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2/19/10 MUTATION JARGON

Science Dec. 2007 http://www.sciencemag.org/cgi/content/full/318/5858/1842

• Will a base pair substitution, addition or deletion cause a phenotype?

• Will a larger scale change such as a gene duplication cause a phenotype?

• What information do you need to address this question?

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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/

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the “cystic fibrosis” gene codes for 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

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• All individuals with sickle cell anemia have the same missense mutation in the B globin gene.

• In contrast, over 1600 different mutant alleles of the CF gene have been discovered world-wide

Cystic Fibrosis Gene: CFTR http://www.genet.sickkids.on.ca/cftr/StatisticsPage.html see statistics, gene, consortium data search database: pull up gene map

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

Sequence variation = non-disease-causing. It is sometimes designated as “polymorphism”, According to the definition on this web site, a “sequence variation” has to reach an allele frequency of 1% to be a polymorphism. When a sequence variation is found in only one single individual, it is not possible to determine if it is a “non-disease-causing” variation.

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

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find most common mutation: http://www.genet.sickkids.on.ca/cftr/ConsortiumDataPage1.html http://www.genet.sickkids.on.ca/cftr/resource/Table1.html

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MUTATION JARGON POINT MUTATION or GENE MUTATION

Scale of mutation is small Alteration of a single base pair or a small number of adjacent base pairs

↓

AT THE DNA LEVEL single base pair substitutions: transitions & transversions

indels: insertion or deletion of a few base pairs other: transposon insertion

AT THE LEVEL OF AT THE PROTEIN GENE EXPRESSION LEVEL promoter mutations nonsense splicing mutations missense regulatory mutations [neutral] silent frameshift

AT THE LEVEL OF GENE FUNCTION

loss-of-function gain-of-function

[neutral] ↑

CHROMOSOME MUTATION

involves segments of chromosomes or whole chromosomes CNVs = copy number variations

alterations in chromosome structure and number (deletions, duplications, translocations and inversions)

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AGT

TCA

TCA

UCA5' 3'

5'

3'

3'

5'

5' 3'

transcription

AGT

splicing and processing in eukaryotes

DNA

UCAAGU

mRNA

serine codon on mRNA

serine anticodon on tRNA

5'

serine serine attached to tRNA at 3' endser

3' 5'

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

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NOTE: code is always in RNAspeak

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What is a missense mutation?

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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?

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Hard to say a priori without additional information on: • the nature of the amino acid substitution • the site of the mutation in the protein 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 • if the mutation affects the folding or stability of the protein • if 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

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Recent study on a protein called human factor VIII which 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

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

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

arginine?

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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: AGA arg AAA lys both are basic amino acids: substitution of arg for lys may not affect protein function arg = arginine lys = lysine

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BUT: don’t assume that a chemically equivalent substitution will always be neutral Example: Protein: Triose-P-isomerase Glu Asp change in active site decreases catalytic activity 1000X glu= glutamic acid asp = aspartic acid

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Silent (same sense) Mutation: a mutation that doesn’t change the meaning of the codon (the same amino acid is specified) CUU (leu) CUC (leu) Degenerate code: many amino acids are specified by more than one codon

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Nonsense mutation: ?

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Nonsense mutation: a mutation that generates an abnormal stop codon It results in premature termination of translation and a truncated polypeptide

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Reading Frame: the codon sequence that is determined by reading nucleotides in groups of 3 from some specific start codon (AUG) How does the translation machinery know where to start?

How does a eukaryotic ribosome identify the correct reading frame?

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Frameshift mutatio ?

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Frameshift mutation: addition or deletion of one or a few base pairs causing a change in the translational reading frame

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BRCA1 database BRCA1 gene plays a role in the repair of double-stranded breaks in DNA About 5% of breast cancer cases are caused by an inherited susceptibility allele. A mutation in a gene called BRCA1 is thought to account for approximately 80% of families with a high incidence of both early-onset breast and ovarian cancer. Table 1 summarizes some of the data that has been collected on BCRA1 mutations in such families. Table 2 shows neutral polymorphisms found in control families (not showning an increased frequency of breast and ovarian cancer). Table 1. Predisposing mutations in BRCA1. Science 266: 66 1994 NA indicates not applicable. ND = not determined Mutation Frequency Kindred Codon # Nucleotide Coding in control change effect chromosomes 1901 24 -11 bp frameshift

0/180

2082 1313 Gln --> Stop 0/170 1910 1756 Extra C frameshift 0/162 2099 1775 T-->G Met --> Arg 0/120 2035 NA ND Loss of

transcript ND

Table 2. Neutral polymorphisms in BRCA1. For the frequency in control chromosomes, the number of chromosomes with a particular base at the indicated polymorphic site is shown (A,C,G, or T). Frequency in control chromosomes Codon Base Name location in codon* A C G T PM1 317 2 152 0 10 0 PM6 878 2 0 55 0 100 PM7 1190 2 109 0 53 0 PM2 1443 3 0 115 0 58 PM3 1619 1 116 0 52 0 *That is, position 1,2 or 3 of the codon.

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Information Containedin the Sequence of a Gene

Proper functioning of a gene requires:

1. Coding Region specifies RNA & amino acid sequence

1. An intact gene product (protein or RNA)

2. Other Sequence Information (signals for generating RNA)

2. Proper expression of the gene:

a. promoter (RNA polymerase binding site) transcription termination site

a. transcript generated from the correct stretch of DNA

b. regulatory elements (operators in prok's; enhancers in euk's)

b. transcript generated in the appropriate amount at the appropriate time in the appropriate cells

c. splice site signals c. transcript spliced correctly

DNA RNA PROTEIN

How do point mutations affect the functioning of a gene?