Bio 251 Exam 3

Nucleic Acid Tests

looking to identify characteristic sequences of nucleotides in your target organism; can you find sequences of DNA or RNA that are present in your pathogenic organisms and not present in people

Ribosomes

rRNA and proteins

18S rRNA

eukaryotic ribosome, small ribosomal subunit

Information for 18S rRNA in ANY organism stored in

gene that holds info for building 18S rRNA; when that gene is transcribed, the final product is 18S rRNA

Polymerase Chain Reaction (PCR)

a protocol that uses the enzyme DNA polymerase to quickly synthesize billions of copies of a target DNA sequence

What is needed to do PCR?

-original source of DNA
-DNA polymerase enzyme (1 or more)
-primers (DNA or RNA)
-nucleotides; complementary to the DNA you're trying to copy

PCR cycles

1) warm liquid close to boiling to break hydrogen bond holding 2 strands of DNA; 2 primers that are chosen will bind with each strand of DNA that has been separated
2)2^2 copies of DNA
3)2^3 copies of DNA

Explain PCR process

researcher chooses what length and location of DNA is going to be copied, builds the appropriate primers, loads into mixture and you get copies; PCR can make billions of copies of part of just the gene that holds information for building 18S rRNA

PCR to detect Plasmodium in a blood sample

-Not present: PCR will not produce copies of part of 18S rRNA gene from Plasmodium
-Present: PCR will successfully produce billions of copies of part of the 18S rRNA gene from Plasmodium

Agarose gel

contains colorimetric of fluorescent dye; how we visualize copies of 18S rRNA gene

Well

teeth you place PCR in

How DNA moves through agarose gel directionally

1) aqueous buffer conducts electron flow, this is a closed circuit
2) electric current runs through the aqueous buffer from the Cathode to the Anode
3) any soluble negatively charged particles (ex: DNA) in a well will move through the gel towards the Anode
4) shorter/smaller particle will move more quickly

For each species of Plasmodium the copies of part of the 18S rRNA...

gene are of different lengths, so if there is a PCR product then the length of the product molecule indicates which species is present

Chloroquine

vaccine for malaria in pill form taken daily only 33% effective

How Chloroquine works

1) can diffuse from blood into the lumen of the digestive vacuole
2) can bind non-covalently to the heme polymerase (HP) and inactivate it
3) increase in ROS randomly break molecules eventually killing parasite

Structure of Hemoglobin

-a tetramer of : 2 alpha-hemoglobin molecules 2 beta-hemoglobin molecules
-each hemoglobin monomer is bound to one heme group
-each Fe2+ ion in a heme can bind to one O2 molecule

Heme

a ringed molecule with iron ion Fe2+ in the center

Free radical

an atom, molecule, or ion that has an unpaired electron in its highest-energy orbitals; HIGHLY chemically reactive

Reactive Oxygen Species (ROS)

a subclass of free radical: different forms of oxygen molecules that are much more chemically reactive than normal O2; EVERY ROS is a free radical but NOT all free radicals are an ROS

ROS molecules are normally present and

oxygen picks up low energy electron at end of ETC; ROS is a normal byproduct of aerobic respiration

Why are ROS molecules harmful to ALL cells?

they randomly take electrons from other atoms in other molecules breaking covalent bonds

The price for aerobic respiration/extra ATP

is ROS; MOST cells survive by producing proteins to neutralize ROS

Why is random breaking of covalent bonds harmful to the cell?

because the function of damaged molecules will be reduced, eliminated, or changed

Antioxidants

neutralize free radicals by giving them electrons

"free heme" not bound to Hemoglobin is

harmful to cells including Plasmodium

'"free heme"

a pro-oxidant; it can generate ROS molecules

How Plasmodium obtains nutrients while inside an erythrocyte (6)

1) Plasmodium pinocytoses Hemoglobin (Hb) from erythrocyte cytoplasm
2) Hb is held in digestive vacuole (DV)
3) Lysosomes carry digestive enzymes including proteases to the DV
4) enzymes CANNOT digest heme
5) Heme is toxic to Plasmodium bc heme can produce ROS
6) Heme waste is polymerized into Hemozoin (HZ) which is an insoluble crystal; polymerized heme is unable to produce ROS

How is Hemozoin polymerized?

Plasmodium synthesizes and uses the enzyme Heme polymerase; if something were to prevent heme polymerase from working then there would be and increase in ROS concentration of Plasmodium

Humans related to fungi

Opisthokonta are a monophyletic group that includes animals, sponges, fungi (1.3 GYA ago)

Fungi characteristics

uni- or multicellular, chemoheterotrophic, digestion of food occurs outside of the cell, cell wall include polysaccharide chitin

Chitin

NAG, located on extracellular membrane

Structure of multicellular fungus

hypha: branched filaments that is one cell wide; EVERY cell in a fungus is in contact with environment

Unicellular fungi

yeasts

How fungi obtain energy and carbon (5)

1) fungal cells make physical contact with organic material
2) fungal cells synthesize digestive enzymes
3) excrete digestive enzymes into to environment where organic material is
4) fungal enzymes digest macromolecules into small molecules
5) small molecules are transported into fungal cells

What do fungi contribute to the environment?

they are decomposers: organisms that convert large complex molecules into simple molecules; these simple molecules can then be used as materials by living organisms to build more cells

Fungi microbiota in humans

majority of symbiotic eukaryotes in/on human skin are fungi

Structure of human skin

epidermis made of 5 layers of living and dead keratinocytes

Stratum corneum

outermost skin layer; contains dead cells filled with keratin

Malassezia furfur (fungi on skin)

-commensal yeast
-about 80% of fungal population of healthy human skin
-unable to synthesize fatty acids independently
-colonizes surface of the stratum corneum, usually near sebaceous glands that produce oils

Malassezia furfur infections

-opportunistic pathogens; when they become pathogenic they change from being unicellular to multicellular (mycelial)
-may cause dandruff and folliculitis
-usually treated with Imidazole; a topical antifungal medicine that is a liquid soap
-extreme condition: catheter-aquired fungaemia in neonate and adult patients undergoing lipid replacement therapy

Medically important Metazoan symbionts

-Chordata: humans
-Arthropoda: Class Arachnida Genus Demodex

Demodex folliculorum (eyelash mite)

-member of Class Arachnida (related to spiders, scorpions, ticks)
-length 279-294 micrometers
-life cycle 15-21 days
-present in 20-80% of healthy people
-they eat dead keratinocytes and sebaceous oils
-commensals, but can be associated with some diseases
-infested follicles usually hold 2-6 mites

Blepharitis symptoms/cause

-inflammation of eyelids
-symptoms: watery red eyes, itchy eyelids, red swollen lids, crusted lashes
-cause: occurs when tiny oil glands near the base of the eyelashes become clogged

Treatment of Demodex blepharitis

-tea tree oil (TTO); extract of leaves of Melaleuca alterifolia found in Australia

T4O (terpinen-4-ol)

the ONLY component of TTO that kills Demodex

How T40 kills Demodex (5)

-targets the nervous system
1) Neurotransmitter ACh that is released by a presynaptic neuron
diffuses across the synapse and binds noncovalently to an ACh receptor in the membrane of a postsynaptic cell. The postsynaptic cell continues transmitting the signal
2) All signals must end: Acetylcholinesterase (AChE) normally hydrolyzes Ach into acetate and choline
3) T4O is an AChE inhibitor (AChEI): it binds non-covalently
to AChE and prevents it from working
4) ACh builds up in synapses, and causes excess ACh signaling
5) This results in muscle paralysis, respiratory failure, increased respiratory secretions, seizures, coma, and death in humans; similar effects happen to Demodex in their nervous system from T40

Living system

any system that uses energy for the purpose of growth, maintenance, and reproduction; if NOT using energy then it is NOT alive

Cell

fundamental unit of life; structural and fundamental unit

Characteristics of viruses

-acellular
-contain nucleic acid DNA or RNA never both
-possess a proteinaceous capsid
-do NOT possess ribosomes or organelles
-CANNOT generate their own ATP
-dependent on cells for reproduction

Viruses are not

cells and are not alive

Virus size

-most are less than 400nm; usually much smaller than a single prokaryotic cell (1-10 micrometers)
-smallest ex: Porcine circovirus (PCV) 17nm; infect pigs
-largest ex: Pithovirus massiliensis 1.4 micrometers; infect amoebae

Capsid structure

hollow shell that is made of multiple non-covalently bound proteins and that encloses viral genetic material (DNA or RNA)

Capsid function and example

-protection of nucleic acids from physical damage and host immune responses
-example: Hepatitis A (HAV): icosahedral symmetry (20 triangular faces); contains RNA; +ssRNA

Classifying Viruses

-Morphology: shape (helical, polyhedral, spherical, complex); presence/absence of an envelope
-Nucleic acid type: DNA or RNA
-Replication mechanism ex: ytic or lysogenic cycles
-Host organisms ex: bacteriophages target bacteria
-Type of disease caused ex: influenza virus

Transcription of a virus gene

1) The product, an RNA molecule, was constructed by enzymes using the antisense DNA strand as a template
2) The nucleotide sequence of the new RNA molecule is complementary (base-pairing roles) to the sequence of the antisense DNA strand
3) The nucleotide sequence of the new RNA molecule is identical to the sequence of the sense DNA strand

Polyhedral example

Human adenovirus (HAdv); icosahedral virus dsDNA

Helical example

Tobacco Mosaic Virus (TMV) +ssRNA

Spherical example

Influenza A (IAV) -ssRNA (8 different RNA molecules); envelope

Complex example

T4 bacteriophage; dsDNA (in head)

Structures external to capsid

viral envelopes are membranes; phospholipid bilayers that include proteins; some include glycoproteins

Enveloped RNA virus example

SARS-CoV-2, HIV

Enveloped DNA virus example

Herpesviridae

Bacteriophage replication process

1) Attachment: phage attaches by tail fibers to host cell
2) Penetration: phage lysozyme opens cell wall; tail sheath contracts to force tail core and DNA into cell; viral DNA is often circularized in host cytoplasm; host chromosomal DNA molecule is digested
3) Biosynthesis: replication of viral DNA and production of viral proteins begins
4) Maturation: assembly of phage particles
5) Lysis: phage lysozyme breaks cell wall

Lytic cycle of a T-even bacteriophage

1) Attachment: bacteriophage binds non-covalently to host cell
2) Penetration: bacteriophage penetrates host cell and injects its DNA; viral DNA is circularized in the host cytoplasm; host DNA is digested
3) Biosynthesis: viral DNA is replicated; viral proteins are synthesized
4) Maturation: viral components are assembled into virions
5) Lysis: host cell is lysed; new virions are released

Recombination process

1) enzymes locate identical (or nearly identical) nucleotide sequences in two parallel dsDNA molecules
2) Enzymes break covalent bonds in existing DNA strands,
and then covalently connect donor and recipient strands

Lysogenic cycle of a bacteriophage (5)

1) attachment
2) Penetration: host DNA is NOT digested
3) Bacteriophage DNA is covalently integrated into the host chromosomal DNA molecule, Bacteriophage is now referred to as a prophage
4)The lysogenic bacterium reproduces normally
5) In certain circumstances, enzymes will excise the prophage from host chromosomal DNA, initiating the lytic cycle

Triggers for a prophages entry into the lytic cycle

-desiccation
-exposure to mutagenic chemicals
-exposure to ultraviolet or ionizing radiation

Both transduction processes

Both process DNA from one host cell being brought into a 2nd host cell and the vector for that transfer is a virus

Generalized transduction (involves lytic cycle)

1) A phage infects the donor bacterial cell
2) Phage DNA and proteins are made, and the bacterial
chromosome is broken into pieces
3) Occasionally during phage assembly, pieces of bacterial
DNA are packaged in a phage capsid. Then the donor cell
lyses and releases phage particles containing bacterial
DNA
4) A phage carrying bacterial DNA infects a new host cell, the recipient cell
5) Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells

How recipient may benefit from transduction

ex: they could obtain a gene that allows it to fight an antibiotic for better survival

Specialized transduction (involves lysogenic cycle)

1) Induction of the lytic cycle
2) Prophage is incorrectly excised from bacterial chromosome
3) Phage DNA incorporating some bacterial genes
4) Phage repilicates
5) Bacterial cell is lysed; phages are released
6) Phage infects new host cell
7)Phage incorporated into chromosome
8) Host cell chromosome acquires both phage DNA and genes from previous host

Similarities of transduction processes

both transfer genomic DNA from one host cell to another; affected by a viral particle

Differences of transduction processes

in terms of numbers

Animal DNA virus replication ex: Human Papilloma Virus (HPV; dsDNA)

1) Attachment: virion attaches to host cell
2) Entry and uncoating: virion enter the cell and its DNA is uncoated; the proteins that make up the capsid disperse in the cytoplasm
3) A portion of the viral DNA is transcribed, producing mRNA that encodes early viral proteins (in the nucleus)
4) Biosynthesis: viral DNA is replicated, and some viral proteins made (in cytoplasm)
5) Late translation; capsid proteins are synthesized
6) Maturation: virions mature
7) Virions are released

Animal DNA virus replication may or may not...

kill the cell immediately and can go on for a long period of time

Animal RNA virus replication ex: SARS-CoV-2 (+ssRNA)

1) Spike protein on the virion binds to ACE2, a cell-surface protein. TMPRSS2, an enzyme helps virion enter
2) The virion releases its RNA
3) SOME RNA is translated into proteins by the cells machinery (ribosomes)
4) SOME of these proteins form a replication complex to make more RNA
5) Proteins and RNA are assembled into a new virion in the golgi complex and...
6) Released

Retrovirus

makes use of an enzyme called reverse transcriptase; can build a DNA strand using RNA as a template

Animal retrovirus replication ex: HIV

1) Retrovirus enters by fusion between attachment spikes and the host cell receptors
2) Uncoating releases the two viral RNA genomes and the viral enzymes reverse transcriptase, integrase, and protease; proteins of capsid disperse
3) Reverse transcriptase copies viral RNA to produce double-stranded DNA
4) The new viral DNA is transported into the host cell’s nucleus, where it is integrated into a host cell chromosome as a provirus by viral integrase. The provirus may be replicated when the host cell replicates.
5) Transcription of the provirus may also occur, producing RNA for new retrovirus genomes and RNA that encodes the retrovirus capsid,
enzymes, and envelope proteins
6) Viral proteins are processed by viral protease; some of the
viral proteins are moved to the host plasma membrane
7) Mature retrovirus leaves the host cell, acquiring an envelope and
attachment spikes as it buds out

SARS-CoV-2

-Severe Acute Respiratory Syndrome- Coronavirus (50nm)
-RNA is 30 kb long
-Includes at least 14 genes that are translated into proteins; one of those 14 genes holds information for building spike proteins and another gene hold information for building Nucleoprotein

Spike protein structure

-1,273 aa long
-S1 and S2 are non-covalently bound to each other
-each spike comprises of three S1/S2 dimers
-S1: 1 to 685 aa

SARS-CoV-2 replication in human cells

1) +ssRNA is translated into polyproteins
2) polyproteins self-cleave into multiple proteins that form a protein complex
3) SOME proteins in this complex build partial -ssRNA; others build viral genomic -ssRNA
4) partial and genomic -ssRNAs are then transcribed into +ssRNAs
5) new genomic +ssRNAs are used to build new virions
6) partial +ssRNAs are translated into viral proteins by host ribosomes
7) viral N is translated in the cytoplasm
8) viral S, E, M are translated at the RER

ACE2 receptors located

nasal epithelial cells, motile cilia of airway epithelial cells, lungs: ciliated cells, type 2 alveolar cells

COVID cell damage can lead to ARDS

acute respiratory distress syndrome, cardiac dysfunction, loss of taste and smell

Symptoms of COVID

-fever
-dry cough
-fatigue
-dyspnea SOB
-hemoptysis (coughing up blood)
-Leukopenia (reduced # of circulating leukocytes)
-lymphopenia (reduced # of circulating lymphocytes)
-bilateral pneumonia
-olfactory/gustatory disorders

US fatality ratio

1.2%

mRNA COVID vaccines

Moderna/Pfizer: antigen- full length S protein stabilized in pre-fusion state by proline substitutions

Vaccine delivery

mRNA is carried within artificially constructed lipid nanoparticles (LNPs)

How mRNA is take up by a human cell

1) LNPs are taken into vesicles
2) LNP and vesicle membrane fuse
3) mRNA is released into the cytoplasm

mRNA vaccine produces immunity (T and B cells are part of our adaptive immune response)

1) LNPs that hold mRNA for viral Spike protein are injected into muscle tissue
2) LNPs are taken into vesicles; Spike protein mRNA is released into muscle cell cytoplasm
3) host ribosomes use mRNA to synthesize viral protein
4) Viral protein induces adaptive immune response
-using receptors muscle cells present viral protein (antigen) to T cells
-viral proteins in plasma membrane of muscle cell makes contact with receptors on B cells

mRNA longevity

median half-life of mRNA molecules is c 10hr
-cells break down mRNA and get rid of it within a few days after vaccination
-the Spike proteins may stay in the body up to a few weeks
-after 100 hrs more than 99.9% of a given mRNA has been digested by the cell

COVID PCR test

looks for pieces of virus in nose, throat, or respiratory tract to check for an active infection (nasal swab, buccal swab, saliva sample)

COVID Antigen Test

looks for pieces of proteins that make up the virus to check for an active infection (nasal swab, buccal swab, saliva sample)

Antibody Test

looks for antibodies that formed to fight off the virus in blood to determine if there was a past infection (blood sample)

PCR tests

amplify 2 short sequences that are part of the viral Nucleoprotein gene and 1 short sequence that is part of a human gene for a ribonuclease

MOST antigen tests

use antibodies to bind to viral N protein in a human sample

Antibody test

look for presence of antibodies that can bind to viral Spike and/or viral N proteins in a blood sample

Mutation

a change of at least one member of a sequence of monomers
-DNA and RNA change in at least one nucleotide
-proteins: a change in at least one amino acid

DNA replication errors

-median of 1 somatic mutation per 3,571,429 bp
-1,697 somatic mutations every time a female cell divides
-1,669 somatic mutations every time a male cell divides

Different variants of COVID

replicable mutations in SARS-CoV-2 RNA:
-result in mutations in amino-acid sequence of S1
-result in changes in properties of S1
-result in changes in infectivity of SARS-CoV-2

Scrapie

a fatal degenerative disease affecting the CNS of sheep and goats