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5Study Guide Exam IV (Chap 18-22) 1. The damaged base in DNA is repaired by base excision repairpathway. Incorrectly paired nucleotides in DNA are repaired by mismatch repairpathway. Bulky DNA lesions can be repaired by nucleotide excision repairpathway. A...

5Study Guide Exam IV (Chap 18-22) 1. The damaged base in DNA is repaired by base excision repairpathway. Incorrectly paired nucleotides in DNA are repaired by mismatch repairpathway. Bulky DNA lesions can be repaired by nucleotide excision repairpathway. Altered nucleotides in DNA can be changed back into their original structures by direct DNA repairpathway. Double-strand DNA breaks can be repaired by homologous recombinationand nonhomologous end joiningpathways. 2. Examples of transition and transversion mutations TRANSITION: is a base substitution mutation where a single purine is changed for a purine (A-G, GA) or a pyrimidine is changed for a pyrimidine (T-C, C-T) ; 4 possible scenarios TRANSVERSION: is a base substitution mutation where they are inverse. A purine can switch for a pyrimidine and so on; 8 possible scenarios Also can be base Substitution change! Examples of missense, nonsense, silent, neutral mutations Missense:CODON CODON OR STOP CODON CODON Base substitution that changes the codon from one amino acid to another UCA (serine) → UUA (leucine) Or Base substitution that changes a stop codon to a codon for an amino acid UAA (stop) → UUA (leucine) Protein sequence will be longer There can be no protein sequence depending if it lands on a start codon! Nonsense:CODON STOP CODON Base substitution that changes a codon coding for an amino acid to a stop codon (for polypeptide termination) UCA (serine) → UAA (stop) Protein sequence shortened Silent: Base substitution that does not change the specified amino acid CUU (leucine) → CUA (leucine) No biological consequence Neutral: CODON CHEMICALLY SIMILAR ONE Missense mutation that changes the amino acid to a chemically similar one CUU (leucine) → AUU (isoleucine) Often has some observable but minimal effect on protein function Examples of reverse mutation and suppressor mutation: (both end result in WILD TYPE) Reverse mutation - alters a mutant phenotype/genotype to the wild-type (by changing DNA seq) (SINGLE Mutation) Suppressor mutation: (DOUBLE mutation) A genetic change that hides or suppresses effect of another mutation Occur at a different site from the original mutation site A double mutant- Basically an original mutation undergoes another mutation to display a normal phenotype. displays wild-type phenotype 3. Examples of loss-of-function and gain-of-function mutations. Loss-of-function Causes a loss of protein function (e.g. cystic fibrosis, fly eye color) Complete loss of function is also called a null mutation Can also be due to a mutation in the regulatory regions required for transcription, splicing or translation Usually, recessive Gain-of-function Create an entirely new trait or cause the trait to appear in the wrong tissue or at the wrong time in development Usually, dominant e.g., overactive telomerase / achondroplasia 4. How does UV affect DNA? Detailed reactions. Purines, pyrimidines absorb UV energy Adjacent pyrimidines are covalently joined to form pyrimidine dimers Pyrimidine dimer distorts DNA helix Block DNA replication the cell usually dies UV induced pyrimidine T:T dimer distorts DNA CUT BY NUCLEOTIDE EXCISION REPAIR ( WITHIN SAME STRAND) 5. What mutation is most likely to cause null loss-of-function? Why? A frameshift mutation NULL LOSS OF FUNCTION: Causes a loss of protein function, can also be due to a mutation in regulatory regions 6. Insertion or removal of one nucleotide base pair in the coding region of DNA results in a frameshift mutation. Insertion or removal of two nucleotide base pairs in the coding region of DNA results in a frameshift mutation. Insertion or removal of three nucleotide base pairs in the coding region of DNA results in a In- Frame deletion or insertion mutation. 7. Use restriction enzyme/restriction endonucleasesenzyme to cut DNA at a specific site. Use DNA ligaseenzyme to fuse two pieces of DNA together. UsePolymerase chain reaction (PCR)technique to amplify trace amount of DNA. Use western blottechnique to detect a specific protein. Use FISH/northern blot/RT/CDNA library/transcriptome, RNA seek technique to study gene expression/transcription. Use CRISPR-Cas9technique to edit a gene/genome in a cell.  Use GWA, genomic library/southern blot/FISH/ PCRtechnology to study the whole genome. Usetranscriptome, cDNA library, RNA seek, Northern blottechnology to study the entire transcription in an organism. Useproteome, western,2D PAGE technology to study all the proteins in an organism. Use reverse transcription. (RT-PCR)technology to detect COVID-19 virus infection. 8. What is CG island or CNG island? (a) 5’-ATCTACGTTATA-3’ or (b) 5’-ATCTACCTTGTA-3’ How do you identify which DNA sequences are more likely to be methylated? Sequence with most CG and CNG (less likely to be transcribed) Repress/Inhibit transcription How do you identify which histone sequences are more likely to be methylated? Sequence with most R and K (can be transcribed or inhibition of transcription) How do you identify which histone sequences are more likely to be acetylated? Sequence with most R and K 9. The set of all RNAs encoded by the genome is called transcriptome (fall under functional genomics) 10. PAGE stands for polyacrylamide gel electrophoresis, what is it used for? 1st Run a 2d gel Technique to separate a mixed sample of proteins to identify according to protein size This is done for proteomics and western blotting SNP stands for single nucleotide polymorphism, what is it used for? These are DNA variation in a single base pair equivalent to different alleles used for GWA. A single-nucleotide polymorphism is a germline substitution of a single nucleotide at a specific position in the genome. CNV stands for copy-number variation, what is it used for? Copy number variation is sections of the genome are repeated and the number of repeats in the genome varies between individuals. Natural mutation due to deletion, insertion or duplication due to certain condition or diseases Exp: Autism The differences in the number of copies of large DNA sequences. Caused by deletions or Insertions (dupes). Derived from studying the human genome. RFLP stands for restriction fragment length polymorphism, what is it used for? Technique used to generate genetic markers for genomic analysis It is the changes in length of DNA fragments created by restriction endonucleases. Can be used for mapping diseases, pedigree and family relation Useful in genome-wide disease association analysis  MAPPING FOR DISEASE ASSOCIATION ALSO, RFLP FOR PEDIGREE GENOTYPING The band that’s most repetitive is most likely linked to the disease! GWA stands for genome -wide association, what is it used for? (GERM-WIDE used to find Sips in order to find genes that are causing disease in a genome. This is done by comparing the genome of a healthy person against that of a sick person. Used for stroke-risk gene FISH stands for Fluorescent In Situ Hybridizationwhat is it used for? Locate or to view a gene on a chromosome Locate view/determines specific mRNA in a different cell/tissue How to see it you need a probe; based on base pairing DNA w/DNA RNA w/RNA You need a microscope to see your probe! One type is FISH: Florescence in situ hybridization Another type: In Situ mRNA (dark color) to study gene expression in tissue CRISPR stands for clustered regularly interspaced short palindromic repeats, what is it used for? Used for gene (DNA) editing, that relies on the action of CRISSP RNA’S and Cas proteins targets genes for DNA deletion, insertion, replacement modification. It is a segment of DNA CHIP stands for CHROMATIN IMMUNOPRECIPITATION, what is it used for? Used for identifying DNA binding sites of a specific protein and the locations of modified histone proteins. Process for studying epigenetics. 11. Haplotype is DNA variation on a SINGLE chromosome, what is it used for? To study SNPS on a single chromosome to have a specific target HapMap project RNA-seq is sequence entire RNA sequence, what is it used for? Determines the expression of genes in a genome, used to analyze a transcriptome RNA is isolated from cells and converted to cDNA and the resulting cDNA fragments are sequenced. 12. In epigenetics, acetylation occurs to where? and results in what? ACETYLATION: occurs in RNA, it is the addition of negatively charged acetyl groups by (acetyl transferase) to the positively charged lysine and arginine tails, now neutral. This loosens DNA that allows transcription to occur, turns on gene expression In epigenetics, methylation occurs to where? and results in what? METHYLATION: occurs to DNA, is the addition of methyl groups by methyltransferase to cytosine bases in DNA. This causes the DNA to tighly coil (deacetylase enzymes) around the histone repressing transcription. Proteins can’t bind, inhibits gene expression. Can lead to phenotypic changes. Can be site specific- Once methylated stays methylated the cytosine reduce and dna example: queen bees eat royal jelly they maintain the same and die if not eaten 13. Epigenetic mechanism of queen honey bee and work bees. All have the same genetic sequences, but display different morphologies and “roles” in the hive due to epigenetics (the change in gene expression due to environmental factors but they don’t change the DNA sequence in itself). All bees are served what we refer to as commoners’ bread which methylates for Dnmt3 genes that silence these queen-like characteristics. The queen be on the other hand is being fed royal jelly which demthylates this Dnmt3 gene allowing these silenced genes to express freely. The jelly has to be given early enough before the bee is fully developed. If not, the bee will believe its a queen bee act nurturing and believe it’s the queen be but phenotypically it will remain a worker bee. 14. You are trying to determine whether a certain RFLP marker is linked to a specific disease gene in dogs. Assume that the disease is an autosomal dominant. Based on the pedigree and RFLP autoradiogram shown below, what band(s) is most likely linked to the disease? What band(s) is most likely not linked to the disease? The band that is linked to disease is 1.5 kb or 4.5 kb So, the 6kb band is most likely not linked to the disease 15. How did black, red and white grapes come about? Transposons/insertions Black Grapes: no transposons are present White/green Grapes: A lot of transposons present Red grape: little transposons are present Color due to transposition RETROTRANSPOSON: was a piece of RNA was transcribed from a DNA transposon, is the copied back by reverse transcriptase Black grape: has full production of pigment anthocyanins (no transposons insertion) Red grape: has partial removal of Gret1 retrotransposon (Class 1) Green grape: full insertion of Gret 1 retrotransposon, mutation disrupts pigment production. How did moth coloration come about? Pollution -more melanin production due to transposons How did tails in mammals come about? Inhibit- short 16. What are Southern blot, northern blot and Western blot hybridizations, PCR, and 2-D gel electrophoresis? When to use them? Detect RNA- northern blot hybridization Detect DNA- Southern blot hybridization Detect protein- Western blot hybridization- Run PAGE 2-D gel electrophoresis: a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. PCR: to amplify DNA fragments-first use PCR use thermal stable DNA poly (cycles of 3 temps: denaturing, annealing and amplification; powerful tool of amplification trace amount of DNA/RNA 17. The set of all proteins encoded by the genome is called proteomes 18. How does plant cloning compare to animal cloning? PLANT CLONING: contain all, three genomes (eukaryotic, mitochondrial, and chloroplast). Under the right conditions cells and redefine and grow into a whole plant wihtout sexual reproduction Genetically identical Single carrot cells can be regenerated into whole plants ANIMAL CLONING: contain two genomes (eukaryotic and mitochondrial). Cloning was thought to be impossible but after the Dolly experiment in 1997 it was proven possible, not yet in humans. Egg cell removed from donor with all the genetic data (cytoplasm, nucleus, DNA, *is unfertilized*), a micropipette removes the nucleus, a donor nucleus is taken and starved in low nutrient culture medium, the donor nucleus is shocked by electrical pulses to fuse into the unnucleated egg cell, is then fertilized, cell division, embryo, and then it is implanted into a surrogate mother. ◦NOT genetically identical 19. What is totipotency? Types of cells vs degree of potency. POTENCY: the ability that cells have to become onto other cells Has four degrees (in mammals) TOTIPOTENCY; ability a cell has to divide and produce all types of cells like for example differentiated cells in an organism. Can develop a whole organism on their own. DIFFERENTIATED CELLS: adult stem cells PLURIPOTENCY: ability a cell has to develop most cells in the body except for placenta cells. Cant produce a whole organism. INDUCED PLURIPOTENT STEM (iPS) cells: derived from adult somatic cells MULTIPOTENCY; ability a cell has to develop some cells of the body, even less than pluripotent cells MONOPOTENCY; ability a cell has to develop into one type of cell Red blood cells. 20. Monozygotic twins are identical in their DNA in embryo, but differ in their Epigenetics, phenotype. The difference is due to epigenetic changes (dna methylation/acetylation/chromatin remodeling) and minimal DNA mutations that happen early in mitosis. They differ more when they are older due to environmental factors, gene expression changes with age. 21. What is genomic imprinting? An example? GENOMIC IMPRINTING; an epigenetic mechanism, occurs by modifying the activation genes that control expression of the genes. ◦Expression of the genes (whether they are silenced or not) can be strongly influenced by the parent from which the genes were inherited Associated with heavy methylation of affected genes (no transcription, methyltransferase) 22. Based on the DNA fingerprints shown below, who is the child’s biological father? Mo = mother; Ch = child; M1–M5 = potential fathers. B1-B10: marker bands present in the child. 23. A particular transposable element generates flanking direct repeats that are 4 bp long. What is the sequence that will be found on both sides of the transposable element at the insertion positions indicated on each of the following sequences? And how do you know? (there’s a question like this on exam) 1st: Cut at arrow put transposable in make repair ADD TGAC 2nd: put a box and add the last ones after the arrow a. 5’-ATTCGAACTGAC TGACCGATCA -3’ b. ATTCGAA CGAACTGACCGATCA-3’ DIRECT REPEATS IN HOST  3 steps 1. Cuts at a chromosome 2. D.S transposon insertion 3. Fill up gaps- host genome will repair 24. Which following pairs of sequences might be found at the ends of an insertion sequence of a transposable element? Inverted is in Transposons a. 5′—GGGCCAATT—3′ and 5′—CCCGGTTAA—3′ b. 5′—AAACCCTTT—3′ and 5′—AAAGGGTTT—3′ c. 5′—TTTCGAC—3′ and 5′—CAGCTTT—3′ d. 5′—ACGTACG—3′and 5′—CGTACGT—3′ e. 5′—GCCCCAT—3′ and 5′—GCCCAT—3′ 25. How much nuclear DNA does each human cell have (in base pairs)? 6.4 DIP – 3.2 HAP A skin cell (diploid-somatic) 6.4 BP A liver cell (Dip-som)6.4 BP A heart cell (Dip-som)6.4 BP A sperm cell (Hap- germ)3.2 BP A fertilized egg cell (Dip-som)6.4 BP An unfertilized egg cell (hap-germ) 3.2 BP 26. How much nuclear DNA does each Arabidopsis cell have (in base pairs)? 250 Dip -125 Hap A leaf cell (dip-som) 250 bp A root cell (dip-som) 250 bp A pollen cell (hap-germ)125 BP A fertilized egg cell (dip-som)250 bp An unfertilized egg cell (hap-germ) 125 BP 27. How much nuclear DNA does each Drosophila cell have (in base pairs)? 340 Dip- 170 Hap A skin cell (Dip-som)340 bp A liver cell (Dip-som) 340 bp An eye cell (Dip-som)340 bp A sperm cell (Hap-germ) 170 bp A fertilized egg cell (Dip-som)340 bp An unfertilized egg cell (Hap-germ)170 bp 28. Same or different of the nuclear genomes? And why? A man’s DNA sequence in skin cell vs liver cell Same, Dip-som cells are alike 3.2bp A man’s DNA sequence in heart cell vs liver cell Same, Dip-som cells are alike 3.2 bp A man’s DNA sequence in kidney cell vs sperm cell Different kidney is Dip-som cells 6.4bp and sperm cells are Hap-germ 3.2 bp A man’s epigenetics in skin cell vs liver cell Same, Dip-som cells are alike 3.2 bp A man’s epigenetics in heart cell vs liver cell Same, Dip-som cells are alike 3.2 bp A man’s epigenetics in kidney cell vs sperm cell Different kidney is Dip-som cells 6.4bp and sperm cells are Hap-germ 3.2 bp but depending on lifestyle choices and his specifc gene expression they might be displayed differently DNA sequence in a queen bee vs DNA sequence in a worker bee (female bee) Same, even though they go through different epigenetic factors their sequences stay the same (epigenetics doesn’t alter DNA). For queen bee the DNA sequence is being demethylated to fully expressed genes, worker bee all systems regular Dnmt3 still is methylated DNA sequence in a queen bee vs DNA sequence in a drone (male bee) Same, even though they go through different epigenetic factors their sequences stay the same (epigenetics doesn’t alter DNA) For queen bee the DNA sequence is being demethylated to fully expressed genes, drone bee all systems regular Dnmt3 still is methylated DNA sequence in a worker bee vs DNA sequence in a male bee Same, no consumption of royal jelly for either Epigenetics in a queen bee’s DNA vs that in a worker bee Different, the queen bee and worker bee aren’t exposed to the same environment meaning they will have different epigenetic effects Epigenetics in a queen bee’s DNA vs that in a male bee Different, the queen bee and worker bee aren’t exposed to the same environment meaning they will have different epigenetic effects Epigenetics in a female bee’s DNA vs that in a male bee Same, they are exposed to same environmental factors and will have similar epigenetic effects. 29. Examples of ortholog and paralog genes. Ortholog: homologous genes, found in different species from common ancestor (monkey, human) Ex: hemoglobin has same functioned all animals, all different species (homologous genes, two different species) Paralog: homologous genes, same organism (same individual) due to gene duplication (homologous genes, within the same species) Protein may be identified by the homology to one or more orthologs 30. You use polymerase chain reaction to amplify a specific gene from a trace amount of DNA. If one copy of the gene is n present in the starting material, after 20 cycles of PCR, how many copies of the gene can be produced? After 30 cycles? 2 copies are produced 20 and after 30 cycles 1 x 230 1x 2 ( 2 is always the number n is the amount of cycles) If two copies of the gene are present in the starting material, after 20 cycles of PCR, how many copies of20the gene can30 be produced? After 30 cycles? 4 Copies & 4 Copies 31. Somatic mutations vs germ-line mutations. Somatic mutations : Occur in somatic tissue Not involved in production of germ cells (e.g. sperm and eggs) Somatic cells divide by mitosis, so the mutation is passed on to daughter cells (but not to daughters/sons!) Most mutations no observable phenotype Some stimulate cell division and growth Basis of nearly all cancers Germ-line mutations: occur in cells that give rise to gametes mostly during meiosis Are inherited by future generations mutation is passed on to daughters/sons Mom’s cells are homozygous for: ATGCCAGCCTGA –Her egg that makes the kid is: ATGCCGGCCTGA After mutation, the new variant exists in her progeny and subsequent generations (unless no more offspring) This new variant can be good, bad or nothing! May affect phenotype / cause lots of genetic variation in human height 32. Difference between apoptosis and necrosis. APOPTOSIS: programmed cell death ALL CELLS ARE MEANT TO DIE., can also be triggered by viral infection, DNA damage or mitochondrial damage. Process: caspases enzymes cleave (split) other caspases to activate more of ◦ the enzyme. These degrade other proteins at specific sites. Once the cell becomes small enough a macrophage attaches which activates DNase, leading to degradation of nuclear genome and then death. Example: frog losing its tail, humans loosing webbed fingers on development. NECROSIS: unprogrammed cell death. Is not genetically controlled. Can be caused by injury, unexpected circumstances ◦Process: the cell swells, causing cell lyses and releases cytoplasmic material, then death. 33. How is a X chromosome in mammals inactivated? Detailed process. RNA effect on epigenetics, process is started by LONG NON-CODING RNA. It begins to cover the chromosome, then once its fully covered methyl groups attach and begin dna methylation that will inactivate the chromosome 34. What is the genetic mechanism of fragile X syndrome? (most common for of inheritable mental retardation) Both Fragile X syndrome and Huntington’s disease are both insert 3 nucleotides Individuals with fragile X syndrome, CGG triplet repeats increased to 100’s or even 1,000’s of copies (insertions) Can’t produce enough FMR protein (fragile X mental retardation) copy number vs severity or onset age What is the genetic mechanism of Huntington’s disease? Germ line mutation where insertion of CAG repeats into the protein sequences occur (excess of glutamine) causes the progressive breakdown of nerve cells in the brain. Homework: 1. Consider a change from UGA to UGG in the coding region of a mRNA. What is this mutation and the likely consequence? Missense mutation from a stop codon to another codon for an amino acid! UGA is a stop codon UGG is tryptophan can be used in making of suppressors. The protein sequence will be longer than usual. 2. What are the genetic causes for fragile-X syndrome and Huntington’s disease? Nearly all cases of fragile X syndrome are caused by a mutation in which a DNA segment, known as the CGG triplet repeat, is expanded within the FMR1 gene. Normally, this DNA segment is repeated from 5 to about 40 times. In people with fragile X syndrome, however, the CGG segment is repeated more than 200 times. 3.Distinction of Southern, northern, western blot and in situ hybridizations. Can you tell them apart? Northern blot hybridization 1. Extract RNA from cells on to a membrane 2. Run agarose gel electrophoresis (from sea weed) 3. Use probe: a piece of DNA that can bind to targeted RNA How to identify covid-19? By using the Western blot hybridization technique 1. Extract proteins from cells 2. Run special gel: PAGE (it’s a compound form a polymer) 3. You use a specific antibody for a protein- to detect signals (NO PROBE USED) 4. If you don’t see the band your sample does not have the protein In Situ Hybridization Locate or to view a gene on a chromosome Locate view specific mRNA in a different cell/tissue How to see it you need a probe; based on base pairing DNA w/DNA RNA w/RNA You need a microscope to see your probe! One type is FISH: Florescence in situ hybridization 5. Gain of function: 4.Use PCR to amplify a gene. If starting material has 2 copies of this gene, after 35 cycles of PCR, how many copies of this gene are produced? 2x235 6. Genomic size of Arabidopsis= 125 Fly= 170 Human=6.4 dip – hap 3.2 7. Genome: Study of all genes 8. Proteome: study of all proteins in an organism 9. Transcriptome: study of RNA- the entire transcription I clicker questions. 1. Transcriptomics directly studies? a. RNA 2. Epigenetic modification can include all of the following EXCEPT: a. Dna methylation, repressing transcription. b. DNA acetylation, activating transcription c. Non coding RNA, recruitment enzymes that modify histones. d. Histone acetylation, increasing transcription  e. Small RNA inhibit specific mRNA’s 3. Souther blot hybridization is used for analysis of a. DNA 4. Uv light is harmfull because a. It can link two bases together to distort DNA structure 5. D- transfer small volumes of liquid Mock questions 1. Which of the following mutations is most likely to cause null-of function? a. FRAMESHIFT 2. A mutation that changes a G base to A is an a. Transition 3. Given the common characteristics of transposable elements, which of the following pairs of sequences might be most likely found at the ends of a transposable element? a. 5’-ATACCCTTT-3’ and 5’AAAGGGTAT-3’ 4. Insertion or removal of two nucleotide base pairs in DNA usually results in a a. Frameshift mutation 5. Huntington disease can strike at an earlier age and bring about a more rapid degeneration and death in successive generations within a family. This degeneration and death in successive generation within a family. This phenomenon can be explained by which mechanism? a. Expansion of trinucleotide repeat in the coding sequence of the gene 6. Which of the following is NOT an example of epigenetics? a. Transposon transposition. 7. Which of the following sequences is likely to be more heavily methylated? CN and CNG look for them 8. Haplotype a. A set of genetic variants on a single chromosome 9. How do epigenetic marks compare to monozygotic twins? a. They are similar early in life but are increasingly dissimilar with age. 10. Diet (food) in methyl donors for the mothers may affect development of their progeny by a. Changing epigenetics modification of DNA or proteins 11. The set of all RNA encoded by the entire genome is called the a. Transcriptome 12. A base substitution may cause all following, except for a. Creation of a polypeptide with an entirely new sequence 13. The cell can repair this mutated DNA as show below using? a. Base excision repair 14. Black grapes are the results of a. Gene expression for pigments synthesis 15. You want to cut a DNA molecule into fragments at specific sites. What should you do? a. Using a restriction endonuclease