Anatomy of the Gene

MUPGRET WorkshopJune 17, 2004

2003: 50 Year Anniversary of the Discovery of The DNA Double Helix

The famous DNA Double Helix paper was published in Nature in 1953!

What Is DNA?

DNA is a chemical contained in every cell of your body.

DNA is a Chemical!?

•We are made up of chemicals, formed from the elements carbon (C), hydrogen (H), oxygen (O), phosphate (P) and others

•DNA is made up of carbon (C), hydrogen (H), oxygen (O),phosphate (P)

•We breathe air which contains oxygen molecules (O2).•We eat food which is composed of chemicals called proteins,

sugars, and fats.•Our bones are made up largely of calcium (Ca)•Our bodies make energy by breaking down chemicals such as

sugar!•We store energy in our body in the form of carbohydrate

chemicals.

Yes!

What other kinds of chemicals are in your body?

DNA Hereditary material. Contains all information to make

proteins. Linear polymer of nucleotide. Each one has sugar, phosphate

and a base.

DNA Base connected to sugar by β-

glucosyl linkage. Nucleotides are connected to one

another by a phosphodiester bond. Bases are perpendicular to helix.

The Double Helix Two strands of DNA run in opposite

directions, complementing each other and pairing with hydrogen bonds.

A and T pair together and C and G pair together.

Helix is most often right-handed (B-form).

3D DNA Strands: Building Blocks are DNA Letters

“Red” DNA Strand

“Green” DNA Strand

Each DNA Strand Contains One Backbone and Many Building Block DNA Letters (Bases)

Gre

en

St r

and

DN

A L

ette

rs

Red

St r

and

DN

A L

ette

rsDNA Letters:

A, G, C, T

How Does DNA Carry Information?

To answer this question we must take a closer look at DNA.

DNA is a biopolymer•Polymers are molecules made of repeating units or building blocks•DNA has four chemical building blocks symbolized by the letters A,G,C,& T•The letters of your DNA are in a specific order that carries information about you!!

So, a DNA polymer can be represented as a string of letters:

A G C T T A G G G T A A A C C C A T A T A

DNA Carries Information in the Sequence of DNA Letters

. . .A G C T T A G G G T A A A C C C A T A G . . .

A gene

• A gene is a length of DNA letters that contain an instruction for a cell to follow.

• The cell uses specially designed protein machines to read the information in genes.

The Order of DNA Letters Encodes the Genetic Information

Example of the DNA letters in a gene:

AGCTTAGGGTAAACCATATAGGGCCATACCCTATCGGTAAGCTT

AGCTTAGGGAAAACCCATATAGGGCCATACCCTATCGGTAAG

The order or sequence of the A, G, C and T letters in the DNA polymer encodes the actual genetic information

The specific order of the DNA letters carriesthe information.

• Changing the order of the DNA letters will change the information carried by the gene.

• We will talk about how this happens later!

Secret of DNA Fingerprinting Lies in the Ability to Detect Small Differences in DNA Letters Among Individual Samples

Look around the room and see how different we all look. Then compare any two human genomes:

•The DNA letters are almost the identical order (sequence) between any two human genomes!

•A very small number (0.1%) of the DNA letters differ between any two human genomes.

•Two plants that look very similar may be close or distantly related becausehumans select for desirabletraits in new varieties.

Genes Can Have Hundreds to Millions of DNA Letters

. . .A G C T T A G G G T A A A C C C A T A G . . .

A gene

It can take hundreds, thousands or even a million or more letters (bases) to “spell out” the instructions in a single gene.

…and what for?

Controlling Gene Expression

The specific order of DNA bases in a gene encode a protein product.

Genes have START and STOP signals that specify the length of the protein chain.

Control DNA region is in front of the “coding region” and controls expression of the gene.

PROMOTER

+1PROTEIN CODING REGION

GENE

mRNA

Control DNA region is called a promoter.

DNA region carrying protein information is called the coding region.

Genes Contain Instructions for Building Proteins

Genes contain instructions for making proteins, one of the major types of the molecules of life, or “biomolecules”

Proteins, like DNA, are polymers

• Protein building blocks are called amino acids

• Amino acids are strung together into long, linear polymers by following the instructions in genes

• In general, a gene encodes the instructions for one protein

When a gene is “misspelled,” the protein made from it

• may be made with an incorrect amino acid

• may not work properly

Review of Gene Expression Pathway in Cells

GENE DNA

mRNA copy of gene

mRNA goes to cytoplasm

Ribosomes translate genetic information encoded in the mRNA into protein building blocks (chains of amino acids)

Protein folds into 3D active structure

Protein functions in cell

Focus on the Genetic Code!

DNA Code Is Copied into a “Portable” Code: mRNA

DNA

RNA POLYMERASE (RNAP: COPIES DNA INTO mRNA)

mRNA

mRNA: AUGGAGUACUAAUAUGUAAAAAAAAAAAAAAAAAAA-3’DNA: ATGGAGTACTAATATGT-3’ TACCTCATGATTATACA-5’

C U A AG A T T

DNA

MFHMAF2001

3’ Note: DNAbase-pairs between backbone strands are not shown here

RNA Code has Different Alphabet Than DNA Code

(RNA has U instead of T)

DNA: ATGGAGTACTAATATGT-3’ TACCTCATGATTATACA-5’

3’-TACCTCATGATTATACA-5’DNA STRAND AUGGAGUACUAAUAUGU mRNA copied from DNA

RNA has U instead of T

When DNA is copied into mRNA (transcription), U is incorporated into mRNA in place of T

DNA Base-PairDNA strand has “T”

DNA Strands “Unzip” so the DNA Letters Can be “Read”

-ATGGAGTACTAATATGT- -TACCTCATGATTATACA-

-TACCTCATGATTATACA- AUGGAGUACUAAUAUGU mRNA copied from DNA

-ATGGAGTACTAATATGT- + AUGGAGUACUAAUAUGU mRNA -TACCTCATGATTATACA-

DNA STRANDS SEPARATE

DNA STRANDS COME BACK TOGETHER BY BASE-PAIRING

DNA HELIX STAYS IN NUCLEUS

mRNA GOES TO CYTOPLASM TO PROTEIN FACTORY

DOUBLE-STRANDED DNA (Region from Chromosome)

Genetic Code is Written in 3-Letter DNA Words (Codons)

CODON MEANINGS:

•A “START PROTEIN” SIGNAL: AUG•A “STOP PROTEIN” SIGNAL: UAA, UGA, UAG•An amino acid building block of a protein•Codons identified in the Genetic Code Table

-TACCTCATGATTATACA- DNA(DNA strands separated) -AUGGAGUACUAAUAUGU mRNA (copied from DNA)

5’-AUGGAGUACUAAUAUGU mRNA

5’-AUG GAG UAC UAA UAU mRNA

mRNA code “read” by ribosome in TANDEM triplets called codons.

Codon adaptors convert RNA letters into the correct amino acid building blocks in the protein chain.

http://anx12.bio.uci.edu/~hudel/bs99a/lecture20/lecture1_6.html

The Universal Genetic Code Table

Name of Building Block Amino Acid: Phe=PhenylalanineLeu=LeucineIle=Isoleucine

AUG CODON: Signal to start making the protein.

STOP Codons:UAAUAGUGA

Genetic Code is Written in 3-Letter DNA Words

CODON MEANINGS:

•“START PROTEIN HERE”: AUG (START) Methionine (Met)•“STOP PROTEIN HERE”: UAA, UGA, UAG•Amino acid building blocks: N-Met-Glu-Tyr-C•Codons are identified in the Genetic Code Table

-TACCTCATGATTATACA- DNA STRAND AUGGAGUACUAAUAUGU mRNA copied from DNA

5’-AUGGAGUACUAAUAUGU mRNA

5’-AUG GAG UAC UAA UAU mRNA Met-Glu-Tyr-STOP

mRNA code is “read” in TANDEM CODONS

Met Glu TyrN C

A SHORT PROTEIN IS A PEPTIDE

Proteins Have Two Ends: The N- And C- Termini

5’-AUGGAGUACUAAUAUGU mRNA

5’-AUG GAG UAC UAA mRNA Met-Glu-Tyr-STOP

Met Glu TyrN C

A short protein (peptide) has only a few amino acid (aa) building blocks.

The first aa in the chain (usually Met) (AUG) is at the N-terminus.

The final aa added to the chain is the C-terminus.

aminoacid

Ribosome Protein Factory Reads the RNA Codons

mRNA

GENE DNA UNZIPS

AA

NUCLEUS

Protein Synthesis

MFHMAF2001

N

Protein Chain Folds

RNA is Copied From DNA (Gene)

Transfer RNA (tRNA): Matches mRNA codon with correct amino acid building blockmRNA

Proteins Fold into 3D Structures

Likes Water

Likes Water

Likes Water

Hates Water

Legend

Polar “Pocket”

Proteins live in a watery environment (living organisms!).

• Chemical parts that hate water fold on inside of protein.

• Chemical parts that love water go to the outside surface of protein.

• Surface of the folded protein interacts with proteins, DNA, RNA, etc.

Small Folded Protein

C

N

Hydrophobic “Pocket”

Human proteins have 20 different amino acid building blocks

Different Protein Chains Fold to Make Proteinswith Different 3D Shapes and Biological Functions

Protein #1

Protein #2

Protein #2

Protein #3

Protein #3

Protein #1

Molecular Structures Related to Protein Function in the Cell EF Hand Binds Calcium

“Syringe”

DNA Intersection: Holliday Junction

Channel

Basket

One Gene-One Protein Archibald Garrod (1902) described

alkaptonuria, a hereditary disorder as an “inborn error of metabolism”.

Proposed that mutations cause specific biochemical defects.

Alkaptonuria defect is dark urine.

A DNA Spelling Mistake Can Alter the Protein Chain

ATG TTC AGG CCA AAT TTT GTC GCG UAA GGA ATTSTART ADD ADD ADDADD ADDADD ADD STOP

TTC to TTT spelling change causes a different protein building block to be inserted in the second position. That is all it takes.

Spelling Mistake The DNA “word” TTC is changed to TTT

ATG TTT AGG CCA AAT TTT GTC GCG

ADD = Codon specifies the amino acid specified by 3-letter “word”ATG/AUG = Codon specifies start and methionine (met)UAA = STOP adding amino acids to protein chain

Mutant Genes Encode Defective Proteins:

(1) WILDTYPE (2) MUTANT

Example: AAA GCT ACC TAT AAA GCT ATC TAT TTT CGA TGG ATA TTT CGA TAG ATA Phe Arg Trp Ile Phe Arg Stop UAG PROTEIN: WT FUNCTION NO FUNCTION

(1) Normal DNA and amino acid sequence makes a wild-type protein.(2) Mutation in DNA changes Trp to Stop to make a short, mutant protein.

Mutations in DNA can be Caused by: • Mistakes made when the DNA is replicated (wrong base inserted) • Ultra violet (UV) light and ionizing radiation (X-rays) damage DNA• Environmental chemical carcinogens can damage DNA• Other factors

A Mutation is a DNA “Spelling Mistake”

DNA Technology: The Awesome Skill, I E Alcamo, Harcourt Academic Press, 2001

Misspelled Genes: 3 Possible Outcomes

A misspelled gene

DNA

Cell may not be able to

follow damaged

instruction

Cell does not make the

protein

X

X

OR Spelling error may be harmless

Functional protein made

by the cell

OR Damaged protein is made

Damaged protein may or

may not be able to function in

the cell.

DNA is Stored in the Nucleus (in Complex Cells)

CELL MEMBRANE Controls entry and exit from cell

NUCLEUS Cell Control Center-contains DNA, acivation of gene send RNA copies out into the cytoplasm.

This is called gene expression.

Complex cells have compartments, bacterial cells do not.

Minimalist Complex Cell

CYTOPLASM The area and material inside the cell, but outside the nucleus and other comparments

DNA

RNA

RIBOSOMESMake proteins from RNA instructions

Gene Structure

Exon 1 Intron Exon2

Introns are removed during translation

Exon 1 Exon2

Intron A noncoding DNA sequence in a

gene that is transcribed but is then excised from the primary transcript in formation a mature mRNA molecule.

Hartl and Jones. Genetics Analysis of Genes and Genomes

Exon The sequences in a gene that are

retained in the messenger RNA after the introns are removed from the primary transcript.

What effect does the presence of introns have on the number of deleterious mutations?

Heterochromatin characteristics

Condensed throughout interphase Late replicating, ie. out of phase

with euchromatin Transcriptionally inactive in the

condensed state. Under-replicated in polytene

chromosomes.

Constitutive heterochromatin Present in all cells at identical

positions on both homologous chromosome and forms a permanent structural characteristic of a given chromosome pair.

Facultative heterochromatin Varies in state between cell types,

developmental stages or homologous chromosome.

Ex. differential state in homologous chromosomes seen in Barr bodies.

Some proposed functions Chromosome level

Stabilize centromeres and telomeres Facilitate chromosome pairing Cause variegation or “sticky” effects

Gene level Modify gene action, penetrance, or

specificity Control mutation

More proposed functions Across chromosomes

Control of chromosome dimensions Regulation of crossing-over, pairing, and

disjunction Control of variegation from genes on other

chromosomes Control of cell size Regulation of growth and differentiation

rates

What is heterochromatin? Composed primarily of repetitive

DNA. Methylated Composed primarily of highly

repetitive and middle repetitive DNA. An example from maize

Mobile element About 50% of genome in

mammals. Up to 90% of genome in plants. If the origin of life was in the “RNA

world”, then they could be early participants in formation of genomes as we know them today.

Kazazian. 2004. Science 303: 1626-1632.

Retrotransposon Special transposon whose

sequence is transcribe to RNA in the cell. After generation of the RNA strand a reverse transcriptase produced by the retrotransposon reconverts the RNA to DNA. This sequence is integrated into the original DNA strand at any position.

http://dvsinfo.weihenstephan.de/genglos/asp/genreq.asp?nr=620

Retrotransposons Specific type of transposable

element. Ancient Ubiquitous Mobility occurs at different rates

depending on class of retrotransposon.

Can effect gene function

Retrotransposons Not all retrotransposons have a

preference for heterochromatin. Ex. MITES preferentially insert into

genes. Important in genome organization. Important evolutionary force.

Genome Evolution Average human diploid genome

has 80-100 active L-1s. L1 insertions account for 1 in 1200

human mutations some of which result in disease.

1 in 50 humans has a NEW genomics L1 insertion occurring in germ cell.

Retroelements stabilize telomeres No telomerase in Drosophila. Drosophila telomeres are long

tandem arrays of two non-LTR retrotransposons HeT-A and TART.

First elements found to have a confirmed role in cell structure by assisting in maintenance of chromosome ends.

Pardue and deBaryshe. 2003. Ann Rev Genet 37: 485-511.

Genome Duplication Evidence is accumulating to

support the idea of both complete or partial duplication within the genome of a number of organisms.

Primates Originally thought chromosome

evolution occurred by random breakage but comparison of mouse and human sequence says: Segmental duplication happened with

syntenic blocks of the genome. 25% of all breakpoints contain > 10 Kb

of duplicated sequence so not random. Duplication is not necessarily causal.

Bailey et al. 2004 Genome Biology 5: R23

Every Cell Has a Complete Copy of Genome DNA:

• Virtually every cell in your body contains its own complete copy of all your DNA

• A single, complete copy of an organism’s DNA is called its genome

• The genome is a set of instructions, like a master plan, written in a molecular language, using DNA instead of paper and ink

But Most of My Cells Don’t Make Melanin-- Right?

• Therefore, each cell in your body has a copy of your genome, which is, in essence, a master plan for making you.

How BIG is 3.2 Billion DNA Letters?

Genome Facts: NOVA OnlineAccess Excellence Cell to Chromosome to DNA

Human Genome

Human Genome Has 3.2 Billion DNA Letters: 3,200,000,000 bp

3.2 billion (3.2 x 109) is the same as:

• 200 (1000 pages each) New York City telephone books

• 3 Gigabyte computer hard drive

• a person typing 60 words/minutefor 8 hours/day, would take more than 50 years to type the entire human genome sequence

• placed end-to-end the DNA in onehuman cell extends almost 6 feet

One DNA base-pair

How Big are Plant Genomes?Plant Genome

Human Genome Has 3.2 Billion DNA Letters: 3200 million bp

Maize (Corn) Genome has 2.5 Billion DNA Letters: 2500 million bp

Arabidopsis Genome has 125 million bp

Rice Genome has 430 million bp

Wheat Genome has 16,000 million bp

One DNA base-pair

Nucleus Executes The Genome Master Plan

Danny Schnell, BMB

PLANT CELL:

CELL WALL

CYTOPLASM

CHLOROPLASTDNA

mRNA

mRNA PROTEIN

NUCLEAR PORE

Genome Master Plan is Executed:

(1) DNA is copied into RNA code (mRNA)

(2) mRNA is transported to the cytoplasm

(3) Translate mRNA code into chain of protein building blocks at the ribosome. CYTOPLASM

RIBOSOME

(1)(3) (2)

CELL MEMBRANE

NUCLEUS

mRNA

DNA is Packaged Into Chromosomes

Adapted from Alberts et al.

Molecular Cell Biology

DNA double helix

GENE (blue)

Several GENES along a length of DNA

DNA is coiled around proteins (more on this later)

Small region of “unwrapped” chromosome

Loosely wrapped DNAin chromosome

Tightly wrapped chromosome

Bacteria usually have one circularchromosome and no nucleus

Organisms with nuclei have variable numbers of chromosomes depending on the species:

• Mosquito 6• Chimpanzees 48• Goldfish 94

How Many Chromosomes Are There?

 

Some plants have few chromosomes like Arabidopsis.

Others, like sugarcane, have many.

How Many Chromosomes Are There?

 

Wheat

Rice

Arabidopsis

Sugarcane

Maize

Potato

Tomato

Cabbage

Carrot

10

20

+100

48

24

24

42

20

18

Human Chromosomes Metaphase Chromosome

Chromosomes are Dynamic Structures

Fruit Fly

CONDENSED EXTENDED

DNA Discovery

Discover DNA: It is Changing OUR Lives…•Human Cloning

(imagine dozens of identical siblings!!)•Designer Babies

(and all of them “perfect”!?!)•Stem Cells

(can we help paralyzed people to walk?)•Gene Therapy

(can we fix “broken” genes?)•DNA Fingerprinting

(nowhere to hide??!!)

Some Questions Students May Have?

Most of us know we have DNA and genes…

What are genes and how much do they influence us?

What do genes and DNA actually do?

What is a genome?

What is the Human Genome Project?

Will the Human Genome Project affect me?

Variations on the Human Theme!

People look very different from each other.Yet we all have features in common, 2 arms, 2 legs, one

head, one nose, etc.

Our DNA Story

Traits are Inherited

Traits are characteristics that vary among individuals.

Simple trait:•Eye color: Blue, brown, green•Seed coat color

Complex traits:•Blood types: A, B, AB, O•Plant height.•Plant disease resistance.

Connection between traits and genes: TRAITS are inherited from parents through GENES!

Genes are Responsible for the Traits You Inherit

Genes determine:

•physical traits and influence personality

•biological characteristics such as blood type

•level of health risk (heart disease, stroke, alcoholism, Alzheimer’s)

•specific genetic diseases (sickle cell, hemophilia, cancer, etc.)

•inherited traits that are passed on to your biological children

However: the environment always affects the result of genetic inheritance.

Example: genes for growth are influenced by nutritionavailable during child development