Bimm 122 Midterm 1_Alistair B. Russell

How do scientists conduct genetic analyses?

a. Observe mutants to infer normal function of a gene and regulatory behavior.

b. Introduce mutations to disrupt a system and observe their effects.

What are the three main principles of evolution?

-Variation exists within populations, subjected to selection, and can be inherited. Ex: Industrial revolution increased # of black moths due to bird predation

Three Domains of Life:

-Archaea: Prokaryotes (no nucleus); include extremophiles (live in extreme environments like hot springs) or methanogens (produce methane).

-Bacteria: Prokaryotes (no nucleus); Gram - or +; important in human health and disease.

-Eukarya: Have a nucleus and can have membrane-bound organelles (ex: Mitochondria & Chloroplast). Includes animals, plants, fungi, and microbial eukaryotes (amoebae, yeast, and algae).

-All three have microbial life (except viruses)

Endosymbiosis (Lynn Margulis):

Eukaryotes likely arose from an archaeal host that engulfed bacteria, which became mitochondria (and later chloroplasts), retaining their own DNA/RNA and ability to replicate.

Gram (+) vs (-):

-Gram (+): Thick peptidoglycan cell wall + inner (plasma) membrane; no outer membrane → stains purple.

-Gram (−): Thin peptidoglycan cell wall + inner membrane + asymmetric outer membrane containing LPS → stains pink/red

-Both are prokaryote b/c they lack a nucleus

Model Organism Characteristics:

-Easy to grow in lab

-Makes genetic analysis easy

-Evolutionarily conserved features

-Ex: Mice, flies

E. coli Characteristics:

-Gram (−) 

-Facultative anaerobe (grow w/ or w/o O2). 

-Uses O2 as e- acceptor if available

-Predominant aerobe (uses oxygen) in gut microbiota

-Easy to grow/metabolically simple

-Fast growth → doubles every ~30 min

-Lab safe and related to human pathogen

Why Study Bacteria?

1. Fast-growing: Many generations quickly

2. Small: Large populations → easier to find rare mutations

3. Haploid: One gene copy → mutations easily seen

4. Clonal: Asexual reproduction → easier to track inheritance

Haploid vs Diploid:

-Haploid: One set of chromosomes (one copy of each gene). Ex: E. coli

-Diploid: Two sets of chromosomes (two copies of each gene, one from each parent). Ex: Humans

What is broth culture? How is growth measured in broth culture? Why is it advantageous?

-Broth culture: Homogeneous liquid culture where bacteria grow, compete, and make broth cloudier.

-OD600: Measures how much light bacteria block/scatter at 600 nm to estimate bacterial growth (cloudier = more growth).

-Advantage: Non-invasive, easy, and automatable measurement for growth curve.

Bacterial growth curve:

-Lag Phase: Little growth (adapting to environment)

-Log/Exponential Phase: Growing faster

-Stationary Phase: Growth levels off (under starvation conditions)

-Death Phase: Cells decrease

-x = time & y = OD600 (optical density at wavelength 600 nm)

Bacteria Exponential Growth Calculation:

Ex 1: We start with 500 bacteria that double every 30 minutes. How many are there after 4 hours?

Ex 2: A bacterial population increases 1024× in 10 hours. What is its doubling time?

-Final bacteria = Starting bacteria × 2^(time/doubling time) 

-Doubling time = time ÷ log₂(final/starting)

-Ex 1: 500 × 2^(4 hours / 0.5 hours) → 500 × 2⁸ → 500 × 256 = 128000

-Ex 2: 10 ÷ log₂(1024) = 10 ÷ 10 = 1 hour doubling time (log₂(1024) → 2¹⁰ = 1024)

What are bacterial colonies?

-Clonal groups of bacteria grown from one original cell.

-Colony phenotype reflects the original cell’s genotype.

Human vs E-Coli vs Bacteria (General) Genome

Metric DNA lengths:

▪ Gigabase (Gb) = 10⁹ bases = 1 billion bases

▪ Megabase (Mb) = 10⁶ bases = 1 million bases

▪ Kilobase (kb) = 10³ bases = 1,000 bases

DNA Compaction in Eukaryotes vs. Bacteria.

-Eukaryote: DNA wraps around histones to form nucleosome → organize into chromatin

-Bacteria: Negative (-) supercoiling using DNA gyrase + histone-like proteins (H-NS, HU, IHF, Fis) + SMC proteins → forms nucleoid for further structure

What happens if DNA gyrase is inhibited in DNA compaction:

DNA supercoiling is disrupted b/c + supercoiling accumulate → impairs DNA replication and transcription

-Plasmid (remove later)

Nonessential, independently replicating DNA that carries adaptive genes (ex: antibiotic resistance), helping bacteria survive in certain environments. Can be small or large (megaplasmid); LOSING IT may result in loss of adaptive traits.

How does genome size relate to bacterial lifestyle?

▪ Smaller genomes → often obligate pathogens/endosymbionts that rely on host resources → lose unnecessary metabolic genes/pathway 

▪ Larger genomes → often in complex environments (ex: soil), requiring more genes for diverse metabolic and adaptive functions

Auxotrophy vs. Prototrophy:

▪ Auxotrophy: Cannot synthesize required compound → must obtain from the environment. Ex: Chlamydia

▪ Prototrophy: Can synthesize required compound itself.

Why do bacterial genomes vary in GC content?

-Differences in codon usage: 3rd position wobble, 2nd position variation, and organism have preferred codon

-Extreme genome reduction leads to low GC content due to loss of DNA error-correction machinery and mutational biases that ↑ AT and ↓GC.

How do preferred codons and tRNA availability affect translation? Ex: A gene from S. coelicolor (72.1% GC) is expressed in E. coli (50.8% GC). mRNA is produced, but little protein is made. Why?

-Preferred codons often match abundant tRNAs → efficient translation; rare codons may have limited matching tRNAs → less protein production 

-Different codon preferences; E. coli may lack sufficient matching tRNAs, reducing translation efficiency despite successful transcription.

Why are bacterial genomes so gene rich?

1. Largely no splicing → fewer introns and less noncoding space.

2. Polycistronic messages (operons) → multiple genes can be encoded by one RNA

3. Genes can overlap and occupy different reading frames.

Gene Number Calculation in Bacteria. Ex: 5 Mb

▪ ~1 gene per 1 kb of DNA 

▪ Ex: 5 Mb = 5,000 kb → approximately 5,000 genes.

Monocistronic vs Polycistronic Messages/Operons:

-Monocistronic (typical eukaryotes): 1 mRNA encodes 1 protein → generally uses 1 reading frame.

-Polycistronic (typical bacteria): 1 mRNA encodes multiple proteins, each with its own start and stop codons → can use all 3 reading frames on one RNA.

Bacterial vs. Eukaryotic RNA Processing:

▪ Bacterial: No nucleus → No 5′ cap, splicing, or poly-A tail; Has 5′ triphosphate 

▪ Eukaryotic: Nucleus → 5′ cap, splicing, poly-A tail; can undergo alternative splicing

▪ Exception: Bacteria DO have RNA processing, specifically for rRNA and tRNA. mRNA is possible but in limited cases.

Why Does Bacterial mRNA Have a 5′ Triphosphate?

The 1st nucleotide retains a 5′ triphosphate b/c bacterial mRNA does not undergo typical eukaryotic 5′ capping.

Translation in Bacteria vs Eukaryote:

▪ Bacteria: No nucleus → transcription and translation are coupled (cotranslation), so ribosomes translate mRNA immediately.

▪ Eukaryotes: Transcription occurs in the nucleus; mRNA is exported to cytoplasm for translation

Why is bacterial DNA replication asymmetric? Why is it important?:

DNA synthesis is 5′ → 3′, so leading and lagging strands are replicated differently. Causes mutational biases, driving GC skew (unequal G vs. C), but can help visualize genome replication patterns.

Why don’t bacteria “curate” their messages?

Because bacteria lacks a nucleus so mRNA is immediately accessible to ribosome, so there are fewer opportunities for alternative splicing, nuclear retention, or extensive error control compared to eukaryotic mRNA

What does collinearity mean in bacterial genes?

Bacterial DNA coding sequences directly correspond to their RNA and protein sequences because bacteria largely lack introns/splicing.

Genome Replication in Humans vs Bacteria:

-DNA polymerase works at similar speed for both

-Human Stem Cells (HSC) can double every ~30 hours vs. ~30 minutes in E. coli (60× difference)

-Humans have ~700× more DNA using multiple origins of replication, while bacteria uses one.

DNA synthesis:

Copying DNA by adding complementary nucleotides 5′ → 3′ to an existing primer strand with a free 3′-OH.

OriC site:

-Site where bacterial chromosome replication begins.

-Contains:

(a) AT-rich region: Easier to melt (2 H bonds vs. GC's 3).

(b) 9-bp repeats: DnaA binding sites that help initiate DNA unwinding.

Theta (θ) Replication:

-Bacterial chromosome replication starts at single oriC and proceed bidirectionally 

-Fork slowed/blocked at directional Ter (termination) site bounded by Tus protein 

-Replication ends at the Dif site opposite of oriC.

Can E. coli start replication before finishing the previous round?

Yes, because during fast growth, E. coli can initiate overlapping rounds of replication (multifork replication), allowing rapid division.

Linear vs. Circular Chromosomes

▪ Linear (Eukarya): End-replication problem b/c lagging strand cannot replicate chromosome ends b/c no free 3’ OH for DNA polymerase. Uses telomeres (repetitive DNA sequences), maintained by telomerase, to protect chromosome ends.

▪ Circular (bacteria): No chromosome ends → no end-replication problem or telomeres needed.

What is OriV?

Origin of replication of a vector/plasmid.

Rolling-Circle Replication:

-Semiconservative, unidirectional plasma replication with 2 origins (double- and single-stranded). 

-A nick made in one DNA strand creates a free 3′-OH for DNA polymerase to extend, displacing the old strand, which circularizes and becomes double-stranded.

Which two DNA polymerases are primarily involved in normal E. coli chromosome replication?

-DNA Pol III: Primary replicative polymerase; synthesizes most new DNA.

-DNA Pol I: Removes RNA primers using 5′ → 3′ exonuclease activity and replaces them with DNA.

-Both contains 3′ → 5′ exonuclease activity for proofreading.

Exonuclease vs. Endonuclease:

-Exonuclease: Removes nucleotides from a free DNA/RNA end (5′ → 3′ or 3′ → 5′; some can do both).

-Endonuclease: Cuts within a nucleic acid strand; does NOT require a free end.

Methyl-directed mismatch repair (MutSLH):

(Study)

-Repairs DNA mismatches after replication by identifying the methylated old strand as correct and fixing the unmethylated new strand.

Purine vs Pyrimidine

-Purine: 2 ring =  AG

-Pyrimidine: 1 ring = UTC

Transition vs. Transversion Mutations. Which one is more common?

-Transition: Substitution within same category (purine ↔ purine: A ↔ G; pyrimidine ↔ pyrimidine: C ↔ T).

-Transversion: Substitution between categories (purine ↔ pyrimidine: A/G ↔ C/T)

-Transitions are more common because bases have similar size and structure.

Why are the genes in methyl directed mismatch repair called Mut?

-Scientists found that inactivating mutations in these genes greatly increases e.coli error rate

What causes mutations besides polymerase misincorporation

Deamination, Oxidation, and UV radiation.

Cytosine deamination:

Normally C pair w/ G. Deamination converts C to U, which pairs with A instead of G. If not repaired before replication, CG permanently becomes AT

Base Excision Repair (BER):

(Study)

-Repairs cytosine deamination by removing uracil from DNA, replacing it with the correct base, and sealing the repaired strand before replication makes the mutation permanent.

Guanine Oxidation

-Oxidative damage converts G → 8-oxoG, which incorrectly pairs with A instead of C. If unrepaired: GC → TA transversion mutation.

-MutM: Removes damaged 8-oxoG from DNA before replication → creates an AP site → repair restores G:C pairing.

-MutY: Removes incorrect A paired with 8-oxoG after replication → repair replaces A with C.

-MutT: Prevents damaged nucleotides from entering DNA by converting 8-oxoGTP → 8-oxoGMP.

What are the main mutations caused by UV rays?

Thymine-thymine dimers: 2 Thymines become covalently bonded, creating a lesion that interferes with DNA replication.

How does E. coli repair UV damage?

Uses photolyase and light (350–550 nm) to break thymine dimers and restore DNA via photoreactivation.

Homologous recombination:

Process that uses an identical or highly similar DNA region as a template to repair DNA breaks.

RecBCD

(Study)

Repairs dsDNA breaks through homologous recombination by processing the broken DNA ends, helping load RecA, and using a homologous DNA sequence as a template to restore the damaged region.

RecFOR

(Study)

-Repairs ssDNA gaps caused by stalled replication by loading RecA onto the exposed ssDNA, allowing homologous recombination to restart DNA synthesis past the lesion.

What is a Holliday junction?

A crossed DNA intermediate formed when homologous DNA strands exchange partners during recombination.

Translesion synthesis:

-Last-resort mechanism where E. coli uses translesion DNA polymerases IV and V to bypass DNA lesions that block normal replication, even when recombination (ex: RecFOR) is impaired

-Lack 3′ → 5′ exonuclease proofreading → allow replication to continue but are more error prone (mutations, ex: RecFOR-deficient survivors); therefore, tightly regulated.