unit 2 genetics

Forward mutation

A mutation that changes the wild type allele of a gene to a different allele.

Effect: usually leads to partial/complete loss of normal gene function; sometimes can create new/altered function (can be beneficial)

• many ways to disrupt a functional protein (nucleotide substitutions, insertions, deletions)

• ex: disruption of brown eye pigment → blue eyes

Reverse mutation

A mutation that causes a mutant allele to revert back to the wild type.

Effect: restores the original function that was previously lost

• true reversion: original nucleotide restores at the exact mutation site

• second-site reversion: mutation elsewhere that repairs the mutation without repairing the original nucleotide

Ames test: uses reverse mutations to detect potential carcinogens by measuring whether a chemical can cause mutated bacteria to revert back to their functional wild-type state

Superscript (+)

The notation used to designate wild type alleles.

Substitution

A mutation in which a base at a certain position in one strand of the DNA molecule is replaced by one of the other three bases

• transitions: purine to purine

• transversions: purine changes to pyrimidine or vise versa

• types: silent, missense, nonsense

• only alter specific codon where swap happens → rest of sequence downstream remains in correct reading frame

Transitions

Base substitutions where a purine replaces another purine.

Transversions

Base substitutions where a purine changes to a pyrimidine or vice versa.

Deletion

A mutation in which a block of one or more nucleotide pairs is lost from a DNA molecule.

• frameshift deletion: if number of deleted bases is not a multiple of three → entire reading frame downstream is disrupted

Insertion

A mutation characterized by the addition of one or more nucleotide pairs.

Inevitable mutation

A mutation that becomes permanent in both strands of the double helix if repair enzymes do not correct the problem before the next round of DNA replication.

Depurination

A DNA alteration in which a purine base is hydrolyzed from the sugar-phosphate backbone

• sugar phosphate backbone remains intact but base is missing

Deamination

The removal of an amino group (NH2) from a base, such as converting cytosine to uracil.

Proofreading function

An editing function of DNA polymerase in the form of a nuclease that becomes active when the polymerase makes a mistake during DNA replication.

Tautomerization

A cause of mistakes in DNA replication related to the fact that each base has 2 tautomers.

Mutagen

Any physical or chemical agent that raises the frequency of mutations; most mutagens are carcinogenic.

Homologous recombination

A method to repair double-stranded breaks accurately by using complementary base pairing.

• Very accurate because it copies directly from an identical template

Nonhomologous end joining

A method to repair double-stranded breaks that brings together DNA ends that were not originally adjacent to each other.

• Directly connects broken ends of DNA back together without needing a matching template

• very error prone

Multicellular organisms have ___ mutation rates per generation tha bacteria

higher

sperm cells have ____ mutation rate than in human eggs

higher

What happens if repair enzymes do not correct proble before next round of DNA replication

mutation becomes permanent in both strands of double helix = heritable mutation

complementation test

A genetic test used to determine if two recessive mutations affect the same gene or different genes

• wild type: mutations in different genes

• mutant type: mutations in the same gene

• (-) no complementation → complementation group

• (+) complementation

Seymour Benzer Experiment w/ Bacteriophage T4

• gene can undergo recombination

• gene produces mutation

• helped define intragenic recombination and deletion mutations

intragenic recombination

Benzer proved that recombination can also happen within the boundaries of a single gene between sites → yields two products

gene expression

The process by which genes info is converted to RNA and then polypeptide {transcription and translation}

transcription

polymerization of ribonucleotides (guided by complementary base pairings) produces an RNA transcript of a gene

• eukaryotes: nucleus , prokaryotes: cytoplasm

initiation

1. RNA polymerase binds to double stranded DNA @ beginning of the gene

2. RNA polymerase binds to promoters

3. After binding to promoter, RNA polymerase unwinds part of double helix → complex formed between RNA polymerase and unwound promoter is called an open promoter complex

4. enzyme identifies template strand and RNA polymerase aligns first two ribonucleotides at the 5’ end of the new RNA

5. RNA polymerase forms phosphodiester bond between first two ribonucleotides

6. RNA polymerase releases a subunit

elongation

• read 3’ to 5’ direction and created 5’ to 3’ direction

1. Region of DNA unwound by DNA polymerase is the transcription bubble

2. sense region: protects from exonucleases and external factors

3. antisense region: serves as the template strand to create mRNA

Termination

• RNA sequences signal the end = terminators

  • intrinsic - cause RNA polymerase core enzymes to terminate transcription

  • extrinsic - require additional proteins to bring termination

terminators

specific sequences in mRNA that are transcribed from the gene

• harpin loops: RNA folds back on itself bc of complementary base pairings

• releases both RNA polymerase and completed mRNA chain from DNA

Eukaryotic RNA Processing

1. RNA splicing

2. 3’ poly A tail

3. 5’ methylated cap

RNA Splicing

exons: sequences found in DNA + mature mRNA (E for expressed and exited out to translation)

introns: regions on pre mature mRNA spliced out of primary transcript

splicesome

small nuclear ribonucleoproteins that make precise cuts @ splice donor and splice acceptor sites

• cleaves introns to be degraded by exonucleases

alternative splicing

transcript spliced in different ways → multiple unique mRNA and protein variants

• allows for adaptability or evolutionary advantages

• some exons not expressed and cleaved out rather than spliced by the spliceosome at each splice acceptor sites

5’ methylated cap

backward G nucleotide added to 5’ end by capping enzyme + modified with methyl groups

3’ poly A tail

Poly-A polymerase adds a tail

RNA editing

recodes codon and expands protein diversity

Reverse Transcription

copies RNA genome into double stranded DNA

Translation

sequence of nucleotides in mRNA directs assembly of amino acid sequence in corresponding polypetide

• occurs in ribosomes (in cytosol) which coordinate movement of tRNA

tRNA

• serves as adapter molecules that mediate the transfer of info from nucleic acid to protein

• short single stranded RNA molecule

• each tRNA carries one particular amino acid

• has two functional units: amino acid attachment site @ 3’ end & anticodon loop

Amino acid attachment site @ 3’ end on tRNA

• located on top of L shape {CCA sequence}

• this end chemically attaches to aminoacyl-tRNA-synthetase

anticodon loop on tRNA

• located on bottom loop

• contains anticodon which pairs complementary w codon on mRNA

aminoacyl-tRNA-synthetase

catalyze the attachment of tRNA to their corresponding amino acid forming tRNA

two step process: covalent bond between an amino acid and the 3’ end of its correspondig tRNA

• enzyme has recognition sites

• synthetase activates amino acid → AMP amino acid → carboxyl group from AMP transfers to hydroxyl group → charged tRNA

wobble

ability of the 5’ most base of an anticodon to pair w more than one type of base at the 3’ of the codon

Ribsomes

• recognize mRNA features to start transcription

• ensure accurate interpretation of genetic code by stabilizing interactions between tRNA and mRNA

• supply enzymatic activity that links amino acids in the growing peptide

• moving along 5’ to 3’ they expose mRNA to codons in sequence, ensuring linear addition of amino acids

• help end polypeptide synthesis by dissociating both from mRNA and polypeptide

structure of ribsomes

1. 30S subunit - intially binds to mRNA

2. 50S subunit - contains peptidyl transferase which form peptide bonds between amino acids

before translation begins = two subunits exist separately; then at the start of translation they come together to form complete ribosome

tRNA binding sites

aminoacyl (A) site: charged tRNA binds here first

peptidyl (P) site: intiating tRNA binds here first and tRNA carrying growing peptide are located here during elongation

exit (E) site: releases tRNA from ribosome

Initiation - Translation

sets stage for addition of nucleotide or amino acid building blocks to create initiation complex

prokaryotes: ribosome binding site

1. shine-dalgarno box: 5’ AGGAGG 3’

2. 5’ AUG 3’: initiation codon (modified methionine)

eukaryotes:

1. small ribosomal subunit recognizes initiation factors

2. complex migrates along mRNA to the initiation site 5’ → 3’

elongation - translation

nucleotides and amino acids are added successively to growing peptide

1. tRNA @ P site and tRNA @ A site = forms peptide bond between amino acids

2. ribosomes move with help of elongation factors → initiating mRNA transferred to E site from P site → other tRNA shifts from A site to P site after tRNA at P site gives amino acid to tRNA at A site

3. empty A site receives another tRNA. → initiating tRNA bumped off to E site and leaves ribosome

4. Peptidyl transferase forms second peptide bond

5. amino acid grows longer and tRNA moves from A to P to E site

elongation factors

proteins that aid in elongation phase of translation

termination - translation

brings polypeptide synthesis to a halt

• stop codons: translation ends when ribosome encounters 1/3 nonsense/stop codons: UAA, UAG, UGA

• release factors: bc no tRNA complementary to stop codons, release factors bind to ribosomes A site

release factors

specialized proteins that

1. signal ribosome to halt peptide synthesis

2. release completed polypeptide from tRNA attached to C terminal

3. tRNA, mRNA, ribosomal subunits then dissociate from each other

Posttranslational Modification

changes such as phosphorylation that occur due to polypeptide after translation

• examples: methylation, acetylation, glycosylation, etc…

eukaryotic vs. prokaryotic processes differences

1. presence of nuclear membrane in eukaryotes

2. eukaryotic complexities

3. variations in way translation is initiated

4. additional trasncript processing in eukaryotes

silent mutation

changes codon into mutant codon that specifies exactly the same amino acid

• usually third nucleotide of codon

• does not affect gene expression nor phenotype

missense mutation

changes codon into a mutant codon that specifies a different amino acid

• conservative substitution: substituted amino acid has similar chemical properties to the one it replaces and has little to no effect on protein function

• nonconservative substitution: substituted amino acid is very different and has very noticeable consequences

nonsense mutation

changes codon to stop codon

• results in shortened truncated proteins

frameshift mutation

insertion and deletion of nucleotides

• if number of extra/missing chromosomes is not divisible by 3 → skews reading frame downstream of mutation and usually results in truncated proteins

loss of function mutation

mutation that reduces or abolishes the activity of a gene

1. null mutation: block function of protein completely → prevents synthesis of inactive mutant protein

2. hypomorphic mutation: produces less of wild type protein or weak detectable mutant protein (usually recessive to wild type alleles)

haploinsufficiency

one wild type allele does not provide enough gene product to avoid a mutant phenotype

gain of function alleles

rare mutations that alter gene function rather than disable it

• almost always dominant to wild type alleles

• often lethal when homozygous because they are pleiotropic → lethality results from absence of normal gene function

1. hypermorphic mutation: mutant allele that generates either more protein than the wild type allele or a more effecient protein

2. neomorphic allele: produces novel phenotype due to production of a protein with a new function or due to ectopic expression of protein

3. antimorphic allele: block activity of wild type alleles of the same gene causing loss of function even in heterozygotes

noncoding genes

genes that are transcribed but not translated

nonsense suppressor tRNA

suppressing effects of nonsense mutations by inserting an amino acid into polypeptide at stop codon

can only survive if

1. other tRNA’s recognize same codon suppressing tRNA

2. other stop codon normally found at ends of mRNA coding regions

biochemical pathway

orderly series of chemical reactions within a cell in which molecules are converted stepwise into a final product

experimental results of one gene, one enzyme

mutation in one gene blocks pathway at one step → supplementing medium with any intermediate that occurs after → restores growth

• supplementation w intermediate that occurs before missing enzyme does not work because mutant cell cannot convert

• auxotroph: nutritional mutant microorganism that requires supplementation w substances not needed by wild type strains

generic amino acid structure

1. amino group

2. C-H-R Group

3. carboxyl group

polypeptides

• Constructed by covalent peptide bonds

• linear polymers of amino acids

• have chemical polarity (nonpolar amino acids)

• N-terminus: end of polypeptide synthesized first (not connected to any other amino acid)

  • synthesized first by ribosome

  • start of polypeptide chain

• C-terminus: free carboxylic acid group

  • synthesized last by ribosome

  • end of polypeptide chain

three dimensional structure

primary, secondary, tertiary, quaternary

primary structure

linear sequence of amino acids

• responsive for other folds/twists

• Peptide bonds between adjacent amino acids

secondary structure

localized regions, repetitive coiling, and folding of a polypeptide

• driven by hydrogen bonds of the backbone

• have alpha helix and beta pleated sheets

tertiary structure

ultimate three dimensional shape

• driven by interactions of the side chains

• various forces: hydorgen bonds, electrostatic bonds, hydrophobic interactions, disulfide bridges

denaturation

disruption of hydrogen bonds by heat, pH, chemicals → proteins unfold

chaperones

protein that helps other proteins fold into their native tertiary structure

quartenary structure

two or more polypeptide chains that associate in a specific way

• three dimensional configuration of subuits

• same interactions like hydrogen bonds, electrostatic bonds but between different various subunits

reading frame

grouping of nucleotides into nonoverlapping sets of three

intragenic suppression

combining a deletion with insertion in the same gene restores proper reading frame → proving codons are triplets

histones

small proteins that bind to and nuetralize negative charged DNA

nonhistone proteins

serve as

• structural

• active in replication

• chromosome segregation

• regulate transcription during gene expression

nucleosomes

basic unit of chromatin

condensins

form rings around DNA organizing chromosomes into loops

• greatest importance in starting the process of chromosome condensation during mitosis

transcription changes

less frequently DNA is transcribed → more compacted → to overcome → opening chromatin through chromatin remodeling complexes with ATP shift/remove nucleosomes and histone acetyltransferases add acetyl groups to histone tails

Euchromatin

lighter staining bc less dense, less condensed containing most genes

• loosely packed, open chromatin

• transcriptionally active

• replicates during S phase

heterochromatin

darker straining, tightly packed, mostly inactive

• block transcription factors and RNA polymerase from binding to promoter entirely

• can spread outward along chromosome

• replicates late during S phase

position effect variegation (PEV)

Occurs when a gene that normally resides in active euchromatin is moved right next to a region of dense heterochromatin.

• In some cells, heterochromatin spreads far enough to cover the nearby gene, blocking transcription factors entirely and silencing the gene.

histone modification: methylation

Histone methyltransferases (HMTs) add methyl groups to lysine or arginine residues on histone tails without altering the overall electric charge of the protein.

• Structural Effect & Gene Expression: Can cause either transcription activation or repression, depending entirely on the specific amino acid residue modified and the number of methyl groups added

histone modification: acetylation

Histone acetyltransferases (HATs) add negatively charged acetyl groups to positively charged lysine residues on histone tails. This neutralizes the positive charge on the histones, reducing their electrostatic attraction to the negatively charged DNA backbone.

• Structural Effect: The DNA unwinds from the histone octamer, transforming dense heterochromatin into open, relaxed euchromatin.

• Gene Expression: Promotes transcription (active). The relaxed structure allows transcription factors and RNA polymerase II unobstructed physical access to promoter regions.

• Reversal: Histone deacetylases (HDACs) remove acetyl groups, restoring the positive charge and re-compacting the chromatin to silence gene expression.

G-banding (giemsa banding)

G-banding is a classic technique used to produce a visible pattern of dark and light bands along metaphase chromosomes

• G-dark bands: tightly packed heterochromatin with lower gene density and stain dark

• G-light bands: loosely packed euchromatin with higher gene density and stain light

Fluorescence In Situ Hybridization (FISH)

uses fluorescently labeled single-stranded DNA or RNA probes to locate the presence or absence of specific nucleic acid sequences directly on chromosomes.

• FISH can target individual genes or sequences as small as a few kilobases.

Xist gene in X inactivation

gene located on the X chromosome itself

• The X chromosome chosen for inactivation turns on its Xist gene.

• Xist produces a special type of RNA (long non-coding RNA) that is not made into a protein.

• Instead, this Xist RNA spreads out and physically coats the entire X chromosome that produced it.

• The RNA coating recruits silencing proteins (like histone deacetylases and methyltransferases) that pack the DNA extremely tightly and add chemical "off" switches (epigenetic marks).

• Because the coated X chromosome is packed so tightly into inactive heterochromatin, it condenses into a tiny, dense lump against the inner wall of the nucleus.

• This shriveled, silenced X chromosome is called a Barr body.

epigenetic phenomena

heritable alteration in gene expression not due to mutation in base pair sequence

DNA methylation

enzymatic addition of methyl groups to basds in DNA in which a methyl group is added to 5’ C in cytosine base

• DNA methyltransferases catalyze methylation of cytosine

methylation of CpG Islands

DNA sequences that may be hundreds/thousands base pairs long

unmethylated CpG island → chromatin is open → transcriptionally active

methylated CpG island → dense heterochromatin → gene silenced

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CpG island

Methylation usually occurs on a cytosine base that is immediately followed by a guanine base on the same strand of DNA, connected by a phosphate bond. These regions are called CpG sites.

Clusters of these sites, known as CpG islands, are commonly found near gene promoters (the region where transcription factors bind to start reading a gene)

Repression of genes by DNA methylation

methylation → gene silencing: when promoter adjacent CpG islands become methylated

• repressors called methyl-CpG-binding proteins bind to methylated islands which causes chromatin to close blocking transcription

Examples of DNA Methylated genes

1. Tumor suppressor genes: methylation of CpG islands near tumor suppressor genes silence them leading to increased tumor aggresiveness

2. Rett syndrome: developmental brain disorder caused by loss of function in MeCP2 gene

3. found also within insulators → CTCF binding site can be methylated → prevets insulator from binding CTCF → changes expression of nearby genes

Genomic Imprinting

gene expression depends on the parent that translates it, caused by sex specific DNA methylation

• copy of one parent is silenced (transcriptionally inactive) while copy from other parent is active

• established in programmed or sex specific manner

• paternally inherited: allele from father from transcribed

• maternally inherited: allele from mother not transcribed

Imprinting control regions

certain DNA sequences that are methylated - imprinting

Insulator Methylation Mechanism

1. insulator mechanism of imprinting

• unmethylated insulator → binds to CTCF → enhancer cant interact with promoter → silenced

• methylated insulator → prevents CTCF binding → promoter and enhancer in same TAD → activated

2. CpG island mechanism

• unmethylated CpG → silenced

• methylated CpG → activated

human pedigree paternally silenced

• gene could pass because childs normal maternal allele expression

• allele from father is not expression and gene is methylated

human pedigree maternally silenced

• children have 50% chance of receiving even if heterozygous

• allele from mother is methylated and is not expressed

hox gene repression

encode transcription factors setting up body plan of animals relying on stable silencing (can either activate or silence gene)

transgenerational epigenetic inheritance

transmission of epigenetic marks (such as DNA methylation, histone modifications, or non-coding RNAs) from parents to offspring across generations without changing the underlying DNA sequence