Lecture 8: Genetic Recombination Flashcards
What is the outcome of mitosis
genetically identical cells
Lottery and Genetic Recombination Analogy
- you wouldn’t put the same number on the tickets you would put different ones for greater success
- cells will genetically recombine to help with success, so that hopefully as environment changes we can increase the amount of winning combos to keep organisms alive
Why are we so diverse (3 reasons)
1) mutation
- alter genes and their outcomes
2) random fertilization
- any sperm+any egg
3) recombination
- reshuffle genes to provide evolutionary advantage to continue species
Mechanism of Genetic Recombination
a) requires 2 DNA molecules that similar but non-identical
b) Homology allows DNA on different molecules to line up and recombine precisely
c) Enzymatic cutting + pasting of both DNA backbones from each of 2 DNA molecules required for recombination
what kinds of DNA are similar but not identical
homologs chromosomes
- we need this DNA bc 2n (mom and dad are similar to each other, dipoles are homologs)
- same genes, same order
Simplified model of genetic recombination
We enzymatically cut and paste backbone of DNA and eventually re-seperate them
- they are then recombined
Genetic Recombination in Bacteria
occurs in E.Coli
- BACTERIAL CONJUGATION: brings DNA of two cells into close proximity
- TRANSFORMATION and TRANSDUCTION provide additional sources of DNA for recombination
- Some bacteria genetically reshuffle as genes are transferred and recombined with existing DNA (genetically identical clones allow for this, we basically worked with them and manipulated them for understanding)
Genetic Recombination in E.Coli
Prototrophs- bacteria grow on minimal media because they make their own a.a (all 20)
Auxotrophs- bacteria with mutations does not grow on minimal medium
- Three letter gene name: + normal, - mutated allele
Complete vs Minimal Medium
Prototrophs ON MINIMAL
- have full complement of nutrients don’t need the complete media
Auxotrophs ON COMPLETE
- missing some nutrients so they need the complete media
Replica Plating
technique to
1) identify prototrophs versus auxotrophs
2) identify+count genetic recombination in bacterial colonies
PROCESS:
In replica plating, a master plate containing a complete medium allows the growth of both prototrophic (photo) and auxotrophic (auxo) mutants because it provides all necessary nutrients, while a minimal medium only supports prototrophic mutants that can synthesize all required nutrients, thus not allowing auxotrophic mutants to grow.
Experimental Evidence for Genetic Recombination in Bacteria
Lederberg and Tatum
demonstrated genetic recombination in bacteria by mixing two strains of E. coli (auxo), leading to the formation of prototrophic colonies that could grow without specific nutrients, indicating that genetic material was exchanged.
important bc it shows that bacteria can exchange genetic information important for antibiotic resistance
Bacterial Conjugation
Bacterial recombination by conjugation:
- bacteria are haploid
- sex pilus connects 2 bacteria
- donor sends DNA via cytoplasmic bridge to recipient
Recipient Undergoes Recombination
Plasmids: Circular, non chromosomal transferable DNA (independent of bacterial chromosomes)
R Plasmids: confer resistance to antibiotics (have specific genes that are resistant to antibiotics)
= HORIZONTAL GENE TRANSFER
VERTICAL TRANSFER
new transferred genes from 1 bacterial cell to another
- from parent to offspring
Bacteria will get homologs chrosomes from
another bacterial cell, donor will send pilus to connect the bacteria
What does the F factor have genes for
genes to encode for sex pilus
- cytoplasmically connects F+ cell to F- cell
- F- cell converts to F+ cell
- No recombination
F Factor + Conjugation
we need this to make sex pilus
- Donor cell must have F factor (fertility plasmid)
F+ cells = donors with F factor
F- cells = recipients w/o F factor
Difference between F+ and F-
F+ = transfers a copy of F- plasmid to recipient to F- to F+ so that is becomes a donor
- Just copying/sending chromosome, no actual genetic recombination occurs *
Transfer of Genetic Mutation During Conjugation
F Factor plasmid backbone cut
- 1 of 2 strands is sent over to recipient and simultaneously is being replicated
- allowing for double strands of DNA in both cells = ROLLING CIRCLE REPLICATION
Is F Plasmid transferred in Rolling Circle Replication
Yes
- no bacterial chromosome has been moved over yet, so recombination cant occur yet
Hfr Cells and Recombination
Hfr integrate F factor into bacterial chromosome through recombination:
- Her cells can conjugate with F- cells
- Recipient becomes partial diploid
Hfr Cells- high frequency cells ex F+ plasmid
How does genetic recombination occur
double-crossing over in recipient
- new generations have recombined DNA
F factor into chromosomal DNA yield
Hfr cell
gene mapping
genes that are closest
- increases likelihood of getting across
- mutated versions of genes are therefore, homologs
= similar but not identical
**The frequency of recombination with all genes on chromosomes. If a particular gene has an increased frequency, its clear to frequency plasmid
- increased likelihood of making it across sex pilus
Partial Diploid
bacteria that possesses 2 copies of some genes, typically due to the presence of an extra piece of DNA, such as a plasmid, along with its chromosomal DNA.
Why isn’t the full chromosome not always sent
- because the sex pilus is deconstructed
= remains a F- cell, because when you cut F plasmid not all of it is transferred
PARTIAL DNA IS TRANSFERRED AND DNA RECOMBINATION OCCURS
Mapping genes by conjugation
- mated Hfr and F- cells that differ in number of alleles
- at regular intervals after conjugation commenced, remove cells and break apart mating pairs
- cultured separated cells and analyzed for recombinants
greater time to conjugate before separation, the greater number of donor genes into recipient
The order and time at which genes were transferred
able to map and assign relative positions of several genes of E. coli chromosome
Transformation
occurs when bacteria take up DNA from disintegrated bacteria
- linear fragments recombine by double crossing
- transformation bacteria usually have DNA protein in wall
Artificial transformation (part of transformation)
- alters cell membrane for DNA penetration
electroporation:
recipient can grab DNA from dead cell/environment
- can be or a natural availability for some bacteria, DNA comes through to make pores
ELECTROPORATION DEFINITION:
technique that uses an electric field to increase the permeability of cell membranes, allowing DNA or other substances to enter the cells; it can involve recipient cells taking up fragments of dead cells through a process called “natural transformation,” where they scavenge for DNA from their environment.
= Horizontal gene transfer
Transduction
- occurs when bacterial phages (which are DNA carriers from donor to recipient) transfer DNA from 1 bacteria to another
- Virus incorporate DNA fragments from host cell:
- if DNA fragments are homologs
- bacteria becomes partial diploid
- Recombination by double crossovers
= Horizontal Gene transfer
3a. Generalized Transduction
1) phage attachment
2) phage enzymes: releases enzymes to poke holes
3) phage DNA replication: viral enzymes will cut up bacterial chromosome
wants to take the energy to build more self-viruses
RECOMBINATION CAN OCCUR
4) phage proteins: reconstruct bacteriophages
5) phage assembly
6) phage release: phage removed to infect new bacterial cells
a piece of bacterial chromosome is put in
- stats alive but on another molecule to reproduce and recombine
3b specialized Transduction
Viral DNA is brought in, and could stay is living cell and lysogenic cycle
can also go Dormant to make no viral products
Prophage
- will grow and divide (multiply DNA by separation)
- continue to reproduce the viral chromosome
- Comes out of lysogenic cycle *
Virulent vs Temperate Bacteriophage
virulent bacteriophage
- uses only lytic cell of infection
- kill host bacteria
temperate bacteriophage
- uses both lysogenic and lytic cycle of infection
- may or may not kill host bacteria
prophage- bacteriophage integrated into host DNA
Genetic Recombination in Eukaryotes : Meiosis
- meiosis occurs in different places in organismal life cycles
- meiosis changes both chromosome number and DNA sequence
- meiosis produces 4 genetically different daughter cells
- several mechanisms contribute genetic diversity
Sexual Reproduction
- produces offspring by union of male and female gametes (sperm and egg)
- meiosis produces gametes with 1/2 chromosome number
gametes are genetically different - evolutionary advantage: genetic shuffling
Fertilization
- fuses nuclei of egg and sperm
= zygote - restores parental chromosome number
Animal Life Cycles
- diploid phase dominates
1) meiosis followed by gamete formation
2) haploid phase is reduced and short, no mitosis
In Males=4 nuclei from meiosis form separate sperm cells
In Females=only 1 nucleus becomes an egg
What must eggs have for zygote formation
large amount of cytoplasm because if its fertilized its ready to be divided to make macromolecules etc etc
Homologs Chromosome pairs
- paternal chromosomes from male parent
- maternal chromosomes from female parent
= sets are homologs to each other, their alleles may be different within homologs pairs
Meiosis 1
- recombination exchanges segments between homologues
- produces two haploid cells with chromatids attached
alleles
version of 1 gene
Meiosis separates homologs pairs
before meiosis: diploid (2n)
after meiosis: haploid (n)
Meiosis 2
- sister chromatids separate into separate cells
- produces 4 recombined haploid cells
2 meiotic divisor produce
4 haploid non-identical nuclei
What are things that occur during meiotic cell cycle
1) prophase 1: sister chromatids condense into chromosomes
2) synapsis: pairing of homologs
3) tetrads: fully paired homologs
4) recombination: mixes alleles across tetrads
Prometaphase 1
- nuclear envelope breaks down
- kinetochores (miroctubules) attach to polar spindles + (not directly) to chrosomes
Metaphase 1 and Anaphase 1
METAPHASE:
- tetrads align on metaphase plate
ANAPHASE:
- homologs segregate move to poles (sister chromatids attached)
for both:
- Nondisjunction creates abnormal chromosome number
RANDOM ALLIGNMENT, 1 pair of homologs doesn’t dictate the arrangement of chromosome
Telophase 1 and Interkinesis
- No change in chromosome
- spindle disassembles
Interkinesis: the pause between meiosis 1 and 2 where no DNA replication occurs
Prophase 2, Prometaphase 2
- chromosome condense, spindles form
- nuclear envelope breaks, kinetochores attach to microtubules
Metaphase 2
- chromosomes align on metaphase plate
independent arrangement
Meiotic Cell Cycle
Anaphase 2 and Telophase 2
- spindles separate chromatids
- spindles disassemble
- new nuclear envelopes form
- 4 GENETICALLY DIFFERENT HAPLOID CELLS FORM *
What is nondisjunction
- both members of pair of homologs chromosomes connect to spindles from the same pole
- following anaphase, one pole then receives both copies of pair and the other pole receives 0
= gametes that have 2 copies of a chromosome
AFTER FERTILIZATION: zygote has 3 copies of chromosome instead of 2
Ex. Trisomy 21 (seperation is inaccurate due to gametes)
Nondisjunction in plants
an irregular number of chromosomes can be beneficial
Sex Chrosomes in Meiosis
- gametes produced by female may receive either X (oogenesis)
- gametes produced by males may receive either X or Y chromosome (spermatogenesis)
Meiosis and Mitosis compared
- both: similar cell divisions, meiosis divides twice
- mitosis: 2 identical daughter cells (diploid)
- meiosis: 4 genetically different cells (haploid)
- premeiotic interphases similar to mitotic interphase (G1,S,G2)
- chromosome copied into sister chromatids
GENETIC VARIABILITY
1) GENETIC RECOMBINATION
2) RANDOM SEGREGATED AT ANAPHASE 1
3) ALTERNATIVE COMBO AT ANAPHASE 2
4) RANDOM FERTILIZATION
Genetic recombination
- recombination (crossing over)
key genetic shuffle of prophase 1
tetrads held together at synaptonemal complex:
- 2 of 4 chromatids exchange alleles
- chiasmata or crossovers are points of exchange
Crossing-Overs
- Occurs at random on CHIASMATA (synaptonomeal complex of proteins that keep the chromosomes back to back)
- occurs between non sister chromatids where they just exchange segments of DNA
Synaptonemal Complex
- how homologs chromosomes are held together
Random segregation
- key genetic shuffle of metaphase 1
- each chromosome of a homologs pair may randomly end up at either spindle pole
- Any combo of maternal and paternal chromosomes= segregated to gametes *
- 2X number of possible combination
At Metaphase 1
- Chromosomes line up randomly
alternative combo at anaphase 2
- attachment of spindle to kinetochore on sister chromatids is random
- therefore alignment is random
- increased variation
random fertilization
random chance of male and female gamete forming zygote
- meiosis allows randomness bc gametes are genetically different necessary for mendelian laws of inheritance
what happens to hydrogen bonds when DNA is separated
they are broken, forming templates to allow for precise replication of genetic material
How is DNA paired
by enzymes that separate H bonds of one double helix and allow the base to reassociate with bases in a homologus helix.
in some types of bacterial recombination, one of the participating cells Is what
DEAD
what were Lederberg and Tatum testing in essence
whether or not bacteria have a kind of sexuality in they reproduction process
- they use E.Coli for their experiment to isolate two strains, where 1 strain could grow only with biotin or methionine and the other didn’t need biotin or methionine but needed leucine, thiamine, and theorenine. When they were mated they carried SOME protortophic alleles
- some form of recombination between the DNA molecule of the two parental types must have produced the necessary combination with prototrophic alleles
T/F auxotrophs are mutant strands
T, and this is why they cant synthesize amino acids
What is Lederberg and Tatums experiment important for?
Antibiotic resistance
-Lederberg’s experiment showed that recombination in bacteria results from pre-existing genetic mutations, not from mutations induced by environmental factors
-showing that resistant bacteria survive and reproduce when antibiotics are introduced.
What do bacterial cells do instead of fusing
Conjugate
- make contact via long tubular sex pilus to make a cytoplasmic bridge
conjugation facilitates what
form of sexual reproduction in prokaryotic organisms
if the F plasmid is passed onto each daughter cell, its what type of inheritance
If the F plasmid is copied and passed directly from the donor to recipient cell, its what type of inheritance
- VERTICAL
- HORIZONTAL
How does F plasmid become part of main bacterial chromosome
- F plasmid gets near main chromosome and lines up in a short region of homology to undergo recombination
- when 2 circular DNA molecules recombine they fuse into a larger circle
Is the integrated Plasmid with 1 cell or is it between the chromosomes of different cells
with 1 cell
- meaning that after recombination the F plasmid is put into a cell ITS NOT put into the chromosomes of different cells
Why are Hfr cells called that
- high frequency cells
- because they can promote recombination of DNA between cells by exporting copies of chromosomal genes to another cell
What happens when the F plasmid is integrated into the bacterial chromosome
- genes are still available for expression
- therefore, Hfr cells make sex pili and can conjugate with an F- cell
Difference between F plasmid transfers alone than when Hfr cells transfer genetic material
F plasmid: recipient cells become F+ with the plasmid
Hfr: origin of transfer is near the middle of the integrated F plasmid, so only 1/2 of the total F plasmid DNA is transferred at the front of the other 1/2 of F plasmid
{BECAUSE SEX PILUS BREAKS BECAUSE INTEGRATION OF F FACTOR MAKES PILUS FORMATION PRONE TO DISRUPTION}
- therefore the cell will become a partial diploid
What happens to incoming alleles that are not recombined onto the chromosome
they’re lost
what do transcription and transduction allow for that conjugation doesn’t
enables recipient cells to recombine with DNA obtained from dead donors
What can a mistake in the infection cycle cause
transfer of bacterial genes from a donor to a recipient cell
What is a prophage essentially
- state of phage (virus) integrated into host chromosomal DNA
What is specific to specialized transduction
In specialized transduction, only bacterial genes near where the phage inserted itself in the bacterial DNA are mistakenly included in the phage DNA due to a recombination error.
THE ERROR:
phage DNA incorrectly cuts out, taking nearby bacterial genes with it.
Two kinds of Differences with Meiosis
- halves chromosome number
- new combinations of alleles arising from recombined DNA sequences
Different alleles of a given gene have similar
BUT DISTINCT DNA SEQUENCES
- Therefore they likely encode different variations of RNA or proteins
Differentiate between
CHROMATIN
CENTROMERE
SISTER CHROMATIDS
CHROMOSOME
CHROMATIN
- stuff chromosomes are made from: nucleic acid + proteins
CENTROMERE
- structure that helps chromosome get oriented during cell division and hold SC together
SISTER CHROMATIDS
- evident after chromosome replicates its DNA
CHROMOSOME
- joined sister chromatids are part of one chromosome
How can we determine the number of chromosomes a cell can have
count the number of centromeres
- two of each type of chromosome are found in a cell-diploid
- one of each-haploid
T/F Chromosomes pair up during mitosis
F
What happens when homologs chromosomes are paired
chromatids physically exchange segments
the synapotmeal complex is a
protein framework
- it disappears when exchange is complete towards the end of prophase 1
spindle attachment during mitosis vs meiosis
MITOSIS:
spindle fibers attach to centromeres of sister chromatids
MEIOSIS:
spindle fibers attach to homologous chromosomes during meiosis I and sister chromatids during meiosis II.
T/F cells from meiosis directly enter cell duplicating again
F, they produce reproductive cells
the nucleus will have (at start of meiosis)
1/2 the number of chromosomes present in meiocyte that began meiotic division
why is it hard for humans to produce genetically identical offpsirng
- so much variability introduction by recombination and juggling at DNA
- TWINS:
- arise from mitotic division of.a fertilized egg
genetic variability arrives from 4 sources
1) genetic recombniation betwn homologs chromosomes
2) differing combinations of mom and dad chromosomes segregated to poles during anaphase
3) differing combinations of recombinant chromatids segregated to the poles
4) sets of male and female gametes that unit
