MBIO 1220

Midterm 2

Central Dogma of Molecular Biology

• Replication: DNA → DNA

• Transcription: DNA → RNA

• Translation: RNA → Protein

Genetics

• Science of heredity

Molecular Biology

Science dealing with DNA and protein synthesis

Genome

• Total DNA contained in cell

  • Consists of chromosomes and any plasmids

    • Chromosomes contain genes

Genes

Sections of DNA that code for a functional product

DNA

Macromolecule made of nucleotides

Nucleotides

• Nitrogenous bases

  • A: Adenine

  • T: Thymine

  • G: Guanine

  • C: Cytosine

• Sugar

  • Deoxyribose (1’ to 5’)

• Phosphate

DNA - Double Helix

• Two strands held together by hydrogen bonds between bases

• Complementary

• Sequence of one strand determines the sequence of the other

Base Pairing Rule

• A always pairs with T

• G always pairs with C

How are nucleotides linked?

• By covalent phosphodiester bonds

• 5’ carbon of one nucleotide is joined to 3’ carbon of the next nucleotide, with a phosphate between them

DNA Direction?

• 5’ to 3’ direction

  • Starting at 5’ end

  • Finishing at 3’ end

• Two strands of DNA run antiparallel

Flow of Genetic Information

1. Replication: DNA is copied before cell division

2. Gene Expression: DNA is used to make proteins

3. Recombination: DNA can flow between two different bacterial cells

DNA Replication

• One parental double stranded DNA molecule is used to make 2 identical double stranded DNA molecules

• Complementary

  • One strand can serve as template for synthesis of the other strand

  • DNA polymerase reads the order of the nucleotides in the template strand to make a complementary new strand

DNA Replication - Step 1

• A small segment of the dsDNA unwinds and the strands are separated

  • Forms replication fork

• Each separated strand serves as template for synthesis of a complementary strand

  • Short RNA primer is produced by the enzyme primate

  • Serves as starting site for nucleotides to new strand of DNA

DNA Replication - Step 2

• Synthesis of Leading strand

• DNA polymerase can only synthesis DNA in one direction - 5’

  • Template must be read in the 3’ → 5’ direction

  • Follows replication fork

  • Synthesis of the leading strand is continuous in the 5’ to 3’ direction

DNA Replication - Step 3

• Synthesis of the Lagging strand

• DNA polymerase can only make DNA in 5’ to 3’ direction

  • The second strand must be made in the opposite direction

• DNA polymerase synthesizes small fragments of DNA: Okazaki Fragments

  • Made in the 5’ to 3’ direction

  • Afterwards, RNA primers are removed and the fragments are joined together by enzyme DNA ligase

Gene Expression

• 2 Parts

  • Transcription: Information stored in DNA is copied into RNA

  • Translation: Information inRNA is decoded to make protein

Transcription

• Synthesis of RNA from a DNA template

  • Sequence is complementary To a gene

    • Except it contains U instead of T

3 Types of RNA

• Messenger RNA (mRNA): Carries information for making a specific protein

• Ribosomal RNA (rRNA): Forms part of the ribosome

• Transfer RNA (tRNA): Transports specific amino acids for protein synthesis

Transcription - Step 1

• Initiation

• RNA polymerase binds to the gene at specific site called the promoter

  • Separates (melts) the two strands

  • Only one DNA strand is copied → the template

  • Template is read in the 3’ → 5’ direction so that RNA can be made in the 5’ → 3’ direction

Transcription - Step 2

• Elongation

• RNA polymerase moves along the template synthesizing new RNA

• Allows DNA to rewind behind it

Transcription - Step 3

• Temrination

• When RNA polymerase encounters the terminator (end of gene) it falls off the template and releases the newly synthesized RNA

Genetic Code

• information in mRNA must be translated to make proteins

• Organized into sets of 2 nucleotides - codons

• Each codons specifies an amino acid to be added during protein synthesis

• Sequence of codons in an mRNA determines sequence of amino acids in the protein

• 3 Codons specify STOP codons:

  • UAA, UAG, UGA

  • Signal end of protein synthesis

Translation - Step 1

• Initiation

• Ribosome assembles on mRNA

• tRNA carrying amino acid formyl-methionine enters P site

• tRNA carrying second amino acid enters ribosome

• Specified by the codon in the A site

• Ribosome joins the amino acids together by peptide bond

Translation - Step 2

• Elongation

• Ribosome moves a distance of one codon down the mRNA

  • Next codon is now in place in A site

• Correct tRNA enters A site, bringing with it the next amino acid to be added

• Amino acid is joined to the chain

• Forms a polypeptide

• Elongation continues until a STOP codon is reached

Translation - Step 3

• Termination

• When a STOP codon enter the A Site, ribosome disassembles and releases the polypeptide

• Polypeptide is folded into the correct shape and becomes a protein

• Ribosome can initiate translation of another mRNA

Genetic Change in Bacteria

• Mutation

• Horizontal Gene Transfer

Mutation

• Change in nucleotide sequence of DNA

• May cause change in protein encoded by gene

Mutation - Base Substitution

• Single nucleotide is replaced by another nucleotide

• When DNA replicates, results in a substituted base pair

• When DNA is transcribes and translated can result in an incorrect amino acid in the protein

  • Missense mutation

Mutation - Frameshift

• Insertion: One or two nucleotide(s) added to the gene

• Deletion: One or two nucleotide(s) removed from the gene

• Changes the reading frame of mRNA

  • Sequence of amino acids changed “downstream” of mutation site

  • Almost always results in a non-functional protein

How do spontaneous mutations occur?

• Occur in a dense of mutagens, due to occasional mistakes during DNA replication

How do induced mutations occur?

• Occur when DNA damaging agents cause changes in DNA sequence - mutagens

• Ex. Radiation, some chemicals

Regardless of origin, mutations can result in…

1. No effect on the protein (remains functional) - Silent mutation

2. Protein with a different amino acid sequence that may have altered function - Missense mutation

3. Premature STOP codon - Incomplete (truncated) protein, usually nonfunctional - Nonsense mutation

Plasmids

• Self replication, dsDNA molecules

• Contain non-essential genes

  • Ex. Genes for toxin production

Plasmids: F Plasmids - Fertility Factors

• Carry Genes to make F pilus (sex pilus)

  • Involved in conjugation (transfer of genetic material between bacteria)

Plasmids: R Plasmids - Resistance Factors

• Carry genes for antibiotic resistance

Plasmids: Vir Plasmids - Virulence Factors

• Carry genes for toxin production

Horizontal Gene Transfer - Transformation

• Pieces of “naked” DNA are taken up by a bacterial cell

• Ie. From dead cells, or from released plasmids

• These pieces can be integrated into the chromosome

  • Recombination

  • Can then be passed to progeny and become a stable part of the genome

Horizontal Gene Transfer - Transduction

• Small fragments of DNA transferred between bacteria by bacteriophage

  • Viruses that infect bacteria

• Phase attaches to bacterial cell wall - injects its DNA into the cell

• Phage DNA is replicated inside bacterial cell

• Phage DNA also encodes enzymes that cut the bacterium’s DNA into fragments

• As new phages are being assembled, some accidentally receive a piece if bacteria DNA instead phage DNA

  • Transducing particle

Transducing Particle

• Can carry bacterial genes to another cell

• Injection mechanism is still fully functional

• But, bacterial DNA is injected into the cell

• If the injected DNA recombines with the existing chromosome, it becomes a stable genetic element

  • Ie. Will be passed to progeny

Horizontal Gene Transfer - Conjugation

• Bacterial mating

• Mediated by genes encoded on an F factor

• Transfer occurs when:

  • Donor cell (F+) forms an F pilus and uses it to attach to recipient cell (F-)

  • Pilus retracts bringing the cells together

  • Donor cell replicated the F factor as a copy is passes to the recipient

  • The recipient becomes an F+ cell

Viruses

• Latin word for poison

• Acellular particles capable of infecting host cells and causing disease

• Not free-living, require a host cell in which to multiply

  • Obligate intracellular parasites

  • Use host metabolic systems and usually disrupt normal host cell function

Virus Features

• Acellular - Do not have a plasma membrane

• Contain a single type of nucleic acid - DNA or RNA

  • Surrounded by protein coat

  • May or may not have additional envelope of lipids

• Have very few of their own enzymes

  • Take over enzymes of host

Host Range

• Viruses can infect animals, plants, fungi, Protozoa, and bacteria

• Most viruses are specific for a single host species

  • To infect a cell, the virus must recognize features on the host cell surface

  • Ex. Some viruses recognize the fimbriae of a certain bacterial species

Viral Size

• Electron microscope is required to view viruses

• Range from 20 - 1000 nm in length

Viral Architecture - Nucleic Acid

• Can have either DNA or RNA as the genetic material - not both

• Can be single stranded or double stranded

  • Linear or circular

• Can be in several pieces - segmented

• Total amount of nucleic acid = a few thousand to 250,000 base pairs

  • E. coli chromosome = ~4,600,00 base pairs

• Nucleic Acid and Capsid - Nucleocapsid

  • Minimum required structure for a virus

Viral Architecture - Capsid

• Protein coat surrounding the nucleic acid

• Made up of individual proteins called capsomeres

Nucleocapsid

• Nucleic Acid and Capsid

• Minimum required structure for a virus

Viral Architecture - Envelope

• Not present in all viruses

• Lipid bilayer (membrane) acquired from the host cell

• External coating around nucleocapsid

• Additional viral proteins inserted into envelope, Spikes

Morphology of Virus

1. Polyhedral

   • Usually icosahedral - shape with 20 triangular faces

2. Helical

   • Long rods- rigid or flexible

3. Enveloped

   • Roughly spherical - dictated by liquid bilayer

4. Complex

   • Polyhedral head with a helical tail

   • Only found in bacteriophages

Classification of Viruses

1. Nucleic Acid Type

   • DNA or RNA

   • Single stranded or double stranded

   • Segmented or single molecule

2. Capsid Structure

   • Polyhedral

   • Helical

3. Presence of Envelope

Naming Viruses

• Family - Ends with suffix - viridae

• Genus - Ends with suffix - virus

• Species - Specific epithets are not used

  • Instead virus species are given a descriptive name

• Ex.

  • Family: Herpesviridae

  • Genus: Simplexvirus

  • Species: Human herpesvirus 2

  • Virus responsible for genital herpes

Multiplication of Animal Viruses - Adsorption

• Attachment to host cell

• Viruses have attachment sites, recognize protein or glycoprotein of host membrane

Multiplication of Animal Viruses - Penetration

• Entry into host cell

• Most enveloped viruses enter by fusion, lipid of envelope fuse with host cytoplasmic membrane

• Makes virus enter the cell via endocytosis

Multiplication of Animal Viruses - Uncoating

• Viral nucleic acid is freed from the capsid

Multiplication of Animal Viruses - Biosynthesis

• Viral nucleic acids are replicated

  • DNA replication occurs in the nucleus

  • RNA replication occurs in the cytoplasm

• Viral proteins (capsomeres) are synthesized in the cytoplasm

• Relies on the host metabolic machinery

• Ex. Replication and transcription enzymes, ribosomes

Multiplication of Animal Viruses - Maturation & Assembly

• New visions are assembled

  • Capsomeres form the capsid

  • Nucleic acid enters capsid, forms the nucleocapsid

Multiplication of Animal Viruses - Release

• Naked viruses burst out, rupture host cell, and host cell dies

• Enveloped viruses bud out, virus pushes through cytoplasmic membrane

  • Steady release of mature viruses

  • Host cell stays alive for a long time

Interactions between Viruses and Animal Hosts

• Host defence plays major role in outcome of viral infection

  • Protects against otherwise lethal infection

• Most healthy humans carry a number of…

  • Viruses

  • Antibodies to viruses

• If virus is transferred from immune host to another individual, can result in infection

Animal Virus Infection - Acute Infection

• Usually short duration

• Disease symptoms result from tissue damage

  • Lysis of host cells - Release and spread of virus particles

• Host defence systems gradually eliminate virus

  • May take days or weeks

• Host Mandy envelop long lasting immunity

• Ex. Mumps, Influenza, Polio

Acute Infection with Late Complications

• After acute period, some non-infectious particles remain

  • Can cause serious disease later

• Ex. Measles → subacute sclerosis panencephalitis

• Fatal brain disorder - occurs up to ten years after recovery from measles

Animal Virus Infection - Persistent Viral Infections

• Virus is continuously present in body, but may or may not cause disease

  • Ie. May be no symptoms

• Infected host can still serve as reservoir

  • Can transmit virus to others

Animal Virus Infection - Chronic Viral Infection

• After acute period, infectious virus remains present at all times

  • May or may not cause noticeable symptoms

• Ex. Hepatitis B (Serum hepatitis virus)

  • Transmitted by blood, or sexually transmitted

  • May have acute period - Fever, nausea, jaundice

  • After acute period, virus numbers stay high for the rest of the patients life

    • May cause cirrhosis or liver cancer after many years

Animal Virus Infection - Latent Viral Infections

• Acute infection followed by symptomless period

• Virus integrates a copy of its DNA into a host cell chromosome and remains dormant

  • Provirus

  • Disease can be reactivated years later

  • Symptoms may be different

• Ex. Varicella-Zoster virus (herpes family)

  • Causes - Chicken pox (Varicella) in children

  • Remains latent for years - No disease

  • Can reactivate late to cause shingles (Herpes-Zoster)

Viruses & Human Tumours

• Tumour: Abnormal growth of tissue

• Benign Tumour: Does not spread

  • Malignant Tumour: Metastasize and inside nearby tissues (Ie. Cancer)

Cell Growth is Controlled by Two Types of Genes…

• Proto-oncogenes: Stimulate cell growth

• Tumour Supressor Genes: Inhibit cell growth

• Mutations in these genes can lead to uncontrolled cell growth, tumour formation and cancer

Cancer Causing Viruses

• Oncogenic Viruses

• Carry oncogenes, genes that interfere with the cells control mechanisms

• Most are DNA viruses

  • Integrate viral DNA into the host chromosome as provirus

  • Oncogenes continue to be expressed

Viruses Associated with Cancers in Humans - Hepatitis B & C

Believed to cause almost all cases of liver cancer

Viruses Associated with Cancers in Humans - Epstein-Barr Virus

• Causes infectious mononucleosis

• May cause lymphoma (cancer of white blood cells) and some cancers of the nose and throat

Viruses Associated with Cancers in Humans - Human Papillomavirus (HPV)

• Sexually transmitted - Genital warts

• Believed to cause almost all cases of cervical cancer

Virus-like Infectious Particles - Viroids

• Naked RNA

• No protein coat

• Results in some diseases in plants

  • Not yet found in animals

Virus-like Infectious Particles - Prions

• Infectious protein particles

• No genetic material (RNA or DNA)

• Linked to several human and animal diseased

  • Transmissible spongiform encephalopathies

  • Sponge-like holes in the brain

Mode of Infection

• seem to be transmitted through food

• Ex. Sheep infection with prions - Scapie

  • Eaten by cows - Mad Cow Disease

  • Eaten by humans - Variant Creutzfeldt-Jakob Disease

• Not usually destroyed by high temperature

  • Can be destroyed by heat (480°C)or a combination of autoclaving in a solution of sodium hydroxide (strong base)

  • of disease in humans occurs several years after infection

• Onset of disease in humans occurs several years after infection

  • Not clear why or how it accumulates in the brain

  • Always fatal - No treatment or cure

Overview of Innate Immunity

• Refers to defences present at birth

• Non-Specific: Act against all (most) microbes in the same way

• No Memory Component: Cannot recall previous contact with an invader

• Always Present: Active before an infection occurs

  • Responds rapidly

Innate Immunity Includes…

• First Line Defences

  • Physical and chemical barriers that prevent microbes from entering the body

• Second Line Defences

  • Component that act to eliminate microbe that have invaded body tissues

  • Cellular defences

  • Molecular Defences

  • Fever and inflammation

First Line of Defences - Physical Barrier: Skin

• Outer surface of skin consists of dead cells and keratin (protective protein)

• Frequently shed - removes microbes

• Dry - Inhibits growth of microbes

  • Skin infections are more common on moist areas of skin, or in moist environments

• Outer layer of skin is an excellent defence - rarely penetrated by microbes

  • Most infection occur under the skin - after skin has been broken

• Some microbes are able to eat dead skin cells and oils secreted by the skin

  • Results in body odour

First Line of Defences - Physical Barrier: Mucous Membranes

• Involves in fluid or gas exchange

• Offer less protection than skin

• Line our “tracts”

  • Ex. Digestive tract

• Secrete mucous - a glycoprotein - keeps membrane for drying (cracking)

  • Traps microbes

• Mucocilliary escalator

  • Cilia sweep mucous

First Line of Defences - Physical Barrier: Fluid Flow

• Saliva, tears, urine, vaginal secretions - move microbes away from body

First Line of Defences - Chemical Barrier: Acidity of Body Fluids and Skin

• Stomach acid - pH of 2

  • Destroys many bacteria and toxins

• Skin - Fatty acids and acid - pH of 3 - 5

• Prevents growth of many microbes

First Line of Defences - Chemical Barrier: Lysozyme

• Enzyme that degrades peptide glycine

• Found in sweat, tears, saliva, and nasal secretions

First Line of Defences - Chemical Barrier: Lactoferrin

• Iron binding proteins in milk, mucous

  • Makes iron unavailable to slow growth of microbes

First Line of Defences - Chemical Barrier: Defensins

• Short polypeptides

• Poke holes in microbial membranes

• Produces by epithelial cells

First Line of Defences - Chemical Barrier: Normal Microbiota

• Acquired shortly after birth

• Prevent growth of pathogens

  • Competitive exclusion and Microbial antagonism

Second Line Defence…

• Cells of the immune system…

  • Leukocytes: White blood cells

    • Always found in normal blood, but increase in response to infection

  • Phagocytes: White blood cells that use phagocytosis to “eat” microbes

Leukocytes - 3 Broad Groups: Granulocytes

• Have large granules in cytoplasm - Visible with light microscopes

• 3 Sub-Groups

  • Basophils

  • Eosinophils

  • Neutrophils

Granulocyte Sub-Group: Basophils

• Weak phagocytes

  • Secretes chemoattractants

  • Release histamine - Causes inflammation, allergies

Granulocyte Sub-Group: Eosinophils

• Destroy large pathogens - Ex. Parasitic worms

• Produce extracellular digestive enzymes to attack parasites

Granulocyte Sub-Group: Neutrophils

• Strong phagocytes

  • Polymorphonuclear

  • Can leave the blood and migrate into tissues to destroy invading microbes

Leukocytes - 3 Broad Groups: Mononuclear Phagocytes

• Also have granules - But they are not visible under light microscope

• 2 Sub-Groups:

  • Monocytes

  • Dendritic Cells

Mononuclear Phagocytes Sub-Group: Monocytes

• Initially non-phagocytic

• Leave blood, enter tissues and change into macrophages - Strong phagocytes

• Often found in organs - Filter out invading pathogens as blood passes through

Mononuclear Phagocytes Sub-Group: Dendritic Cells

• Phagocytize foreign material and bring it to the adaptive immune system for “inspection”

  • Antigen presentation

Leukocytes - 3 Broad Groups: Lymphocytes

• 3 Types

  • Natural Killer Cells

  • T Lymphocytes

  • B Lymphocytes

Lymphocytes Sub-Group: Natural Killer Cells

• Responsible for killing infected body cells and tumour cells

• Attack any body cell that displays unusual proteins in the cytoplasmic membrane

Lymphocytes Sub-Group: T Lymphocytes

• Part of adaptive immunity alongside B Lymphocytes

Lymphocytes Sub-Group: B Lymphocytes

• Part of adaptive immunity alongside T Lymphocytes

Molecular Defences

• Compliment System

  • About 30 proteins that circulate blood

  • Work together in cascade

    • Action of one protein triggers action of the next

  • Complement can be triggered several ways:

    • Small molecules being to the surface of invading microbes

Result of Activating the Complement Cascade - Opsonization

• Attach to microbes and act as a flag to attract phagocytes

  • Increases phagocytosis by 1000x

Result of Activating the Complement Cascade - Enhancing Inflammation

• Increase blood vessel permeability

• Attract phagocytes to infection site

Result of Activating the Complement Cascade - Lysis of Foreign Cells

• Formation of membrane attack complexes (MACs)

  • Pokes holes in membranes

  • Kills Gram negative, but not Gram positive bacteria

inflammatory Response

• In response to tissue damage: Blood vessels dilate, fluids leak and leukocytes migrate into tissues

  • More blood reaches are

  • Allows phagocytes to enter tissues - Increase phagocytosis

  • Brings platelets to form blood clots, and nutrients for faster repair