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Molecular and Cell Biology
MCB 2400
Colleen Spurling

DNA Structure ❖ What is the hereditary material? ➢ Proteins- 20 amino acid building blocks ➢ DNA- 4 nucleotide bases building blocks ▪ Thymine, Guanine, Cytosine, Adenine ❖ Scientists that identified and described genetic material ➢ Federick Griffith- discovered DNA as genetic material when studying, demonstrated the principle of “Transformation”, transformed “R” bacteria into “S” bacteria ➢ Avery, Macleod, McCarty- discovered DNA is responsible for transformation in bacteria, used DNAse and protase to try and identify genetic material ▪ Avery- the “transforming principle” is destroyed by enzymes that cleave DNA ➢ *Hershey and Chase-Found that phosphorus containing components are the genetic material of bateriophages. (e.coli bacteriophage) ➢ *Erwin Chargraff- Showed that in several species contained equal amounts of the base (A)(T) and equal amounts of (C)(G) making it a 1:1 ratio of purines and pyrimidines ➢ Rosalin Franklin- Used x-ray diffraction to deduce the overall structure and the double helix of the DNA ➢ James Watson and Francis Crick- discovered that there are covalent phosphodiester bonds between nucleotides, hydrogen bonds between nucleotides of base pairs, and sugar phosphate backbone ❖ General Structure of a nucleotide ➢ Sugar- deoxyribose ➢ Phosphate group- phosphorus atom bonded to four oxygen atoms (PO4) ➢ Nitrogenous base- Thymine, Guanine, Cytosine, Adenine ▪ Purines- (2 circles) Adenine, Guanine ▪ Pyrimidine- (1 circle) Thymine, Cytosine ❖ Types of bonds ➢ Hydrogen bonds- hold bases together and the individual strans of the DNA ➢ Covalent (phosphodiester) bonds- hold the nucleotides in one strand together ❖ DNA Structure ➢ 2 nucleotide chains- backbone formed by alternating sugar and phosphate groups ➢ Complementary base pairing- • Joins the two chains together • Uniform width- One purine base pairs with one pyrimidine • 2 hydrogen bonds between A and T • 3 hydrogen bonds between G and C • Coiled like helical staircase with 10 bases/per turn ❖ Calculations ➢ 5’- A T G C G C A A T A C G -3’ 3’- T A C G C G T T A T G C -5’ ▪ How many hydrogen bonds are in the above double stranded DNA? • ANSWER: • A=T 6 base pairs x 2 hydrogen bonds= 12 • C=G 6 base pairs x 3 hydrogen bonds= 18 • 12+18= 30 ▪ If a double stranded DNA molecule is 15% thymine, what are the percentages of all other bases? • Thymine = 15% • Adenine= 15% • Cytosine= 35% • Guanine= 35% ▪ A DNA molecule 10 base pair long consists of 30% thymine, how many cytosine nucleotides are in this molecule? • 10 base pairs x 2= 20 nucleotides • Thymine=30% • Adenine=30% • Guanine=20% • Cytosine=20% x 20 = 4 nucleotides that are Cytosine DNA Synthesis ❖ DNA Replication ➢ High fidelity- no errors! Can be trusted! ➢ Once and only once per cell cycle- avoids re replication ➢ Complete- the entire genome must be copies ❖ DNA replication is semi-conservative ➢ Each strand serves as a template ➢ Each of the original strands remains intact ➢ DNA molecule is half (semi) conserved during replication ➢ Have multiple origins of replication because of how large the genome is ❖ Steps in DNA replication 1. Double helix unwinds at replication origin ▪ Helicase- enzyme responsible for the unwinding of the double stranded DNA into a single stranded DNA by breaking hydrogen bonds ▪ Hydrogen bonds break: ▪ Binding Proteins- prevents hydrogen bonds from reforming by stabilizing the separate strands 2. Single stranded DNA 3. Replication bubble ▪ Primase- enzyme that adds RNA primer to provide a free 3’OH end to allow DNA polymerase to add new nucleotides and lays down primers needed to initiate DNA synthesis • Specialized kind of RNA polymerase • Used only in DNA replication • Consists of a short RNA stretch 10-12 nucleotides long • Can start copying a template without a free 3’OH 4. Primer bound to DNA Template ▪ DNA Polymerase- synthesizes new nucleotides to unwind double stranded DNA into single stranded DNA/ adds dNTPs to an existing 3’0H ▪ dNTPs-the building blocks of DNA, its polymerization form phosphodiester bonds 5. 5’3’ DNA synthesis 6. Leading strand(continuous) & lagging strand (discontinuous) ▪ Okazaki fragments- fragments of the lagging strand formed during DNA replication ▪ Lagging strand lags behind in a 5’3’ direction ▪ Leading strand progresses continuously without restarting 7. RNA primers Replaced with DNA ▪ Ligase- enzyme that repairs nicks in the sugar phosphate back bone between Okazaki fragments catalyzing the formation of a phosphodiester bond between two fragments 8. Two double stranded-semiconservative DNA molecules!! ❖ Challenges of DNA replication 1. DNA is packaged in nucleosimes- ▪ Nucleosomes are displaced by the replication fork and are rapidly reassembled onto DNA. New and old histone proteins are randomly assembled on the 2 daughter DNA strands. 2. Chromosomes are linear ▪ Removal of RNA primers at the ends of chromosomes leaves unreplicated cap ▪ Telomerase- activity is needed for replication of telomeres • Differentiated cells in multicellular organisms have little or no telomerase activity - > telomeres progressively shorten (aging) • Mutations affecting telomerase function cause premature aging diseases • Telomerase is expressed in 90% of cancers (cells that divide indefinitely) ❖ Polymerase Chain Reaction ➢ Makes possible the amplification of a particular fragment ➢ Number of copies of the target sequence doubles in each round of reps ➢ Taq polymerase- special DNA polymerase isolated from bacterial Themophiles ➢ Primers- are complementary to the ends of the target sequence and on opposite strands ❖ Ingredients of PCR 1. Template DNA- containing the gene of interest to be amplified 2. DNA polymerase- heat-stable Taq DNA polymerase 3. dNTPs- to make the new DNA strand dATP, dCTP, dGTP, dTTP 4. Buffer- provides the correct pH salt conditions for DNA polymerase 5. Primers- Hybridize/bind/anneal the complementary sequence on template strand/ provide double stranded DNA polymerase attachment; provide exposed 3’OH ❖ Steps of PCR 1. Double stranded targeted DNA sequence ▪ Denaturation(HOT)- hydrogen bonds holding a double stranded DNA molecule must be broken to allow taq polymerase to access the sequence information and synthesize the new strand 2. Single stranded DNA ▪ Annealing(COOL)- the temperature of the reaction to allow base pairing to occur again • Because design to complementary base pair to the sequence of interest are much smaller than the long strands of the parent DNA molecule, they will more readily anneal to the single stranded parent DNA 3. Primer bound to DNA template ▪ DNA Primers 4. 5’3’ DNA synthesis ▪ Elongation(WARM)- taq polymerase activity has an optimum temperature of –70C, by bringing the temperature back up after annealing, taq polymerase becomes activating and begin synthesizing in 5’3’ direction off the end of the primer 5. Two double stranded-semiconservative DNA molecules!! Quiz ➢ PCR Primers  5’- A A T ( C G T A T C A G C A G C A G T G) A C T -3’ 3’- T T A ( G C A T A G T C G T C G T C A C) T G A -5’  Primers: ▪ 5’- C A C T G C –3’ ▪ 5’- C G T A T G –3’ ❖ Comparing PCR and replication PCR Replication In Vivo or In Vitro In Vitro In Vivo Catalyzing enzyme Taq Polymerase DNA Polymerase Initiation Site (Begins) Primer Binding site Origin of Replication Primers DNA RNA Number of Primers 2 Many Template DNA DNA End Product DNA DNA Sanger Sequencing and Transcription ❖ Sanger Sequencing/ Dideoxy Sequencing Steps 1. Use of PCR- You first need to isolate the fragment sequence from the rest of the genome • Enzymes used: taq polymerase • Types of nucleotide triphosphates: dNTPs • Cycle steps: Heat (denature), Cool (anneal), Warm (elongation) 2. Perform Dideoxy Sequencing- Once fragments is isolated you can perform the dideoxy sequencing reaction where multiple fragments of varying lengths will generate • Types of nucleotides needed:  Regular dNTPs  Fluorescently labeled chain terminating ddNTP- the absence of a 3’OH group on the ddNTP prevents the addition of another nucleotide • Each tube contains:  Billions of DNA molecules in a reaction tube  Strand can be terminated at any position 3. Capillary electrophoresis- when fragments elutes from capillary tube, it is excited by a laser, the wavelength emitted by flourphore is recorded and appears as a peak on a computer print out. Wavelength color of the peak indicates which base is present at that specific location in DNA sequencing ❖ Next Generation sequencing vs. Dideoxy sequencing ➢ Next generation: ▪ Can assess the entire genome simultaneously (whole genome sequencing) ▪ DNA fragmet and bound to flow cell (hundreds of millions of cells) ▪ Sequencing by synthesis; signal is detected as nucleotide is incorporated while synthesizing complementary strand to one bound cluster ➢ Dideoxy sequencing: ▪ Shot sequence (200-700bp) is isolated and amplified using PCR ▪ Sequencing reaction is performed to generate fragments of varying lengths ▪ Fragments are the result of labeled chain terminating ddNTP’s Transcription ❖ The central dogma of molecular biology proposes information flows from DNA to RNA to protein ➢ Replication- information is transferred from one DNA molecule to another ➢ Transcription- information is transferred to an RNA molecule from segment of DNA that constitutes the gene ▪ Components: • ssDNA template • rNTPs • RNA polymerase ▪ Pre-mRNA processing: • Intron/exon splicing • 5’cap • 3’polyAtail ➢ Translation- information is transferred from mRNA to a protein through a code that specifies the amino acid sequence ❖ Gene expression ➢ Gene expression- decode information in genes to produce molecules that determine phenotypic traits of organism ❖ Classes of RNAs in Eukaryotes ➢ Serve different important roles as well including structural roles, role in processing pre- mRNA and roles in silencing of other mRNAs ❖ mRNA ➢ mRNA- Carries information the specifies a particular protein ➢ Codon- Three mRNA bases in a row which specifies a particular amino acid ➢ Transcripts- differentiated cells produce certain mRNA molecules, this information is used to manufacture the encoded proteins ❖ mRNA Synthesis ➢ Sequence is complementary and antiparallel to DNA template strand sequence ➢ NTPs- sugar-phosphate bonds between nucleotides ➢ Does not require a primer ➢ Needs RNA polymerase ❖ Transcription unit components ➢ Promoter- initial binding site of RNA polymerase and transcription initiation factors ▪ Promoter recognition by RNA poly. II is a prerequisite for transcription initiation ➢ RNA-coding region- ▪ RNA transcript is 5’3’ ➢ Terminator ❖ TATA binding protein ➢ TATA binding protein- binds to the minor groove of DNA straddling the double helix of DNA like a saddle ❖ Transcription Factors ➢ Interact and form an apparatus that binds DNA at certain sequences ➢ Initiate transcription at specific sites on chromosomes ➢ Respond to signals from outside the cell ➢ Ling the genotype to the environment ❖ RNA synthesis ➢ Complementary and antiparallel to template strand ➢ RNA synthesis: new nucleotides always added to 3’ and of mRNA strand ➢ RNA polymerase II- enzyme that read the DNA template and generates an mRNA transcript ❖ Transcription Steps 1. Initiation- TATA binding proteins unwind DNA and Transcription factors bind and recruit/ position RNA polymerase II ▪ Promoter- special sequence that signals the start of the gene (binding protein)/ initial binding site of RNA polymerase and transcription initiation factors • **Promoter will NOT be read and incorporated into the synthesized mRNA transcript ▪ Promoter(binding protein) unwinding DNA  Single strand 2. Elongation- Transcription bubble forms, polymerase II constructs primary mRNA transcript ▪ Free RNA nucleotides bond with exposed complementary bases on the DNA template strand ▪ Transcription factors left behind on the promoter ▪ RNA polymerase II moves along the DNA template 3. Termination- mRNA transcript released into cytoplasm and Polymerase II dissociates from DNA ▪ Polymerase II sometimes continues to transcribe past the end of a gene ▪ Cleavage of pre-mRNA at specific site occurs ▪ **RAT 1, 5’3’ exonuclease, degrade the extra RNA ▪ Once RAT I, reaches Polymerase II transcription terminates ▪ Ends when an exonuclease enzyme (RNA II) causes RNA pol II to dissociate from DNA ❖ Simultaneous transcription of mRNAs ➢ Several mRNAs may be transcribed from the same template DNA strand at a time Quiz Template is complementary to product! Primer becomes incorporated into synthesized fragment! ❖ How to determine the coding strand vs. the template strand 1. Find the start codon 5’—ATG—3’ 2. Mark off codons from start codons 3. Find a stop codon in frame • 5’—TAA—3’ • 5’—TAG—3’ • 5’—TGA—3’ Translation ❖ Concept of Gene ➢ Not all DNA contribute to the mRNA molecule and protein ➢ Gene is now defined as the total DNA sequence required to encode an mRNA molecule ➢ This includes coding portions and noncoding portions ❖ Pieces of genes ➢ Exons- DNA that encode polypeptides (upper case letters) ➢ Introns- DNA intervening between exons that does not encode polypeptide ❖ Splicing ➢ Intron Splicing- Removal of introns in nucleus of the primary RNA transcript ▪ Splicing modifies transcripts or building blocks ▪ Genome complexity ▪ Eukaryotic specific character ▪ Different splice patterns in different tissues ▪ Increases complexity of the genome ➢ Sequences at exon-intron boundary specify locations for splicing ❖ Alternative splicing ➢ Increases complexity ➢ Many outcomes from one gene ❖ mRNA modifications ➢ transcription takes pace in the nucleus but translation takes place in the cytoplasm ➢ before the mRNA molecule extas the nucleus it is modified 1. Splicing- removal of introns • Removes all noncoding information in RNA transcript 2. 5’ methyl cap- 5’ end of the mRNA is capped • Essential for initiation of translation • Added immediately during transcription by enzyme coupled with RNA pol II • Stabilizes mRNA, prevents degradation, and helps recruit the ribosome • Triggers intron removal machinery • NOT added to non-coding RNA’s 3. Poly-A tail- the 3’ end is usually modified • Helps with mRNA stability • 50-250 A’s added to the end of transcript • NOT coded in the DNA- post transcriptional modification • Stabilizes mRNA, the longer the Poly A the more translation occurs ❖ Translation ➢ The synthesis of every protein molecule in a cell is directed by and mRNA originally copied from DNA ➢ Information-transfer process- RNA base sequence determines an amino acid sequence ➢ Chemical process- links amino acids to polypeptide chain ❖ Translation: building a protein ➢ 3 bases specify one amino acid ➢ linear nucleotide sequence determines linear amino acid sequence ❖ Types of RNA needed 1. Processed mRNA transcript • RNA pol II derived- translated • Rapidly turning over • Heterogeneous size (different genes code for different messages for different proteins) • Different cells have different populations 2. Ribosomes (derived from rRNA) • RNA pol I derived- not translated • Abundant and complex organelles • >50 different proteins and RNA molecules • consist of two subunits: 1 large and 1 small • Catalyze protein synthesis • Very stable, 3. Transfer RNA (tRNA)- transports specific amino acids to the ribosome for protein synthesis • RNA pol III derived- not translated • One for each codon • Carries charged amino acids • Molecular adaptor- nucleotide to amino acid • Cloverlead shape • Amino acid at 3’OH • Anticodon loop- set of three bases that align the mRNA codons ❖ The Genetic Code ➢ Universal- same in all organisms ➢ Start and stop codons: ▪ Start Codon- AUG ▪ Stop Codons- UAA, UAG,UGA ➢ Non-overlapping- read 3 bases at a time linearly ➢ Unambiguous- each codon=1 amino acid ➢ Degenerative- 1 amino acid coded by more than one codon ❖ Translation steps 1. Initiation- takes place by scanning mRNA for a codon ▪ 5’ methyl cap is instrumental 2. Polypeptide formation ▪ mRNA translated in 5’3’ direction ▪ **The polypeptide is synthesizes from the amino end 5’ { (N) terminus } to the carboxyl end 3’ {(C)terminus} ▪ Polypeptide chains- linear polymers of amino acids ▪ Peptide bonds- link the carboxyl group of one amino acid to the amino group of the next amino acid 3. Termination- ends at the stop codon ▪ UAA, UAG, OR UGA specify binding of a release factor ❖ Translation ➢ Multiple copes of a protein are made simultaneously ❖ Information Transfer ➢ Coding capacity of DNA is explained by its great length ➢ 4 letter base specified 20 amino acid alphabet ➢ one allele results in many mRNA molecules via transcription ➢ One mRNA results in many protein molecules via translation 3’ T A T A C C T A A C A/G A C T C G 5’ DNA Double- Helix 5’ A T A T G G A T T G T/C T G A G C 3’ Coding Strand mRNA 5’ 3’ 5’ MethylR5’ A U A U G G A U U G U/C U G A G C 3’ UTR Poly A Tail Cap Anticodon tRNA 3’ U A U A C C U A A C A/G A C U C G 5’ Polypeptide N-- | M e t | A s p | C y s| S T O P| --C Chain Epigenetics ❖ Structure of Hemoglobin ➢ O2/CO2 transport ➢ Tetramer ➢ Adult hemoglobin: ▪ HbA ▪ 2 alpha chains ▪ 2 beta chains ➢ Fetal Hemoglobin ▪ HbF ▪ 2 alpha chains ▪ 2 gamma chains ❖ Developmental patterns of globin gene expression ➢ Hemoglobin synthesis begins in the first few weeks of embryonic development ➢ Shortly before birth there is a smooth switch from fetal gamma-globin gene expression to adult B-globin gene expression ➢ Globin chains are expressed from gamma-globin and b-globin gene clusters ❖ Sickle cell anemia ➢ Reactivation of the fetal form of the hemoglobin complex would potentially solve or decrease the sickle problems since the fetal hemoglobin gamma could replace the mutated hemoglobin beta ❖ Transcriptional Regulation ➢ Binding of transcription factors to regulatory elements ➢ Altering gene accessibility through chromatin modifications ❖ DNA is packaged into nucleosome forming chromatin ➢ Nucleosome- 146 base pairs wrapped around the core histone ➢ Nucleosome remodeler complexes= large multi-protein complexes, use ATP to provide energy to “move” the nucleosome ❖ Chromatin ➢ Chromatin represents both an obstacle to gene expression (makes DNA less accessible), but it also provides many opportunities to regulate gene expression ➢ Changes in chromatin that can affect gene expression ▪ Chromatin remodeling- remodeling complexes reposition nucleosome to provide rapid access to DNA sequence ▪ Histone modifications ▪ DNA methylation ❖ The “Histone Code” ➢ Many post-translational modifications are known to occur at the N terminus ▪ Acetylation ▪ Methylation ▪ Phosphorylation ➢ Alter rates of transcription ➢ Acetylation of specific lysines can release “grip” on associated DNA ▪ S-Serine ▪ K-lysine ❖ Acetylation ➢ Acetylation of positively charged Lysine (K) amino acid residues ➢ Enzymes involved: ▪ HATs- add acetyl group ▪ HDACs- remove acetyl group ➢ Acetylation reduces the over all basic (positively charged) charge of histone tails and as a result the interaction between tails and DNA becomes more loose ➢ Activation ❖ Acetylation- 1. Addition of acetyl groups to lysine (k) at the N terminus of histone tails Enzyme: HAT 2. Neutralized positive charge lysine 3. Loosens DNA around histones and gene is TURNED ON 4. Weak interactions with DNA ❖ Deacetylation- 1. Removal of acetyl groups from lysines of N terminal tails of histone ENZYME: HDAC 2. Positive charge that comes due to NH 3t end of the lysine’s R group 3. Histone tails tightened around DNA and histones 4. Gene is OFF 5. Strong interactions with DNA 6. An excess would inhibit transcription ❖ Methylation= epigenetic modification 1. Addition of methyl group to cytosine of 5’—CG--3’  ENZYME: DNA methyl transfrase DNMT 2. Transcription factors and RNA polymerase blocked from gene 3. The gene is OFF/ transcription DOES NOT OCCUR 4. Recruit methyl binding proteins which in turn recruit histone deacetylase enzymes responsible for removing acetyl groups from histone N- terminal lysines resulting in Transcriptionally silenced chromatin ❖ Demethylation- 1. Removal of methyl cap from cytosine 5’—CG—3’ ➢ ENZYME: DNMT 2. Transcription factors and RNA polymerase not blocked from genes 3. The gene is ON/ transcription OCCURs ❖ Gene expression requires 1. Transcription factors and RNA polymerase 2. Open Chromatin (accessible for transcription machinery) ❖ cDNA microarrays ➢ cDNA Microarrays- simple way to examine levels of gene expression and changes in gene expression ❖ Epigenetics ➢ Epigenetics- heritable changes not caused by changes in the DNA sequence ▪ Affect which parts of the genome are accessible to transcription factors ❖ Genomic Imprinting ➢ Genomic imprinting- characteristics encoded by autosomal genes whose expression is affected by the sex of the parent transmitting the genes ▪ Genomic imprinting of the lgf2 gene in mice and humans affects fetal growth. (a) The paternal lgf2 allele is active in the fetus and placenta, whereas the maternal allele is silent ▪ Silencing genes from one parent=differential DNA methylation in male vs. female germ lines ▪ Methyl (CH3) groups bind to DNA and silence in a sex specific pattern ▪ “Blocked: genes replicated during mitosis and silencing maintained in somatic cells ▪ X-inactivation- turns off entire chromosomes ▪ Imprinting- turns off specific genes ❖ Genomic imprinting in human disease ➢ Two distinct syndromes result from a small, spontaneous deletion of 15q11, depending on the gens paternal origin ➢ 3 imprinted genes in the 15q11 region – 2 silenced maternally and 1 silenced paternally ▪ Parder-Willi syndrome- deletion inherited from father ▪ Angelman syndrome- deletion inherited from mother ❖ Gene expression can be controlled at multiple levels/ transcription regulation 1. Chromatin structure level 2. Transcription level- high transcription (producing lots of RNA molecule) will increase the levels of that particular gene product. Protein levels are tightly regulated: no point in expressing too much or too little. Fine balance. 3. **Alternate splicing- generates different mRNA transcripts from the same transcription units 4. mRNA stability- 5’ cap and polyA influence RNA turnover. Stable RNAs will be continuously translated, whereas unstable RNAs will undergo “pulses” of translation as soon as they are exported’ 5. MicroRNAs (block protein synthesis) 6. Posttranslational modifications- Translation depends on a number of regulatory proteins and RNAs, but also on aminoacid availability and tRNA availability ▪ Translational regulation- alters the rate of protein synthesis, microRNAs, small RNA molecules only 20-24 bases long, complementary base pair to mRNA molecules, target mRNA for degradation, control of timing developmental genes • RNAi- technology utilizes this process to alter gene expression ❖ RNA Interference (RNAi) ➢ Post transcriptional gene silencing ➢ Clinical research targets include: macular degeneration, cancer and antiviral therapy, asthma, hypercholesterolemia, and inherited genetic diseases ❖ Only 1.5% of genome encodes protein the rest… ➢ Telomere repeats ➢ Centromere repeats ➢ Pseudogenes- sequences very similar to known genes but are not translated ➢ Transposons ▪ Transposable element (retrotransposon) ▪ Most abundant repeat sequence of DNA ▪ 45% of genome, can move ▪ Most commin in primates: Alu element • Alu element is transcribed • Reverse trancriptase produces dsDNA molecule • dsDNA molecule is inserted into genome ▪ May serve important evolutionary role ➢ Viral DNA ▪ Retroviral sequences comprise 8% of genome ▪ HERVs- human endogenous retrovirus ▪ Reverse transcriptase copes viral RNA to DNA ▪ Intergrade inserts viral DNA into chromosome ❖ Genes are now classified as DNA sequences that contribute to a phenotype of function, plus sequences, both in the gene and outside it, that control gene expression ➢ Gene regulation can occur at many levels ▪ RNA ▪ DNA ▪ Protein (modification post translation) ➢ Essential to determine when, where and how much od gene product to make ➢ Complexity higher vertebrates comes from more sophisticated control of this process Sequence Mutations ❖ Mutations ➢ Mutations- heritable change in genetic material ▪ Somatic- mutation occurs in pre-mitotic cell or…. ▪ Germline- mutation occurs in pre-meiotic cell ❖ Three basic types of mutations: substitutions, insertions, deletions: ➢ Base substitution mutation ▪ Base Substitution- a base substitution alters a single codon • Transition- substitution of pyrimidine to pyrimidine or purine to purine • Transversion- substitution of purine to pyrimidine or pyrimidine to purine  Silent mutation- does not effect the protein product  Missense mutations- affects the protein product ➢ Effect: region effected, disrupt crucial amino acid, replacement of amino acid similar or dissimilar chemically  Nonsense mutations- creates a new stop codon ad protein production will be truncated or nonfunctional ➢ Effect: location in protein ➢ Base insertion/ Deletion Mutations ▪ Severe, frameshift mutations ▪ Affects the codon and all downstream codons ▪ Base Insertion- adding a base alters the reading frame and may change many codons ▪ Base deletion- deleting a base alters the reading frame and may change many codons ❖ Mutations: Effect on gene function 1. Altered level of expression 2. Loss-of-function- gene inactivation or nonfunctional gene product ▪ Recessive mutation 3. Gain of function- gene activation when or where it should not be ▪ Dominant mutation ❖ Transitions vs. Transversions ➢ Spontaneous point mutation biased in favor of transitions ➢ Ration of transitions to transversions 2:1 ❖ What causes mutations ➢ Spontaneous mutations- random, unpredictable= errors in replications ▪ Tautomeric shifts- positions of protons change ▪ Rare unstable forms of the 4 bases ▪ Abnormal pairing ▪ Wobble base pairing- If base is in rare form at moment of replication inserts wrong paring partner ➢ Induced mutations- chemical mutagen/radiation ▪ Less than 1 error in a billion nucleotides arises in the course of DNA synthesis! ▪ Chemical: • Base analogs- chemicals incorporated into DNA look like bases but pair wrong • Cause chemical changes in the DNA which result in mis-pairings: alkylating agents, deamination, oxidative reactions • Cause insertions or deletions by distorting the 3D structure of the helix: intercalating agents  Intercalating agents produce mutations by “sandwiching themselves between adjacent DNA base ▪ Radiation: • Ionizing radiations- breaks covalent bonds by dislodging electrons from atoms, result in DS breaks • UV causes formation of abnormal covalent bonds and bulges in DNA • Thymine dimers- induced by UV light block DNA replication- inhibition of cell division ❖ Wobble base pairing ➢ Wobble- due to flexibility of DNA helical structure ➢ Wobble in translation- ▪ Degeneracy ▪ Mutation of 3 base in codon may not lead to amino acid change ❖ DNA polymerase proofreading ➢ Competition: polymerase activity vs. 3’5’ exonuclease activity ❖ Mismatch Repair ➢ The most important role of mismatch repair is as a last chance error correcting mechanism in replication ➢ Repair is super efficient ➢ 1 mutation per 1 billion base pairs in replication ❖ Excision Repair ➢ Multistep enzymatic process ➢ Starch of a damaged DNA strand is removed ➢ Replaced by resynthesis using the undamaged strand as a template Forensics ❖ HGP goal: Obtain a complete catalog of our gene ➢ Completed in 2003 ➢ Variability in human genotypes exist in the form of: ▪ SNPs (Single Nucleotide Polymorphisms) ▪ STRs(2-9bp Short Tandem Repeats) ▪ VNTRs- 10-100 base pair sequence repeated in tandem, while the repeated units themselves are usually the same between individuals the number of times they are repeated varies. ❖ STRs(Short Tandem Repeats) ➢ STRs(2-9bp Short Tandem Repeats)- very short DNA sequences repeated in tandem in the genotype ➢ Differences between persons occur in the number of repeats ❖ SNPs (Single Nucleotide Polymorphisms) ➢ SNPs (Single Nucleotide Polymorphisms)- Single base change in a region of DNA ❖ How does expansion occur? ➢ Strand slippage and hairpin look formation during replication ➢ The number of copies of a trinucleotide may increase in replication owing to the formation of hairpins ❖ Allele and locus ➢ Locus- a particular position or location on a chromosome ➢ Allele- alternate sequence at a particular locus ❖ Genotype designations ➢ AA- homozygous dominant ➢ Aa- heterozygous ➢ aa- homozygous resessive ➢ Genotypes can represent ▪ Genes ▪ Alleles of non-protein coding segments for the DNA ❖ What is a DNA profile ➢ For autosomal genes, each human has 2 copies for every gene, one from mother one from the father ➢ Together they make up a genotype for that particular gene or locus ➢ The DNA profile is a unique representation of a specific individual ❖ Possible outcomes ➢ Match ➢ Exclusion ➢ Inconclusive ❖ Huntington Disease: Autosomal Dominant ➢ Progressive degeneration of nerve cells, with death 10-20 years after initial symptoms ▪ Child of an HD parent has a 50-50 chance of inheriting the HD gene ▪ A person who inherits the HD gene will sooner or later develop the disease ▪ Disease worsens in successive generations ➢ Huntington Disease and Trinucleotide repeats: ▪ Normal Huntington Disease (htt) gene has (CAG)n repeats within an exon. • As the altered HD gene is passed from one generation to the next, the size of the CAG repeat expansion often increases in size. Larger repeat expansions are usually associated with an earlier onset of signs and symptoms. This phenomenon is called anticipation • CAG repeat # determines phenotype  Threshold of normal alleles= 35,36,37  Larger the repeat #, earlier age of onset & more severe disease  <34= Normal phenotype ▪ Normal Huntington protein has polyglutamine repeats • Huntington protein interacts with other proteins regularly found only in the brain • Large polyglutamine tract causes protein aggregates in neurons • Kills the neuron • Deaths of different neurons = different clinical features ▪ Normal allele has small number of copies & is stable. ▪ Mutant (mhtt) allele has larger number of copies & is highly unstable Chromosomal Abnormalities ❖ Non-disjunction ➢ Non-disjunction- the failure if homologous chromosomes to properly segregate ➢ Very serious consequences-proper number & kind of chromosomes required for proper development ➢ Monosomy (loss of one member of the homologous pair) almost always lethal ➢ Trisomy (one additional member of an homologous pair) almost always lethal ❖ Non-disjunction of autosomes ➢ Trisomy 13: Patau Syndrome (extreme malformation of organ systems, survival < 3 months) ▪ 1/6000 live births ➢ Trisomy 18: Edward Syndrome (slow growth & multiple abnormalities, survival 2-4 months) ▪ 1/8000 live births ➢ Trisomy 21: Down’s Sydrome (characteristic facial features & retardation, survival into adulthood) ▪ 1/660 live births ▪ range of mental impairment from mild to severe ▪ most well studied- results from over expression of genes in chromosome 21 ▪ Less devastating because the gene is poor ❖ Non-disjunction of sex chromosmes ➢ X and Y act as pairing partners and normally segregate ➢ Homogametic- Females- XX ➢ Heterogametic- Males- XY ➢ Imbalance in sex chromosomes is less damaging than imbalance in autosome. ❖ Sex chromosome dosage in mammals ➢ Random X-inactivation-only one X is in an active state; all others are inactive & condensed into Barr bodies ➢ Easy assay is Barr body (small, darkly staining body in the interphase cell of normal females) ➢ # Barr Bodies = # supernumerary X chromosomes ➢ X-inactivation allows for survival of individuals with sex chromosome aneuploidy ❖ Sex Chromosome Aneuploidy ➢ Klinefelter Syndrome (XXY) (2n+1)- 1 Barr body, abnormal male ➢ Turner’s Syndrome (XO) (2n-1) (45,X)- 0 Barr bodies, abnormal female ❖ Chromosomal mutations/ Abnormalities ➢ Chromosomal rearrangements: lead to abnormal gametes ▪ Duplications • Important source of new genes with novel functions ▪ Deletions ▪ Translocations ▪ Inversion ➢ Aneuploidy- individual chromosomes ➢ Polypoidy- entire sets of chromosomes ❖ Chromosome duplications and deletions ➢ Duplication : ➢ Deletion: ➢ EFFECTS: Unbalanced gene dosage leads to developmental abnormalities. ❖ Translocations ➢ Translocations- movement of genetic material between nonhomologous chromosomes ➢ Reciprocal: ➢ Nonreciprocal- same as reciprocal except change in only 1 chromosome ➢ Robertsonian ❖ Translocation trouble ➢ Acute Promylociytic Leukemia ▪ Reciprocal and balanced mutation ▪ Fuses part of PML gene (15) with part of RARA gene (17) ▪ Resulting protein is PML-RARα ▪ Normal RARα protein controls genes for differentiation ▪ Normal PML blocks cell growth and stimulates apoptosis ❖ Chromosome inversions ➢ May lead to an alteration in gene function and regulation ➢ Caused by Inversion Loop ➢ Rec 8 Syndrome ▪ At least one parent has a chromosome 8 inversion ▪ Heart and urinary tract abnormalities ▪ Moderate to severe intellectual disability ▪ Distinctive facial appearance ▪ Many do not survive past early childhood ▪ Deletion of piece of short arm ▪ Duplication of piece of long arm ❖ Effect of age one women’s reproductive potential ➢ The older the female gets the more incidence per 1,00 births could happen ❖ Effect of age one men’s reproductive potential ➢ 20 rare but serious genetic disorders are less rare in children born to men beyond young adulthood. ➢ Men over age 40 are six times more likely to father autistic children as those under 30
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