BM1110 Lectures 2 - 8

The Scientific Method

The scientific method ensures that there is rigour and reproducibility in the field. Testing hypotheses requires controlled experimentation because:

1) you need to make sure you are only testing one parameter at a time (e.g. no confounding effects) and

2) you need to ensure all your experimental conditions/instruments/reagents are working properly.

 

Positive controls: Give the expected effect (eliminates false negatives) and makes sure that the reagents, conditions etc. used in an experiment are working. Ask yourself "How will I know that the conditions of the experiment are set up correctly?"

Negative controls: Give the baseline effect (eliminates false positives) and make sure that the observed effects are due only to the parameter being tested. Ask yourself "How will I know that what I'm measuring is due only to the parameter being tested?"

 

The Cell Theory of Life

Cell Theory states that:

  1. All living things are made of cells

  2. Cells are the basic units of life

  3. Cells can only be generated from other cells

 

Microscopy

 

Important terms:

Magnification - the increase in the specimen's apparent size

Resolution/ Resolving power - the ability of an instrument to show two objects as being separate

 

Cells can be unicellular (single celled) or multicellular. All cells are surrounded by a plasma membrane that separates the cell from its environment. The membrane is composed of phospholipids and proteins. The phospholipids form two layers (the phospholipid bilayer), with hydrophobic tails facing together and hydrophilic heads facing the extracellular and intracellular aqueous environments.

 

There are two types of cells:

  • Prokaryotic (e.g. bacteria) lack a nucleus and organelles

  • Eukaryotic (e.g. humans, plants, fungi) possess membrane-bound nucleus and organelles

 

Sizes of cells:

  • Prokaryotes (e.g. bacteria) lack organelles and are usually smaller in size (1 - 10 µm)

  • Eukaryotes (e.g. humans, plants) possess membrane-bound organelles and are usually larger and more complex (10 - 100 µm)

 

Eukaryotic Cell Structure

All cells are bound by a plasma membrane.

However eukaryotic (animal, plant, fungi) and prokaryotic (bacteria, archaea) cells have different structures:

  • Animal cells do not have a cell wall. They contain lysosomes and sometimes cilia/flagella. Animals use centrioles to form spindle fibres during cell division.

  • Plant cells possess a cell wall. They contain chloroplasts. Large vacuoles are important for maintaining cell turgor

  • Bacterial cells do not possess organelles. They have a cell wall made of peptidoglycan

 

Eukaryotic cells possess membrane-bound organelles that each have a specific function:

Nucleus - contains the DNA encoding the cell’s genome. Site of DNA replication and RNA transcription.

Smooth endoplasmic reticulum - joins directly to the nucleus. Site of production and transport of lipids/steroids.

Rough endoplasmic reticulum - joins directly to the nucleus. Site of production of proteins (the roughness comes from the ribosomes on the outer membrane).

Golgi apparatus - stores and distributes cell products.

Lysosome - contains digestive enzymes to degrade food, damaged cellular parts, harmful bacteria.

Vacuoles - store food and waste. Maintains pressure and size of cell by storing water.

Mitochondria - site of cellular respiration (electron transport chain).

Chloroplasts - in plants. Site of photosynthesis (conversion of light energy into chemical energy).

Cytoskeleton - provides mechanical support to maintain shape and assist in cell movement. Integral to the process of cell division.

Cilia/flagella - motile appendages that allow unicellular eukaryotic cells to move. For cells with tissue, cilia enable liquid to pass over epithelial linings, e.g., in digestion.

Centrioles - pairs of cylinder-shaped organelles that make up the centrosome, which is involved in forming the spindle fibres during cell division of animal cells.

Animal cells

Organelles found in animal cells, but not plant cells, include centrioles, lysosomes and flagella

 

Plant cells

Organelles unique to plant cells include: large vacuoles, chloroplasts and other plastids, and glyoxysomes (used by seeds to convert fats into sugars)

 

Nucleus

Largest organelle, enclosed by a double membrane (nuclear envelope)

Contains the DNA, in the form of chromatin (DNA wrapped in proteins), that carries the code to make the cell's proteins

Contains the nucleolus, which is the site of ribosome synthesis

 

Ribosomes

Structures that function as sites of protein synthesis in the cytoplasm.

 

The Endomembrane System

This is a system of interconnected membrane-enclosed compartments that carry out a range of metabolic activities, including protein post-translational modification, protein degradation, protein secretion and lipid synthesis. Consists of the endoplasmic reticulum (ER), transport vesicles, Golgi apparatus, lysosomes and vacuoles.

 

Mitochondria

Mitochondria are the "powerhouses" of the eukaryotic cell, involved in the production of ATP under aerobic conditions. They have their own genome, separate from the nuclear genome, and are the result of a bacterial endosymbiosis event early in eukaryotic evolution.

 

Chloroplasts

These are the organelles in plants responsible for harvesting the energy from light for the generation of glucose. As with mitochondria, chloroplasts have a separate genome from the nucleus and evolved from a bacterial endosymbiosis event.

 

Composition of the Plasma Membrane

The plasma membrane is a phospholipid bilayer.

Phospholipids are made of:​

• Hydrophilic “heads” that associate with water ​

• Hydrophobic “tails” that form the membrane core​

 

Bilayer forms a membrane about 8 nm thick 

 

The physical properties of cell membranes depend on their components: the lipids, proteins and carbohydrates.

The fluid mosaic model describes the membrane as a mobile (fluid) lipid phase in which a variety of discrete components (a mosaic) such as protein float freely.

 

Membrane proteins may be:

  • integral - span across the membrane or are partially embedded in the membrane

  • peripheral - non-covalently attached to one side of a membrane

  • anchored - covalently bound to a lipid moiety such as glycosylphosphatidylinositol (GPI) that is inserted into the membrane

 

The fluid property allows for rapid repair and is important for many of the cell membranes properties (e.g. vesicle formation, phagocytosis, cell division).

 

Membrane fluidity is affected by factors such as:

  • Lipid composition: Cholesterol and long-chain, saturated fatty acids pack tightly beside one another resulting in a less fluid membranes. A higher proportion of shorter-chain fatty acids, unsaturated fatty acids, or less cholesterol results in a more fluid membrane.

  • Temperature: Molecules move more slowly and fluidity decreases at lower temperatures. In some organisms the lipid composition of their membranes change with temperature, with unsaturated fatty acids and fatty acids with shorter tails replacing saturated fatty acids in colder temperatures in order to maintain membrane fluidity.

 

Membranes function as barriers: Compartmentalisation

Some compounds simply diffuse through membranes ​eg. O2, CO2, H2O, glycerol, fatty acids, steroid hormones​

​Others are blocked​ eg. glucose, inorganic ions, proteins, nucleic acids, large lipid aggregates ​

 

Electrochemical gradients can also be established across membranes

 

Allows for compartmentalisation - the separation of functions in different parts of a cell on either side of a membrane

 

 

Types of Membrane Transport

There are two basic processes through which substances can be transported across membranes:

  • passive - does not require energy input

  • active - required energy input

 

Passive membrane transport - Simple Diffusion and Facilitated Diffusion

Simple Diffusion

Diffusion is the random motion of solutes that leads to a state of equilibrium

Some small molecules, or hydrophobic molecules such as some hormones, are able to diffuse across cell membranes through simple diffusion

 

Facilitated Diffusion

Polar or charged molecules are less likely to be able to diffuse across membranes but may still cross the membrane passively with the aid of protein channels.

Two main types:

  • channel proteins - integral membrane proteins, spanning the membrane, that form "tunnels" through the membrane to permit particular substances to pass through e.g., gated channel proteins.

  • carrier proteins - these bind to particular substances and increase their diffusion through the membrane e.g., glucose transporter.

 

Diffusion aided by either of these types of proteins is called facilitated diffusion. It is a passive process since energy from ATP is not required.

 

 

Active membrane transport 

Active transports requires the input of energy and is directional

Three types of membrane proteins carry out active transport:

  • symporters

  • antiporters

  • uniporters

 

 

Primary Active Transport

Involves direct hydrolysis of ATP to provide the energy needed to carry out the transport e.g., the Na+ - K+ pump; an antiporter

 

 

Secondary Active Transport

Energy from ATP is not used directly. The energy from ATP is used by a separate primary active transport protein to set up an ion gradient and it is the energy from this that drive secondary active transport. e.g., Na+ symporter used to transport glucose​

  • Na+ gradient set up by Na+/K+ pump (active transport)

 

 

Transport of larger macromolecules and particles - Endocytosis and Exocytosis

Some molecules, such as proteins and nucleic acids, are too large to be handled by membrane protein transport systems.

These larger molecules, and large particles, are handled by endocytosis (transport into the cell) or exocytosis (transport out of the cell).

Three types of endocytosis will be covered:

3 types of endocytosis transport macromolecules​

  • ​Phagocytosis: from Greek for “cellular eating” ​- for large particles: macrophages and neutrophils do this​

  • Pinocytosis: from Greek for “cellular drinking” - smaller dissolved substances, e.g. proteins or fluids, used by secretory cells to retrieve membrane material​

  • Receptor-mediated endocytosis: mediated by cell surface molecules​

 

 

DNA and RNA

DNA is the material that carries an organism's genetic code, although some viruses have an RNA genome (eg. HIV, Ebola, influenza, SARS)

 

DNA and RNA nucleotides are made up of three molecules:

  • a phosphate

  • a sugar (ribose in RNA or deoxyribose in DNA) and

  • a nitrogenous base (A, T/U, C, or G).

 

The carbon skeleton of the sugar is the reference point for describing the structure of the nucleic acids. From the O group, the carbons are counted around the ring starting with the C attached to the base (e.g. C1’, C2’, C3’, C4’ and C5’), because the nitrogen has the highest precedence over the other substituents on the carbon ring. The 1’ carbon is where the base is covalently attached; the 2’ carbon possesses an OH (hydroxyl group) in RNA but no OH group in DNA (hence, deoxyribonucleic acid: a nucleic acid with no oxygen on the ribose group); the 3’ carbon is attached to the phosphate group of the next nucleotide; the 4’ carbon has no functional groups; and the 5’ carbon is bound to the phosphate of the same nucleotide.

 

There are four types of bases in DNA:

  • Adenine (A)

  • Cytosine (C)

  • Guanine (G)

  • Thymine (T)

Due to their structures, A can only pair with T and G can only pair with C

In RNA, the thymine is replaced with Uracil (U).

 

G-C bonds are stronger than A-T bonds because the former have 3 hydrogen bonds, while the latter only have 2.

 

 

Chargaff’s Rule: 

You can derive these conclusions independently just knowing that A pairs with T and G pairs with C, but they have been formalised into "Chargaff's Rule", which states that in DNA:

1) the total amount of purines is equal to the total amount of pyrimidines

2) the total amount of G equals C; and the total amount of A equals T

 

To calculate the percentage of each base:

1)   Number of G + A (PURINES) = Number of C + T (PYRIMIDINES)

2)   Number of Guanines = Number of Cytosines

3)   Number of Adenines = Number of Thymines

4)   Number of G + A + C + T = 100% of DNA bases

 

DNA Structure

DNA is a right-handed, antiparallel (complementary strands run in opposite directions) double helix, with the two strands held together by based pairing.

 

The helix is not symmetrical but has major and minor grooves. Specific sequences are recognised by DNA-binding proteins that make contact with the edges of the DNA bases through the major and minor grooves.

 

 

DNA Replication

In cells, nucleic acids are synthesised in a 5’ to 3’ direction. This directionality is named due to the 5’C and 3’C on the sugar backbone.

A DNA polymerase is responsible for DNA replication, by adding the correct nucleoside triphosphate precursors to match the complementary base in the template DNA.

DNA is replicated semi-conservatively, so that each daughter cell ends up with one old and one new copy of the double stranded DNA

 

 

EUKARYOTIC GENE REGULATION

There are several ways in which eukaryotes can regulate gene expression:

 

Chromatin structure (epigenetics): DNA modification can affect the coiling of chromatin, which affects how accessible genes are for transcription. Covalent modifications can be made to the DNA directly (e.g., methylation) or to the histones that package the DNA (methylation, acetylation). These modifications are not genetic (they do not affect the DNA sequence, but are heritable - they can be passed on to daughter cells, and even between generations of the whole organism!

 

Transcription initiation: transcription is regulated via transcription factors and enhancers, which are proteins that help form the transcription initiation complex. The rate of transcription initiation (and therefore how much mRNA and protein is produced) is controlled via these small regulatory proteins.

 

RNA processing and export: pre-mRNA is processed by capping, splicing, polyadenylation and export into the cytoplasm. The mRNA cannot be translated into protein until the mature mRNA is exported from the nucleus, so this transport to the cytoplasm can control the rate of translation.

 

mRNA degradation: RNA is relatively unstable, which enables cells to rapidly stop producing proteins via translation when they are no longer required. The polyA tail length directs mRNA to be degraded. Over time the ~200 polyA tail degrades. Once ~30 polyA nucleotides remain, the mRNA is rapidly degraded by exonucleases. MicroRNAs also regulate gene expression by binding specific mRNAs and targeting them for degradation.

 

Translation initiation and tRNA availability: Translation initiation can be inhibited by:

  • inhibitory RNAs that bind mRNA and repress translation (and can also cause mRNA degradation), which results in “gene silencing” (a reduction in gene expression). 

  • capping of mRNA with an unmodified GTP, which prevents translation

  • binding of repressors (proteins) that block ribosome binding to mRNA

 

tRNA availability can also affect translation rates. Codons corresponding to tRNAs with low abundance can stall translation, while the abundance of codons corresponding to common tRNAs results in high rates of translation (processivity).

 

Post-translational modifications: can alter the function of proteins after translation. This means that some proteins can be inactive until they are transported to their correct cellular site, where chemical modifications (e.g., glycosylation, phosphorylation, methylation etc.) or proteolysis can occur to form the active protein.

 

Protein degradation: proteins that have lost functionality or are no longer required are degraded via the lysosomes or via the ubiquitin/proteasome pathway.