Microbiology Test Claude #3

Describe DNA and the building blocks that make it.

• DNA = two polynucleotide strands spiraling around an imaginary axis, running antiparallel (5’→3’ and 3’→5’).

• Nucleotide = pentose sugar + phosphate group + nitrogenous base (sugar + base = nucleoside).

• Bases pair by hydrogen bonds: A–T (U in RNA) and G–C.

Describe the process of replication. Where does it start and how does it occur?

• Starts at the origin of replication (oriC in E. coli). DnaA binds OriC and the A-T-rich DNA unwinding element opens.

• Replication is bidirectional, making two replication forks on the circular chromosome. DNA polymerase copies parental strands into daughter strands, 5’→3’, starting from an RNA primer.

• Forks meet at the termination region.

DnaA:

binds oriC to start unwinding

Describe synthesis of the leading versus lagging strand. How and why are they made differently? What does 5’→3’ have to do with it?

DNA pol can only add nucleotides in the 5’→3’ direction, but the strands are antiparallel. | Leading strand: runs the same way the fork opens, so it is made continuously. | Lagging strand: runs opposite, so it is made discontinuously as Okazaki fragments, each needing its own RNA primer. | Primers are then removed and fragments joined.

How is replication finished and how do you resolve issues at the end?

Forks meet at the termination region of the circular chromosome. RNA primers are removed and Okazaki fragments joined into a continuous strand. | ★ Topoisomerase separates the interlinked daughter chromosomes. In linear eukaryotic chromosomes the lagging-strand end can’t be primed, so telomerase extends the ends.

Describe the process of gene expression.

Step 1 Transcription: DNA → mRNA by a single RNA polymerase (often polycistronic mRNA in bacteria/archaea). | Step 2 Translation: ribosome reads mRNA codons and builds a protein. | In bacteria the two are coupled (polysomes form).

What are the start and stop signals?

Transcription start: promoter (Pribnow box; sigma factor helps). Stop: terminator, either intrinsic (RNA stem-loop) or Rho-dependent. | Translation start: start codon with fMet-tRNA, aided by the Shine-Dalgarno sequence. Stop: stop codon recognized by release factors (RFs).

Identify the molecular participants and important sequences for transcription.

RNA polymerase; sigma factor; promoter (sequences before the start point, e.g. Pribnow box); DNA template (transcription bubble with a temporary RNA:DNA hybrid); terminator sequences (stem-loop); Rho factor for Rho-dependent termination.

Identify the molecular participants and important sequences for translation.

mRNA with leader (Shine-Dalgarno) and start codon; ribosome (16S rRNA binds S-D; 23S rRNA is the ribozyme forming peptide bonds); tRNAs (anticodon + amino acid acceptor end); fMet-tRNA; stop codon; IFs, EFs, RFs.

Describe the protein factors needed for translation. Where is energy used?

Initiation factors (IFs): fMet-tRNA binds start codon, 16S rRNA binds S-D. | Elongation factors (EFs): new tRNA binding, transpeptidation, translocation. | Release factors (RFs): recognize stop codon, disassembly. | ★ Energy: GTP used by IFs/EFs/RFs; ATP used to charge tRNAs with amino acids.

What is the genetic code and how does coded information flow from nucleic acid to protein?

Triplet codons in mRNA specify amino acids; tRNA anticodons pair with codons. The code is redundant, non-ambiguous, and universal. Wobble at the third position means fewer tRNAs than codons are needed. | Flow: DNA → (transcription) mRNA → (translation) protein.

What is an operon and what are the general required molecular participants?

A cluster of structural genes controlled together (one mRNA). | Participants: operator (DNA regulatory site); repressor (protein from a separate regulatory gene that blocks transcription); inducer or corepressor that modulates the repressor; ★ promoter and structural genes.

How are operons used for regulation: inducible vs repressible?

Negative control (repressor, specific to one operon): | Inducible: usually off; catabolic needs; inducer inactivates the repressor. | Repressible: usually on; anabolic needs; corepressor activates the repressor. | Positive control (activator protein) gives overall metabolic regulation across multiple operons.

Describe in detail how catabolite operons like the lac operon function.

Three structural genes for lactose uptake and metabolism. The lac repressor (lacI) binds the operator and blocks transcription, so enzymes are not made unless lactose is present. Lactose (inducer) binds the repressor, releasing the operator. Also needs CAP/cAMP (glucose low) for high expression.

Describe in detail how catabolite operons like the ara operon function.

AraC acts both positively and negatively: with arabinose present it promotes transcription; with arabinose absent it blocks transcription.

How were mutant strains used to identify the components of the lac operon?

Mutants showed which component was damaged: | I−: repressor altered/absent, can’t bind operator → genes always on. | Oc: operator sequence altered, normal repressor can’t bind → always on. | Is: repressor always binds (superrepressor) → always off.

Explain the role of CAP as a positive regulator of gene expression.

All catabolite operons have a CAP binding site. CAP (activated by cAMP) must bind before RNA polymerase can bind the promoter. cAMP level is inverse to glucose: adenyl cyclase is active only when glucose is limiting, so with no glucose CAP is active.

Describe in detail how the trp operon functions.

Structural genes synthesize tryptophan. Negative control by the trp repressor: operon functions only in the absence of tryptophan (repressible, anabolic). Tryptophan acts as the corepressor.

How is the trp operon regulated with attenuation?

Attenuation = termination of transcription within the trpL (leader) region. It depends on stem-loop structures that form in the mRNA according to trp level.

What are riboswitches?

Specialized transcription attenuation: the mRNA leader sequence (the riboswitch) folds differently when an effector molecule binds, which can terminate transcription. They can also regulate translation by changing leader folding.

Explain how different forms of RNA govern genetic processes.

Riboswitches (sensory RNAs) change folding on ligand binding. Small noncoding RNAs bind complementary mRNA sequences to control translation. ★ Also: rRNA catalysis (23S ribozyme), tRNA decoding, crRNA/gRNA guiding CRISPR.

What is a mutation?

A stable, heritable change in the base sequence of DNA. Point mutations are substitutions or insertions/deletions; larger mutations can also occur.

How do mutations occur?

Spontaneous (arise naturally, e.g. replication errors) or induced (by mutagens such as chemicals or radiation).

What effects might a mutation have on a protein? How are they classified?

Classified by the change to the protein: ★ silent (same amino acid), missense (different amino acid), nonsense (premature stop), frameshift (insertion/deletion shifts the reading frame).

Describe the three main mechanisms that transfer DNA between prokaryotic cells.

Horizontal gene transfer by: | 1. Conjugation: plasmid transfer by direct contact via sex pilus, unidirectional. | 2. Transformation: uptake of naked DNA by a competent cell (Com proteins). | 3. Transduction: transfer by bacteriophages.

Where does recombination happen?

Recombination is the exchange of genes between two pieces of DNA. ★ In the recipient cell, donor DNA recombines with the recipient chromosome (or plasmid) after transfer.

What is the F plasmid and how are cells categorized by it?

F plasmid = conjugative plasmid (autonomously replicating DNA) encoding the sex pilus. | F+: has free F plasmid (donor); F−: lacks it (recipient). F+ × F−: pilus contact, rolling circle replication, recipient becomes F+. | Hfr: F factor integrated in chromosome; donor genes transferred, complete F usually not. | F′: F factor incorrectly excised, carries host genes that can be transferred.

What is the difference between generalized and specialized transduction?

Generalized: lytic cycle of a virulent phage; host DNA fragments mistakenly packaged into the phage head; random part of the genome transferred. | Specialized: temperate phage after lysogeny; prophage incorrectly excised; only a specific portion of the genome transferred.

What is the CRISPR system and how does it naturally work?

Clustered Regularly Interspaced Short Palindromic Repeats: a prokaryotic immune system. Pieces of viral DNA are integrated into the CRISPR array; ★ their RNA guides Cas nucleases to cut matching viral DNA on reinfection.

How is CRISPR used for gene therapy?

Cas9 nuclease plus a guide RNA (gRNA/crRNA) targets a chosen DNA sequence and cuts it. Eukaryotic DNA repair mechanisms then fix the cut, allowing targeted gene editing/therapy.

What is gene cloning?

Recombinant DNA technology: inserting a gene into a vector (plasmid), putting it in a host cell, and copying it as the cell divides.

How are restriction enzymes and plasmids used for cloning?

Restriction enzymes bind a restriction site and cut DNA into restriction fragments. Cut the gene and the plasmid vector with the same enzyme, join them, and insert the recombinant plasmid into cells by heat shock or electroporation (transformation). Vectors can be cloning vectors or expression vectors.

What components does a plasmid need to be used in research?

Ori (origin of replication); MCS (multiple cloning site); selectable marker/reporter gene; sequencing sites.

How can you identify cells that received a plasmid using blue-white screening?

★ The MCS sits inside lacZ. Plate on antibiotic + X-gal: cells with no plasmid don’t grow. Blue colonies = plasmid without insert (lacZ works). White colonies = plasmid with insert (lacZ disrupted) → recombinant cells.

Identify the divisions of the immune system. What parts are specific or non-specific?

Innate (non-specific): 1st line (mechanical/chemical barriers); 2nd line (cells, phagocytosis, complement, interferons, inflammation). | Acquired (specific): humoral (B cells/antibodies) and cell-mediated (T cells).

How does the whole system communicate?

Cytokines (chemical permission to activate and attack), interferons, complement and inflammation mediators; PAMPs recognized by PRRs (TLRs, NLRs); antigen-presenting cells show antigen on MHC to T cells.

Describe some innate mechanical and chemical barriers.

Mechanical: skin, mucous membranes, ciliary escalator, fluids (tears, saliva, urine). | Chemical: lysozyme, transferrins, defensins, bacteriocins, sebum, low pH.

Identify the different cells of the immune system. What jobs do they perform?

Neutrophils (60–70%), lymphocytes (20–25%), monocytes (3–8%), eosinophils (2–4%), basophils (0.5–1%); also dendritic cells, macrophages, mast cells, NK cells. Phagocytes: neutrophils, macrophages, dendritic cells. ★ Eosinophils: parasites; mast cells/basophils: histamine; NK cells kill infected cells.

Describe phagocytosis.

Main phagocytes: neutrophils, macrophages, dendritic cells. Common pathogen components (PAMPs) are recognized non-specifically by PRRs such as TLRs and NLRs; binding changes gene transcription. ★ Then engulf, form phagosome, fuse with lysosome, digest.

Describe inflammation.

Nonspecific response to tissue injury: redness, pain, heat, swelling. Mediators (cytokines, histamine) start a cascade; WBCs (neutrophils, monocytes/macrophages) emigrate to the site; then tissue repair.

Describe complement activation.

Serum proteins activated in a cascade via classical, lectin, or alternative pathways. Results: opsonization/immune adherence (enhanced phagocytosis), inflammation, and membrane attack complex (MAC) causing cytolysis.

Define antigen, antibody, epitope, and hapten.

Antigen: anything that elicits an immune response. | Antibody: ★ immunoglobulin made by B cells that binds a specific antigen; it is also the B cell receptor. | Epitope: antigenic determinant that antibodies recognize. | Hapten: partial antigen, too small to elicit a response alone; becomes antigenic when combined with a carrier protein.

Differentiate classes of lymphocytes and their roles.

B cells: humoral immunity, make antibodies against extracellular pathogens. | Helper T cells (CD4+): activate other cells via cytokines. | Cytotoxic T cells (CD8+): kill infected/cancer cells. | ★ NK cells are the innate lymphocytes.

Where are lymphocytes educated?

Primary lymphoid organs: bone marrow and thymus. ★ B cells mature in bone marrow, T cells in the thymus. Activated in secondary organs (lymph nodes, spleen).

How are lymphocytes activated?

Need antigen-specific activation: clonal selection followed by mitosis. Naive helper T cells are activated by an APC; B cells are activated by their specific antigen. Both give effector and memory cells.

What is the role of T cell receptors and co-receptors?

TCRs are membrane-bound, have a variable binding region, and bind antigen fragments presented on MHC. Co-receptors: CD4 (helper T) binds MHC II; CD8 (cytotoxic T) binds MHC I.

What types of T cells are there and what do they do?

Helper T cells: class (Th1, Th2) depends on signaling/genetic changes; release cytokines to activate the response. | Cytotoxic T cells: bind infected/cancer cells and trigger apoptosis of the target. | ★ Memory T cells persist for the 2nd exposure.

Describe the role and types of MHC.

MHC molecules mark self and present antigen. | Class I: on almost all nucleated cells; presents to CD8+ cytotoxic T cells. | Class II: only on antigen-presenting cells; presents to CD4+ helper T cells.

Describe the role of B cells. How are they activated and what do they do?

B cell receptor = antibody, typically antigen-specific. Antigen-specific activation (with helper T cell cytokines) triggers clonal selection, proliferation and differentiation into plasma cells (secrete antibodies) and memory B cells.

Describe the different types of antibodies.

IgM: pentamer, first Ig made. IgG: 80% of serum Ig. IgA: secreted across mucosal surfaces. IgE: lowest level; parasites, mast cells (allergy). IgD: part of the B cell receptor complex.

How do antibodies function?

Secreted antibodies bind antigens, neutralizing pathogens or making them better targets for phagocytes and complement proteins (opsonization, complement activation).

List and describe the types of hypersensitivity.

Type I (anaphylactic): IgE; localized (hives, asthma) or systemic shock; skin-test diagnosis, desensitization. | Type II (cytotoxic): IgG/IgM + complement; ABO, Rh, hemolytic disease of the newborn. | Type III (immune complex): soluble antigen-antibody complexes deposit in basement membranes. | Type IV (cell-mediated): delayed, T cells and cytokines/macrophages; contact dermatitis, tuberculin test.

Identify the modes of disease transmission.

Contact: direct (close association), indirect (fomites), droplet (airborne droplets). | Vehicles: food, water, air, body fluids, fomites. | Vectors: arthropods (fleas, ticks, mosquitoes); mechanical (carried on feet) or biological (pathogen reproduces in vector).

Identify the stages of a disease.

★ Incubation → prodromal → illness (acme) → decline → convalescence.

List and describe pathogen virulence factors.

Structural or soluble components that magnify pathogenicity, often on chromosome or plasmid (pathogenicity islands). Types: adherence; enzymes (coagulase, proteases, collagenase); toxins (exotoxins, endotoxins).

Classify toxins.

Exotoxins: secreted diverse proteins. AB toxins (B binds cell, A disrupts function, e.g. protein synthesis); pore-forming; superantigens (overstimulate T cells → cytokine storm). | Endotoxins: ★ lipid A of gram-negative outer membrane, released on lysis; trigger cytokine release.

DNA polymerase

adds nucleotides 5’ → 3’

RNA primer

initiates synthesis; later removed

Replisome

complex of replication proteins at the fork

Helicase

unwinds double stranded nucleic acid

Single-strand binding proteins (SSB)

Keeps strands apart

DNA gryrase/ topoisomerase

• cuts backbone

• relieves supercoils

Primase

Lays down