MUTATIONS Izzati Adilah bt Azmir 02 039 OBJECTIVES

MUTATIONS Izzati Adilah bt. Azmir 02 -039

OBJECTIVES • Learn how the DNA structure can ultimately change into a new structure and permit a new phenotypic development. • Study types of mutation that could be inherited for the next generation onwards. • Determine the process and mechanism of mutation depending on the type of mutagens.

LECTURE CONTENT 1. TYPES OF MUTATION AND THE EFFECTS ON ORGANISM 2. PHENOTYPIC VARIATION 3. FACTORS CAUSING MUTATIONS 4. THE REPAIR OF DNA DAMAGE 5. THE STUDY OF MUTATIONS

TYPES OF MUTATIONS 1 Gene Mutation • Point Mutation – Silent – Missense – Nonsense • Frameshift Mutation – Addition – Deletion 2 Chromosome Mutation • • Deletion Duplication Inversion Translocation

TYPES OF MUTATIONS 3 Genome Mutation • • Aneuploidy Polyploidy Autopolyploidy Allopolyploidy

1 GENE MUTATION Point Mutation Frame shift

GENE MUTATION • Involve insertion or removal of 1 or more base pairs • Gene mutation is a change in single base pair within DNA sequences

EFFECTS OF GENE MUTATIONS • Most mutations are neutral - they have no effect on the polypeptide. • Some mutations result in a less active product; • Less often an inactive product; • Very few mutations are beneficial.

EFFECTS OF GENE MUTATIONS(cont) • Affects molecular changes in the DNA sequence of a gene • Alter the coding sequence within a gene • Causes permanent change in DNA sequence

BODY (SOMATIC) AND GAMETE (GERM)MUTATION • Body cell mutations can cause cancer. – only the individual is affected • Gamete cell mutations affect the egg and the sperm. – all offspring of the individual can be affected. In multicellular organisms (plants or animals) mutations may occur in the somatic cells of the organism. Somatic cells are the cells involved in growth and repair and maintenance of the organism. A mutation in these cells may lead to cancer and certain of chromosomal mutations may be involved in aging. Other mutations happen in the germ cells and these mutations may appear in the gametes and then in the offspring produced by sexual reproduction. These sorts of mutations are called germ cell mutations.

GENE MUTATION: The Types 1. Point mutations - a one base change in DNA. 2. Frame Shift Mutations - the addition or deletion of 1 or more bases. These are due to powerful mutagens; chemical or physical.

1. POINT MUTATION (PM) 3 TYPES: • silent mutation - single base substitution in the 3 rd base nucleotide position of a codon. This results in NO change in amino acid. Note that the first 2 letters of the genetic code are the most critical. • missense mutation - single base substitution in 1 st or 2 nd base nucleotide position. This results in changed amino acid. • nonsense mutation - single base substitutions that yield stop codon. Note: there are 3 nonsense codons in the genetic code = NO PROTEIN

PM: Silent mutation single base substitution in the 3 rd base nucleotide position of a codon. This results in NO change in amino acid. Note that the first 2 letters of the genetic code are the most critical.

PM: Missense mutation Single base substitution in 1 st or 2 nd base nucleotide position. This results in changed amino acid. This is equivalent to changing one letter in a sentence, such as this example, where we change the 'c' in cat to an 'h': Original Point Mutation : The fat cat ate the wee rat. : The fat hat ate the wee rat.

PM: Nonsense mutation single base substitutions that yield/become stop codon. Note: there are 3 nonsense codons in the genetic code = NO PROTEIN

2. FRAME SHIFT MUTATIONS Gene addition or deletion One or more bases are added or deleted, the equivalent of inserting or removing letters in a sentence. But because our cells read DNA in three letter "words", adding or removing one letter changes each subsequent word. This type of mutation can make the DNA meaningless and often results in a shortened protein & non-functional.

An example of a frame-shift mutation using our sample sentence is when the 't' from cat is removed, but we keep the original letter spacing: Original Frame Shift : The fat cat ate the wee rat. : The fat caa tet hew eer at.

2. FRAME SHIFT MUTATIONS Additions Normal gene GGTCTCCTCACGCCA ↓ CCAGAGGAGUGCGGU Codons ↓ Pro-Glu-Cys-Gly Amino acids Addition mutation GGTGCTCCTCACGCCA ↓ CCACGAGGAGUGCGGU ↓ Pro-Arg-Gly-Val-Arg

2. FRAME SHIFT MUTATIONS Deletion Normal gene GGTCTCCTCACGCCA ↓ CCAGAGGAGUGCGGU Codons ↓ Pro-Glu-Cys-Gly Amino acids Deletion mutation GGTC/CCTCACGCCA ↓ CCAGGGAGUGCGGU ↓ Pro-Gly-Ser-Ala-Val

2. FRAME SHIFT MUTATIONS

2. FRAME SHIFT MUTATIONS

MUTATIONS CAN BE NEUTRAL • They may have little or no effect on the survival of an organism or on its ability to reproduce. • They may result in the same kind of organism meaning that the change still tells the cell to do what it should, so there is no difference. • It is estimated that the average human has 50 -100 mutations within their DNA - most (if not all) are neutral or beneficial

MUTATIONS CAN BE NEUTRAL (cont) • Bacterial resistance to antibiotics • Insecticide resistance in bugs • Rapid mutation rates in virus’s proteins allowing them to adapt to new “hosts”

MUTATIONS CAN BE BENEFICIAL • In humans, it can be a different set of circumstances… Here’s an example: • Sickle-Cell Anemia is a genetic disorder in which there is a defect in the structure of red blood cells. This leads to fatigue and anemia when not treated. • However, it has been found that people who are carriers for Sickle-Cell Anemia also has some genetic protection against another disease, malaria.

MUTATIONS CAN BE BENEFICIAL (cont) • In evolutionary studies, scientists have connected the presence of a brain chemical microcephalin (a proposed mutation) with the human’s development of art, music, and complex toolmaking practices • This same research indicates that the human brain is still evolving and becoming more and more capable of more complex tasks • Some humans have been found to have mutations that protect them from other diseases, such as AIDS

2 CHROMOSOME MUTATION Deletion Duplication Inversion Translocation

CHROMOSOME MUTATION Chromosome structure become influenced by; 1. Change in amount of genetic information in chromosome because of – Deletion – Duplication 2. Similar amount of genetic information but the materials are rearranged – Inversion – Translocation

CHROMOSOME MUTATION (cont) • Deletion – Loss of chromosomal segment • Duplication – Repetition of chromosomal segment. Gain of segment. • Inversion – A change in the direction of the genetic material along a single chromosome. Reversal of region. • Translocation – A segment of one chromosome becomes attached to a different chromosome – Simple translocation • One way transfer – Reciprocal translocation • Two way transfer

Duplications • In this mutation, some genes are duplicated and displayed twice on the same chromosome. Gain of segment of DNA. • Insertion of an extra copy of a region of a chromosome into a neighboring position. • Zygotes produced from gametes involving duplications are often viable and may or may not have any serious problems. • Various sorts of duplications are related to color vision conditions many of which are quite subtle in their effects e. g certain anemias involving abnormal hemoglobins called the thalassemias.

Duplications (cont) • Charcot-Marie-Tooth disease is a group of disorders passed down through families that affect the nerves outside the brain and spine. These are called the peripheral nerves. • Symptoms usually begin between mid-childhood and early adulthood. They may include: – – – – Foot deformity (very high arch to feet) Foot drop (inability to hold foot horizontal) Loss of lower leg muscle, which leads to skinny calves Numbness in the foot or leg "Slapping" gait (feet hit the floor hard when walking) Weakness of the hips, legs, or feet Later, similar symptoms may appear in the arms and hands, which may include a claw-like hand.

Duplications (cont) • Problems in at least 40 genes cause different forms of this disease.

Deletions • Deletions result when a gene is mistakenly removed from a chromosome, as a result of unequal crossing over. • Often zygotes produced by gametes involving deletions are not viable since they do not have the full compliment of genes.

Deletions (cont) • Cri du Chat results from a very rare mutation caused by the loss or deletion of a significant portion of the genetic material from chromosome number five which is vital to cell growth. • The cry is caused by an abnormal development of a child’s larynx

Translocation • Movement of part of a chromosome to another part of the genome. • May happen with the same chromosome. – translocation is an intrachromosome translocation. Other translocations involve transfer of a region of a chromosome to a non homologous chromosome. For example certain types of Down syndrome involve translocations between chromosome 14 and chromosome 21. This type of translocation between non homologous chromosomes is called an interchromosomal translocation.

Inversion • Inversions happen when a whole region of genes on a chromosome gets flipped around. • 2 types of inversions. – paracentric inversions the centromere is not included in the inversion. – pericentric inversions, the centromere is involved in the inversion. Both these types of inversions lead to abnormalities in crossing over and meiosis resulting in some chromosomes which are not viable, while others are viable but have new combinations of genes. These sorts of inversions are thus important in reshuffling genes on a chromosome.

TRANSLOCATION INVERSION

Inversion (cont) Inversion mutations, also, only affect a small part of the gene Normal gene GGTCTCCTCACGCCA ↓ CCAGAGGAGUGCGGU Codons ↓ Pro-Glu-Cys-Gly Amino acids © 2010 Paul Billiet ODWS Inversion mutation GGTCCTCTCACGCCA ↓ CCAGGAGAGUGCGGU ↓ Pro-Gly-Glu-Cys-Gly

CHROMOSOME MUTATION

Sickle Cell Anemia • Name of Gene Product: hemoglobin, beta • Protein Function: Hemoglobin molecules, which reside in red blood cells, are responsible for carrying oxygen from the lungs to various parts of the body for use in respiration.

• Sickle-cell anemia is a blood related disorder that affects the haemoglobin molecule, and causes the entire blood cell to change shape under stressed conditions. • In sickle cell anaemia, the haemoglobin molecule is defective. • After haemoglobin molecules give up their oxygen, some may cluster together and form long, rod-like structures which become stiff and assume sickle shape.

Sickle Cell Anemia Mutation of hemoglobin gene

3 GENOME MUTATION Aneuploidy Polyploidy Autopolyploidy Allopolyploidy

• Normal organism is euploid with exact chromosome number that is multiple of chromosome set (2 n). • E. g Drosophila melanogaster normally with 8 chromosome. The species is diploid, having two sets of 4 chromosomes each. Rare occasion where abnormal fruit fly produce 12 chromosomes, containing 3 sets of 4 chromosomes each. This alteration is called triploid fruit fly with 12 chromosomes. What about triploid individual?

Chromosome numbers can vary in 2 ways Polyploid – An increase in the number of the complete sets of chromosome – In animals and plants Aneuploid – Abnormal number of chromosomes within a set – Variations are less common

CHANGES IN CHROMOSOME NUMBER a. b. c. d. Aneuploidy Polyploidy Autopolyploidy Allopolyploidy

a. Aneuploidy • Normally 2 N (haploid individual) ends up either with extra copies of homologous chromosomes or fewer than the normal diploid number. • Happens when homologous chromosomes fail to segregate properly during meiosis (non disjunction). • Monosomy (2 n-1) in which the diploid individual has only one member of a certain homologous chromosome. • The other common type of aneuploidy is called trisomy (2 n+1) because the individual has three copies of the particular chromosome.

a. Aneuploidy (cont) • Happens when homologous chromosomes fail to segregate properly during meiosis (non disjunction).

a. Aneuploidy (cont) • Aneuploidy leads to a number of syndromes in humans. For example trisomy 21 leads to Down syndrome, characterized by mental retardation and other abnormalities. • Aneuploidy involving the sex chromosomes is common. XYY males are normal but… • XXY males and XXXY males have a syndrome called Klinefelter syndrome. These males are often actually intersexed or hermaphroditic with partially developed sexual organs of both genders. These individuals are sterile and are often subjected to hormones and surgery to bring them into conformance with social gender roles.

a. Aneuploidy (cont): Klinefelter syndrome

a. Aneuploidy (cont): Klinefelter syndrome • As XXY males enter puberty, they often don't make as much testosterone as other boys. • Can lead to a taller, less muscular body, less facial and body hair, and broader hips than other boys. • As teens, XXY males may have larger breasts, weaker bones, and a lower energy level than other boys. • By adulthood, XXY males look similar to males without the condition, although they are often taller. They are also more likely than other men to have certain health problems, such as autoimmune disorders, breast cancer, vein diseases, osteoporosis, and tooth decay. • XXY males can have normal sex lives, but they usually make little or no sperm. Between 95% and 99% of XXY males are infertile because their bodies don't make a lot of sperm.

Edwards Trisomy 18

The ‘XYY’ Jacob’s syndrome men • 47, XYY ; an extra copy of the Y chromosome • Taller than average, but typically causes no unusual physical features. Most have normal sexual development and are able to father children. • Associated with the risk of learning disabilities and delayed development of speech and language skills. Delayed development of motor skills (such as sitting and walking), weak muscle tone (hypotonia), hand tremors or other involuntary movements (motor tics), and behavioral and emotional difficulties are also possible. • A small percentage of males with 47, XYY syndrome are diagnosed with autistic spectrum disorders, which are developmental conditions that affect communication and social interaction.

The ‘XYY’ Jacob’s syndrome men

b. Polyploidy • 3 N/sets or more of chromosomes in a nucleus. • Can happen because of a failure of the spindle fibers in mitosis or meiosis to segregate chromosomes into separate groups. • Many organisms have specialized polyploid tissues even organisms we typically consider as diploid. – For example in plants a so called double fertilization leads to the genesis of a diploid zygote from the union of two gametes produced by the haploid gametophytes, but also a specialized triploid tissue (3 N) called endosperm. This tissue is produced when a male gamete fertilizes special diploid tissue from the flower. In mammals, cells of the liver are typically polyploid.

b. Polyploidy (cont) • Believed to be an important mechanism in the development of new species and a common pattern in plants is to find populations of two species both of which might be diploid. Where the species overlap a series of localized polyploid populations are often found. These polyploid populations are often effectively reproductively isolated from the parent species and thus can be considered species in their own right. • E, g plant species and some fish and amphibians; – domestic wheat is hexaploid(6 N). ‘ – Seedless plants are usually triploid (3 N). Consider a tetraploid plant (4 N). The gametes of this plant are going to be effectively diploid (2 N) and if they are fertilized by a normal haploid gamete (N), the result is a triploid plant. Since triploid plants have an odd number of chromosomes, typically the gametes have variable number of chromosomes are usually not viable. This is why triploid plants are used to produce seedless plants. Since most plants can self fertilize, the tetraploid plant can breed with itself and produce viable tetraploid populations.

b. Polyploidy (cont)

b. Polyploidy (cont) • Individuals with triploid syndrome have three of every chromosome for a total of sixty-nine rather than the normal forty-six chromosomes. • Babies with Triploid Syndrome usually are lost through early miscarriage. However, some infants have been born and survived as long as five months. Affected infants are usually small and have multiple birth defects. • Those that survive are usually mosaic, meaning that some cells have the normal number of 46 chromosomes and some cells have a complete extra set of chromosomes.

c. Autopolyploidy • Autopolyploidy is polyploidy in which all the chromsomes originate from the same diploid parent species. • Domestic banana and various seedless plants are often triploid autoployploids.

d. Allopolypoidy • Allopolypoidy is a polyploidy in which the sets of chromosomes are from different species. Usually hybrid plants (N 1 + N 2) from such crosses are not fertile since proper pairing of chromosomes does not occur in meiosis. • But sometimes the chromosome number spontaneously doubles leading to tissues with 2 N 1 + 2 N 2. If this tissue is germ tissue, tissue that can give rise to haploid tissue via meiosis, the result can be gametes with the N 1 + N 2 chromosome complement. • When two of these gametes fuse, the result is an allopolyploid plant with a viable chromosome complement (2 N 1 + 2 N 2).

FACTORS CAUSING MUTATIONS

Factors that causes mutation 2 Factors that contribute to mutation – Error in DNA replication. – Damaging effects of mutagens • CHEMICALS: Alkylating agents like nitrosoguanidine, nitrosamine, etc. • RADIATIONS: X-rays, U. V. rays, etc.

Error in replication

Factors that causes mutation Chemical mutagens - used in research to study mutagenesis. There are 3 kinds of chemical mutagens. 1. 2. Alkylating agents. – Adds alkyl group, such as methyl group CH 3 Cn. H(2 n+1), result in mispairing bases in DNA replication – Pairing with wrong bases; methyl group bond with G, it will pair with thymine instead of cytosine. – Eg. formalin, nitrogen, mustard, and ethylene oxide (reacts with G changing it to bind with T). Intercalating agents. – Inserts into DNA and pushes bases apart. – Eg. AFLATOXIN - a chemical produced by peanut and grain molds. The mold is Aspergillus flavus (fungus) causing framshift mutation. – Eg. Benzopyrene – from smoke causing frameshift mutation.

3. Base analogs. – Mimics a nitrogenous base. Eg. AZT is a modified sugar that substitutes for T. – Eg. 5 - bromouracil binds with A or G.

Factors that causes mutation (cont) Physical mutagens: 1. Nonionizing radiation – Causes the formation of T= T dimers. UV light @ 260 nm. – Affecting formation harmful covalent bonds between pyrimidine (T and C). – Forming gap in in DNA strand = no pairing, no replication = cell death

2. Ionizing radiation – damages DNA by causing the formation of “free radicals” leading to mutations. – Eg. X-rays. Gamma rays from radioactive fallout penetrates the body. Alpha rays from inhaled dust containing radioactive fallout.

Possible effects of Mutagens

STUDY OF DNA REPAIR Types of repair: 1. Dimer repair • • Light repair Dark Repair 2. Other types • Methylases

Light Repair • Also known as photoreactivation • When bacteria that previously exposed to UV light, they should be later exposed to visible light. • The visible light will induce the bacteria to produce an enzyme to repair the mutation. • Phytolases – light repair enzyme • Helps in separating the dimers of two thymine • Using visible energy

Light Repair (cont)

Dark Repair • Nucleotide excision • Repair mutation from any causes including dimer • The enzyme will cut off the incorrect bases and fill it with newly synthesized DNA • The enzyme occur in either present or absent of light.

Methylases • Discover by Hamilton Smith, explain how the abnormal DNA sequences that is not obviously show the different such as dimer, could be detected. • The methylases will bound with all normal bases that following the parents strands. • Endonuclease then cut the bases that doesn’t have the methylases bond.

THE STUDY OF MUTATIONS The fluctuation test Technique of replica plating

Why This Study Was Introduced? • Problems faced by scientists have led to the study of mutation – costly and long period. • Objective of the study are: – To differentiate between spontaneous mutation and induced mutation – To isolate particular mutant from culture that containing both mutation and normal microorganism.

The Fluctuation Test • Introduced by Salvador Luria and Max Delbruck (1943) – E. g. Penicilin • The mutation might occur in early culture naturally or have been induced by its environment during replication. • Ames use their idea and invent new test. • This test is to differentiate.

Ames Test

The Replica Plating • Introduced by Joshua and Esther Lederberg (1952) • Similar reason as the fluctuation test. • The particular mix culture m/org was prepared in master plate. • A sterile velveteen pad was then gently pressed on the master plate. • Then it will be pressed to 2 other plates.

Reference • Replica Plating http: //www. sumanasinc. com/webcontent/anim ations/content/replicaplating. html