Lectures #13-19

Mendel’s Principle of Segregation

• each diploid organism has two alleles for each gene (locus)

• these alleles separate during meiosis, so each gamete receives only one allele

• explains why offspring inherit one allele from each parent

Mendel’s Principle of Independent Assortment

• alleles at different loci (different genes) assort independently during gamete formation

• The separation of one pair of alleles does not affect how another pair separates

• leads to genetic recombination: new combinations of traits

Recombination

• Def: the process of sorting alleles into new combinations

• In F1, (AaBb) individuals

  • Parental gametes (nonrecombinant): AB and ab

  • Recombinant gametes: Ab and aB

• Recombination occurs when crossing over happens between homologous chromosomes

• Result: new combinations of alleles that differ from those found in the parent (now have recombinant gametes)

• Recombination frequency helps geneticists measure the distance between genes on a chromosome

Recombinant Gametes

Gametes that contain new combinations of alleles

Non-Recombinant Gametes

Gametes that contain only the original combinations of alleles that were present in the parents

Connecting Mendel to Chromosomes

• Walter Sutton (1903): proposed that genes are located on chromosomes

  • explains the physical basis for Mendel’s principles

    • Segregation: homologous chromosomes separate during meiosis I → each gamete gets one chromosome with one allele per gene

    • Independent Assortment: each pair of homologous chromosomes aligns and separates indepdently of others during meiosis

• HOWEVER: most organisms have fewer chromosomes than genes, so some genes must share the same chromosome

  • If genes are on the same chromosome, they can violate independent assortment and tend to be inherited together

Linked Genes

• genes located close together on the same chromosome (likelihood of crossing over between these two genes is very low)

  • tend to be inherited together

  • these genes form a linkage group and do not assort independently

• During meiosis:

  • linked genes usually travel together into the same gamete

  • only crossing over can separate them and create recombinant gametes

• Degree of linkage

  • Close together → strong linkage, few recombinants

  • Far apart → weaker linkage, more recombinants

Notation for Linked Genes

BE / be

Linkage in Sweet Peas (Lathyrus odoratus)

• parental cross

  • P1: Purple flowers, long pollen

  • P2: Red flowers, round pollen

• F1: all purple and long (dominant traits)

• F2: ratios did not match expected Mendelian 9:3:3:1

• Conclusion: the genes for flower color and pollen shaped are linked on the same chromosome

  • they did not assort independently

  • recombination between them was limited

How Crossing Over Breaks Linkage

Complete Linkage

• genes very close together on the same chromosome that do not cross over

• Effect: only parental (nonrecombinant) gametes are produced

Crossing Over and Linked Genes

• crossing over occurs during Prophase I of Meiosis: an exchange of genetc material between non-sister chromatids of homologous chromosomes

  • produces recombinant gametes with new combinations of alleles

• With a single crossover

  • two chromatids remain unchanged → nonrecombinant

  • two chromatids exchange segments → recombinant

  • result: 50% nonrecombinant and 50% recombinant

  • only two out of four chromatids in a homologous pair are affected

Calculating Recombination Frequency

• Recombination Frequency (RF): the percentage of recombinant progeny produced in a cross

• Formula: RF = (Number of recombinant progeny) / (Total number of progeny) x100%

Visual Map of the Autosomes: chromosomes 1-22 from a single sperm cell

Coupling and Repulsion: Australian Blowfly example set-up

In linked genes, the arrangement of alleles on homologous chromosomes affects which phenotypes appear most often

• thorax color gene (p)

  • p+ = green (dominant)

  • p = purple (recessive)

• purparium color gene (b)

  • b+ = brown (dominant)

  • b = black (recessive)

• Testcross carried out with heterozygote (p+p b+b) and homozygous recessive (pp bb)

Coupling (Cis) Configuration

Arrangement of linked genes in which wild-type alleles of two or more genes are found on one chromosome and mutant alleles are on the homologous chromosome

• each chromosome “carries the same type” of alleles: one chromosome has all the normal versions (wild) and the other has all the mutant versions: p+b+ / pb

• most gametes will contain the original nonrecombinant (parental) because crossing over is rare between linked genes

• when a gametes is produced without crossing over, it will inherit either all wild-type alleles or all mutant alleles: no mixed combinations

Repulsion (Trans) Configuration

Arrange of two linked genes in which each of a homologous pair of chromosomes contain one wild-type (dominant) allele and one mutant (recessive) allele

• Chromosome arrangement: p+b / pb+

• Most numerous offpsirng

  • green thorax, black puparium (p+b)

  • purple thorax, brown puparium (pb+)

• these nonrecombinant phenotypes different from the parental phenotypes

• only different between the two crosses is allele arrangement, not genotype

Predicting Outcomes of Crosses with Linked Genes

• knowing the allele arrangement on a chromosome + the recombination frequency allows prediction of prpgeny types and proportion

• Example:

  • if given recombination frequency of 16%, know that each recombinant gamete type is 8% each, leaving the nonrecombinant games at 42% each

  • can use this to predict the offspring proportions if multiple the two frequencies of the parents together

Testing for Independent Assortment

• genes may appear linked or independent depending on the proportion of nonrecombinant vs. recombinant progeny

  • slight deviations from 1:1:1:1 ratio could be due to chance or linkage with crossing over

• how to test:

  • calculate expected probabilities assuming independent assortment

  • use chi-square goodness-of-fit to compare observes v. expected numbers

Chi-Square Goodness-of-Fit Test

Test whether observed data match a predicted ratio (ex. Mendelian ratio)

STEPS:

1. State hypothesis (H₀ (null hypothesis): observed =expected

2. Write the observed and expected counts

   1. Mendel’s Laws for monohybride cross expect 3:1 phenotype ratio

   2. E = Total Progeny x Expected Proportion

3. Compute Chi-Square:

   1. Complete for each category and then add them

   2. (Observed-Expected)² / Expected

4. Find Degrees of Freedom

   1. Number of categories - 1

5. Compare to the critical value, using p=0.05 and your degree of freedom

   1. If Chi-square is SMALLER than critical value, the difference between observed and expected is NOT significant (fits Mendel ratio)

   2. If Chi-square is LARGER than critical value, difference is significant

Limitations of the Chi-Square Goodness-of-Fit Test

• a significant chi-square results tells you something is different from expectation but doesn’t tell you why

  • causes could be linkage, lethality, penetrance, or environmental effects

• Why a deviation might have occured

  • Genes are linked

    • two loci are not independent, violating independent assortment

  • expected single-locus ratios are correct

    • some genotypes may have lower survival

    • incomplete penetrance: not all individuals with the genotype show the phenotype

    • environmental factors or smpling errors can also affect ratios

Chi-Square Test of Independence

To test whether two traits are inherited independently (Mendel’s second law) or they are linked

STEPS:

1. Carry out a testcross between individuals differing in two characteristics (would be given this, the observed ratio)

2. Construct a contingency table

   1. place one trait’s genotypes along the top and other trait’s genotypes along the side

   2. fill in observed counts for each cell and compute row totals, column totals, and grand total

3. Calculate expected values

   1. For each cell E = (Row Total x Column Total) / Grand Total

4. Compute Chi-Square for each cell

   1. Complete for each category and then add them

   2. (Observed-Expected)² / Expected

5. Determine Degrees of Freedom (# rows -1) x (# columns -1)

6. Compare to critical value and interpret

   1. If chi-square is GREATER than critical value, there is a signficant difference and traits are NOT sorting independently (likely linked)

Hypothesis of Chi-Square Test of Independence

• Null Hypothesis (H₀)

  • two traits sort independently according to Mendel’s second law

  • there is no associate between trait 1 and trait 2

  • knowing the genotype for one trait does not change the probability of the other trait

    • X has no effect on Y

• Alternative Hypothesis (H_a)

  • the two traits do NOT sort independently

  • there is an association between trait 1 and trait 2

  • knowing the genotype for one trait changes the probability of the other trait

    • X influences Y, the traits are likely linked

Genetic Maps

• discovered by Thomas Hunt Morgan and students, found that recombination frequencies reflect the physical distances between genes on a chromosome

  • genes far apart are more likely to recombine than genes close together

• Genetic Maps: Map of the relative differences between genetic loci, markers, or other chromosome regions determined by rates of recombination; measures in recombination frequencies or map units

• Map units (m.u. or cM): units of measure for idstances on a genetic map; also called a centiMorgan (cM)

  • 1 map unit = 1% recombination rate

• Physical Maps: Map of physical distances between loci, genetic markers, or other chromosome segments; measured in base pairs. Units: Megabases (MB), actual DNA length

Genetic Map v. Cytogenetic Map v. Physical Map

• Genetic Map

  • units: centiMorgans (cM) which measure recombination frequency

  • shows the relative positions of genes based on how often they recombine during meiosis

    • do NOT indicate physical length

• Cytogenetic Map

  • visual representation of the chromosome under a microscope

  • shows

    • p arm (short arm) and q arm (long arm)

    • bands (dark and light regions) created by staining techniques

    • labels like 11.32, 11.31, 12.1, 21.1 which correspond to band positions using karyotyping

  • Physical Map

    • units: Megabases (Mb), actual DNA lenth

    • shows the physical distance in base pairs between genes or markers

Mapping: Pairwise recombination frequencies and gene order

• when mapping genes on a chromosome, we often only have pairwise recombination frequencies (RFs): the percentage of offspring showing recombination between two genes

  • smaller RF: genes closer together

  • larger RF: genes farther apart

Mapping: Identify the two genes at the ends

• Largest RF always occurs between the two genes at the ends of the segment, because recombination is additive along the chromosome

Mapping: Identify the middle gene

• remaining gene must lie between the two ends (the ones that had largest recombination frequency)

• we cannot tell if B is closer to A or C beyond what the RFs show, but additive rule confirms its in-between

Constructing Genetic Maps: adding a fourth gene

• adding a fourth gene (D) requires examining RFs between D and existing genes

• highest RF indicates genes that are furthest apart

Limits and Considerations in Genetic Mapping

• 50% Recombination Limit

  • when two genes show 50% recombination, we cannot tell whether

    • they are on different chromosomes (unlinked)

    • they are on same chromosome but very far apart

  • genes that are far apart of the same chromosome behave as if they are unlinked bc crossing over occurs so frequently that parental and recombinant gametes are produced equally

• Double Crossovers

  • Def: two crossover events occur between the same two genes

  • Effect: the second crossover reverses the effect of the first, restoring the og parental combination of alleles

  • Consequence for mapping:

    • some recombinant events go undetected, leading to underestimation of the true distance between genes

  • Frequency: rare, but double crossovers are more likely when genes are far apart

Two-Point Testcrosses for Gene Mapping

Cross between an individual heterozygous at two loci and an individual homozygous for recessive alleles at those loci

STEPS:

1. Identify linked genes

   1. find all instances where the RF < 50%

   2. RF tells us the distance of the linkage group

2. Compare Possible Positions

   1. Try positions until find the one where RFs in action are consistent with determined RFs

Crossover Types

• Genotype: Aa Bb Cc (heterozygous at 3 loci)

• Coupling Configuration:

  • Chromosome 1: ABC (all dominant alleles)

  • Chromosome 2: abc (all recessive alleles)

• Single Crossover

  • produces 2 recombinants and 2 nonrecombinants

• Double Crossover (crossover between A&B and B&C)

  • produces 2 recombinants and 2 nonrecombinants

  • only the middle gene changes compared to nonrecombinants (making it harder to detect on a two-point cross)

Why Use a Three-Point Testcross?

• Limitations of Two-Point Crosses

  • Mapping pairs of genes is inifficient

  • many crosses are needed to determine gene order

  • double crossovers often go undetected → inaccurate map distances

• Three-Point Testcross Advantages

  • Maps three linked genes at once

  • detects double crossovers, providing more accurate distances

  • determines gene order from a single progeny set

Three-Point Testcrosses for Gene Mapping

Objective: determine gene order and genetic distances for linked loci

STEPS:

1. Create F1 heteozygotes

   1. Cross WT homozygotes (st+ e+ ss+ / st+ e+ ss+) x triple recessive homozygotes (st e ss / st e ss)

   2. Get F1 heterozygotes: st+ e+ ss+ / ss e ss

2. Perform the testcross

   1. Cross F1 heteozygous females with triple recessive males (when using Drosophilia bc crossing over only occurs in female Drosophilia)

   2. Outcome: 8 possible phenotypic classses for the progeny 2³=8

   3. Interpret Testcross Progeny

      1. all progeny express alleles from homozygous parent

      2. recombination events in heterozygous parent determine the genetic map

      3. rare phenotypes → indicate double crossovers and gene order

      4. traits that appear together most frequently → nonrecombinant (parental) types

Using Three-Point Testcross Data to Map Genes

• Mapping principle:

  • Traits that appear together most frequently → nonrecombinant types

  • Rare phenotypes → double crossovers reveal middle genes

  • use recombination frequencies to calculate map distances (m.u.)

STEPS:

1. Determining Gene Order

   1. Identify nonrecombinant and recombinant types

   2. nonrecombinant progeny → most frequent phenotypes

   3. single crossover progeny → intermediate frequency phenotypes

   4. double crossover progeny → rarest phenotypes

   5. NOTE: when frequencies are relatively even (phenotypes appear at similar rates, then genes are likely not linked, either on dif chromosomes or far apart)

2. Test Possible Gene Orders

   1. Three possible orders, figure out which produces the observed double-crossover progeny

   2. For double crossover events, only the gene in the middle changes relative to the nonrecombinants

3. Determining Locations of Crossovers

   1. Rewrite progeny genotypes in the correct order to identify locations of recombination events

4. Calculating Recombination Frequencies and Map Distances

   1. Recombination Frequency (RF): (# of recombinant progency / total # progeny) x100

   2. include all recombinants: single AND double crossovers

   3. RF % = m.u.

Effects of Multiple Crossovers

Double crossovers can involve different numbers of chromatids

• Two-strand double crossover: only 2 of 4 chromatids swap: 0% recombinant gametes

• Three-strand double crossover: 3 chromatids swap: 50% recombinant gametes

• Four-strand double crossover: all 4 chromatids swap: 100% recombinant gametes

  • ends up with similar result of one crossover event

Notes:

• not all double crossovers create new allele combinations

• average effect of all double crossovers: 50% recombinant gametes

Limits of Genetic Mapping Accuracy

• Undetected crossovers

  • some multiple crossovers produce the same gametes as single crossovers

  • these go unnoticed in progeny → map distances appear shorter than actual physical distances

• Effect of gene distance

  • genes closer together: few multiple crossovers → genetic map = physical map

  • genes far apart: more multiple crossovers → genetic map underestimates true distance

    • recombination frequency plateaus at 50% even if the physical distance is much larger

• Correction

  • Use mathematical mapping functions (based on Poisson distribution)

  • Estimate actual distances more accurately

Implications for Genetic Mapping: Undetected Crossovers

• When we measure recombination frequency, we assume that each crossover creates a detectable recombinant gamete

• Problem: multiple crossovers (ex. two crossovers between the same two genes) can restore the original allele arrangement, making it look like no crossover

• Result: these events go unnoticed in progeny → we underestimate recombination frequency

Observed v. Actual Map Distance

• Recombination frequency does not increase linearly with distance

  • Starts proportional at short distances

  • Flattens near 50% because multiple crossovers restore the original arrangement

• Genes far apat → recombination frequency maxes out at 50%

  • Cannot distinguish very distant genes from genes on different chromosomes

• Result: Genetic map underestimates true physical distance

• Solution: Use mapping functions to correct for undetected errors

Haplotype

A specific set of linked genetic variants or alleles on a single chromosome or on part of a chromosome

Linkage Disequilibrium

Nonrandom association between alleles in a haplotype

SNPs

A site in the genome where individual members of a species differ in a single base pair

Mapping Function

Mathematical function that relates recombination frequencies to actual physical distances between genes

The Role of Bacteria and Viruses in Our World

Bacteria and viruses are everywhere, viruses infect all organisms and are the most abundant biological entities on earth

• Global Impact

  • Ocean bacteria produce 50% of earth’s oxygen

  • Remove 50% of atmospheric CO2

• agriculture

  • Pathogens of crops and animals

  • Provide nutrients like nitrogen and phosphorous to plants

• Human Health

  • Natural bacteria live in the mouth, gut, and skin, aiding digestion, immunity, and disease prevention

  • Many infectious diseases are caused y bacteria or viruses but can be controlled with antibiotics and vaccines

Bacteria and Viruses in Medicine and Genetics

• Medical and Industrial Importance:

  • bacteria produce drugs, hormones, food additives, and chemicals

  • viruses used in gene therapy to deliver healthy genes

• Genetic significance:

  • they have simple genetic systems = ideal for studying heredity and gene function

  • They share core genetic features with humans and other organisms

• Studies of bacterial and viral genetics have led to:

  • discovery of DNA as genetic material

  • Gene regulation models (ex. lac operon in E. coli that allows cells to digest lactose)

  • Tools for biotechnology and molecular biology

Advantages of Using Bacteria and Viruses for Genetic Studies

1. Reproduction is rapid

2. Many progeny can be produced

   1. 1 and 2 together allow lots of large generations in a short amount of time

3. Haploid genome allows all mutations to be expressed directly

4. Asexual reproduction simplifies the isolation of genetically pure strains

5. Growth in the laboratory is easy and requires little space

6. Genomes are small

7. Techniques are available for isolating and manipulating their genes

8. They have medical importance

9. They can be genetically engineered to rpoduce substances of commercial value

Diversity of Prokaryotes

Prokaryotes: unicellular organisms with a relatively simple cell structure. Prokaryotes include bacteria (eubacteria) and archaea

Two Main Groups:

• Archaea: unicellular organisms with prokaryotic cell structure that are found in all environments

• Eubacteria (Bacteria): most familiar bacterial species

Notes:

• DNA sequencing of uncultured bacteria has transformed our understanding of microbiology

• Bacteria and Archaea are genetically distinct from each other and bacteria/ eukaryotes are also genetically distinct

Bacterial Shapes and Sizes

• Bacteria exhibit a wide variety of shapes

  • Cocci (spherical)

  • Bacilli (rod-shaped)

  • Spirilla (helical)

• Size varies greatly: many are very very small but a few are visible to the naked eye

Bacterial Structures

Functional Diversity!

• Photosynthetic bacteria capture sunlight and produce oxygen

• Spore-forming bacteria survive extreme conditions

  • resistant to heat, cold, radiation, drought, chemicals, etc

• Stalks or filaments superficially resemble fungi

  • Stalks allow bacteria to anchor to surfaces, like rocks, plant roots, or sediments in aquatic environments, which allow them to stay in nutrient-rich environments

Bacterial Complexity

• Proteins like FtsZ help bacterial cell division, similar to eukaryotic tubulin in mitosis

• Bacteria have proteins that:

  • Condense DNA (Like histones in eukaryotes)

  • Maintain cell shape and cytoskeletal support

• Chromosome replication is coordinated with cell division, ensuring each daughter cell receives one copy of the genome (an exact copy)

NOTE: Bacteria have round DNA, don’t have the crossing over we’re used to, so the FtsZ ring helps separate bacteria (bacteria cannot just to cytokinesis)

Studying Bacteria Genetically

• Bacterial heredity is similar to other organisms, but:

  • they are haploid (only one copy of each gene)

  • cells are tiny, making phenotypes hard to observe directly

• Implication: scientists must use special lab techniques to study their genetics

• Key tool: Culture media: nutrient mixtures that allow bacteria to grow under controlled conditions

Growing and Analyzing Bacteria: Types of Media

• Minimal medium: only nutrients required by wild-type (prototrophic) bacteria

• Complete medium: includes all nutrients needed for growth, including supplements for mutants (auxotrophs)

Growing and Analyzing Bacteria: Growth Methods

• Broth culture: liquid medium in sterile test tubes

• Agar plates: solid medium poured into Petri dishes

Growing and Analyzing Bacteria: Plating

• Spread bacteria on agar → each cell grows into a colony (genetically identical)

• Colonies allow scientists to isolate pure strains and count individual bacteria

Studying Bacterial Phenotypes

• Microbiological study bacterial phenotypes: traots that can be onserved or detected chemically

• Colony appearance: color, shape, texture

• Observing phenotypes helps identify mutant strains for further genetic study

  • phenotypes can be difficult to tell apart

  • only works if phenotypes are visible to the human eye

Auxotrophs

Auxotrophs are mutant bacteria that have lost the ability to make a specific compound that they need to live, so much acquire that nutrient from environment

Comparison with wild-type (prototrophs)

• Wild-type bacteria: can grow on minimal medium because they make all necessary nutrients

• Auxotrophs: require supplemented medium to provide missing nutrients

Allows scientists to identify missing genes and study metabolic pathways

Auxotroph Example: Detecting Leucine Auxotrophs

1. Spread bacteria on medium containing leucine → both wild-type (leu+) and mutant (leu-) grow

2. Use replica plating to transfer colonies to:

   1. Plate with leucine (supplemented medium)

   2. Plate without leucine (selective medium)

3. Compare growth:

   1. leu+ bacteria grow on both plates

   2. leu- mutant grow only on supplemented medium

4. Colonies that grow only in the supplemented medium are leucine auxotrophs and can be cultured for further study

Modern Genomic Methods in Bacterial Research

Genomic methods: isolate and analyze DNA sequences from bacteria

• Key advantage: can study bacteria that cannot be grown in the lab

• Insights gained from genomics:

  • Bacterial diversity: discover new species and strains

  • Bacterial evolution: track how bacteria change over time

  • Gene organization: see how genes are arranged on the chromosome

  • Gene function: understand what different genes do

• Complements traditional methods like colony observation and replica plating, giving a more complete picture of bacteria genetics

The Bacterial Genome

• bacteria are unicellular and lack a nuclear membrane

• most bacterial genomes are single, circular chromsomes of double-stranded DNA

  • Example: E. coli: 4.6 million base pairs

• some bacteria have multiple chromsomes

  • vibrio cholerae: 2 circular chromosomes

  • rhiobium meliloti: 3 chromosomes

• rare cases have linear chromosomes

• protein-coding DNA

  • 90% of bacterial DNA encodes proteins (E. coli)

  • Only 1% of human DNA encodes proteins

Vibrio cholerae

• two circular chromosomes are double-stranded DNA loops

• each chromosomes carries different sets of genes, which together provide all the information the bacterium needs to survive and cause disease (cholerae)

• having multiple chromosomes can allow for specialization of gene functions and faster adaptations

• each chromosome may carry distinct functions, for example: one for essential metabolic functions, another for symbiosis with plants, and another for accessory functions

Plasmids

Small, usually circular DNA molecule that is distinct from the bacterial chromosome

• Plasmids replicate independently of the bacterial chromosome

Can be single-copy or multiple-copy per cell (even if only have 1 chromosome, can have many plasmids)

• Not essential for survival, but can:

  • promote gene transfer between bacteria

  • carry antibiotic resistance genes

  • aid in genetic engineering

Plasmid Replication

• replication begins at the origin of replication (ori)

• DNA strands separate, and replication proceeds around the circle

• results in two daughter plasmids, each with one new and one old DNA strand

Episomes

Episomes: plasmid cpable of replicating freely and able to integrate into a bacterial chromosome

• like retroviruses, episomes can integrate their DNA into the host genome

• however, episomes are bacterial DNA, note viral RNA, and don’t need reverse transcriptase (unless engineered in labs)

• both systems show how DNA can move between “extra” DNA elements and chromosomes

Episomes

Example: R Plasmid (Resistance Plasmid)

• An R plasmid is a plasmid that carries genes for antibiotic resistance

• Like other episomes it can exist independently in the bacteria cytoplasm or integrate into the bacteria chromosome

• r-determinants

  • these are genes that confer resistance to various antibiotics and toxic substances

  • ‘in the image, the resistance genes are labeled Tc^R: resistance to tetrocycline

• RTF segment (Resistance Tranfer Factor)

  • this segment contains genes necessary for plasmid replication and transfer between bacteria

Gene Exchange in Bacteria

• genetic exchange in common in bacteria and contributes to evolution

• all mechanisms involve:

  • DNA transfer

  • recombination with the recipient chromosome

• Three mechanisms:

  • Transformation: uptake of DNA from the environment

  • Transduction: DNA transfer via bacteriophages (viruses)

  • Conjugation: direct transfer from donor to recipient

Transformation

• Bacterium takes up naked DNA from the surrounding medium

• Transferred DNA can recombine with the bacterial chromosome

• Can create recombinant bacteria with new traits

• Transformation frequency varies among species; lab techniques can increase uptake

Famous Bacterial Transformation Experiment

• Frederick Griffith (1879-1941) was a British bacteriologist who is famous for his classic experiment in 1928 demonstrating bacterial transformation

  • S strain: smooth, virulent (causes disease)

  • R strain: rough, non-virulent (does not cause disease)

• He killed S strain bacteria by heat and mixed them with live R strain bacteria

• Some R strain bacteria “transformed” and became virulent S strain, causing those mice to die

Transduction

• bacteriophage infects a donor bacterium, accidentally packaging bacterial DNA → take up some bacterial DNA

• bacterial chromosome is sometimes partially broken down to make nucleotides for replicating viral DNA

• Virus infects a new bacterium, delivering recombinant DNA

• Recombination can creat recipient bacteria with new genes

• Host range limits transduction to same or closely related species

Transduction in Salmonella Example

P22 Phage in Salmonella

• Organism: Salmonella enterica (bacterium)

• Virus: P22 bacteriophage

• Process:

  • P22 infect a donor Salmonella cell

  • During viral replication, some bacterial DNa fragments are accidentally packaged into new viral particles

  • The donor DNA can join with the recipient’s DNA creating a recombinant bacterium with new traits

Key Concept:

• Bacteriophages act as vectors for gene transfer

• Transduction can transfer genes for traits such as toxin production or antibiotic resistance

Transduction v. Reserve Transcription

Transduction

• a bacteriophage (virus that infects bacteria) transfers DNA from one bacterium to another

• Steps:

  • virus infects a donor bacterium

  • virus accidentally packages bacterial DNA into its capsid

  • Virus infects a new bacterium and delivers the DNA

  • the transferred DNA can recombine with the recipient’s chromosome

• Key point: DNA moves via a virus; no RNA or reverse transcription is involved

Reverse Transcription

• involved copy RNA into DNA using the enzyme reverse transcriptase

• typical in retroviruses (ex. HIV)

• viral RNA is converted into DNA, which can then integrate into the host genome

• Key point: RNA → DNA, not just DNA transfer

Conjugation

• requires cell-to-cell contact; a cytoplasmic bridge (pilus) forms

• DNA transferred: plasmid or part of the chromosome from donor → recipient

• after transfer, crossing over may occur, creating a recombinant chromosome

• directionality: DNA moves only from donor → recipient (no reciprocal exchange, so donor does not get anything new)

• Note: not all bacteria can do this, need the F Factor to form a pilus (bridge)

Bacterial Conjugation and the F Factor

• The F Factor (fertility factor) is a plasmid episome found in some E. coli and other bacteria that carries genes that allow a bacterium to conjugate

• F+ cells (with the F factor): can donate DNA to F- cells (without F factor)

• During conjugation: the F plasmid is copied and transferred to the recipient cell, making it F+ and capable of donating DNA to others

• Importance of F factor:

  • enables horizontal gene transfer, spreading genes like antibiotic resistance

  • can integrate into the chromosome (Hfr), transferring chromosomal genes to the recipient

  • helps map bacterial genes and study gene function

Discovery of Genetic Exchange in Bacteria

Lederberg and Tatum’s 1946 Experument

• Before 1946, bacteria was believed to reproduce only by binary fission (dividing of organism by small polyp that breaks off and grows on its own), no genetic exchange was known

• Loshua Lederberg and Edward tatum challenged this idea using E. coli mutants

• Their experiment provided the first evidence of recombination in bacteria

Lederberg and Tatum: Experimental Design

• Auxotrophs: bacteria that cannot grow without certain nutrients because of gene mutations

• Prototrophs: can grow on minimal medium (make all nutrients themselves)

• Strains Used (auxotrophs for complementary mutations) DON’T NEED TO MEMORIZE STRAIN INFO

  • Y10: thr- thi- bio+ phe+ cys+

    • needs threonine, leucine, and thiamine added to grow

  • Y24: thr+ leu+ thi+ bio- phe- cys-

    • needs biotin, phenylalanine, and cysteine added to grow

• Hypothesis: if bacteria can exchange genes, mixing Y10 and Y24 might allow them to complement each other’s mutations, producing some offspring that can grow without supplements

Lederberg and Tatum: When Y10 and Y24 Were Mixed

• when mix ed and plated on minimal medium, a few colonies grew

• each strain alone produced no growth, therefore colonies could not have arisen by random mutation: too many simultaneous mutations would be required

• interpretation:

  • colonies were prototrophic recombinants with the genotype thr+ leu+ thi+ bio+ phe+ cys+

  • means that genetic exchange and recombination occurred between Y10 andY24

Does Gene Transfer Require Direct Contract?

• Bernard Davis (1950) designed a U-tube with a fine-pore filter separating two bacterial strains

  • Allowed liquid medium to pass

  • Blocked bacterial cells (to see if DNA would pass through filter if bacteria could not)

• Results:

  • After incubation, no colonies grew when plated on minimal medium

  • DNA did NOT pass through the filter

• Conclusion:

  • Gene transfer requires direct contact between bacterial cells

• Why did this not work?

  • cell has to be dead and break apart for DNA to be free-floating

  • this bacteria may just not be able to do transformation

The Fertility (F) Factor and Conjugation

• Conjugation depends on a plasmid present in the donor cell

  • In E. coli, plasmid is called the fertility (F) factor

• F factor carries genes to form a pilus, which allows transfer of DNA to another cell

• Cells with the F factor are called F+ (donor); cells lacking it are F- (recipient)

• F factor is an episome (can integrate into the chromosome bc it is a plasmid)

• Contains:

  • Origin of replication (oriV): site where plasmid replication begins

  • Origin of transfer (oriT): site where DNA transfer begins

  • Conjugation genes (tra genes): encode the proteins that form the pilus

• Once the entire F factor is transferred, the recipient (F-) becomes F+

  • both cells have full copy of F plasmid and can be donors in future conjugations

• Direct transfer is defined: the oriT site always enters the recipient first

• Only F plasmid genes are transferred in this type of conjugation - not chromosomal genes (yet)

  • could NOT explain the transfer of chromosomal genes discovered by Lederberg and Tatum

The Fertility (F) Factor and Conjugation: Steps

1. Contact: F+ cell forms a pilus that attaches to an F- cell

2. Connection: the pilus pulls the cells together, forming a cytoplasmic bridge

3. Nick and Transfer: One DNA strand of the F plasmic is nicked at oriT and begins to move into the recipient

4. Replication: as transfer occurs, rolling-circle replication replaces the transferred strand in the donor

5. Completion: The recipient replicates the incoming strand, forming a new double-stranded F plasmid

Note: now both cells can continue to pas on F+ (but chromosomal DNA is yet to be transferred)

Hfr Cells

• Hfr (High-Frequency Recombination) strains: where the F factor integrates into the bacterial chromosome through crossing over

  • creates a single, continuous DNA molecule with chromosomal genes and F factor sequences

STEPS:

1. Cells connected via pilus. F factor nicked at oriT in Hfr cell; 5’ end enters F- cell first

   1. takes some of the chromosomal DNA with it (which will be copied over)

2. Transferred DNA strand replicates as it enters the recipient

3. Pilus bridge disappears. Transferred Hfr DNA (F factor + chromosomal segment) crosses over with F- chromosome

   1. occurs since F factor is no longer a plasmid, crossing over can occur

4. F- cell now has recombined chromosome (but not F+ because it didn’t get the F factor). Non-integrated DNA is degraded (didn’t copy the plasmid or F factor). Hfr cell remains unchanged. This cell can now be replicated and passed on to later generations by binary fission (cell division)

Consequences and Frequency of Hfr Events

• the recipient cell rarely becomes F+ or Hfr

  • the entire F factor must be transferred

  • rarely happens before the cells separate

• Hfr formation occurs about 1 in 10,000 f+ cells

• This low frequency explains the rare recombination events observed by Lederberg and Tatum (why only some bacteria grow)

• Occasionally, the F factor is excised from the chromosome, converting an Hfr cell back into F+

F’ Cells

• Excised F factor is removed from the chromosome

  • natural process during DNA replication or recombination events

  • helps the plasmid return to an independent form

• When it excises:

  • becomes a separate circular plasmid again

  • may carry nearby chromosomal genes (ex. lac genes)

    • plasmid would be F factor with one other gene

• Resulting plasmid = F’ plasmid

• F’ plasmids can transfer both F factor and chromosomal genes (that were on the F’ plasmid) to F- cells

  • this creates partial diploids (merozygotes); cells with two copies of some genes, one on the bacterial chromosome and one on the newly introduced F plasmid

    • useful for studing gene function and dominance

Merozygotes

A partial diploid (merozygote) is a bacterial cell with two copies of some genes: one on the chromosome and one on an F’ plasmid

Uses in Studying Genes:

• Gene Function

  • Scientists can compare the activity of the plasmid-borne gene vs. the chromosomal copy

  • ex. if the chromosomal copy is mutated and the F’ plasmid has a WT gene, the cell can regain the function. this allows researchers to test which gene copy is functional

• Dominance

  • in a merozygote, you can see which allele is dominant:

    • if the plasmid gene “overrides” the chromosomal allele →

    • id the plasmid gene does not restore function → chromosomal allele is dominant

    • especially important in recessive v. dominant gene studies in bacteria

Cell Types (F factor)

Mapping Bacterial Genes with interrupted Conjugation

• Purpose: Determine the order of genes on a bacterial chromosome

• Process:

  • Conjugation occurs between Hfr donor and F- recipient

  • Full chromosome tranfer takes 100 minutes

  • If conjugation is interrupted early, only part of the chromosome is transferred

  • Genes that enter the recipient first are located closer to the F factor integration site

• Transfer always begins at the integrated F factor and moved in a defined direction

• By interupting at different times, you can see which genes entered the recipient (and thus determine which genes are located closest to the origin of transfer)

• Today, DNA sequencing is the preferred method for bacterial gene mapping, so this method is not used as much anymore

Transformation in Bacteria

Transformation = uptake of DNA from the surrounding environment

• DNA can integrate into bacterial chromosome or plasmids

• Occurs naturally when dead bacteria release DNA fragments

  • important in soil and marine environments as a genetic exchange route

• Key role in discovering DNA as the genetic material

  • Used in laboratory techniques to transfer genes into bacteria

  • helps scientists study gene function and engineer bacteria

Competence and DNA Uptake

• cells that can take up DNA through their cell membranes are said to be competent

• competence is influenced by

  • growth stage

  • DNA concentration in the environment

  • environmental factors (temperature, nutrients)

• DNA uptaken can be bacterial and non-bacterial

• during transformation, one DNA strand is degraded, while the other may pair with homologous regions in the chromosome (but needs a lot of things to go correct in order for this to happen)

• Integration:

  • integration requires two crossover events to insert single-stranded DNA into the bacterial chromosome

  • remaining single-stranded DNA is degraded by bacterial enzymes

  • in some species, double-stranded DNA can enter and integrate directly

  • resulting cells:

    • transformants: received and integrated new DNA

    • non-transformed: retained only original bacterial DNA

Cell division: one daughter may be transformed, the other non-transformed

Laboratory Techniques

• bacterial geneticists can increase transformation efficiency using lab techniques:

  • calcium chloride treatment: makes membranes more permeable

  • heat shock or electroporation: DNA uptake facilitated uses an electric pulse to create transiet pores in the membrane

• special competent strains of bacteria developed for research

• enables transformation of bacteria like E. coli, which are not naturally competent

• essential for gene transfer, plasmid introduction, and genetic

Using Transformation for Gene Mapping

• Requires

  • Donor stran: prototrophic (has functional genes)

  • Recipient strain: auxotrophic (lacks genes for certain nutrients)

• Process

  • Donor DNA is isolated, purified, and fragmented

  • Recipient cells are treates to become competent

  • DNA fragments enter recipient cells and may recombine with homologous regions

Using Transformation for Gene Mapping: Cotransformation

• After DNA enters the recipient

  • Donor DNA undergoes recombination with the recipient chromosome

  • generates transformants with different combinations of genes

  • example: genes a, b, c

    • single transformants: a+, b+, or c+

    • multiple gene transformants a+b_, etc

• genes closer together are more likely to be cotransformed

  • process in which two or more genes are taken up together during cell transformation

• frequency of cotransformation directlly proportion to proximity of genes

  • genes closer together → likely on same DNA fragment → cotransformed

  • genes far apart → unlikely on the same fragment → rarely cotransformed

• Conclusion: Cotransformation frequencies can be used to map relative positions of genes on bacterial chromosomes

Horizontal Gene Transfer Definition

• Horizontal gene transfer (HGT): genes passed between species without reproduction

  • mechanisms: conjugation, transformation, transduction

  • contrast: vertical transmission = genes passed from parent to offspring

• genome sequencing has shown that many bacteria have large portions of DNA that originated in other species

  • in E. coli, 17% of its genome appears to have been acquired by HGT, not inherited through ancestors

    • recombination can occur through HGT

Horizontal Gene Transfer: Medical Importance

Virulence (Disease-Causing Ability)

• HGT can give harmless bacteria the genes need to become pathogenic

• example

  • Vibrio cholerae became dangerous after acquiring a toxin gene via transduction

Antibiotic Resistance

• many antibiotic resistance genes are located on plasmids, which can be easily exchanged between bacteria, even across species

• example

  • methicillin-resistant Staphylococcus aureus (MRSA) strains can transfer resistance genes to other bacteria in hospitals → making infections harder to treat

Viruses Definition

• noncellular replicating structure consisting of nucleic acid surrounded by a protein coat (not considered life)

• can only replicate within a host

• infect all forms of life: bacteria, plants, animals, and fungi

  • come in many different shapes and sizes

Viruses: Non-Living

Do NOT meet most of the standards for life

• No cellcular structure (not made of cells)

• No metabolism or energy production (no mitochondria or ATP production)

• Cannot reproduce independently

  • do not have the proteins needed for transcription and translation (no polymerase, etc) so must replicate using host cell

• Inert outside a host

  • can only survive brief periods outside of host

Virus Components and Genome

Consist of:

• nucleic acid genome (DNA or RNA)

• protein coat (capsid) surrounding the genome

• some viruses also have a lipid envelope derived from host cell membrane

Viral genomes vary widely:

• may be DNA or RNA

• Single or Double-stranded

  • dif shapes and structures have dif functions

• Linear or circular

Viral Structure

• Structure is related to how they infect and what hosts they target

• Enveloped:

  • Outer structure: lipid membrane around capsid

  • Entry strategy: fuses with host membrane

  • Example: HIV or Influenza A (see below)

    • enveloped virus containing RNA genome segments; infects vertebrate respiratory cells

• Non-Enveloped:

  • Outer structure: protein capsid only

  • Entry strategy: often enters by injecting or endocytosis

  • Example: bacteriophage 4

    • complex structure with protein head and tail fibers; infects bacteria

Host Specificity (Tropism)

• Viruses infect only specific hosts and cell types

  • Determined by receptor binding on host cells (lock and key ides)

  • A virus can only infect cells that have the correct receptor

• Host range = types of organisms a virus can infect

• Tropism = specific cells/tissues a virus targets

  • the preference of a virus to infect specific host species: significantly influencing its distribution, transmission, and potential public health risk

  • examples:

    • bacteriophages: infect bacteria only

    • influenza virus: infects respiratory epithelial cells

    • HIV: infects immune cells

    • avian influenza prefers birds, but has now also impacted cows

Steps of Virus Replication in Host Cells: Using Bacteriophages

1. Attachment: the T4 phage uses its tail fibers to bind to specific surface proteins on an E. coli cell that act as receptors

2. Entry of phage DNA and degradation of host DNA: the sheath of the tail contracts, injecting the phage DNA into the cell and leaving empty capsid outside. Cell’s DNA is hydrolyzed

3. Synthesis of viral genomes and proteins: The phage DNA directs production of phage proteins and copies of the phage genome by host and viral enzymes, using the host cell’s machinery

4. Self-assembly: Three separate sets of proteins self-assemble to form phage heads, tails, and tail fibers. The phage genome is packaged inside the capsid as the head forms

5. Release: The phage directs production of an enzyme that damanges the bacterial cell wall, allowing fluid to enter. The cell swells and finally bursts (killing it) and releasing 100-200 phage particles

Steps of Virus Replication in Host Cells: Using Regular Capsid

1. Glycoproteins on the viral envelope bind to receptors on the host cell. For some viruses, the envelope fuses with the plasma membrane; others enter by endocytosis

2. The capsid and viral genome enter the cell. Digestion of the capsid by cellular enzymes releases the viral genome (which looks like RNA so the cell doesn’t destroy it)

3. The viral genome (red) functions as a template for synthesis of complementary RNA strands (pink) by a viral RNA polymerase (hacks RNA transcription)

4. New copies of viral genome RNA are made using the complementary RNA strands as templates

   1. similar to reverse transcription

5. Complementary RNA strands also function as mRNA, which is translated into both capsid proteins (in the cytosol) and glycoproteins for the viral envelope (in the ER and Golgi apparatus)

6. Vesicles transport envelope glycoproteins to the plasma membrane

7. A capsid assembles around each viral genome molecule

   1. when it leaves, the cell is often killed

8. Each new virus buds from the cell, its envelope studded with viral glycoproteins embedded in membrane derived from the host cell

How Cells Block Viruses v. How Vaccines Work

• Glycosylation (Cell’s Defense)

  • Sugars added by your cell’s enzymes cover and change receptor sites (your body changing/blocking the lock)

  • Acts like a physical shield → virus cannot attach

  • Determined by DNA and evolve over time

• Vaccines (Immune System Defense)

  • introduce harmless viral parts or instructions

  • teach your immune system to recognize and block the virus

  • antibodies act like “guards “ that stop the virus before it reaches the receptor

• Glycosylation modifies the lock (receptor)

• Vaccines neutralize the key (virus)

Glycosylation (Cell’s Defense)

• viral infection depends on binding to specific host cell receptors (lock-and-key interaction)

• mutations in receptor genes can change their shape, preventing virus attachment

• glycosylation (sugar molecules added to receptors) can physically block the virus from binding

• these changes may arise from evolutionary adaptation or random variation

• Result: determines which cells or species a virus can infect (host range/tropism)

• Example:

  • MERS-CoV used DPP4 receptor to enter cells, but in some species glycosylation physically blocks the virus-binding site, preventing infection entirely