genetics unit 2

tetrad analysis is used in

fungal genetics

ascomycetes

products of meiosis (spores) contained within a sac (ascus)

products of fungal meiosis

spores (sister cells)

sac in fungal genetics

ascus

fungi are a phyla by

reproductive strategy

how do fungi grow

through mycelia & multinucleases, no cell wall/membrane

alternates between 2C and 1C but always haploid

mating groups

in fungi, force outbreeding

e.g. A cannot fuse with A, alpha cannot fuse with alpha, but alpha and A can fuse

many species are _____ for most of life

haploid (syncticial)

what happens when two individuals meet

fuse to form heterokaryon (2N)

two main types of mold

neurospora and sordaria

found in leaf litter and break down cellulose

reproductive unit in fungi

perithecium

what will an ascus do after two mycelia fuse

synthesize thicker cell wall, undergo gene expression, turn into spores

how are the spores arranged in neurospora and sordaria?

linearly in order of meiosis

tetrads

linear array of 8 spores

can see segregation of spore color, texture, size

asci

formed in parithecum

sac like structure

each ascus =

one meiosis

middle of ascus =

anaphase I plane of division

middle of each ½ ascus =

anaphase II plane

for tetrads, anaphase II pattern

2, 2, 2, 2

for tetrads, anaphase I pattern

4+4

how to calculate map distances with tetrads

(Anaphase II segregations/total asci)/2

order of spores reflects

segregation of chromosomes/chromatids

how can you map two genes relative to one another or one gene vs. centromere

by looking at pattern of spores

auxotrophic mutants

cannot synthesize a specific essential compound due to a genetic mutation and require it from their environment for growth

steps for ascospore development

1. cells from different strains fuse to form a diploid zygote

2. after chromosome duplication, the diploid nucleus undergoes first meiotic division

3. each daughter nucleus undergoes the second meiotic division, producing two haploid nuclei

4. each haploid nucleus divides mitotically to produce two twin nuclei

5. the resulting eight nuclei are partitioned into separate cells, each developing into an ascospore

why do we divide by 2 in calculating map distances with tetrads?

each ascus represents all 4 chromatids

whole ascus isn’t recombinant, only half of offspring in ascus are

gene conversion

one allele actively changed into other

result of DNA repair

what pattern is a 3 + 5 tetrad

neither Ana I or II, gene conversion

gene conversion process

Spo11 homodimer

5’ to 3’ resection

strand invasion (strand from other homologue as template to repair)

further processing

F

large plasmid, codes for sex pilus

sex pilus

tube for DNA transfer

Hfr+

high frequency of recombination

bacteria (typically E. coli) with fertility factor, F, integrated into main chromosome

how many base pairs does a bacteria’s core genome have

2-4 million

how many base pairs do plasmids have

10k-low 100k

plasmids can

jump from cell to cell

plasmid genes are often

advantagous

plasmids can recombine with

main chromosome

genes transfer in a

linear order

how long does it take to go around the circle/transfer chromosomes

100 minutes

prototrophs

can make everything they need

replica plating

use velvet or grid to produce identical plates with different growth environments

can grow the same bacteria on plates with different conditions

conjugation

exchange of genes via plasmids

transduction

exchange of genes via plasmids incorporated into bacteria virus

transformation

pick up DNA from environment

fertility factor F

plasmid in bacteria that enables the transfer of genetic material through conjugation, converting F− recipient cells into F+ donor cells.

F’ contains

chromosomal loci

can take over host loci, F can recombine with full circle chromosome → some of full circle chromosome taken through bridge

Hfr strains

contain F origin of transfer on chromosome: chromosome transfers

E. coli maps in what unit

minutes

co-transfer frequency

closer together, more likely to be co-transferred

high co-transfer frequency = high likelihood of co-transfer

genes that transfer first are

closest to origin of transfer

interrupted mating experiment

mix donor strain Hfr+ with recipient strain Hfr- of opposite genotypes at time 0

grow in liquid culture with gentle swirling

sample every 10 minutes, take aliquots and vortex

spread plate on media to genotype

resolution is _____

5-10 minutes

genes close together will appear at same time, asynchronous transfer

gene transfer is mapped in

minutes

time to transfer marker genes

distinguish phenotype between recipient and recombinants

bacteriophages

generally small

have a nucleic acid genome

viruses

no cell physiology

must infect cell to replicate

nucleic acid surrounded by coat material (usually protein but can be membrane from host)

lambda virus

DS DNA virus, ~50,000 base pairs, linear molecule with sticky ends (12 bp), comp. SS projections

virus life cycles

lytic

lysogenic

lytic cycle (more typical)

1. circularize

2. replicate

3. when phage head coat, rolling circle replication, stuffs lambda head at one site like factory assembly line. last set of enzymes made, chews cell from inside by wall, lambda phage go out and attack cells. timed, cannot leave early or late

transduction in bacterial and phage genetics

host DNA transferred via bacteriophage

specialized transduction

phage lambda and allies

lambda inserts into host genome, when excises can carry flanking DNA

lysogenic life cycle

virus integrates its DNA into the host genome, remaining dormant while the host cell continues to live and reproduce.

generalized transduction

any area of host genome transferred

e.g. phage P2, lyses host, but first fragments host genome

P2

lytic, but one of the first genes transcribed is nuclease (chops up host genome randomly, stops E. coli defense rxn

max distance for 2 genes to be transferred together is 2 minutes

head capsule is about

2 minutes worth of E. coli DNA

in the lytic cycle,

phage genes are transcribed and translated, new phage particles are produced, and the host cell is lysed.

when was DNA first isolated

in 1869 by Friedrich Miescher from white blood cells

DNA has a high

phosphorus content

nucleic acids are high in

phosphorus

Griffith experiment 1928

transformation- direct uptake of genetic information by cell from environment

used virulent (S) and attenuated (R) Streptococcus pneumoniae

what did Griffith find?

virulent into mouse caused dead mouse

attenuated into mouse, mouse survived

heat treated virulent into mouse, mouse survived

dead virulent and live attenuated into mouse, virulent was cultured, mouse died

Avery, MacLeod, McCarty, 1944 experiment

fractionated S strain strep into proteins, nucleic acids, and lipids

exposed R strain to different fractions

repeated with refinements over several years

what did Avery, MacLeod, and McCarty find

unclean results that indicated nucleic acids were genetic material

Hershey Chase experiment

produce radioactive isotopes to label and track nucleic acids and proteins through systems

32P is beta emitter used to mark DNA

35S is weak beta emitter used to mark nucleic acids

grow T2 in E. coli fed with both isotopes

harvest T2, wash

infect unlabeled E. coli

allow to go partway through life cycle but not to lysis

pellet cells

scintillation counter

detects and measures ionizing radiation by converting radiation-induced light flashes into electrical signal

findings of Hershey Chase experiment

35S remains in liquid

32P remains with cell

nucleic acid DNA is genetic material

how long is DNA

cm to meters per chromosome

composition of nucleotide DNA

nitrogenous base (unique portion) carbon nitrogen oxygen circle, off of C1

sugar (deoxyribose) pentose 2’ deoxyribose (C1-C5)

phosphate groups off of C5 which is out of ring

OH on C3

what makes uracil different

it is missing a methyl group. over time, uracil turns into cytosine which is ok in short term RNA but not long term DNA

how to tell the difference between C, U, and T

if there is a methyl group single ring, T

no methyl group single ring, C

C=O instead of C-NH2, U

ribose

OH groups on 2’ C and 3’ C

3’ OH is essential for

sugar-phosphate backbone formation

new nucleotides can only be added to 3’ end

Fred Sanger’s experiment

modified nucleotide to 2’3’ dideoxynucleotide that is missing both 2’ and 3’ OH

INHIBITS SYNTHESIS, no 3’ OH to form next phosphodiester bond

applications in DNA sequencing

what are the medical applications of Sanger’s experiment

chemotherapy agents

cancer cells are force fed dideoxynucleotide triphosphates

cancer cells die during S phase because DNA synthesis cannot be completed

35S analogues

35S analogues refer to substances that mimic the properties of sulfur-35 (35S) in various applications.

In molecular biology, 35S-labeled nucleotides are used in DNA sequencing methods, allowing for more detailed information per reaction.

fluorescently labeled

modified nucleotides that emit light when exposed to specific wavelengths, widely used in molecular biology for various applications such as DNA sequencing, qPCR, and imaging.

chemically labeled nucleotides

allows researchers to visualize otherwise invisible processes

inosine nucleotides

central purine nucleotide that serves as a precursor for the synthesis of adenine and guanine nucleotides and plays key roles in RNA function and cellular metabolism.

chargaff’s rule

[purines]=[pyrimidines]

[A]=[T], [G]=[C]

holds for dsDNA only

crystal

core group of atoms repeated over and over

x-ray interacts with electron cloud, can disperse or focus it

Pauling model for DNA

triple helix, phosphates inside, no model for forces holding strands together

does not work, DNA and phosphates repel

Fraser model for DNA

triple helix, phosphates outside, H bonds linking strands

does not account for Chargaff’s rule

how do you build up 3D placement of atoms

by scatter pattern of Xrays

what did Cochrane do

solved math for helix

periodicity

spacing between adjacent bases 0.34 nm

10 bases gives one complete helical turn

diameter of 2 nm

puts constraints on structure, need correct bond lengths and angles

Watson and Crick :/

double helix with bases inside

two strands accounting for Chargaff’s rule

had correct bond lengths from Cambridge

DNA structure

linkage between adjacent bases is phosphodiester

2 strands: antiparallel and complimentary

one strand is 5’-3’ linkages, other is 3’-5’

right-handed helix, side facing you rises to the right

Guanine and Cytosine

3 H bonds between

adenine and thymine

2 H bonds between