UNIT VII – MOLECULAR GENETICS Big Campbell – Ch 17, 18, 20 Baby Campbell – Ch 10, 11, 12

UNIT VII – MOLECULAR GENETICS

Big Campbell – Ch 17, 18, 20

Baby Campbell – Ch 10, 11, 12

UNIT OVERVIEW• Protein Synthesis• Regulation of Gene Expression

o Prokaryoteso Eukaryotes

• Mutationso Chromosomalo Geneo Cancer

• DNA Technologyo DNA Testing Techniqueso PCRo Recombinant DNAo Extensions

I. PROTEIN SYNTHESIS

• Genotype → phenotype• Central Dogma

I. PROTEIN SYNTHESIS, cont• History

o Archibald Garrod First to suggest genes dictate phenotype through production of

enzymes Made in 1909 after studying disease known as alkaptonuria

o George Beadle & Edward Tatum Worked with bread mold, Neurospora Caused mutation of mold’s DNA through repeated X-ray exposure Mutated Neurospora required enriched medium Concluded DNA was no longer producing functional enzyme for

metabolic pathways Their work led to one gene → one enzyme hypothesis Eventually modified to one gene → one protein Then, one gene → one polypeptide Now, one gene → one ??

I. PROTEIN SYNTHESIS, conto Working Models of Study of the Central Dogma

C. elegans

I. PROTEIN SYNTHESIS, cont

II. IMPORTANCE OF RNA

• Ribonucleic Acido

o

o

II. IMPORTANCE OF RNA, cont• Types of RNA

o mRNA (_____________________)

Disposable copy of gene “Coding RNA” Exits nucleus via ___________

o tRNA (______________________) Transfers amino acids to

ribosome according to recipe contained in mRNA

o rRNA (_______________________)

Primary component of ribosomes

Synthesized in _____________

II. IMPORTANCE OF RNA, cont

o Non-coding RNAs …

III. TRANSCRIPTION• Each gene contains a promoter - a specific sequence of nucleotides that marks the

beginning of a gene

• RNA polymerase unzips the DNA and begins moving in nucleotideso Nucleotides added in a _____________ directiono No primer is requiredo Only one side of the double helix is transcribed; known as the template strando o

• Transcription continues until a termination signal is reached

IV. TRANSLATION

• mRNA is read in groups of 3 nucleotides known as a codon

o Sequence of three nucleotides that code for an amino acid

o This is also known as the reading frame

o Redundancy

o AUG

o Stop Codons

IV. TRANSLATION, cont• Transfer RNA

o Function

o Anticodon

• Ribosomeso Function is to facilitate coupling of

mRNA codon and tRNA anticodon during protein synthesis.

o Made up of 2 subunits

o Prokaryotic vs Eukaryotic

o rRNA is transcribed from DNA, then ribosome is constructed in_______________

IV. TRANSLATION, cont• tRNA must bind to an amino

acido Cytoplasm of every cell stocked

with all 20 amino acids required for protein synthesis

o Each amino acid is joined to the correct tRNA through action of an enzyme known as aminoacyl-tRNA synthetase

o There are 20 aminoacyl-tRNA synthetases

Active site fits a specific amino acid

ATP provides the energy needed to form covalent bond between tRNA & corresponding amino acid

IV. TRANSLATION, cont

o Each tRNA anticodon must match up with the mRNA codon to insure the correct amino acid has been delivered to the ribosome.

o Occurs according to base pairing rules, however there are more mRNA codons than there are tRNAs.

o Certain nitrogen bases in the third position of the anticodon will base pair with more than one corresponding nitrogen base in a codon. Known as wobble.


• Ribosome has 3 binding “sites” for tRNAo A Site – Holds the tRNA

carrying the next amino acid to be added to the polypeptide chain

o P Site – Holds the tRNA carrying the growing polypeptide chain

o E “Site” – Site where tRNAs exit the ribosome

• Newly added amino acids form peptide bond with carboxyl end of growing polypeptide

IV. TRANSLATION, contInitiation

IV. TRANSLATION, contElongation

IV. TRANSLATION, contTermination


• Polyribosomeso Multiple ribosomes that translate the same mRNA multiple

timeso Found in both prokaryotic & eukaryotic cells

V. PROKARYOTIC GENE EXPRESSION• Protein Synthesis

In transcription, RNA Polymerase recognizes and binds to the promoter sequence

Transcription & translation occur virtually simultaneously

VI. REGULATION OF GENE EXPRESSION IN PROKARYOTES

• Important adaptation for bacteria

• Two basic mechanisms for metabolic controlo Regulation of Enzyme

ActivityFeedback Inhibition

o Regulation of Gene ExpressionOperons

VI. PRO GENE EXPRESSION REGULATION, cont

• Operon Modelo Operon = Promoter + Operator + all genes required for a given

metabolic pathway o Operon acts as a single transcription unito Promoter → Binding site for RNA polymeraseo Operator → “On-off” switch located either close to or within the promoter

Operator controls whether or not RNA polymerase can bind to the promoter region

Therefore operator determines whether operon genes are transcribed & translated

VII. PRO GENE EXPRESSION REGULATION, cont• Operon Control

o Operon can be turned off by a protein known as a repressoro Repressor binds to operator and prevents attachment of RNA

polymerase to promotero Repressor is a protein controlled by a gene known as a regulatory

gene in a different location on chromosome; not part of operonExpressed continuouslyAlways a small supply of repressor protein present

VII. PRO GENE EXPRESSION REGULATION, cont• Types of Operons

o Inducible OperonsOperons that are usually off;

that is, not usually transcribedCan be stimulated when a

specific molecule interacts with regulatory protein

Example is the lac Operon Regulates transcription of

genes required for breakdown of lactose

Typically off; bacterium is metabolizing glucose, other carbs; lactose is not present

VI. PRO GENE EXPRESSION REGULATION, contInducible Operons

lac Operon, cont When lactose is

available, lactose itself binds with repressor; inactivates it by changing its shape

Repressor cannot bind to regulator

Therefore, RNA polymerase is able to bind to promoter; operon is “on”

3 enzymes required to metabolize lactose are synthesized

VII. PRO GENE EXPRESSION REGULATION, conto Repressible Operons Transcription normally occurs Can be inhibited when a specific

molecule binds allosterically to regulatory protein

Example is the trp Operon Operon controls production

of 5 enzymes required to synthesize amino acid, tryptophan when it is not available to bacterium in surrounding

Operon normally on; repressor inactive

VII. PRO GENE EXPRESSION REGULATION, contRepressible Operons

When tryptophan is present, it binds to the repressor of the trp operon, activating the repressor, and turning off enzyme production.

Tryptophan acts as a co-repressor, a

molecule that works with a repressor protein to switch an operon off.

VII. PRO GENE EXPRESSION REGULATION, cont

VI. PRO GENE EXPRESSION REGULATION, cont• Positive Gene Regulation

o In addition to repressors, some operons are also under the control of proteins known as activators

o Essentially the opposite of repressors o They “turn up” an operon by making it easier for RNA polymerase to bind to

DNA, therefore facilitating transcription of operon geneso In the lac operon . . .

If both glucose and lactose are available, bacterium utilizes glucose until its supplies are depleted

As glucose ↓, concentration of cyclic AMP (cAMP) ↑Increase in cAMP triggers release of activator protein known as CAP;

CAP binds to promoter, facilitates binding of RNA polymerase to promoter of operon to enhance synthesis of enzymes of lac operon

When glucose concentration is high, decrease in cAMP results in decrease in CAP → RNA polymerase has very low affinity for lac operon promoter so lactose metabolism does not occur

VI. PRO GENE EXPRESSION REGULATION, cont

VII. EUKARYOTIC GENE EXPRESSION

Transcriptiono Within the promoter is a

DNA sequence known as the TATA box – repeated Ts and As that identify the transcription site

o Proteins known as transcription factors recognize the TATA box, bind, and allow for attachment of RNA polymerase

VII. EUKARYOTIC GENE EXPRESSION, cont

• Transcription, conto Transcription continues

until polyadenylation signal (AAUAAA). mRNA is released 10-35 nucleotides downstream from polyadenylation signal although transcription continues

o At this point, RNA strand is known as the RNA transcript or pre-mRNA


• Transcription, conto Editing the mRNA

Each gene has long segments of non-coding DNA known as introns

Introns must be cut out of mRNA, remaining regions known as exons are spliced together, exit the nucleus, and are expressed in the translated proteins

VII. EUKARYOTIC GENE EXPRESSION, cont• Transcription, cont

o Modifying the mRNA5’ end of mRNA is “capped” with a guanine nucleotide

Known as 5’ cap3’ end has an additional 50-250 adenine nucleotides added

after polyadenylation signalKnown as poly A tail

Both modifications appear to help mRNA leave the nucleus, protect the mRNA, and facilitate the attachment of ribosomes to the 5’ end of the mRNA



VIII. REGULATION OF GENE EXPRESSION IN EUKARYOTES

• Early in development, eukaryotic cells are totipotento Mammalian embryos remain totipotent until 16-cell stage

• Cells are described as pluripotent once extra-embryonic membranes (placenta, etc) are formedo AKA embryonic stem cells

VIII. REGULATION OF EUK GENE EXPRESSION, cont

• As development continues, cells of multicellular organisms differentiateo Differentiation due to differential

gene expression in each cell, not different genes

o Some organisms can de-differentiateRegeneration in animals In plants, root cells can grow

into mature plant IPS – Induced Pluripotent

Stem Cells

VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont

• Gene expression is regulated by three mechansimso Regulation of

chromatin structure

o Regulation of initiation of transcription

o Post-transcriptional regulation

VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contRegulation of Chromatin Structure

2-3 m of DNA per cell is elaborately folded DNA wraps around proteins called histones. Charge attraction holds

DNA to histones. Cluster of histones forms nucleosome. Stretches of DNA between nucleosomes are known as linkers


• Folding of DNA is highly specific• Generally, the more condensed the

DNA is, the less likely it is to be transcribed. o

• During interphase, DNA is visible as irregular clumps of chromatin. Two types:o Heterochromatin

o Euchromatin


• Modification of Histones o Acetyl (-COCH3) group added to N-end of histone “tail” o Neutralizes + chargeo Histone less attracted to nucleosome, coil loosens, DNA becomes

more transcribable.• DNA Methylation

o Addition of methyl groups to certain bases in DNA Most often involves cytosine

o Deactivates DNA o For example, in females, inactivated X chromosome is highly-

methylated

VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contTransciptional/Translational Regulation

• Regulation of Initiation of Transcription o Transcription Factors

Bind to TATA boxForm Transcription Complex that allows RNA Polymerase to bind to DNA

o Enhancer SequencesDNA sequences May be located up to 20,000 bp “upstream” from the promoterBind activator proteins

o SilencersBind repressor proteins

o Work together to determine rate of transcription

VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contTransciptional/Translational Regulation

• Post-Transcriptional Regulation Alternative RNA Splicing

VIII. EUKARYOTIC GENE EXPRESSION REGULATION, contTranscriptional/Translational Regulation

• Post-Transcriptional Regulation, cont

Degradation of mRNA

Translation

Protein Processing & Degradation


• Post-Transcriptional Regulation, cont“Other” RNAs

MicroRNAs (miRNAs) Formed from longer RNA strand that folds onto itself to create a hairpin

loop Enzyme called Dicer trims it into a short double-stranded fragment One strand is degraded; the remaining strand can bind to any

complementary mRNA Blocks translation

Small interferring RNAs (siRNAs) Similar in mechanism to miRNAs Original RNA strand longer, more “hairpins”; generates many more siRNAs


• miRNA

IX. MUTATIONSo Change in the nucleotide

sequenceo May be spontaneous mistakes

that occur during replication, repair, or recombination

o May be caused by mutagens; for example, x-rays, UV light, carcinogens

o Two categories Gene Mutations Chromosomal Mutations

IX. MUTATIONS, cont• Gene Mutations

o Point mutations – change in a gene involving a single nucleotide pair; 2 types

Substitution – Further subdivided into . . . SilentNonsenseMissense

Frameshift – due to addition or deletion of nucleotide pairs

X

Normal mRNA

IX. MUTATIONS, cont• Gene Mutations & Phenotype

o Traits may be described as dominant, recessive, etc . based on the effect of the abnormal allele on the organism’s phenotype

o Vast majority of proteins encoded in genes are enzymeso Abnormal allele → Defective enzyme

If the enzyme produced by the normal allele is present in sufficient quantities to catalyze necessary reactions,

No noticeable effect on phenotypeDefective allele is classified as recessive

If the lack of normal enzyme production by defective allele cannot be overcome by normal allele,

Organism’s phenotype is affectedDefective allele is classified as dominant

IX. MUTATIONS, cont• Chromosomal Mutations

o Chromosome Number Mutations/Disorderso Alterations in Chromosome Structure

Often due to mistakes made during __________________

X. A CLOSER LOOK AT CANCER• In early 1900s, scientists realized there are viruses that can cause

cancer, including Human Papilloma virus, Epstein-Barr virus, and HTLV.

• Research led to discovery of cancer-causing genes called oncogenes

• We now know there are two important categories of genes in which mutations may lead to cancero Oncogenes/Proto-oncogenes

o Tumor Suppressor Genes

X. A CLOSER LOOK AT CANCER, cont• Oncogenes

Amplification – Increases number of copies of proto-oncogene; will increase protein production Point mutation in the promoter for an proto-oncogene, or in the gene itself Movement of DNA - May change the rate at which gene at which gene is transcribed,

therefore, translated Translocation Transposons

“Jumping Genes”Genes that are moved due to folding of DNA, cut (or copy) & paste mechanism

X. A CLOSER LOOK AT CANCER, cont

Oncogenes & Transposons



• Tumor-Suppressor Genes o Encode for proteins that inhibit cell division therefore any mutation that inhibits

activity of tumor-suppressor gene may lead to abnormal cell growth and formation of tumors.

o Act by producing proteins that repair damaged DNA, control density-dependent inhibition & anchorage dependence, or act as CDKs

o Gene that is most often defective in human cancers codes for transcription factor known as p53Known as the “guardian angel of the genome”Serves as the master brake on the cell cycle when DNA damage has occurred


• Tumor Suppressor Genes, p53 cont. When stimulated by DNA

damage, p53 activates several genes with multiple effects Genes activated to halt

cell cycle DNA repair genes

turned on If DNA damage cannot

be repaired, “suicide genes” are activated; results in apoptosis



• Tumor-Suppressor Genes, cont o BRCA 1, BRCA 2 geneso BRCA 1• Women who inherit one

mutant allele have ~ 60% chance of having breast cancer by 50

• Individuals with two normal alleles have ~ 2% chance


DNA TECHNOLOGY & GENOMICS

I. TECHNIQUES IN DNA TECHNOLOGY• Restriction Enzymes

o Used by bacteria to “chop up” viral DNA

o Bacterial DNA protected by _________o Very specific

Each enzyme recognizes a particular nucleotide sequence

Called a restriction sequence or restriction site

Palindromic Cuts made at specific points May create “sticky ends”

o Used in gel electrophoresis o Also used to form recombinant DNA

Fragments may be pasted together with DNA ligase to form recombinant DNA

I. TECHNIQUES, cont

• Polymerase Chain Reaction (PCR)o In vitro method of

amplifying small amounts of DNA DNA is heated to

separate the double helix.

Mixture is allowed to cool, DNA primers attach to target

Heat-stable polymerase is used to extend the primers in the 5’–3’ direction.

I. TECHNIQUES, cont

• Gel Electrophoresiso Separates DNA

fragments based on sizeo Restriction fragment

analysis DNA treated with

restriction enzymes Resulting fragments

migrate based on size Produce a pattern

characteristic of original DNA and restriction enzyme used

I. TECHNIQUES, cont

• Southern Blotting Designed by Dr.

Southern Detects particular

DNA sequences

• Northern Blotting Detects particular

mRNA sequences

• Western Blotting Used to detect

proteins

II. EXTENSIONS IN DNA TECHNOLOGY • Recombinant DNA

DNA containing nucleotides from other sources

Process utilizes restriction enzymes that make jagged cuts in DNA; creates sticky ends

When DNA from different sources treated with same restriction enzyme, sticky ends “mix & match”

Often use reporter genes to determine success; for example, ampicillin resistance

II. EXTENSIONS, cont

• cDNA - complementary DNAo Procedure for “cloning DNA” that uses mRNA, reverse transcriptaseo

• STRs – short tandem repeatso Short segments of DNA that are highly repetitive, polymorphico Repeat patterns are inheritedo Useful for identifying individuals

• SNPs – single nucleotide polymorphismso Single base-pair that shows variation in a significant % of populationo SNPs that alter the fragment length following exposure to restriction

enzymes called RFLPs (restriction fragment length polymorphisms)o Genetic markers


• DNA Microarray Assayso AKA DNA

Chipso Test used to

determine gene function, gene interactions

o May be used to determine agressiveness of cancers, method of treatment, etc

II. EXTENSIONS, cont• Gene Cloning

o Process of preparing multiple copies of a particular segment of DNAo Requires host and vectoro Hosts

Initially done using bacterial cells Now eukaryotic hosts are used

YeastPlants

o Vector Should have 4 characteristics

Ability to replicate independently of host cell DNARecognition sequenceReporter geneSmall size

Possible vectors includePlasmidsVirusesYAC = Yeast Artificial Chromosome

II. EXTENSIONS, cont• Gene Cloning

Use of plasmid as vector

Plasmid isolated from bacterial cell

Foreign DNA inserted into plasmid

Plasmid returned to bacterial cell; described as recombinant bacterium

Foreign gene is cloned as bacteria reproduce

Common bacterium used for plants is Agrobacterium tumefactiens


A CLOSER LOOK AT

GENECLONING


• Reproductive Cloning Nuclear Transplantation Process of using unfertilized

egg cell & replacing nucleus with DNA

In 1997, scientists were able to produce first reproductive clone, “Dolly”, by culturing somatic cells in a nutrient-poor medium to de-differentiate them and force them back to totipotency.

Reproductive cloning in animals has enjoyed limited success.

II. EXTENSIONS, cont• Gene Silencing

o Knockout GenesUse of genetic recombination

to create an inactive , “knocked out” gene

Mutated allele introduced into embryonic stem cells

Forms chimerasOften used in mice to study

gene expression


o RNAiBased on principal of

microRNASmall-interfering RNA

(siRNA) synthesized complementary to mRNA

Base-pairing occursTranslation is blockedHas been used to block

production of growth factors in certain cancers

III. GENOMICS• Human Genome Project

International government effort begun in 1990 Goals

o identify all the approximately 20,000-25,000 genes in human DNA, o determine the sequences of the 3 billion chemical base pairs that

make up human DNA, o store this information in databases, o improve tools for data analysis, o transfer related technologies to the private sector, and o address the ethical, legal, and social issues (ELSI) that may arise

from the project.

Celera Genomics o Shotgun sequencing

Completed early and under-budget in 2003 Genomics has given rise to proteonomics

Documents

UNIT VII – MOLECULAR GENETICS Big Campbell – Ch 17, 18, 20 Baby Campbell – Ch 10, 11, 12