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Chapter 3- Mutation and environment.pdf

  1. Chapter 3 Genetic Polymorphism T. Abed Al Kareem Noureddine 1
  2. Activity 1: Mutations and Environment • What are the different types of mutations? • Chromosomal mutation (Chapter 5) • Gene mutation T. Abed Al Kareem Noureddine 2
  3. Gene mutation: • Definition: is the accidental changes of DNA (nucleotide sequence) that may lead or not to change in phenotype. Note: new allele is called mutant allele. • Causes: 1. Spontaneous mutation: due to error in natural biological process such as DNA replication 2. Induced mutation: due to environmental factors that causes mutations (called mutagens) UV radiation- polycyclic aromatic hydrocarbons (PAH)- free radicals- smoking.. T. Abed Al Kareem Noureddine 3
  4. Transmission and impact of mutation • Are all mutations could be transmitted to off springs? The transmission of a mutation depends on the type of cell where mutation had occurred. Somatic cells: mutation can not be transmitted to off springs Germline sex cells: mutation can be transmitted to off springs. • Why mutations in somatic cells may be significant although they are not transmitted to offspring? Mutations in somatic cells may develop cancers such as cancer in respiratory system (due to PAH), skin cancer due to UV radiations… T. Abed Al Kareem Noureddine 4
  5. Consequence of mutation • Consequence of mutation: Are all mutations harmful? Mutation can be: 1. Harmful: lead to new disease. Example: thalassemia 2. Favorable: mutations lead to new allele that results in new favorable phenotype. This cause diversity of living things (example dark moth) 3. Silent: change in DNA that has no change in phenotype T. Abed Al Kareem Noureddine 5
  6. Introduction to Genetic Polymorphism Introduction: • Definition: Genetic polymorphism is presence of 2 or more variants in a certain sequence of DNA. • Cause: Genetic polymorphism is due to mutation. • Note: Genetic polymorphism could be in coding regions of DNA (genes) or non-coding regions (document 1) • Coding regions of DNA: is portion of DNA that codes for protein • Non-coding region: portion of DNA that does not code for protein (example: telomere, centromere, introns in the gene, satellites...) • Note that terms coding and non coding regions of DNA different than terms coding and coding strand of DNA T. Abed Al Kareem Noureddine 6
  7. Document 2: Mutations and multiple alleles Revision: • Relation between gene and phenotype: DNA (gene) → protein → phenotype normal allele → normal protein → normal phenotype Mutant allele → altered protein → altered phenotype T. Abed Al Kareem Noureddine 7
  8. DNA protein synthesis G A A T T T A T G C C C G C T T A A A T A C G G G C Triplet Double Stranded DNA • Transcribed Strand • Template • Non-coding • Used in protein synthesis • Non- Transcribed Strand • Non- Template • coding • Not used in protein synthesis C U U A A A U A C G G G C mRNA Codon • Complementary to transcribed • Identical to non transcribed but T replaced with U Transcription Translation Polypeptide Leu__ lys __ tyr __ Gly Note: when writing an aminoacid sequence of polypeptide don’t forget to draw the peptide bond between each 2 consecutive a.a T. Abed Al Kareem Noureddine 8
  9. Remarks regarding protein synthesis • Remarks: • RNA contains uracil (U) instead of thymine (T) • Stop codons: UAA /UAU/ UAG where translation stops • Change in DNA leads to change in mRNA that leads to change in amino acid sequence and thus change in 3d structure of protein leading to a new phenotype. T. Abed Al Kareem Noureddine 9
  10. Normal DNA of gene Normal mRNA Normal protein (functional) Normal phenotype mutant DNA of gene mutant mRNA altered protein (functional) abnormal phenotype mutation Diagram showing relation between gene and phenotype in normal and mutated gene T. Abed Al Kareem Noureddine 10
  11. Types of genetic mutation G A A T T C C T T A A G G A T T C C T A A G G A A T T C C T T A A G G A G A T T C C T C A A A G G A A T T C C T T A A G G A C T T C C T G A A G Deletion addition substitution T. Abed Al Kareem Noureddine 11
  12. Consequences of different types of mutations • Substitution mutations are point mutations that affect one nucleotide thus may lead or not to a change in the sequence of 1 amino acid. • Addition (insertion) or deletion mutations are frameshift mutations disturb the DNA reading frame, so it changes the whole sequence after the mutation T. Abed Al Kareem Noureddine 12
  13. Consequences of different types of mutations Type of mutation Normal Mutant Consequence DNA producing aminoacid DNA producing aminoacid Substitution CCA GAG ACT CCA GTG ACT Missnese mutation Pro __ Glu__Thr Pro __val__Thr CCA GAG ACT CCA GAC ACT Silent mutation Pro __ Glu__Thr Pro __ Glu__Thr CCA GAG ACT CCA TAG ACT Non-sense mutation Pro __ Glu__Thr Pro stop deletion TAC ACC ACG A.. TAC CCA CGA.. Frame shift mutation Tyr __ Glu __ Thr Tyr__pro_Arg insertion TAC ACC ACG A.. TAC GAC CAC GA.. Frame shift mutation Tyr __ Glu __ Thr Tyr__ Asp__ His T. Abed Al Kareem Noureddine 13
  14. Note: If you were asked to explain how the change of nucleotides leads to certain disease presented in text. Your answer should include: 1. site and type of mutation 2. Consequence of mutation (at level of mRNA and at level of polypeptide) 3. The effect of change in the amino acid sequence in polypeptide (causes a change in the 3d structure of protein ( or in case of non- sense mutation: a truncated protein is resulted) that becomes inactive 4. Go back to text: indicate the function of protein and relate the effect of inactivation of protein to the disease T. Abed Al Kareem Noureddine 14
  15. T. Abed Al Kareem Noureddine 15 2013 2nd- exercise 2
  16. Answer: We establish the mRNA sequence by replacing T by U • Normal mRNA: UAU ACC CCC GAA CCU GAC AUC • Amino acids sequence : Tyr-Thr-Pro-Glu-Pro-Asp-Ile • Diseased m RNA: UAU ACC CCC AAA CCU GAC AUC • Amino acids sequence : Tyr-Thr-Pro-Lys-Pro-Asp-Ile 2. The mutation by substitution at the level of the first nucleotide of the 280th codon of the DNA where G is replaced by A is transcribed at the level of mRNA by a new codon which is translated into a new amino acid, lysine instead of the glutamic acid. This new amino acid sequence affects the tridimensional structure of the enzyme PAH which becomes inactive (nonfunctional). Since this enzyme is responsible for the transformation of phenylalanine into tyrosine. This transformation doesn’t occur any more leading thus to the accumulation of phenylalanine which in high amount becomes toxic and causes phenylketonuria. T. Abed Al Kareem Noureddine 16
  17. Genes and multiple alleles • An example of multiple alleles in humans is ABO blood group. • 3 different molecules (A, B, o). • Each molecule is made up of: substance H with presence or absence different type of sugar. • Antigen A: substance H + N-acetyl galactosamine • Antigen B: substance H + galactose • Antigen o: substance H • The type of sugar motif added depends on the type of transferase enzyme present. • Different enzymes are due to presence of different alleles as result of different types of mutations occurred from wild type allele T. Abed Al Kareem Noureddine 17
  18. Draw out the definition of genetic polymorphism and its possible causes. • Gene is polymorphic when it has more than 2 alleles. The diversity of alleles are due to mutations. T. Abed Al Kareem Noureddine 18
  19. Document 3: Polymorphic genes in population • Define polymorphic genes: Gene is said to be polymorphic when it has more than 2 alleles of frequency (>1%) • Examples of polymorphic genes • Blood group • Major Histocompatibility Complex • Beta-globin gene T. Abed Al Kareem Noureddine 19
  20. Major Histocompatibility complex MHC Structure and Function: • MHC is also called HLA (Human Leukocyte Antigen) because these proteins were initially described on surface of leukocytes • Structure: large glycoprotein carried by all nucleated cells. • Function: • Determines graft acceptance and rejection • Other function will be discussed in Immune System T. Abed Al Kareem Noureddine 20
  21. Major Histocompatibility complex MHC Transmission: • MHC is coded by 6 genes (haplotype) carried by chromosome 6. • Each gene has many alleles and alleles of each gene are codominant. • That’s why these gene are highly polymorphic • Haplotype: set of genes very close to each other that are inherited as one block T. Abed Al Kareem Noureddine 21
  22. • Define a wild type allele. A wild type allele is the allele that codes for the most common phenotype. No- 1 p: 63 Every individual has 6 genes that are involved in coding for MHC (A B C DP DQ DR) existing in 2 copies (one of paternal origin and one of maternal origin). Each gene has many different alleles and all are codominant. This makes it practically impossible for 2 indviduals to have exact combination of the all 6 HLA loci. On the other hand identical twins have same genetic information so have the same alleles at each of the loci in the MHC complex T. Abed Al Kareem Noureddine 22
  23. Beta globin gene • Hemoglobin contains 2 kinds of polypeptides • 2 alpha • 2 beta • Beta globin gene is coded by gene that has 150 alleles • Most of them are normal • Few of them codes for abnormal beta globin leading to disease. • Solve number 3 and 4 p: 63 T. Abed Al Kareem Noureddine 23
  24. 3) polymorphic gene of B- globin gene is due to the presence of diverse alleles in the human population. These alleles are the results of different types of mutation insertion, deletion, or substitution of DNA nucleotide. 4)The severity of B- thalassemia depends on the site, type and extent of mutation of beta globin gene. In general, substitution mutations are less severe than deletion or insertion, especially if the substitution leads to an amino acid characteristically similar to that of the original amino acid (example lysine and arginine). However deletion of a long stretch of beta globin gene leads to severe thalassemia. Moreover, one must note that the severity of disease depends whether the individual carries one or 2 mutant alleles T. Abed Al Kareem Noureddine 24
  25. Document 4: Detection of Genetic Polymorphism • Introduction: • As defined in previous document, genetic polymorphism is the presence of 2 or more alleles of the same gene. • And we’ve seen that the origin of different alleles is mutation and thus having different sequences of nucleotides in a given locus • So how can we detect the existence of change in nucleotide sequence? T. Abed Al Kareem Noureddine 25
  26. Detection of Genetic polymorphism • Genetic polymorphism could be assessed by: 1. Study phenotypic variations: (for genetic polymorphism at level of coding regions) (ex: presence of different blood groups shows that there are mutations at level of gene responsible of blood group) 2. Restriction Fragment Length Polymorphism (RFLP) that could detect genetic polymorphism at level coding or non-coding region T. Abed Al Kareem Noureddine 26
  27. Principle of RFLP technique: • A mutation in certain site leads to creation or deletion restriction site of specific restriction enzyme leading to variation in number and size of fragments of obtained by restriction enzyme. The obtained fragments are separated according to size using gel electrophoresis In certain cases, southern blot is done using radioactive probe to observe only specific part of DNA fragments. • These will be explained in details in the following slides. T. Abed Al Kareem Noureddine 27
  28. Restriction Enzymes • Restriction enzymes: • Bacterial enzymes • Recognize specific nucleotide sequence (recognition site) • Cuts a specific location in it (cleavage site) • Different enzymes may have different recognition and cleavage sites. T. Abed Al Kareem Noureddine 28
  29. • Example: Restriction Enzymes T. Abed Al Kareem Noureddine 29 1. Determine the number and the length of the restriction fragments obtained as a result of cutting allele A by Hae III enzyme. ( Given Hae III restriction site doc. 2) 2. Determine the consequence of mutation on the produced restriction fragment upon using Hae II enzyme on allele 2 Document 1 Document 2
  30. Answer: 1. Allele A has 2 restriction sites of enzyme Hae III where it will cuts after nucleotides 198 and 240. Therefore, the enzyme cuts the allele into 3 fragments. The length of each fragment is: • Fragment 1 of 198 base pairs (bp) (before the site 198), • Fragment 2: 240-198= 42 bp • Fragment 3: 470-240 = 230 bp 2. Hae III enzyme cuts the DNA when encountering the sequence GGCC. The cutting is done between GG and CC (document 2). Document 3 shows that the restriction site at the level of the nucleotide 240 does no longer exist for allele B due to the mutation by substitution. Instead of the GGCC sequence for allele A there is a GGCT sequence for allele B. As a result, the enzymatic treatment of allele B will give 2 fragments instead of 3: • a fragment of 198 base pairs (bp) (before the site 198), • a fragment of 272 bp instead of the two fragments (42 and 230 bp) T. Abed Al Kareem Noureddine 30
  31. The restriction process and the obtained fragments are obtained in a test tube, so how can we detect these fragments? Gel Electrophoresis T. Abed Al Kareem Noureddine 31
  32. Gel Electrophoresis • How can we detect these fragments? ➢By Gel Electrophoresis • Principle: • DNA is –ve charged due to phosphate group • During electrophoresis bands migrate from –ve pole to +ve pole • Migration depends on size: smaller fragments migrates further • Role of Ethidium Bromide (Et-Br) ➢Dye that binds to DNA and fluoresces under UV light. • Restriction map: is the generated pattern of bands • Note: The enzyme that gives 2 different restriction maps of the 2 alleles is able to detect the genetic polymorphism - + migration T. Abed Al Kareem Noureddine 32
  33. • Application 1: Given the following sequences of 2 different alleles of same gene Allele N:……GGCCTGAATTCGT…..TGACGGCCT..A Allele d:……GGCCTGAATTCGT…..TGACGGGCT..A 1 147 513 1 147 513 T. Abed Al Kareem Noureddine 33 600 600 Document 2 1) By referring to the table of restriction enzymes (in previous slides) , compare the action of Hae III and Eco RI on the 2 different alleles (doc. 1). 2) Deduce the enzyme that is able to detect the genetic polymorphism in this gene. 3) Given the following results of electrophoresis of 3 individuals A, B and C under the effect of Hae III enzyme (document 2). Specify the genotype of each individual. Document 1 For Teacher: Read the notes in slide notes!
  34. • Answer: 1. Hae III has 2 restriction sites (at nucleotides 148 and 518) on allele 1 and gives 3 restriction fragments more than that of allele 2 that has 1 restriction site (148) giving only 2 fragments. One of fragments obtained from allele 1 is equal to that obtained from allele 2. The sum of other two fragments obtained from allele 1 is equal to that obtained from the other fragment of allele 2. On the other hand, EcoRI has same number of restriction sites (1 at nucleotide 153) and restriction fragments obtained (2) on both alleles. 2. Since obtained results in the 2 different alleles by Hae III are different (3 fragments in allele 1 and 2 fragments in allele 2). Then separating them by gel electrophoresis will show 3 different bands for allele 1 and 2 different bands of allele 2 thus obtaining different restriction maps. Therefore we can detect genetic polymorphism for this gene by Hae III. On the contrary, EcoRI shows same number of fragments for the 2 alleles of same size, thus separating them through gel electrophoresis will give 2 bands for each in same position, thus same restriction map is obtained. For that, EcoRI is not suitable for detecting genetic polymorphism for this gene. T. Abed Al Kareem Noureddine 34
  35. Answer (continue) • Individual A: Nd since it has band 452 that is present only in the d allele and bands 370 and 82 that are specific for the N allele. • Individual B: dd since he only has 452 thick band (that corresponds for the d allele) which indicates presence of 2 copies of this allele and lacks the band that correspond for N allele (370 and 82) • Individual C: NN since he has thick bands of 370 and 82 that corresponds for the normal allele (N) which indicates existing 2 copies of this allele and lacks band corresponding for d allele (452 bp) T. Abed Al Kareem Noureddine 35
  36. Restriction Fragment Length Polymorphism (RFLP) • Objective of technique: Detect genetic polymorphism in coding or non-coding regions • The mutation at level of non-coding region of DNA does not alter the phenotype but changes the restriction map. • Therefore, the restriction map is independent of gene function. • The variation at level of non-coding regions is used to determine genetic identity of individual! • DNA fingerprinting (document 5) is an application of RFLP technique T. Abed Al Kareem Noureddine 36
  37. • What two ways could be used to assess genetic polymorphism in population? • Study the phenotypic variation (for coding regions) • RFLP (for coding and non coding regions) • Note: RFLP technique allows to determine the exact genotype of individual in case he had dominant phenotype (he could be homozygous or heterozygous) T. Abed Al Kareem Noureddine 37
  38. Document 5: Genetic Identity of individual • Introduction: Because of the large number of polymorphisms observed in humans, it is virtually certain that each of us is genetically unique (except identical twins). How can we determine genetic identity of individual? Determination of Genetic identity is made by RFLP followed by southern blot. T. Abed Al Kareem Noureddine 38
  39. Southern blot • General Terms: • Probe: a known sequence of radioactive or fluorescent DNA used to hybridize the denatured DNA molecule by complementarity • Denaturing: separation of DNA strands by high temperature or using NaOH • Hybridization: binding of probe to studied bands/ gene by complementarity. • Blotting: transferring T. Abed Al Kareem Noureddine 39
  40. T. Abed Al Kareem Noureddine 40
  41. Southern Blot 1. DNA is cut with restriction enzymes 2. DNA fragments separated by gel electrophoresis 3. Fragments then transferred (blotted) and fixed on filter paper. 4. On this membrane the DNA are denatured then a radioactive 32P probe is added to hybridize specific sequence by complementarity 5. Hybridization of DNA with probe is visualized by autoradiography. T. Abed Al Kareem Noureddine 41
  42. Notes • There are two types of probes that could be used: • Mono-locus Probe: A probe that binds to a sequence that exist only at one locus. It is used in case of detecting a certain gene. • Multi-locus Probe: A probe that binds to a sequence that occurs frequently throughout genome. This type of probe is used in DNA fingerprinting. • Among all fragments obtained from the action of restriction enzyme, only the one where probe can bind partially or totally would appear in results T. Abed Al Kareem Noureddine 42
  43. Example: • In the following example the restriction enzyme Hae III cuts in 3 position leading to 4 fragments of sizes 198, 52, 230 and 30 bp. In gel electrophoresis the 4 bands would appear (unless given in text that there is minimum size for band to appear in the gel electrophoresis). However upon blotting, only the zone where the probe binds partially or totally, the bands would appear. For that in the final results only bands appear (42 and 230 bp that corresponds for allele A as there is restriction site cute at level of 240) and fragment of size 272 bp that corresponds for allele B. T. Abed Al Kareem Noureddine 43
  44. DNA Fingerprinting • DNA fingerprinting is an application of RFLP (using restriction enzymes, gel electrophoresis, southern blot) and it is used for: • Paternity Test • Forensic science (crimes, victims after plane crush..) • Probe used is multi-locus (complementary to sequence occurs frequently throughout the genome in non coding regions) T. Abed Al Kareem Noureddine 44
  45. DNA fingerprinting in paternity test • Each band of child’s DNA fingerprint should be shared with either the father’s or the mother’s DNA fingerprint. • Solve exercise IV + V p: 73 T. Abed Al Kareem Noureddine 45
  46. • P:73 official book: Exercise IV • The DNA isolated from blood traces left at the scene of the crime and the DNA of the suspect are cut with the same restriction enzyme, electrophoresed and blotted onto a membrane. The membrane is then hybridized to a 32P labelled probe which is complementary to a repetitive sequence. The two DNA fingerprints are compared. If the two patterns are identical, then one can safely conclude that the traces of the blood at the scene of the crime are those of the suspect. T. Abed Al Kareem Noureddine 46
  47. • Exercise V No since every band of the child’s DNA finger print is present either in the mother or the father’s DNA fingerprint so the child belong to this couple. T. Abed Al Kareem Noureddine 47
  48. Fluorescent In Situ Hybridization (FISH) • Aim: • Locate locus of gene on metaphasic chromosome using fluorescent probe • Detect chromosomal mutations (deletion, translocation..) • Detect certain types of viral infection • Steps: • Denaturation of DNA • Hybridization: add monolocus probe • Observation: The chromosome shows a fluorescent dot where the studied gene (or sequence of interest) is located. • FISH technique and other mapping techqniques allowed to construct genetic maps T. Abed Al Kareem Noureddine 48
  49. Genetic Map • Genetic map representation of arrangement of genes on chromosome • It can be constructed by using many different mono-locus probes Chromosome X genetic map T. Abed Al Kareem Noureddine 49