At the Level of Gene Function: CHROMOSOME MUTATION

Home , Point mutation

EFFECT OF MUTATIONS ON PROTEIN FUNCTION GENE MUTATION = POINT MUTATION (scales of mutation is small and is localized to a specific region, a single nucleotide or a few adjacent base pairs)

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at the DNA level: Ë single base pair substitutions: transitions & transversions Ë single (or a few) base pair addition or deletion: indels Ë gene mutation by transposon insertion

at the level of at the protein gene expression: level: promoter mutations nonsense splicing mutations missense CHROMOSOME MUTATION regulatory mutations [neutral] • involves segments of chromosomes or silent whole chromosomes or whole genomes frameshift • alterations in chromosome structure and number • deletion, duplications, translocations and inversions at the level of gene function: • CNVs: copy number variations loss-of-function gain-of-function [neutral]

1 Finding your way around a eukaryotic gene

ç upstream = 5’ of…. downstream = 3’ of… è

2 Conventions for displaying gene sequences:

• Only the mRNA-like strand is displayed (complementary strand not shown) • Sequence reads 5’ to 3’ • A cDNA sequence will reflect the sequence of the spliced mRNA and will therefore not include intron sequence • A genomic sequence will include introns and exons and adjacent regulatory regions – sometimes the introns will be indicated in lower case and the EXONS in uppercase (see pg 8 of this lecture)

3 Genomic DNA sequence display

LOCUS NG_011751 7897 bp DNA linear PRI 05-FEB-2012 DEFINITION Homo sapiens sex determining region Y (SRY), RefSeqGene on chromosome Y. TGACCTTCATTTTATGGAGAGAAACAAGCTATAACATGTAGTATCTAAGCTGATTAGAAGAACTAAAAAG AGAAGCTCATACTTGTGCATCAGAAGGTAAATGAAAGAGTGAAGTTACCTCTTTGTTTTAAGGAAGAAAG GAAAATTGTGGATGTCATCTGTTTTCTGTTTACATATTTCAGGCATGGATAGCCACAATGTGATTTTAAG ACGGTTAGTTACAACTGATTTGAAAAAAAAAAAAAATGCTTCACTCTATGAGAAATTTCTTCCCAAGTAT GAAACCTTGTTTTTACAGGCAATTTCCTATACTTTGAAAAAATCAAAATAATAAAGTAAAAGAAAAATAA TTCAGGTGAAGTTAGAGAAAAAAACAGGCAGCATTATTTTAAAGTTGTAAACTATTTTGTTTACTTATAG TTTAATTTACATGTAGTAGATATGCATTTGTAAGGTTCTTCGGCTCAGGTAGGAGATCATTCTATTTCCC ACTGCACCCTACTTCATCCTCCCACTGGCAAATAATTAGATTATCCCTGGGAAAAAAAGATGCCAGTAAA ATTGATCATGTTTAAATGCATCAGTTGCTAGGTGATTTATCTGATTAAGTCTTGAAACAGTAGAACCTAG CAATTAAAGTGAGCATTAACTTCTACCTACCAAATCAGAAGACTATTCTAACTTTTTGAGAATTAGATGT TGAAAATATGGCCCATGAATTTAGCATGGTTAAAATAAAAAACATGCAAACAAAACAAACCCAACATCTT GAAAGGACATTTGACTCTAAAGTCCCAAAAATAATCACAAGTCTAAAAATCCTAAGTTTAGTGTTACTCT ATTACACCTTTTTATTTGTAAGTGTCCTTTCACAAAAGTTTTAAATTTTGCTCTTGTGCATTTTATTTAC CTTTTCTTTTGTTGTTTGTGTCTTTGGTGACCTGCCAACCATTAGACTTCAAAAAACAGCCTATAGCCAA GCTGCAGGATAAATGAACACATAAGTTGACTTAGAATAGTCAACTCTGTCTAGTATACAATTTATGGGGG ATGGTTTATGACCACATATATTTCTACTTTGATGGGAATATCTTGAGATAAAATTAGAGAGAATGAGTGG AGTAATATTCACAACATTTTTGCTGCATTCATCCCTGAATTTGAAGAAATACCAAAGTACATCTTGTGAG GAGAAAAAATAAATAAATTCATATAAAATGTTGTGGGTTTTATTCTTTATGCAGTGGTAAACTGTGTTTG CATACACCATAGCAATTAAATTAGGGCTACAAAGGGTATTTAACTAATGAGCATAAAATACCTTAATGTA CCTCAAATGCAATTAATTGCATTGGACCAATCTAAGTTACTATTCTTCAGTTTTCATTTTTATTTCATTA TTCATTTCATTTTTATTCTGATATAAAAATGAACCAGGATCTGTGTGAAATTATTTGAATCTAATGTCTT TGAACATTTTTCTTACCATACCTTAAGATTAAAAAAACAAAAAAAAATCCCTTAGTTTGGCAACTTTTGC TGTTGGTTAAGCCCGTTTGGATTTAACATTGACAGGACCAGCTAACTTCCTACCAGTTAACATTGCTTGT …………… etc

4 cDNA/mRNA sequence display

LOCUS NM_003140 897 bp mRNA linear PRI 17-DEC-2011 >gi|4507224|ref| Homo sapiens sex determining region Y (SRY), mRNA GTTGAGGGGGTGTTGAGGGCGGAGAAATGCAAGTTTCATTACAAAAGTTAACGTAACAAAGAATCTGGTA GAAGTGAGTTTTGGATAGTAAAATAAGTTTCGAACTCTGGCACCTTTCAATTTTGTCGCACTCTCCTTGT TTTTGACAATGCAATCATATGCTTCTGCTATGTTAAGCGTATTCAACAGCGATGATTACAGTCCAGCTGT GCAAGAGAATATTCCCGCTCTCCGGAGAAGCTCTTCCTTCCTTTGCACTGAAAGCTGTAACTCTAAGTAT CAGTGTGAAACGGGAGAAAACAGTAAAGGCAACGTCCAGGATAGAGTGAAGCGACCCATGAACGCATTCA TCGTGTGGTCTCGCGATCAGAGGCGCAAGATGGCTCTAGAGAATCCCAGAATGCGAAACTCAGAGATCAG CAAGCAGCTGGGATACCAGTGGAAAATGCTTACTGAAGCCGAAAAATGGCCATTCTTCCAGGAGGCACAG AAATTACAGGCCATGCACAGAGAGAAATACCCGAATTATAAGTATCGACCTCGTCGGAAGGCGAAGATGC TGCCGAAGAATTGCAGTTTGCTTCCCGCAGATCCCGCTTCGGTACTCTGCAGCGAAGTGCAACTGGACAA CAGGTTGTACAGGGATGACTGTACGAAAGCCACACACTCAAGAATGGAGCACCAGCTAGGCCACTTACCG CCCATCAACGCAGCCAGCTCACCGCAGCAACGGGACCGCTACAGCCACTGGACAAAGCTGTAGGACAATC GGGTAACATTGGCTACAAAGACCTACCTAGATGCTCCTTTTTACGATAACTTACAGCCCTCACTTTCTTA TGTTTAGTTTCAATATTGTTTTCTTTTCTCTGGCTAATAAAGGCCTTATTCATTTCA A sequence logo showing the most conserved bases around the initiation codon from all human mRNAs. The larger the LETTER at a given location, the greater the importance of a the specific base

LOCUS NP_003131 204 aa linear PRI 17-DEC-2011 >gi|4507225|ref| sex-determining region Y protein [Homo sapiens] MQSYASAMLSVFNSDDYSPAVQENIPALRRSSSFLCTESCNSKYQCETGENSKGNVQDRVKRPMNAFIVW SRDQRRKMALENPRMRNSEISKQLGYQWKMLTEAEKWPFFQEAQKLQAMHREKYPNYKYRPRRKAKMLPK NCSLLPADPASVLCSEVQLDNRLYRDDCTKATHSRMEHQLGHLPPINAASSPQQRDRYSHWTKL

Amino acid sequence reads from the N (amino) to the C (carboxyl) terminus

6 Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die. This adage, from northern European folklore, is an early reference to the common genetic disease recognized today as cystic fibrosis. As the saying implies, the disorder once routinely killed children in infancy and is often identifiable by excessive salt in sweat.. (Scientific American Dec. 1995)

Cystic fibrosis: most common severe recessive monogenic disorder affecting people of European descent

Info about cystic fibrosis http://www.nlm.nih.gov/medlineplus/cysticfibrosis.html http://ghr.nlm.nih.gov/condition=cysticfibrosis http://www.ygyh.org/

7 the “cystic fibrosis” gene codes for the CFTR protein which is a transmembrane protein involved in chloride transport

(note gene is named for its mutant phenotype and not for the protein that it specifies)

CFTR= cystic fibrosis transmembrane conductance regulator

8 http://www.genet.sickkids.on.ca/cftr/GenomicDnaSequencePage.html

9 http://www.genet.sickkids.on.ca/cftr/MRnaPolypeptideSequencePage.html

10 The first questions a researcher interested in exploring the molecular genetics of a disease state addresses generally are 1. Does everyone affected with the disease have a mutation in the same gene – in other words, is the disease genetically heterogeneous? 2. For a given gene, what is the mutational spectrum for individuals with this disease—does every affected person have the same mutation or are there lots of different mutations? 3. How are the mutations distributed in the gene and how do they affect gene function?

Cystic fibrosis is not genetically heterogeneous but it shows extensive allelic heterogeneity • Only mutations in the CF gene (see next page) cause CF, BUT over 1900 different mutant alleles of the CF gene have been discovered world-wide • In contrast All individuals with sickle cell anemia have the same missense mutation in the B globin gene.

11 http://www.genet.sickkids.on.ca/cftr/StatisticsPage.html

12 CF mutations are distributed throughout the gene http://www.genet.sickkids.on.ca/cftr/PicturePage.html

Retrieval of Genetic Information: Central to any information storage system is the ability to access and retrieve the information and to convert it to a usable form. In addition to the sequence information that will be translated into protein via the triplet code, a gene also contains sequence information that specifies 1. where transcription starts and stops on a given stretch of DNA and which strand of DNA is transcribed 2. where splicing occurs (exon/intron boundaries) 3. where, when and at what level the transcript will be produced

14 NOTE: code is always in DNA TCA 5' 3' RNAspeak AGT

transcription

TCA

5' 3' 5' 3' 3' UCA 5' AGT

splicing and processing serine codon in eukaryotes on mRNA mRNA UCA AGU serine anticodon 3' 5' on tRNA

serine serine attached to tRNA ser at 3' end

Chemical conversion of TCA into serine. Accuracy of translation depends on precise matching: (1) of an amino acid with its cognate tRNA (2) of the anitcodon of a charged tRNA with its corresponding codon on the mRNA http://en.wikipedia.org/wiki/Genetic_code

http://en.wikipedia.org/wiki/File:GeneticCode21-version-2.svg

16 What is a missense mutation?

17 Missense mutation: a mutation that alters a codon so that a different amino acid is specified

How will any given missense mutation affect the functioning of a protein?

18 Hard to say a priori without additional information on: • the nature of the amino acid substitution • the site of the mutation in the protein • whether the change is in a highly conserved amino acid

A missense mutation may 1. have virtually no affect on protein function – especially if a chemically similar amino acid is substituted

2. partially or completely inactivate the protein if • the amino acid substitution is in the active site or another site critical for function • the mutation affects the folding or stability of the protein • the mutation affects the processing of the protein or interferes with its transit to the appropriate cellular compartment. See interesting example: In Sex Reversal, Protein Deterred by Nuclear Barrier http://fire.biol.wwu.edu/trent/trent/sexreversal.pdf

3. result in a gain-of-function (see cancer genetics lecture)

19 A protein called human factor VIII has a critical role in blood clotting (Nature November 25, 1999)

• Factor VIII is a glycoprotein that has a critical role in blood coagulation • This protein circulates as a complex with other proteins • Gene coding for clotting factor VIII is mutated in the X-linked disease state hemophila A

21 different amino acid residues in factor VIII are known to be sites of deleterious mutations in patients with hemophila • A number of these are in the hydrophobic protein core • Other mutated amino acids are involved in hydrogen bonding networks that clearly stabilize protein folding • Still others are on the exposed surface of the protein and presumably are important for the interaction of factor VIII with other proteins

A "ribbon diagram" of the structure of the hemophilia domain of human factor VIII

• In this figure, the positions in the protein fold that are found to be mutated in patients with hemophilia A are shown by spheres. • Dark spheres are sites that display severe defects in clotting when mutated • Light spheres are sites that display milder defects. • The atoms at the bottom of the protein are amino acids thought to embed themselves into exposed membranes at sites of blood-vessel damage.

http://depts.washington.edu/mednews/research/hemop hilia.html

21 The enzyme lactate dehydrogenase catalyses the following reaction: pyruvate + NADH à lactate the NAD+

What would the effect be of substituting a different amino acid for arginine?

22 BUT: don’t assume that a chemically equivalent substitution will always be neutral

Protein: Triose-P-isomerase Glu à Asp change in active site decreases catalytic activity 1000X glu= glutamic acid asp = aspartic acid

23 Neutral Mutation: • a mutation that has no effect on the Darwinian fitness of its carrier: an allele that has a negligible effect on the ability of the organism to survive and reproduce

Neutral Missense Mutation: • a subset of missense mutations in which the effect of the amino acid change on protein function is negligible or is not deleterious to the organism for example: codon AGA specifies arg à codon AAA specifies lys arg = arginine lys = lysine both are basic amino acids: substitution of arg for lys may not affect protein function

NOTE: you are responsible for frameshift, nonsense and silent mutations even though we will not cover these terms in class.

26 How do point mutations affect the functioning of a gene?

DNA RNA PROTEIN

Information Contained Proper functioning in the Sequence of a Gene of a gene requires:

1. Coding Region 1. An intact gene product specifies RNA & amino acid sequence (protein or RNA)

2. Other Sequence Information 2. Proper expression of the (signals for generating RNA) gene: a. promoter (RNA polymerase a. transcript generated from binding site) the correct stretch of DNA transcription termination site b. regulatory elements b. transcript generated (operators in prok's; in the appropriate amount enhancers in euk's) at the appropriate time in the appropriate cells

c. transcript spliced correctly c. splice site signals