Biol 121 Lecture Notes PDF

LECTURE 1 Introduce the diversity of microorganisms and the impact of microbes on humans. ● Microorganisms are some of the most metabolically and genetically diverse organisms on the planet ● Microbes affect food availability ○ It can destroy crops, but preserve food ● Microbial diseases change hist...

LECTURE 1 Introduce the diversity of microorganisms and the impact of microbes on humans. ● Microorganisms are some of the most metabolically and genetically diverse organisms on the planet ● Microbes affect food availability ○ It can destroy crops, but preserve food ● Microbial diseases change history Be able to describe the theory of spontaneous generation and how it was disproved ● “Spontaneous generation” is the theory that living creatures could arise without parents ● Oxygen alone does not enable spontaneous generation ● Liquid remained sterile until microorganisms were introduced from the outside. ● Combined sterilization via boiling with access to air and whatever ever life force was in it Understand how Koch’s Postulates are used to describe the role of microbes in diseases (Germ Theory) ● Postulate 1: the suspected organism should be present in ALL cases of the disease and ABSENT from healthy animals ○ It must be found in diseased animals and not in healthy ones ● Postulate 2: The suspected organism should be grown in PURE CULTURE ○ Must be culturable ● Postulate 3: Cells from a pure culture of the suspected organism should CAUSE DISEASE in a healthy animal ○ Make healthy animals sick ● Postulate 4: the suspected organism should be REISOLATED (the same organism as before) ○ Reisolate ● The germ theory of disease ○ A specific type of microorganism causes a specific disease LECTURE 2 Describe the basic differences between models of abiogenesis and expansion in the pool of biological molecules available ● Small organic molecules arise abiotically from simply reduced chemicals sparked by lighting- a generation of organic building blocks Explain what 16S is, how it is used as a molecular clock for bacterial evolution, and why it is suited for this ● The molecular clock is the temporal information contained in a macromolecular sequence ○ Must be able to align molecules to determine genetic relatedness ○ Universal molecules found in all organisms ○ Has conserved functions in all organisms ○ Strictly vertically transferred (generation to generation) ■ Can’t be picked up from the environment or different cells ○ Constant substitution rate – sequence divergence proportional to time (slow evolving) ● The most widely used molecular clock is the gene encoding the small subunit rRNA ○ 16S rRNA (bacteria) or 18S rRNA (eukaryotes) ○ The proteins in organisms of different species are similar to each other. ○ Ribosomal DNA is a good choice because genes that show the most consistent measure of evolutionary rime encode components of the transcription and translation apparatus Describe the endosymbiont theory for the evolution of mitochondria and chloroplasts ● Endosymbiont theory ○ Mitochondria WERE bacteria ○ Chloroplast WERE cyanobacteria ○ Infected or eaten by other species ○ They ended up living together inside ● Endosymbionts are microbes living symbiotically inside a larger organism ● It implied a polyphyletic ancestry of living species, instead of the long-held assumption that species evolve only by divergence from a common ancestor (monophyletic ancestry) LECTURE 3 Compare and contrast eukaryotic and bacterial genomes ● Bacterial and archeal chromosomes range in size from 490 to 9400 kilobase pair (kb) ○ For comparison, eukaryotic chromosomes range from 2300 kb ○ Bacterial genomes have relatively little noncoding DNA (untranscribed) ■ Typically >90% in eukaryotes, <15% in prokaryotes genome ○ No introns ○ Fewer repeated sequence ○ More compact genomes Distinguish between genes and operons ● A gene can operate independently of others or be expressed in tandem with other genes in a unit called an operon ● Operons are usually polycistronic (many genes transcribed together on a single transcript) Distinguish monophyletic vs polyphyletic ancestries for bacteria ● Prokaryotic genetic information is not simply passed from generation to generation ● Genomes change by mutation or acquisition of genetic material within generations ● Monophyletic ○ Descended from a common evolutionary ancestor or ancestral group, especially one not shared with any other group ● Polyphyletic ○ Derived from more than one common evolutionary ancestor or ancestral group and therefore not suitable for placing in the same taxon Compare and contrast mechanisms of horizontal gene transfer and gene acquisition, vertical and horizontal transmission ● Horizontal gene transfer ○ Genomic island - regions of the genomes with the sound of horizontal transfer ○ The movement of genetic information across normal mating barriers, between more or less distantly related organisms ○ Transfer of other species ● Gene acquisition ○ Genetic information can be transferred from the environment ○ Some events are beneficial and initiated by bacteria themselves ○ Some events are harmful and impose on bacterial cells ● Vertical transmission ○ From parent to child ● Horizontal transmission ● Transfer of small pieces of DNA from one cell to another Describe experiments leading to the discovery of transformation ● Many cells are capable of natural transformation ● First demonstrated with streptococcus pneumonia by Griffith (1928) ○ Mortality experiment with mice using two kinds of the same bacterium, distinguished by colony morphology ■ Smooth (with capsule, caused disease) ■ Rough (no capsule, did not cause disease) ■ Rough bacteria mixed with heat-killed smooth bacteria cause diseases to become smooth bacteria ■ Identified a transforming principle that made rough bacteria virulent (åble to cause disease) ● Avery and McLeod (1941) ○ Tested polysaccharide, protein, RNA, and DNA, and only DNA from smooth strain conferred the ability to kill on rough strain ○ The smooth strain DNA made the rough strain able to kill -> change the phenotype Describe mechanisms of defense against foreign DNA in bacterial cells ● Restriction/modification system ● Bacteria cut foreign DNA to pieces using restriction endonucleases ○ Cut at specific DNA sequences (restriction sites) ○ “Endo” – cuts within a DNA sequence ● Bacteria add methyl groups to their own DNA using matching methylation enzymes ○ Protects restriction sites ● Foreign DNA without native DNA methylation pattern is destroyed ● CRISPR ○ Clustered Regularly Interspaced Short Palindromic Repeats” ● Bacterial immune system against viral DNA ● On infection, bacteria cut up invading viral DNA and insert pieces (“spacers”) into their genome ○ “memory” against infection ● Spacers are transcribed and the Cas9 enzyme uses these to monitor DNA sequences complementary to these transcribed spacers ● Matching sequences are then degraded to prevent infection LECTURE 4 Describe the process of chromosomal and plasmid replication in bacteria ● Nucleoid is replicated via replisome complexes, beginning at the origins of replication ● Replication of cellular DNA in most is semiconservative ○ Each daughter cell receives one parental and one newly synthesized strand ● Replisome ○ DNA helicase- unwinds the two strands of DNA ○ SSBs (single-stranded DNA binding proteins) - keep the single DNA strands apart during DNA synthesis ○ Clamp loader and sliding clamps Recruit DNA polymerase II and tether it to the DNA ○ DNA polymerase III _ Synthesizes new DNA strands ○ DNA primase _ produces RNA primers ● RNA primers are required for DNA replication ○ DNA primase is an RNA polymerase ○ The primase synthesizes small RNA molecules (10-12 nucleotides in length) that act as primers (providing the 3’-OH group) for DNA polymerase III to synthesize new DNA strands ○ Evolutionary remnant: RNA was the first genetic material ● Replication Forks ○ Leading strands: Steady growth following DNA Helicase ○ Lagging strand: Okazaki fragments ■ DNA primase is loaded to new sites as the replication fork move ■ DNA Polymerase III synthesizes ~ 1 kb DNA pieces following each RNA primer ● Completion of DNA replication ○ RNase H removes the RNA primers (recognizes DNA/RNA heteroduplexes and removes RNA) ■ on e primer for each leading strand ■ Many primers on the lagging strand- one per Okazaki fragments ○ DNA polymerase I Fills the gaps in her DNA strand ○ DNA Ligase seals the junctions ● Termination of replication ○ Replication ends at defined termination (ter) sites located opposite to the origin ○ Topoisomerase IV catalyzes a breaking and rejoining event that passes the chromosomes through another Describe evolutionary strategies for plasmid maintenance ● Plasmid curing: ○ Loss of plasmid if it no longer confers a benefit ○ Selective pressures: availability of certain nutrients or antibiotics LECTURE 5 Describe the functions of RNA polymerase and sigma factors ● RNA Polymerase ○ A multi-subunit protein complex that uses a nucleic acid template to generate RNA polymer ● Sigma Factor ○ Only needed for initiation of RNA synthesis (Not Elongation) ○ Recognizes promoters by binding to -10(Pribnow box) and -35 regions of genes ○ Guide the core enzyme to initiate transcription ○ Holoenzyme ■ Core enzyme plus sigma factor Describe the general role of transcriptional regulators ● Regulatory protein ○ Help a cell sense environmental/internal changes and alter its gene expression to match ● Regulatory proteins come in two forms ○ Repressors ■ Bind to regulatory sequences in the DNA and prevent transcription of target genes ○ Activator ■ Bind to regulatory sequence in the DNA and stimulate transcription of target genes ● Some regulatory proteins must first bind small ligands to be active ● The transcription of Transcriptional regulators can be regulated as well Describe open reading frames ● Orientation of consecutive, non-overlapping triplets of nucleotides that represent amino acids/stop signals in translation ○ Triplets of nucleotides- codon ● ● ● ○ 61 condoms -> 20 amino acids (degenerate; multiple codons can encode the same amino acid ○ 3 stop codons Contained within mRNA and located between the translation start codon (AUG) and stop codon Each transcript has three possible reading frame The stop codon is the same frame as the start codon Contrast Rho-dependent and Rho-independent transcriptional termination ● Rho-dependent termination ○ Rho is a helicase protein (unwinds nucleic acids) ○ Binds mRNA and moves along the transcript ○ When Rho Resched RNAP/RNA/DNA complex, it unwinds the RNA/DNA duplex and causes RNAP to fall off of DNA ● Rho-independent termination ○ Polymerase slows at the pause site ○ GC-rich sequence (strong base-pairing) forms stem-loop (Rna Secondary structure) ○ Stem-loop causes RNA polymerase to pause ○ Pause site followed by poly U site ○ DNA-RNA UA base pairs are the least stable (even less stable if polymerase is stalled) ○ mRNA breaks off of DNA polymerase released Contrast eukaryotic vs prokaryotic translation ● Prokaryotic translation ○ Faster than the eukaryotic process ○ Different ribosome structure ○ Different initiation and release factors to begin and end translation ○ Less stable mRNAs compared to eukaryotes ○ Coupled with transcription Explain the role of the Shine-Dalgarno sequence in translation ● Ribosome-binding site (RBS aka Shine Dalgarno sequence) on mRNA allows binding to the 30s Subunit ● Shine-Dalgarno site is complementary to sequence at the 3’ end of the 16sRNA LECTURE 6 Identify different types of mutation and mechanisms of different mutagens ● Mutant: an organism that is the direct offspring of a normal member of the species (the wild type,) but is different- containing mutations ● Mutation: Heritable changes in the nucleic acid bases in the genome of an organism ● Mutations CAN provide novel functions- become fixed in populations when they are selected for by natural selection - conferring a fitness advantage ● Spontaneous mutations ○ Arise at a low rate in any cells in the absence of any added agent ○ Errors in DNA replication ● Induced mutation ○ Created by treating the organism with added mutagens_ process of mutagenesis ● Mutagens: agent causing mutation ○ Physical: Radiation or heat ○ Chemical: compounds interfere with DNA chemistry - many are carcinogens ○ Biological: Insertion of transposons. ○ ● Can be classed based on base pair changes ■ Point mutation: change in a single base ■ Transition: purine → purine or pyrimidine → pyrimidine ■ Transversion: purine ↔ pyrimidine ■ Insertion (addition) and deletion (subtraction) of one or more bases ■ Inversion: DNA is flipped in orientation ■ Reversion: DNA mutates back to the original sequence Mutations can be categorized into several information classes based on their effects on the downstream products of DNA (proteins) ○ Silent mutation: does not change the amino acid sequence ○ Missense mutation: changes the amino acid sequence to another ○ Nonsense mutation: changes the amino acid sequence to a stop codon ○ Frame-shift mutation: changes the open-reading frame of the gene Describe how transposons and their mobility in DNA can mutagenize bacteria ● Transposons (transposable elements): ○ “jumping genes” discovered by Barbara McClintock in corn (Nobel Prize in 1983) ● Segments of DNA that can hop from one place in DNA to another – transposition ● Simplest transposons contain transposases: enzymes that promote transposition, flanked by (terminal) inverted repeats (TIR/IR) Describe the Ames Test as a method for testing the strengths of mutagens ● The Ames test examines the ability of a chemical substance to cause Mutations, or the strength of the mutagen tested ● Because cancer is frequently caused by mutations, the Ames test is commonly used to determine if a chemical has the potential to cause cancer ● Uses bacterial Mutant within the hisG gene) that can not synthesize histidine - can’t grow without added histamine ● Place on defined medium (without histidine ) + a chemical with unknown mutagen activity ● Most cells retain the mutant hisG gene meaning no growth on medium without histidine ● Mutagen causes reversion mutation of hisG back to functional form ● More Colonied = stronger mutagen Describe DNA repair mechanisms in bacteria ● Proofreading: correction of mismatch by DNA polymerase III during DNA replication ○ 3’ to 5’ exonuclease activity that removes a mismatch during polymerization ● Photoreactivation: Photolyase enzyme binds to a pyrimidine dimer caused by UV radiation and cleaves the cyclobutane ring ● Excision Repair ○ Nucleotide excision repair ■ Recognizes damage that distorts DNA structure ■ An endonuclease removes a patch of single-stranded DNA containing damaged bases, including dimers ○ Base-excision repair ■ Recognizes and repairs bases that (mostly) do not distort DNA structure ■ In general, recognition is at damaged bases, not a distorted DNA helix ○ • Methyl mismatch repair ■ Recognizes mismatches missed by proofreading ■ Use DNA methylation as an indicator for the newer strand – containing the error ● ● SOS Response ○ A coordinated set of responses to DNA damage ○ Extensive DNA damage induces SOS response – i.e. high radiation that breaks the DNA backbone leaving single-stranded regions ○ • LexA represses (prevents) the expression of SOS genes ○ • RecA (single-strand DNA binding protein) inactivates LexA when DNA ○ Damaged Double Stranded brake repair ○ Double-strand breaks in the genome are dangerous to genome stability ○ Several mechanisms for repairing these, depending on the species ■ Non-homologous end joining (NHEJ) ● NHEJ ● Does not require homology ● Ku-family proteins bind broken ends ● Other proteins including ligases recruited to fix the break ■ Recombination ● Recombination – two regions with homology (i.e. replication fork) ● RecA mediated ● Restores replication fork LECTURE 7 Distinguish prokaryotic from eukaryotic cells by cellular characteristics ● Prokaryotes have no defined organelles ○ DNA is not bounded by a membrane (the nucleus in eukaryotes) ● Prokaryotes are generally much smaller in size ○ Equivalent to a mitochondrion in eukaryotes Distinguish different forms of bacterial cells ● Cocci (coccus) - spheres ● Bacilli - rods ● Vibrios - bent rods ● Spirochetes - helical structure ● Pleomorphic: cells have multiple shapes ● Morphogenesis: change in cell shape Distinguish Gram+ from Gram-bacteria via their envelope characteristics ● Gram Positive Envelope ○ Capsule (Polysaccharide coat) (not all species) ○ S Layer (made of proteins) (not all species) ○ Thick cell wall (amino acid cross-links in peptidoglycan and teichoic acids for strength) ○ Plasma membrane ● Gram Negative Envelope ○ Capsule (Polysaccharide coat) (Not all species) ○ Outer membrane ○ Thin cell wall (4-amino acid crosslinks in peptidoglycan) ○ Thick periplasm ○ Plasma membrane Describe the steps of the Gram stain and what the results show about the structure of the bacterial envelope 1) Add methanol to fix cells to surface air dry a) Cells are fixed to a microscope slide 2) Add crystal violet stain (1min) 3) Add iodine which binds stain to gram-positive cells a) Crystal violet and iodine are added which stains the peptidoglycan in the cell wall purple 4) Wash with ethanol (20sec) a) Alcohol is added, which removes the stain from gram - but not from gram + cells i) Alcohol is thought to strip the cells of their outer lipid membrane allowing the crystal violet-iodine complect to leave the cell wall ii) May also be caused by dehydrating the cell wall of gram-positive bacteria, resulting in the crystal violet iodine complex becoming trapped in the cell 5) Add safranin counterstain a) Cells are counter-stained with the lighter stain safranin b) This colors all gram cells pink c) Gram+ cells retain their darker crystal violet stain and remain purple ● Gram-positive bacteria are stained purple ● Gram Negative Bacteria are stained red/pink LECTURE 8 Understand how chemical gradients across membranes can be used to do work ● The cell membrane acts as a barrier against the environment ● Chemical gradients across membranes can store energy ● The gradient inherently tried to equilibrate ● When this gradient is released, energy can be used to drive cellular processes ● Transport van following chemical gradient (passive) or being against the gradient, requiring energy input (active) Explain the differences between active and passive transport 1) Active: moving molecules against the concentration gradient while using energy a) Active transport requires energy! b) Pumps (transporters requiring energy input) can Move material against the gradient c) Primary active transport i) Direct use of energy (ATP hydrolysis) d) Secondary active transport i) Uses the energy of one gradient to aid the transfer of another substrate against its gradient (coupled transport) ii) symporter: transport system that mobe two molecules across their concentration gradients together iii) Antiporter: transport system that actively transports a molecule in the opposite direction to the driving ion 2) Passive: follows the gradient of material, doesn’t require cellular energy a) Small uncharged molecules, such as O2 and CO2 easily pass across the membrane by diffusion b) Water tends to diffuse across the membrane in a process called osmosis c) Weak acids and bases exist partly in an uncharged from the can diffused across the membrane Explain the differences between facilitated diffusion, ABC transporters, PTS transport ● ABC Transporters: ○ ATP- Binding Cassette ■ Solute-binding protein (SBP); substrate-specific (escorts specific substrate to transporter) ■ Membrane-spanning transporter (channel) ■ ATP-Hydrolyzing protein (provides energy for final transport via conformational change of transporter ● PTS transport ○ Also called the phosphotransferase system for sugar transport into the cell ○ Combines active energy use with facilitated diffusion ● Facilitated diffusion ○ Specific transporters allow for diffusion of substrates across the membrane ○ Distinguish Sec-dependent and Sec-independent secretion systems in bacteria ● Sec system (transport of unfolded proteins) ○ Multi-component protein complex ○ Requires ATP hydrolysis to move protein out ● Sec-dependent secreted proteins have a special amino acid sequence at the N-terminus signal peptide ● The signal peptide is cleaved by the signal peptidase enzyme during secretion ● Secretion systems are generally can be grouped into dec-dependent and sec-dependent systems that use the sec complex to move signal-peptide-containing proteins into the periplasm at least