Genetics and Genomics

Genetics

• The study of inheritance/heredity in living organisms, from parent to offspring

Genomics

• The study of an organism's entire genome including interactions with the environment

Genome

• The sum of all genetic material in an organism Most are chromosomes, some are plasmids, mitochondria and chloroplasts
• Cell genomes are only composed of DNA
• Viral genomes can be either RNA or DNA

Chromosomes

• Distinct structures of neatly packaged DNA molecules
  • Eukaryotic chromosomes feature DNA wrapped around histone proteins, found in the nucleus, are linear, and are diploid or haploid
  • Bacterial chromosomes are condensed into packets using histone-like proteins, with one, two or sometimes more chromosomes

Gene

• Basic unit of heredity
• Made up of DNA
• Carries instructions for building proteins

Genotype

• The sum of all gene types, an organism's unique genetic makeup

Phenotype

• The expression of the organisms genotype
• Creates specific traits/functions

Semiconservative Replication

• Method of DNA replication
• Each new DNA molecule has one old and one newly synthesized strand

Categories of Genes

• Structural genes: encode proteins
• Regulatory genes: control gene expression
• Genes coding for RNA: code for RNA machinery

Location of Genomes

• Nucleus (Eukaryotes)
• Cytoplasm (Prokaryotes)
• Mitochondria (matrix) and Chloroplasts (stroma) (Eukaryotic organelles)
• Viruses (DNA or RNA) enclosed in a protective protein coat (capsid)

DNA Strands

• 5'→3' and antiparallel
• Antiparallel arrangement of helix has strands running in opposite directions
  • One side runs from 5'→3', other from 3'→5'
• DNA polymerase adds nucleotides to a growing chain, synthesizes 5' to 3'
• Covalent bonds link the nucleotide backbone

Purines and Pyrimidines

• Nitrogenous bases attach by covalent bonds at sugar's 1' position
• Purines and pyrimidines join with complementary bases via weak hydrogen bonds
• Purine: shorter name, larger ring structure (two rings)
• Pyrimidine: longer name, smaller ring (one ring)
  • Purine + purine is too wide
  • Pyrimidine + pyrimidine is too narrow
• A pairs with T
• G pairs with C
• Hydrogen bonds hold base pairs together

Enzymes Involved in DNA Replication

• Helicase: Unzips the DNA helix
• DNA Polymerase III: Adds new nucleotides, proofreads
• Primase: Adds RNA primer
• Ligase: Seals gaps
• Gyrase: Untangles DNA supercoils
• DNA Polymerase I: Removes primer, closes gaps, repairs mismatches
• Topoisomerase I and II: Supercoiling and untangling

Transcription and Translation

• mRNA (messenger RNA): Carries genetic information
• tRNA (transfer RNA): Transports amino acids to ribosomes
• Ribosome: Site of protein synthesis (small subunit binds mRNA, large subunit forms peptide bonds)
• Codon: Group of three nucleotides dictating amino acid addition to peptide chain consisting of 64 triplet codes for 20 amino acids, allows insertion of correct amino acids
  • Wobble: First two nucleotides needed to encode an amino acids, the third doesn't change it
  • Anticodons: tRNA loop complementary to mRNA codon, tRNA "anti-codon" complementary to the mRNA codon

Operons

• Found in bacteria and archaea
• Consist of a coordinated set of genes regulated as a single unit
• Inducible or repressible
  • Catabolic operons: Induced by the substrate for which the structural genes code for
    • Produce the enzyme when the substrate (nutrient) is present
  • Repressible operons: Anabolic enzymes
    • Turned off by the synthesized product

Recombinant DNA

• Artificially made DNA strand from combined gene sequences

Horizontal Gene Transfer

• Conjugation: Direct DNA transfer via pilus
  • The donor cell contains Fertility plasmid
  • Bridge forms between cells to transfer DNA
  • Commonly transfers drug/metal resistance, enzyme adherence molecules, and toxin production
• Transformation: Uptake of foreign DNA from environment. Requires a live, competent recipient cell and a free donor DNA
  • Indirect and Polysaccharide capsule
• Transduction: Bacteriophage carries DNA from donor to recipient
  • Donor cell is lysed
  • Defective bacteriophage carries donor DNA
  • Live recipient cell is the same species as donor
  • Transfers toxins, enzymes for sugar fermentation, and drug resistance

Transposons

• Jumping genes
• Transposable elements that shift from one part of the genome to another
  • Can transfer from chromosome to plasmid, or vice versa; from one cell to another (bacteria/eukaryotes)
  • Some replicate before jumping, others simply move
• Involved in:
  • Changes in traits such as colony morphology, pigmentation, and antigenic characteristics
  • Replacement of damaged DNA
  • Intermicrobial transfer of drug resistance (bacteria)
• Transposons Integrate into host cell chromosome, excise and move to another genome location (single copy)
  • Can replicate before moving, increases copy number and genome effect
  • Can jump to plasmid for transfer to other bacterial cells

Mutations

• Spontaneous vs. induced
  • Spontaneous: Random change in DNA from replication errors
  • Induced: Caused by known mutagens (physical/chemical agents)
    • Radiation: UV light, X-rays
    • Chemicals: nitrous acid
• Point mutation: Addition, deletion, or substitution of single bases
  • Wild type: Normal version of gene
  • Missense mutation: Code change leading to a different amino acid placement, including a faulty protein, altered function, or no significant change.
  • Nonsense mutation: Normal mutation becomes a stop codon
  • Silent mutation: Base altered, but amino acid unchanged
  • Back-mutation: Mutated gene reverts to original base composition
  • Frameshift mutation: One or more bases are inserted or deleted, changes mRNA reading frame, nearly always leads to nonfunctional protein

Restriction Enzymes (Endonucleases)

• Recognize foreign DNA, break phosphodiester bonds
• Protect bacteria against incompatible DNA from bacteriophages or plasmids
• Enable biotechnologists to cleave DNA at desired sites, necessary for recombinant DNA technology
• Cuts at palindromes, (DNA sequences read identically from 5' to 3' direction on both strands)
• Restriction fragments are the pieces of DNA produced

Other Enzymes

• Ligase: Seals sticky ends/splices genes into plasmids/chromosomes
• Reverse transcriptase (RT): Replicates AIDS virus, converts RNA to cDNA
• Complementary DNA (cDNA): Made from messenger, transfer, ribosomal, and other RNA types; synthesizes eukaryotic genes from mRNA transcripts (free of introns)
• PCR rapidly increases DNA amount in a sample, sensitive enough to detect cancer or infection from a single gene copy amplifies target DNA in hours
• Primers: DNA strands (15-30 bases) marking the start of DNA amplification
• DNA polymerases from thermophilic bacteria: Taq polymerase remains active through PCR temperature
• Thermal cycler: Automates temperature changes
  • Denaturation- at 94°C: Separates doubled DNA strands
  • Priming- 50-65°C: Primers bind cooled DNA
  • Elongation- at 72°C: DNA polymerase adds nucleotides and doubles

Gel Electrophoresis

• Separates DNA fragments by length
• Requires electrical current and produces fragments of different lengths that migrate through digested samples in different individuals
• Samples are placed in soft agar gel compartments and subjected to electrical current
• Phosphate groups have a negative charge and move DNA toward the positive pole
• Smaller fragments migrate more quickly
• Position of fragments determined by staining gel

Protein Structure and Folding

• Folding equals function, function depends on shapes
• Protein folding
  • Primary structure: Sequence of amino acids held by peptide bonds
    • Genes determines the order and number of amino acids
  • Secondary structure: Primary structure sequence folds in alpha helix or beta-pleated sheet formations.
  • Tertiary structure: More 3D folding occurs, influenced by hydrophilic/hydrophobic R groups (side chains)
  • Quaternary structure: More than one polypeptide chain

Antimicrobial Controls

• Sterilization: Destroys viable microorganisms (including viruses); used on inanimate objects like surgical instruments and commercially packaged food:
  • Methods include heat (autoclave) and sterilants
• Disinfection: Destroys vegetative pathogens, not endospores; used on inanimate objects, boiling food utensils, and examining tables:
  • Methods include bleach, iodine, and heat
• Decontamination/sanitization: Mechanical removal of microbes/debris to reduce contamination like restaurants and breweries:
  • Methods include soaps, detergents, and dishwashers
• Antisepsis/degermation: Chemicals applied to body to destroy/inhibit vegetative pathogens and reduce microbes on skin, involves scrubbing skin or immersing it in chemicals:
  • Methods include alcohol antiseptic rubs hand scrubs

Relative Resistance

• Primary target of microbial control: Organisms causing infection/spoilage
  • Includes mixed bag of microorganisms, some more dangerous and harmful than others
  • Endospores are most resistant
  • Sterilization eliminates endospores and less resistant forms
• Endospores are more resistant than vegetative cells
• "Static"/"stasis": Inhibits/stands still
  • Bacteriostatic, fungistatic, antiseptics (microbistatic)
• Sterilization and disinfection: The process (kills endospores like sterilants)
• Agents used in process: Bactericide, fungicide, virucide, sporicide,
• Sepsis: Growth of microorganisms in blood/tissues
• Asepsis: Prevents entry into tissues
• Antiseptics: Agents applied to skin
• Microbial death: Permanent termination of vital processes

Physical Controls

• Heat
  • Moist: Boiling water for disinfection
    • Coagulation and denaturation of protein
• Dry: hot air/open flame ranges from 160 degrees to several thousand deydrating and denaturing proteins and incinerating

Cold

• Slows growth
• Most aren't affected but preserves at −70°C to −135°C

Desiccation

• Removal of water
• Some killed, some preserved

Lyophilization

• Combination of freezing and drying
• Preserves viability for years
• Exposure to vacuum during freezing removes water

Radiation

• Energy emitted from atomic activities (high velocity within matter/space)
• Gamma and X-ray ionizing sterilizes heat/chemical-sensitive materials
• UV, non-ionizing
  • Optimal is 100 to 400 nm, 240-280nm
  • Pyrimidine dimers

Filtration

• Removes microbes from air and liquid
• Used for heat-sensitive liquids like serum, blood, vaccines, drugs, IV fluids, and enzymes

Action of Heat and Chemicals on Proteins

• Native state: Normal state
  • Denatured state does not revert
  • Different shape changes block substrates by binding active sites

TDT and TDP

• Thermal death time (TDT): Shortest time to kill test microbes at specified temperatures
• Thermal death point (TDP): Lowest temperature in 10 minutes

Chemical Controls

• Germicide Qualities include rapid action, even in low concentrations, water/alcohol solubility, and affordability
  • Factors that affect activity are the nature of microorganisms/materials treated, the degree of contamination, the time of exposure, and the germicide's strength/action
• Chlorine (TB), Ethyl Alcohol (S. aureus/E. coli), Hydrogen Peroxide (S. aureus), Quaternary Ammonium Compound (S. aureus), Ethylene Oxide Gas (S. faecalis)
• Mode of Actions by targeting bacteria, viruses, and fungi
  • Cell wall: Chemicals/detergents/alcohol
  • Cytoplasmic membrane: Detergent/alcohol
  • Cellular synthesis: Radiation/ethylene oxide
  • Proteins: Heat (moist)/alcohol
  • Halogens (chlorine): Kills endospores, denatures enzymes; iodine interferes with metabolic activity
  • Gaseous sterilant/disinfectant (ethylene oxide): Kills endospores, reacts with proteins and interferes with DNA
  • Phenol (carbolic acid): Disrupts cell wall/membranes
  • Alcohol: Dissolves lipids in cell membranes
  • Detergents: disrupt cytoplasmic membrane
  • Acids and alkalis-some bacteria, viruses, fungi- change pH

Antimicrobial Chemotherapy Goals

• Administer drugs to infected patient that target and destroy infective agent without harming the host
• Goals include disrupting structures/functions that halt function

Characteristics

• Easily reach infectious agent, toxic to infectious agent, remains active to be safely broken down/excreted

Drug Categories

• Inhibition of cell wall synthesis
• Inhibition of nucleic acid (RNA and DNA) structure & function
• Inhibition of ribosome in protein synthesis
• interference with cytoplasm function
• Inhibition of folic acid synthesis

Terminology

• Prophylaxis use of drugs to prevent infection in persons at risk
• Antimicrobial chemotherapy use of drugs to control infection
• Antimicrobials all-inclusive term for any antimicrobial drug
• Antibiotics inhibit/destroy organisms; target bacteria
• Semisynthetic, drugs isolated and chemically modified
• Synthetic drugs produced entirely by reactions
• Narrow-spectrum, antimicrobials with limited microorganism array
• (Extended Spectrum), antimicrobials effective against both positive & gram-negative

Kirby Bauer

• An agar plate is swabbed with bacteria is used to measure effectiveness
• Zone of inhibition of antibiotics is measured/compared

Therapeutic Index

• Compares toxic dose to minimum effective dose
  • Lower ratio is more toxic, higher is safer to infection
• For example numbers
  • TI = 1.1 is risky, TI = 10 is safer
• The drug with the highest therapeutic index has the widest margin of safety.

Goals of Antimicrobial Drugs

• Selective toxicity kills/inhibits microbial cell but not damages host tissues
• Disrupts cell processes/structures of bacteria, fungi, or protozoa
• Inhibits virus replications
• Interferes with enzymes required to assemble macromolecules or destroys previously formed structures

Mechanisms of Drug Action

• Targeting drug action types
• Cell Wall: Penicillins/Cephalosporins/Carbapenems
• Protein misreading: Aminoglycosides - protein synthesis by misreading mRNA/translation/block A site and disrupt cytoplasm
• Sulfonamides block tetrahydrofolate
• Quinolones inhibit DNA helicases and transcription
• Polymyxins (colistins)- interacts with cell membrane, leakage of protein and targets cytoplasmic organisms

Spectrum

• Broad: Effective against more than one group of bacteria such as tetracycline
• Narrow: Target a specific group such as polymyxin

Fungal Infection Treatment

• High Human/Fungal similarity means difficult drug specificity and targets
• Drug group example: Azoles

Antimalarial/Antiprotozoal

• Can cause host and host infections, drugs and metabolic toxicity
• Now has treatment such as GIARDIA LAMBLIA

Antihelminthic

• Has very complex parasitic worm targeting

Antiviral

• Targets inhibit viral entry, acid and assembly/release

Drug Resistance

• Organisms become hard to treat drugs to be adaptive or slow/inhibit

Resistance

• High New resistance to occur from spontaneous mutations, or genes being persistent
• Or slower metabolism where micro organisms are harmed

Threats

• Hard to treat microorganisms require nature helping
• Probiotics/Prebiotics transplants are necessary

Toxicity

• Drugs can cause the following to organs liver/nervous tissue