 >24,, still it is mutagenic. 4- Drug X 35 > 24!!, it was NOT mutagenic, but by the effect of liver enzymes it became a mutagen!! شيبس 500>>>>24, still it is a mutagen,, notice that its mutagenicity is أبو 5 قروش 5- very highly increased!!! Lna Malkawi
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