Case 1: Medical Biochemistry 1

List the stages of Wound Healing


  1. Haemostasis
  2. Inflammation/Immune response
  3. Migration
  4. Proliferation
  5. Revascularisation
  6. Maturation/Re-modelling:
  • Collagen Synthesis
  • Scar Formation

What complex processes are involved in Tissue Repair ?


Tissue repair involves complex processes such as:


  • Cell Division
  • Migration
  • Differentiation
  • Synthesis and remodeling of extra-cellular matrix



Healing involves tissue repair


Growth and Repair is associated with cell division (mitosis) and more specifically DNA replication

Describe the structure of DNA (Deoxyribonucleic Acid)


There are about 30 x 10^12 cells in the body, with 200 cell types.


DNA is stored in the nucleus (10 microns)


Genomic DNA is 2m long ( 3 billion Base Pairs per cell)


Eukaryotic DNA is highly organized into many linear chromosomes 946 chromosomes).


DNA is a Double Helix structure: Made up of two stranded, with complementary Base Pairs.


What is the purpose of DNA ?


DNA is the molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses.



Describe the structure of DNA


DNA is an Anti-parallel Dimer


We know that DNA is a double Helix structure.


DNA is made up of two strands.


The DNA is a polymer made up of 4 types of monomers also known as nitrogenous bases:


Adenine - Thymine
Guanine - Cytosine


The Nitrogenous bases are complementary.


The complementary nitrogenous bases are held together by strong Hydrogen Bonds


The DNA strands are also made up of an alternating Sugar - Phosphate backbone.


Sugar-Phosphate backbone gives DNA its overall negative charge.


  • Sugar is a Deoxyribose


  • Phosphate is the molecule between the sugars




What is a Nucleotide ?


A Nucleotide is made up of:


  1. A Sugar (Deoxyribose or Ribose)
  2. A Phosphate Group
  3. Nitrogenous Base

There are two types of nucleic acids


  1. DNA (Deoxyribonucleic Acids)


  2. RNA (Ribonucleic Acids)



What 3 components are Nucleic Acids made of ?


Nucleic acids are made up of the following components:


Phosphate Group


Sugar Molecule (Deoxyribose or Ribose)


Nitrogenous base


Describe the structure of the Deoxyribonucleotide


  1. Deoxyribonucleotide

Deoxyribonucleotide is a DNA nucleotide


Deoxyribonucleotide is made up of:


  • Phosphate Group
  • Deoxyribose Sugar
  • Thymine (T)

Phosphate molecule is negatively charged, thus making the DNA molecule negatively charged.


Deoxyribose Sugar is a 5-membered carbon sugar ring


Phosphate Group is attached to Carbon 5 on the Sugar group, it is located outside the ring


This forms a Carbon 5 - Oxygen - Phosphate bond.


Carbon 1 and Carbon 4 on the sugar molecule are attached to each other by an oxygen atom


The nitrogenous base is ALWAYS linked to Carbon 1 on Sugar

  • The link is a Carbon-Nitrogen Bond



Describe the structure of the Ribonucleotide


  1. Ribonucleotide

Ribonucleotide is an RNA nucleotide


Ribonucleotide is made up of:


  • Phosphate Group
  • Ribose Sugar
  • Uracil (U)

The Phosphate Group is negatively charged, thus making the whole RNA molecule negatively charged.


Ribose Sugar is a 5-memebered carbon sugar ring


Phosphate Group is attached to Carbon 5 on the Sugar group, it is located outside the ring.


This forms a Carbon 5 - Oxygen - Phosphate Bond


Carbon 1 and Carbon 4 on the Sugar are attached to each other by an Oxygen Atom


The Nitrogenous Base is ALWAYS attached to Carbon 1 on Sugar

  • The link is a Carbon-Nitrogen Bond

What are the differences between Deoxyribonucleotide and Ribonucleotide ?


  1. Nitrogenous bases are different

  • Deoxyribonucleotide has Thymine


  • Ribonucleotide has Uracil




  1. Difference in Sugar Structure

  • Deoxyribose Sugar: Hydrogen molecule is attached to Carbon 2


  • Ribose Sugar: Hydroxyl Group is attached to Carbon 2




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List the Two Types of Nitrogenous Bases


  1. Purines: Adenine and Guanine
    Both Purines are found in DNA and RNA


  1. Pyrimidines: Thymine, Uracil and Cytosine
    DNA has Thymine and Cytosine
    RNA has Uracil and Cytosine

Describe the differences between Purines and Pyrimidines


Purines have two rings:


  • A 6-membered ring on the left
  • A 5-memebred ring on the right

Pyrimidines have are smaller and ONLY one ring:


  • A 6-memebred ring



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The formation of a Nucleotide


A nucleotide can undergo a forward reaction of Condensation,


A condensation reaction is a reaction that involves the loss of Water.


As a result:


Carbon-Nitrogen Bond becomes a Glycosidic Bond


Carbon-Oxygen-Phosphate Bond becomes a Phosphoester Link


NOTE: This can be reversed by a Hydrolysis reaction


A Hydrolysis Reaction is the addition of water.

List the types of Nucleotides according to the number of Phosphates attached to Carbon 5


  1. Nucleotide Monophosphate - 1 Phosphate Group
    Eg: Adenosine Monophosphate (AMP)


  1. Nucleotide Diphosphate - 2 Phosphate Groups
    Eg: Adenosine Diphosphate (ADP)


  1. Nucleotide Triphosphate - 3 Phosphate Groups
    Eg: Adenosine triphosphate (ATP)

Adenine is the nitrogenous base present



What are the names of the bonds between the Phosphate and Oxygen Molecules in the Phosphate group ?


Bonds between the Phosphate and Oxygen Molecules in the Phosphate group are known as High Energy Bonds



Why are they called high energy bonds ?


There is a high energy currency in the molecule


What is Adenine ?

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The structure of a Polynucleotide


What is a Nucleoside ?


A nucleoside is made up of:


  1. A Nitrogenous Base


  2. Pentose Sugar



What is the difference between the Nucleotide and Nucleoside ?


A Nucleotide contains a Nitrogenous Base, pentose Sugar and a Phosphate Group


A Nucleoside contains a Nitrogenous Base, and a Pentose Sugar.


  • Nucleoside does NOT have a Phosphate Group

Structure of the Polynucleotide


3' Carbon
5' Carbon
Phosphate


3' Carbon
5' Carbon
Phosphate


3' Carbon
5' Carbon
Phosphate


3' carbon
5' carbon
3


What are the ends ?


There is an Hydroxyl Group attached to the Carbon 3 at the 3' Carbon end of the molecule - 3' (Hydroxyl) End



There is no nucleotide attached to the Phosphate Group at the 5' Carbon end of the molecule - 5' (Phosphoryl) End


What is Polynucleotide Chain?


Polynucleotide chain is made up of nucleotides attached to each other.


Polynucleotides have directionality


A polynucleotide is when a condensation reaction occurs between the Hydroxyl Group at C3 and the Phosphate on C5 of the nucleotide below.


Will form a 3'-5' Phosphodiester Bond

Which of these DNA sequences will melt at the lowest temperature ?



  1. GCGTCCGGG
    CGCAGGCCC



  1. TTAGTAACT
    AATCATTGA



  1. GGCTAAGCTTGCAAGGCGC
    CCGATTCGAACGTTCCGCG


Answer:


Strand 3


Why ?


The shorter the DNA sequence, the lower the temperature required to separate the strands.


The longer the DNA sequences the higher the temperature required to separate the strands.



What is the difference between Guanine-Cytosine Pairs and Adenine-Thymine Pairs ?


G-C pairs have 3 Hydrogen Bonds


A-T pairs have 2 Hydrogen Bonds



DNA - Genetic Information
What is a Gene ?


Information contained in linear sequences of the bases in the polynucleotide chain


What are the essential properties of genes ?


Due to the anti-parallel structure of DNA, Genes are able to undergo precise self-replication


Genes are complex enough to contain a lot of information

Describe the process of DNA Replication


A Model DNA Strand with 5 base pairs - forming a double helix.


DNA replication occur in the S Part (Interphase) of the Cell cycle.


  1. The basic requirement of DNA replication is the separation of the DNA strands

The hydrogen bonds are broken, and the nitrogenous bases are exposed.



  1. After separation, each parental DNA strand becomes a template.


  1. Complementary pairing will take place as new nucleotides bind to the complementary nucleotide on the parental strands.


  1. Each "daughter" molecule has one parental strand and one new strand.


  1. This results in the formation of 2 new DNA strands/ Molecules


NOTE: This process is known as semi-conservative mechanism of replication.


Both daughter DNA molecules have 1 strand from the parent DNA.


The conservative mechanism of replication results in two daughter DNA molecules where on has two parent strands and the other has two new strands.

DNA Synthesis


How is DNA synthesized ?


New DNA is synthesized by adding one nucleotide at a time.


The nucleotide is added in as a Triphosphate


The type of nucleotide triphosphate added depends on which one is needed to attach on the nucleotide on the parental strand.


Eg:


dATP
dTTP
dGTP
dCTP


The "d" stands for Deoxy

Leading Strand, DNA synthesis will occur in 5' to 3' direction.


Lagging Strand, DNA synthesis will occur in 5' to 3' direction, BUT in the opposite direction to Leading strand


List the enzymes involved in DNA Replication


Helicase


Primer


DNA Polymerase 1 & 2


Ligase



Outline the functions of the Enzymes of DNA Replication


  1. Helicase
  • Helicase is responsible for the separation of the DNA double helix strand
  • This results in the formation of a Replication Fork


  1. Primer
  • Primer is responsible for the formation of a RNA primer to prime DNA synthesis


  1. DNA Polymerase 1 and 2

  • DNA Polymerase 1 is responsible for DNA elongation


  • DNA Polymerase 2 is responsible for removing the RNA Primer in the Lagging Strand, and replacing it with DNA




  1. Ligase
  • Ligase links the 3' Hydroxyl and 5' Phosphate groups of DNA fragments



What are Okazaki Fragments ?


Okazaki fragments are the DNA fragments between two RNA primers in the lagging Strand

Can DNA Replication take place at any point ?


Yes, DNA replication can be initiated at many points


Many replication bubbles will occur


These replication bubbles will then expand towards each other, and eventually join to form a daughter DNA molecule.



It is not possible for DNA replication to occur till the end of DNA strand


Due to the presence of RNA Primers


As a result the Okazaki fragments in the lagging strands will be joined by Ligase


NOTE: Every time we replicate DNA we lose a few nucleotides from the End of the Lagging Strands


Due to the DNA Polymerase not being able to reach end of Lagging Strand




Telomeres


What are Telomeres ?


Telomeres protect the chromosomal ends, to prevent the loss of nucleotides


However, if the DNA undergoes replication multiple times


The telomere will begin to shorten, losing DNA nucleotides


As a result the cell will go into Senescence, where it will not undergo replication.

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Regeneration vs Fibrosis

What is Regeneration ?


  1. Regeneration is the replacement of damages cells by the same kind of cells


  • In order for regeneration top occur, the cells need to be continuously dividing. Eg: Epithelial tissue, Fibrous Connective Tissue and Bone Marrow Cells


  • Some cells have a low baseline level of proliferation. Eg: Liver, Kidney, Pancreas, Endothelial Cells, Smooth Muscle, Bone

What is Fibrosis ?


  • Fibrosis is the replacement of destroyed tissue with dense connective tissue


  • This is done by non-dividing cells. Eg: cardiac, Skeletal Muscle Cells and neurons


  • Bigger/ Serious Damage


  • DNA Replication and Cells Division and Transcription and Translation