Biology (SBI4U) - Unit 2: Molecular Genetics

Gene

A sequence of DNA that can be decoded into Protein

Genome

The collection of all genetic material within an organism

Plasmid

Small circular loop of DNA containing one of a few genes. Extra chromosomal DNA found in prokaryotes.

Homologous Chromosome

Chromosome pairs composed of one maternal and one paternal chromosome. These chromosomes contain the same types of genes but the alleles may vary.

Diploid

2n - 2 sets of chromosomes

Haploid

n - 1 set of chromosomes

Prokaryotic Genetics

• Location: Nucleoid Region

• Amount: 1 chromosome

• Structure: 1 circular chromosome

• Packaging: Chromosomes are supercoiled

• Other Organelles: N/A

• Extra Chromosomal DNA: Present in Plasmids

• All DNA is coding; no repetitive sequences

• Number of sets: Usually haploid

Eukaryotic Genetics

• Location of DNA: Nucleus

• Amount: Several chromosomes

• Structure: Several linear chromosomes

• Packaging: Chromatin is condensed into chromosomes with the use of proteins

• Other Organelles: membrane-bound organelles

• Extrachromosomal DNA: mitochondrial DNA & Chloroplast DNA

• Not all DNA is coding; Introns are non-coding regions of DNA; Contains repetitive sequences

• Number of set: Usually diploid, but varies

DNA Structure

• Overall negatively charged

• Double stranded

• Completely base-pairing between strands through H-Bonds

• Antiparallel

  • One strand runs in an opposite direction compared to the other strand, this is the only way stability of the two strands can be achieved

  • One DNA strand has the hydroxyl (-OH) group of the 3’ carbon opposite to the phosphate attached to the 5’ carbon

• Helical

  • The two strands twist in a clockwise direction

  • One foil turn of the helix is 10 nucleotides (3.5 nm in length)

RNA Structure

• Single stranded

• Negatively charged

• RNA can exist in various shapes and sizes as it can fold over and complementary base pair itself

DNA Function

• This is the genetic code

• Stores hereditary information in code form

• Stores the instructions to assemble protein in code form — specifically code can be “decoded” into RNA

RNA Function

• This is the protein code

• Stores the information to assemble amino acids into a polypeptide in code form

• Assists in the actual synthesis of proteins

• Some viruses are this as their genetic/hereditary information

Gregor Mendel

• Performed a statistical analysis of pea plants to study inheritance

• Formed two laws of inheritance:

  • Parents each contribute one copy of genetic information to offspring (“factors”), that were responsible for inheritance

• Contributed to the understanding that DNA is the hereditary material

Hammerling

• Observed that regeneration of new appendages was driven by the nucleus containing part of green algae

• Hypothesized that hereditary information is stored in the nucleus

• Contributed to the discovery that hereditary information is found in the nucleus which led to the understanding that DNA is hereditary information

Friedrich Miescher

• Studied bloody bandages

• Extracted an acidic substance from the nucleus of white blood cells, found that the substance was high in phosphorus, called in “nuclein”

• Contributed to the understanding of the structure of DNA

Griffith - “Transformation”

• “Transforming principle”

• Studied pneumonia bacteria, two different strains:

  • S-strain or “smooth strain” was covered in a capsule (disease-causing)

  • R-strain or “rough strain” did not have a capsule (non-disease-causing)

• Found that the dead smooth strain transferred some substance to the non-virulent rough strain, which transformed the rough strain into a more virulent smooth strain

Avery, Mcleod, McCarty - “Transformation confirmed”

• Futhered Griffith’s work and identified DNA as the transforming agent using Streptococcus Furthered

• The goal was to determine which part of the bacteria was responsible for virulence. What was the transforming principle?

• Could be DNA, RNA or protein

• Although results were very conclusive, they were hesitant to report their findings:

  • They thought protein was still the better candidate to be genetic material

• Some enzymes might not have destroyed all the DNA or protein

Hershey-Chase Experiment

• Set out to find out if the genetic material was DNA or protein

• Used bacteriophage virus: Infects bacteria (E. Coli)

  • Basically, just a protein coat and DNA

• Some phages were labelled with phosphorus, some with sulphur

• They allowed these phages to infect bacteria, then tested the bacteria to see what became incorporated into the bacteria

• Phosphorus was found inside the host, determined that DNA contained the instructions for new viruses

Levene

• Found DNA contains 3 major components:

  • Deoxyribose sugar

  • Phosphate groups

  • Nitrogenous bases

• Structural finding

Chargaff

• Purified DNA and broke it down into the nucleotide subunits: (A) adenine, (T) Thymine, (G) Guanine, (C) Cytosine and measured the amount of each type

• Found A-T and G-C

• Lead to understanding complementary base pairing

Franklin and Wilkins

• Used X-ray crystallography to deflect x-rays off DNa onto a photographic plate, pattern was analyzed to help determine the molecular structure

• Wilkins produced images that suggested a helical structure

• Franklin suggested a sugar phosphate backbone faced inwards, a double-helix which rotated clockwise

Watson and Crick

• Found the five structures we know to be true about DNA:

  • Antiparallel

  • Alternating sugar phosphate backbone

  • Complementary base pairing of nitrogenous bases

  • Purines bonded to pyrimidine through hydrogen bonds

  • Phosphate group of each nucleotide is acidic and gives DNA an overall negative charge

  • Two strands of polymers of nucleotides

Messelsen-Stahl Experiment

• Used E. Coli bacteria which replicated approx. every 20 minutes

• Any newly replicated DNA would have light isotope incorporated into it

• Radio labelled parent DNA with a “heavy” N15. Then the DNA was moved to a new environment containing an N14 light DNA. This would ensure that only new synthesized DNA contained N14/

Messelsen-Stahl Experiment results

• After 1 round of replication:

  • DNA formed one intermediate layer

  • Light (contains only N14 DNA)

  • Intermediate (contains some N14 and some N15)

• Round 2:

  • 1 layer of intermediate and light layer

  • This was predicted for semi-conservative replication

DNA Replication

• Happens before mitosis and meiosis

• To make an identical copy of the eukaryotic genome, the following must occur:

  • Unpacking of the chromosomes

  • Strands to be unwound and then unzipped

  • Base pairing at the replication fork of the leading and lagging strand

  • Repair errors

DNA replication - Step 1: Strand Separation

• Separation occurs at the replication origins (special segments) along the DNA

• Helicase recognizes replication origins and acts to break H-bonds, causing strands to separate and unwind

• This creates a replication fork

• A replication bubble forms when the space between two forks contains replicated DNA

• The replication bubble consists of:

  • One strand oriented in the 5’→3’

  • One strand oriented in the 3’→5’

• After helicase recognizes the replication origin and creates forks, single-stranded binding proteins (SSBs) bind to separated strands to stabilize and keep the strands from re-annealing

• The unwinding within the replication bubble creates tension ahead of the replication fork

• Topoisomerase enzymes (ie gyrase) cut strands to relieve tension and untangle DNA, then seal them back up

• The replication bubble continues to expand/open along a DNA molecule until it merges with one of the other many bubbles (eukaryote)

DNA replication - Step 2: Adding complementary bases (part 1)

• Nucleoside triphosphates are used to build the new strand of DNA

• The phosphates are required to provide the energy necessary to add the bases to the growing strand

• The nucleosides are added to the growing strand via DNA polymerases

• RNA primase adds a small sequence of RNA nucleotides complementary to the parent strand at the beginning of the replication fork

• DNA polymerase III binds to the primer and adds nucleotides, but only in the 5’→3’ direction of the new strand

• The parental 3’→5’ strand can continue to have complementary bases added adjacent to it in a continuous fashion

• This complementary strand is called the “leading strand.”

DNA replication - Step 2: Adding complementary bases (part 2)

• The 5’→3’ parental strand cannot have complementary bases continuously added adjacent to it

• As DNA polymerase III moves away from the fork, eventually it will run into another primer from another replication bubble

• As the replication fork opens, exposing more parental DNA, new primers are continually added so that the polymerase III can continue to add complementary bases

• This strand is called the “lagging strand” as it is synthesized in segments

• Each segment is called an “Okazaki fragment.”

• The lagging strands grow away from the replication fork

• DNA polymerase I replaces RNA primers with DNA nucleotides

• DNA ligase links Okazaki fragments by catalyzing the reaction that forms phosphodiester bonds

DNA replication - Step 2: Adding complementary bases (part 3 - the last primer on the lagging strand)

• The linear nature of the eukaryotic chromosomes presents a problem with the lagging strand

• The last RNA primer near the end of the newly synthesized lagging strand has no DNA adjacent to it

• When the RNA primer on the last Okazaki fragment is removed, it is not replaced because DNA polymerase II has nothing to attach to

• Enzymes cleave the segment of the parental DNA that does not have a copy to remove the overhang

• This results in one of the new copies having less DNA

• Every time the chromosome replicates, the DNA strand shortens

DNA replication - Step 3: Fixing Errors

• As DNA polymerase add new bases, they proofread and correct errors as they go

• If an incorrect base is added, hydrogen bonds cannot form and the strand becomes unstable, the polymerase cannot continue so it backs up and replaces the base

Repair complexes:

• Protein complexes composed of proteins, DNA polymerase I and II works to repair any missed errors along the strand

• When a distortion is found, enzymes remove the error, the appropriate base is filled in an the strand is sealed with ligase

Telomeres

Segments of non-coding DNA composed of repetitive sequences at the end of linear chromosomes

Telomeres function to…

• Keep chromosomes from fusing

• Protects DNA from nucleases (enzymes that function to cut up DNA)

• Assists DNA repair mechanisms by signalling the end of a chromosome

• May determine the lifespan of an organism

The Hayflick Limit

The total number of times that a cell can divide without the loss of function due to the loss of coding DNA

• Different in every species

  • In human cells, approximately 50 times

• Some cells can add base pairs to their telomeres using the enzyme “Telomerase”

Telomerase

An enzyme that adds base pairs to telomeres to prevent loss

• white blood cells and cells that will undergo meiosis

• Cancer cells produce very high quantities, making them essentially immortal

• Telomerase has many implications for cancer research and studies on aging

Cell Sentence (After Hayflick limit is reached)

• As cells continue to divide and the DNA gets progressively shorter, the telomeres will eventually be completely lost

• During subsequent replications, important regions of the DNA may be lost, causing the cell to lose its ability to metabolize, grow or divide

• Cell senescence may account for loss of functions during aging

Telomeres and Aging

• Although scientists have found many correlations between telomere length and aging, it is not the only factor that determines life expectancy

• Generally:

  • Cells divide at divide at different rates and will reach the Hayflick limit at different times

  • As individuals get older, more and more of their cells reach senescence reducing normal functioning