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OLEMISS / Biology / BIOL 336 / How do vectors help replicate inserted dna?

How do vectors help replicate inserted dna?

How do vectors help replicate inserted dna?

Description

School: University of Mississippi
Department: Biology
Course: Genetics
Professor: Ryan garrick
Term: Fall 2016
Tags: Genetics
Cost: 50
Name: Genetics Test 2 Study Guide
Description: Re-upload. Not as detailed as the last one due to a busy schedule, still includes most everything mentioned in class/on slides!
Uploaded: 10/11/2016
60 Pages 125 Views 4 Unlocks
Reviews


Lecture 10


How do vectors help replicate inserted dna?



9/16

Recombinant DNA

- Combining DNA that is from different organisms  

o Do not occur naturally

o Not a product of crossing over

 Ex: gene from fruit fly in bacteria  

• Gene from two different species that would not normally mate to create  mosaic-like chromosomes  

- Can also be used to isolate and copy specific DNA

Recombinant DNA

• ________________: use recognition sites to cut DNA into fragments • ________________: takes up the cut DNA and incorporates it into its own by  sealing the sticky ends

• ________________________________: have the ability to take up the  vectors/plasmids


What makes e. coli a good bacteria?



- Receptor sites – take plasmids up and bring them into cell so that DNA  replication can proceed inside of the competent cell  

Restriction Enzymes (REs)

- Original source from bacteria as a defense mechanism against  ________________.  

• ______ and _______DNA at both strands

- Cuts in zigzag fashion

• The fragments are now called ________________

- Single stranded DNA overhang creates “sticky ends”

Vectors (= plasmids)

• Transports the small DNA fragment into a competent cell  - Helps replicate inserted DNA  

Good vectors… (specific properties for easier cloning) - ________________– region that would be cut by enzymes so that it opens  up to insert DNA fragment of target gene in it


What are the advantages and limitations of cloning?



o it is versatile and can be cut by many different enzymes o the region is a functional gene called Lac Z gene and is involved  in breaking down food and changes color of cell when it is  function ing properly Don't forget about the age old question of What do you call an increase rate of chemical reactions without being consumed?

 only works when NO inserted DNA in genes  

 bacterial cell with this plasmid will break down food and  turn blue  

- _________________________– bacteria takes this up and will be able to  grow on a medium  

o allows bacteria cells to survive on a growth medium

• Replicate

• Are versatile because they have many cut sites for RE

• Transformed hosts can survive on the growth medium/go on to make more  copies

Sticky ends and DNA ligase

FIG. 17-2

• Start with cleavage with ________________from eukaryotic gene and one  from bacterial plasmid (vector)

• Fragments with complementary Tails

• Annealing allows recombinant DNA molecules to form by complementary  base pairing  

• DNA ligase seals the gap  

A recombinant DNA molecule

• If the DNA molecule took up the DNA successfully, blue colonies will form  - if unsuccessful or broken, white colonies will form  

• host cell needs the ampicillin resistant gene to survive ampicillin treated  growth medium and divide so that colonies will form  

What would happen if one of this vector failed to take up the plasmids at all?  - They would die before they’d even be able to be seen because they  would not be resistant to ampicillin and would die from the growth  medium  

Transforming the Host Cell

• E. coli is a good bacterial host/competent cell  We also discuss several other topics like What are the purposes of patrol?

• competent cells will take up the DNA when electrically or heat shocked  - Heat shocked in lab?  

• yeast cells can be used as host cells to study eukaryotic genes

Polymerase Chain Reaction (PCR)

• ________________________________is the DNA polymerase used - Needs high temps  

- Very accurate  

• 3 steps – ________________________________________________ - Makes lots of copies of the DNA

• primers help set boundaries  

PCR: first cycle

• stress the dsDNA with high temperatures to ________________and split the  strands into 2 ssDNA

- 95°C

- each strand acts as template strand

• Primers ________________ to each ssDNA strand at around 50°C

- Primers are fairly short and polar

o 3’ and 5’ ends

o what TAQ polymerase adds base pairs to  

• Taq adds base pairs to the 3’ of the primer (5’ of DNA complementary  strand) (________________)  

• Each cycle doubles the number of templates for the next cycle  - start with 2 strands and go through 8 cycles  

o 28 = 256 strands

 exponential increase We also discuss several other topics like What are the processes involved in the rock cycle?

Advantages (v. Cloning)

• host cells are not needed

• takes way less time  

• More versatile due to primers because it creates more cut sites for DNA  polymerase

• Not a lot of DNA is necessary  

Limitations (v. cloning)  

• primers need some baseline info about the organism’s DNA  • Contamination is easy  

- Can accidentally clone DNA from a different source; need to be  cautious you are not amplifying your own DNA

Lecture 11

9/19

Cloning: recap

• cloning isn’t always perfect, sometimes the bacteria fails to take up the  inserted target DNA

- If something goes horribly wrong – all those bacterial cells will fail to  grow because they lack the gene that makes them resistant to  ampicillin  Don't forget about the age old question of What is the formula for 1-dimensional error measure?

Steps are involved in PCR and in correct order:  

- Denature –> annealing –> extension

More applications…  

• organism identification for illegal activities such as whale meat sales to  food industry  

• Chromosome walking  

- Essentially start at a particular place on a chromosome and extend  outward; generating an entire chromosome sequence  

• finding DNA mutations like when you use PCR

Following cloning or PCR

• ________________________________

- Many different restriction enzymes to cut specific DNA into desired  fragments

- separate fragments using electrophoresis and other methods • Now can find out information from the cut sites

- how many cut sites

- order of cutting

- distance between the cutting

• restriction fragment length polymorphism (RFLPs) helps us identify DNA  variation If you want to learn more check out What does germ line denote in the context of hereditability of mutation?

Electrophoresis

• Technique for separating DNA fragments according to differences in size  using a gel to act as a viscous matrix as well as an electric charge to pass  through this gel

- Send electric current from negative –> positive end; DNA migrates  towards positive end (Anode) We also discuss several other topics like Why do people use twitter?

- Bands with longer fragments will take more time to move through the  gel

- Bands with shorter fragments will take less time  

Medical Diagnosis  

• RFLP analysis can help find disease-associated alleles in a genome  • Ex: ________________l

- A substitution mutation causes a different amino acid sequence which  leads to a ß-globin protein and  

- Homozygous: detrimental; heterozygous: effects show later  • A change in restriction enzyme recognition sites cause a new banding  pattern which can be used for diagnosing the disease the pattern represents  

Pharmacogenomics

• A lot of medications have common and/or fatal side-effects because  medication is variable to work for everyone…usually on works for about 60%  of the population

• Pharmacogenomics: selecting drugs and dosage based on the individual’s  genetic makeup  

- Individuals with same disease metabolize 6 MP differently  - More dependable than trial and error

- Differences due to genetic make-up (TPMT genotype)

- Genetic assay allows customized treatment

Complimentary DNA (cDNA)

• cDNA: only the DNA that codes for some specific protein  1. Extract mRNA

2. Convert mRNA back into its complimentary DNA using Reverse  transcription PCR

Reverse Transcription PCR

• In Reverse Transcription, mature mRNA is what we use to make templates  for coding  

• Oligo-DT primer will anneal to the 3’ end of the double stranded molecule • Reverse transcriptase enzyme makes complementary DNA by extended  from the Oligo-DT primer

• a hybrid molecule of both mRNA and DNA is created so RNAse H enzyme  will digest that mRNA strand

• DNA polymerase will be used by any of the remaining mRNA  • the now double-strand of DNA acts as a snapshot of all the coding DNA in  the genome

DNA Sequencing

• How to determine how genes are organized by nucleotides  - Help determine genetic differences between organisms • Used to only be done by chain termination sequencing, or the Sanger  method.  

Chain Termination Sequencer  

• lots of colored peaks, one for each nucleotide, from a chromatogram  

• reads roughly 800 bp at a time

• a slow and expensive method compared to those newer methods

Lecture 12

9/21

“Next-generation” Sequencers

• ________________________________

- Faster and cheaper

o Reads around 150 bp

• ________________–

- Also faster and cheaper and reads around 400 bp per read.  ** relevant to align and assemble nucleotide sequences**

Ch. 18

Genomics and Bioinformatics

New-ish fields

** Review genomics, proteomics, and bioinformatics  

Genomics

• The study of the complete genetic information of small creatures that live  in the depths of the earth, who guard buried treasure

Karyotype is highly variable

Organism

Diploid number (2n)

African Wild Dog 

78

Badger

32

Carp

104

Adders-Tongue

1262 (!)

Cotton

52

Organismal “complexity”

• Organsimal complexity is not proportional to the  

________________________________________________ or to ________________size  (absolute number of base pairs)

- Genome size – total amount of DNA (base pairs) within a haploid  genome  

Genome size also variable  

Organism

Billion bp (1000 million)

African Wild Dog 

2.66

Snapping Shrimp 

15.45

Carp

1.61

Poison Dart frog

8.70

Hugh-man

(human)

3.03

relate to absolute size of genome?

How does number of chromosomes  

- Size of African wild dog genome is quite larger than carps’ - Chromosomes are of different size (African Wild Dog’s are larger than  carps)  

- Frogs have a very large genome size compared to humans

Sequencing a Genome

• ________________– complete __________ set of all the DNA in a cell - Not consistent size across organsism  

- Viruses have the smallest genomes, prokaryotes have median size,  and eukaryotes usually have the largest genomes  

• DNA sequencing  

- “shotgun” sequencing

o great for whole genome sequencing

 Fragment the genome using restriction enzymes

 Sequence the fragments

 Assemble/order the pieces using some sort of  

electrophoresis  

Genomic Libraries

• Basically a whole genome that has been fragmented that can be inserted  and stored inside of vectors

• Does not involve PCR  

• Just get one copy of each sequence using the fewest number of fragments - BAC or YAC clones

Shotgun Sequencing Technique

• Fragment the genome

- Use specific REs one at a time  

• Get combinations of fragments  

• Computationally sequence the fragments

• End up assembling a whole chromosome sequence

Assembling “contigs”

• ________________– stretch of DNA sequences within one gene and across  adjacent genes that overlap

HP Computing (align, assemble)

• de novo alignment and assembly

- Not a lot of baseline data  

• Resequencing  

- Some baseline information  

Diploid Genomes

• heterozygous genotypes are possible so it may be necessary to align the  gene variants  

- Allele 1 v. Allele 2

DNA Sequence Alignment

• Scenario 1 – the reads do not start in same place

- Align sequences to see a match-up

- Mismatch between 2 nucleotides – point mutation/DNA sequence  substitution

- They are not identical  

Gene Pools

• ________________– a gene pool can contain many allelic variants  • Might have to align the different variants of a gene

- Look out for different kinds of polymorphisms  

DNA Sequence Alignment

• Scenario 2 – many individuals and probably many alleles - Align sequences to see a match-up

o At least 5 different alleles  

 Take multiple sequence alignment and count the number of different sequences that exist

• Bioinformatics allows us to figure all of this quickly  

Annotating a Genome

• Need to identify genes with names and locations to interpret

Protein-coding genes have some hallmarks:

- Start codons for mRNA include AUG and for DNA = TAC o T pairs with A, A pairs with U, and C pairs with G <– in RNA - No stop codons until necessary so that a whole reading frame is  created

- Regulatory region upstream from primer  

o ________-box

o Binding sites – often have conserved genetic DNA sequences

Can also compare to annotated sequences in databases:  

- ________________searches can help identify sequences using unknown  stretch of DNA or amino acid sequences using the NCBI database to  find the closest match

o Query data base with stretch of DNA or amino acid sequence and find the “best” match that exists in data base… can infer the  likely chromosome or origin or function of the portion of DNA that is included in the search

Public databases

• National Center for Biotechnology Information (NCBI)

- Contains DNA sequences, annotated DNA sequences from whole  organisms and model organisms  

• Human chromosome maps, including known polymorphisms - Blue text – protein coding genes  

• Role of bioinformatics is not just to align and assemble existing short reads  of DNA sequences to generate whole genomes, but also in comparison of  whole genomes across individuals within same species or across different  species

BLAST Searches

• Query sequence –> stretch of sequence in which we generated and don’t  know its chromosomal origin or its function; going to compare it to its closest match in the blast search

- Best match highlighted in blue

- FIG 18-3

• Mouse sequence already annotated

- look for the matching portion

 an insulin receptor gene on chromosome 8

- Mouse = subject

• Rat (query) sequence was from an unknown genomic location • Now those identity and function can now be inferred based off of mouse  sequencing  

- Rat = query  

- Can infer that the 280 base pair sequence from the rat probably  originated from the same genomic location in the rat genome  - Can also infer that the 280 rat sequence does not contain stop codons  where they don’t belong if we expect this to be a protein coding  sequence…

o or if we were able to identify an AUG start codon or even an  upstream regulatory region  

Comparative Genomics

• covered so far

- how a whole genome sequence is produced

o shotgun sequencing – fragmenting, sequencing, and aligning and assembling

o annotate genome by identifying through inference what the  identity and function of DNA sequence is  

o once fully genome’d, compare it to the whole genome of a  second, third, or fourth species

• Compare different organisms and their genomes whether they are closely  related or not

- considerable similarity among organisms you would not have thought  • Helps scientists understand structure and function and expression of  human mutant diseases  

o comparative genomics provides a way to identify useful model  organisms that can help in experimental crosses that may inform us how to treat human genetic diseases

• Important evolutionary insights from comparative genomics - direct comparison between genome sizes between different classes of  organisms  

o prokaryotes, eukaryotes, viruses

- Viruses – genome sizes small relatively; moderately strong correlation  between genome size and number of protein coding genes - Prokaryotes – strong linear relationship between genome size and  number of protein coding genes in the genome

- Eukaryotes – diffuse relationship (but still correlated) but not that tight; some large genomes with relatively few protein coding genes (large  portion of genome is comprised of non-coding DNA)

Lecture 13

9/23

From Article:  

• Adaptive change evolved rapidly (w/in 6,000 years)

• Predation rate increases for white mice in dark backgrounds and dark mice  in lighter backgrounds  

- Suggests that phenotype is adaptive and depends on the context of  the environment in which the mice lives

• Single DNA sequence change in protein coding portion – amino acid  replacement

- Different protein variant that functions differently

- How they figured out allelic variant with white coat colored is derived –  A SNP is 1 nucleotide along a stretch of protein coding region… this is  the only variant of focus that exists in natural populations…derived or  inherited?

o Used phylogenic approach  

o Derived because most mice in lineage is dark, only every now  and then a white phenotype will arise

o Tells us what the gene does

• Describes details to controlled cross and gives indication of what the  results mean

- Dominance/recessiveness

- 1 gene or more than 1

- gene is pleiotropic and RR is dark and CC is light so RC is intermediate  - copy of normal allele and copy of mutant allele

o look at figure 4

- proportion of chromosomes that carry the allelic variants - mosaic chromosomes – different phenotypes  

o 75 dominant phenotype and 25 recessive – standard Mendelian  assumption

 does not hold in this case (varying phenotypes)

 not always equal fitness

- Table 1 – shows results of the controlled cross focusing on the F2  generation and their phenotypes; genotypes have been sequenced o PVE is main column of interest because it shows the percentage  of variancts explained

• Figure 2 – basically what we get is 2 cell culture lines identical in every way except for which allelic variants of MC2R gene they have

- Alleles were expressed

- Figure shows how the 2 different cell lines perform with respect to  generating melanin

- MC1R has an activator which shows dark pigment and an antagonist  which produces a lighter color  

o Lots of activating protein – normal allele produces lots of melanin while mutant allele does not produce as much  

 Shows that DNA sequence mutation that alters amino acid  is encoded to the protein produced –> respond differently  in cell  

• The light coloration on the Gulf Coast is due to the MC1R allele while the  light coloration of the mice on Atlantic coast is not due to the MC1R allele.  This suggests that there are different ways to obtain that phenotype  depending on the environment of the population  

- White population on Atlantic coast evolved differently – nothing to do  with MC1R allele  

• Does evolutionary change proceed gradually through many small  mutational steps or can adaptation occur via a few large leaps? • Does adaptation generally proceed through dominant or recessive  mutations? Any of the above

• Do beneficial mutations tend ot affect protein function, or ists spatial or  temporal expression?  

• Are same genes and mutations responsible for similar traits in different  poulations or species  

Lecture 14

9/26

** will be exam questions about the Research Experiment we read and studied **

Ch. 19 –> Applications and Ethics of Genetic Engineering and  Biotechnology

Modified Organisms

• ________________: Recombinant technology to add and remove genes in an  organism  

- Ex: making crops such as cotton insect resistant by inserting a bacteria gene into their genome

• ________________: using living organisms to make some product better - Genetically engineered species  

- Number of jobs in this field is constantly increasing  

- Allows rapid growth of a product

Insulin-Producing Bacteria

• ________________: proteins used by one organism, that probably has  recombinant DNA, to create pharmaceutics for another

- Ex: _________: hormone that regulates carbs and fat metabolism - Can make large quantities of insulin in a pure way due to it being the  first human protein that can be made by a genetically engineered  organism  

• Polypeptide chains A and B are bonded together in insulin  - Originally produced A and B separately and then fused them together  • Human genes encode A and B separately and those genes are put next to  the bacterial lacZ gene

- After transcription and translation, a fusion protein is formed o Promotor region: where polymerase is binded  

o lacZ is bacterial gene not relevant to producing insulin

o insulin gene is important and of eukaryotic origin  

o white colonies are the ones that have taken up the plasmid  o fusion protein – string of amino acids encoded by insulin gene A

• Extract the proteins

• chains separated from lacZ

- first step  

• Subunits bond creating a fully functioning insulin molecule  - first widespread application of genetic engineering for human health  - no risk of passing on diseases  

Transgenic Animals

• Bacteria is useful but does not consistently work well

- can’t have successful translation of mRNA transcript generated by  electrolytic gene when happening with prokaryotic machinery  o bacterial machinery for transcription and translation doesn’t  always work well when dealing with mRNA transcripts from  eukaryotic DNA  

• Cannot modify eukaryotic genes directly

- May have reduced activity or be completely inactive

• So eukaryotes are more often used as a “biofactory”

Vaccines from GE organisms  

• Cause our immune systems to make antibodies that will make us resistant  to the disease  

• Normal vaccines are injections of weak or dead pathogen - weak but still living: ________________

- dead: ________________

- Heat kill a virus – virus still has all its surface proteins  

• Can make subunit vaccines with genetically modified organisms

- Contain only surface proteins of the pathogen

- Still stimulates immune response  

- This approach can slow the rate at which a virus evolves  

Subunit Vaccines

• __________: protects against HPV, help prevent cervical cancer • immune protection against HPV as long as the infection has not been  caught yet

• FDA recommends to get this vaccination before adolescence - Political resistence in sense that in some states that STDs aren’t a  problem for young kids

- FDA argues it’s better safe than sorry

- Argument of possible side effects – are birth defects possible?  

GE and Agriculture

• ________________________________– bringing gene from different species into  an organism  

• phenotypic variants  

• Selective breeding: cross organisms that could create a desirable  phenotype in offspring

- Corn –> thousands of generations of selective breeding causes shift of  phenotype  

- Has been going on forever

• Recombinate technology can speed up process of selective breeding  • GE is good to create organisms for insect resistance, herbicide resistance,  and gaining nutritional enhancements  

Herbicide-resistant crops

• Roundup seems like itd be good buuuut…  

- Inhibits photosynthetic pathways so must be put directly on weeds not  the crop  

• Roundup inhibits EPSP synthase production

- Shuts down a gene crucial for photosynthesis  

• Create Roundup-resistant gene from a virus

- Agrobacterium from soil goes into roots of plants and spreads  throughout; genes that reside on chromosome in bacteria are  translocated into the main chromosome of the plant’s nucleus o How EPSP is reintroduced into the plant cell

• Recombinant plasmid to be transported into the crop plant through the soil - Increase amount of EPSP in plants so that when they are sprayed with  Roundup the crops don’t die because they have more of those EPSP

- Problem: Roundup is not as harmless as originally thought – female  frogs turned into male frogs in fields sprayed with roundup; lots of  human health problems as well linked to Roundup  

- Aka should not be spraying lots of Roundup anyways

- GE did exactly what it needed to do but the consequences of the  herbicide caused problems

Plant Resistant Crops

• Bacillus thuringiensis (Bt) produces a protein that kills some insect  herbivores when they eat the crop and it’ll form crystals in their stomach - Cotton growers  

- Cause of declines in Monarch butterfly (non-target)?

o Pollen plants produced travel far and wide but the protein is still  on the pollen, killing butterflies due to the GE cotton

o Accidental non-target kill

Lecture 15

9/28/16

Learning Goals

• Describe applications of genetic engineering to agricultural crop plants and livestock  

• Recall how crossing-over frequency relates to physical distance between a  pair of genes  

Ch. 19 –> Applications and Ethics of Genetic Engineering and  Biotechnology

Recap

- Extra copies of EPSP gene introduced into genome of crop plants  - Negative consequences of overuse of Roundup NOT crop GE - Protected 24/7 because they contain a bacterial gene to protect it from insect attack

- Non-target kill of insects that do not try to feed on crop o Monarch butterflies killed off due to pollen that also had the  bacterial gene

Nutritional Enhancements

•In certain parts of the world such as Asia and Africa, people are commonly  vitamin A deficient  

- Malnutrition is a worldwide problem  

• Rice is a commonly eaten food in these countries  

- 3rd world countries have lots of vitamin A deficiencies  

• Golden rice can now contain ß-carotene which is a vitamin A precursor  - ethical consideration: companies that put a lot of money in research  and development want to recoup their expenditures

o farmers need to buy this golden rice specifically from one  company every time  

o paten on genes unethical?  

o Using humans as research subjects for effects of golden rice is  not good  

Transgenic Cows

• Utters easily can obtain the Staphylococcus aureus infection - block ducts

- contaminates milk  

- Also leads to death of cow itself  

• introduce lysostaphin gene from S. simulana, expressed in milk  - Natural enemy of staphylococcus aureus because it produces a protein  detrimental to the growth and production of protein  

• This enzyme breaks down the cell wall, acting as a natural antibiotic in milk to wards off infection  

- Helps in longevity of dairy cows  

Genetic Engineering in the News

• Species – homo sapiens

• Habitat – everywhere

• editing genes in human embryos: using CRISPR/Cas for introducing  mutations that are resistant to HIV

- Shouldn’t really be messing with genomes of humans

- Global leaders got together and agreed to not use this CRISPR  technology for modification of humans  

- Some Chinese researchers defy the ethics and experimented by editing human embryos; adding a mutation that damages a gene linked to  natural HIV resistance  

o Moderate success in the possibility of producing fetuses immune  to HIV

CRISPR-Cas System

• Bacteria create a “database” of viral infections and use it as a form of  prokaryotic immunization  

• database: a set of spacer DNA regions on the bacterial chromosome,  matching parts of viruses genome  

• immunization: targets and modifies those parts of the virus DNA sequences (editing), renders it inviable  

- Releases enzymes that edit DNA of virus that is attacking bacteria,  making that virus ineffective  

- Can specify what kind of editing  

Ch. 7 –> Linkage and Mapping in Eukaryotes

Physical Linkage

• Genes on the same chromosome, if they are close together, may not be  separated during Meiosis I

Basic Expectations of No Linkage

FIG 7-1a

• Genes on different chromsomes may be independently assorted without  crossing over due to random combination of alleles in the gametes  

Basic Expectation: Strong Linkage

FIG 7-1b

• The two genes on the same chromosome are close together so they will be  ________________________________________________

• same combination of alleles as parent

Basic Expectation: Weak Linkage

FIG 7-1c

• if two genes are on the same chromosome and are far apart, there is a  ___________linkage

• will probably be separated during crossing over  

• combination of alleles not seen in the parent

- outside chromatids ________________

Recombination and Mapping

• The further apart two genes are together on a chromosome, the __________  probability they have for crossing over *

** need consider multiple meiotic events; not just 1!**

• measured by how many recombinant gametes are formed - helps determine distance between the two genes as well  

Hypothetical example:

- Pair of homologous chromosomes; same size, same genes,  heterozygous

- Physical distance  

- 8 separate meiotic events – 8 different points of crossing over per  chromosome  

- crossing over effects top portion of chromosome  

• Probability a cross-over point lands over certain genes is based on distance between those genes

- the further apart the genes are, the probability of cross-over points  increases  

Lecture 16

9/30/16

Ch. 7 –> Linkage and Mapping in Eukaryotes

Linkage and Mapping Recap

• physical linkage alters phenotypic ratio

- not 9:3:3:1

• Gametes can be parental, with unchanged chromosomes or recombinant,  when crossing-over occurs

• About a 50/50 ratio between the two  

- unmodified chromosomes

- proportion increases the further apart they are and decreases the  closer they are

** Need consider multiple meiotic events, not just one**

Linkage and Recombination

Following Meiosis, several outcomes possible:

• Only parental gametes

- occur 2 genes are very close together

- a cross-over point in between is unlikely  

• Mostly parental gametes

- 2 genes are fairly close together

- some crossing over occurs, but rare

• 1:1 parental vs. recombinant gametes:

- 2 genes are far apart, always decoupled by crossing-over

Mapping

• How far apart two genes are determines how often they’ll be affected by  crossing over

- can measure the map distance in map units to predict frequency of  crossing over  

o 1 mu = 1% recombination between a pair of genes

Single Crossovers

During prophase I:

• crossing over is limited to one pair of non-sister chromatids and locations  are random  

- When A and B X a and b are close to each other, segments of two non sister chromatids are exchanged, but the linkage between the A and B  alleles and a and b alleles is unchanged  

- Could cross over at any point along the chromosomes

Multiple Crossovers

During prophase I:

• multiple crossovers are possible

- Can occur concurrently during same round of meiosis  

o Aka simultaneously at 2 locations at the same time

• must follow inheritance of minimum 3 different genes and their alleles • 2 independent and simultaneous crossing over events

- Takes probability of each event and puts it together –> smaller  probability  

Mapping Accuracy

• crossing over frequency is positively correlated to crossing over location  • So many deviations can arise and look like original state even if its  recombinant

- AB X ab –> non-detectable recombinant bc it essentially restored  original state

- Assume they are close together but in reality they are quite far apart  because double cross-over did occur

o Leads to decrease in accuracy  

• This can cause error because they don’t look like a recombinant but they  are

• Solution: focus on genes that are fairly close instead of further apart  - When distances are small, the theoretical v. actual relationship are  pretty spot on, but when distances are larger, the relationship can be  skewed  

Lecture 17

10/03/16

Ch. 8 –> Genetic Analysis and Mapping in Bacteria and Phage

Bacteria and Phage

Bacteria = Prokaryotes 

• unicellular

• 1 usually circular haploid chromosome

- May or may not have a plasmid  

Bacteriophages = Viruses 

• infect prokaryotic and eukaryotic cells

- Original source of Restriction Enzymes (used as a defense mechanism)  - Genetics in the news – CRSPR gene editing system isolated from  bacteria – natural mechanism for protecting from viruses  

Bacteria, Mutations, and Growth

•DNA mutation characteristics:

- Spontaneous  

- Constantly arise

o High rates of mutation in human genomes in places like  Chernobyl due to high radiation, etc.  

• How populations get new genetic variances  

- Why mutations important in evolutionary process – what natural  selection acts on

- Ex: beach mice  

- Usually detrimental to the individual  

• because the organisms are haploid, phenotype clearly expresses the  genotype

- No dominant allele overriding recessive

• __________________________– where nutrients can be manipulated for  experiments  

- Important: minimal medium has just bare essentials

- Enriched media – has lots of nutrients  

Synthesizing Compounds

Different strains of same species of bacteria:

• ________________

- Can biosynthesize its necessary amino acids  

- Just needs carbon and ions

• ________________

- Have sustained these mutations

- Growth medium must be spiked so that they get the right nutrients  they need

• Both prototrophs and auxotrophs can grow on enriched media • prototrophs can grow on minimal media

- Enriched media spiked with whatever amino acids are needed

Recombination via Conjugation

All inform us about gene order

• ________________– one bacterial cell passes on its genetic makeup to  another bacterial cell to allow genetic diversity.  

• Followed by ________________________________

- One chromosomes cuts out a set of its genetic material and puts in a  new set  

- One remains unmodified whereas in crossing-over, both homologous  chromosomes are modified  

• altered genotypes and phenotypes

- Changing genetic makeup of chromosome in recipient cell  - The giving cell shares only one of the two complimentary strands so it  does not lose any information when sharing with recipient cell

Lederberg and Tatum’s Experiment

• Strains A & B = auxotrophs 

• + functions like it should  

• - non functional  

• Two different strains, both auxotrophic but in their own ways • Mixture of strain A and B  

- Minimal media  

- Strain A or B alone –> no growth which means no prototrophs - No growth in Strain A or B

o Gain of function mutation –> ruled out

o Assume it must be something about the mixture of the 2  autotrophic strain to create the prototrophic strain  

 Did they need to come into contact with each other?

Q: Why are gain-of-function mutations unlikely?

- Mutations strike randomly, should also be seen in controls - Need a particular and complex combo of gains (product law)

Davis’s Experiment

• Release of DNA directly into the media does not occur because bacterial  cells must come in contact with each other

- Not enough for the two strains to be in close proximation –> must have contact

Conjugation, Donors, and Recipients

• DNA is transferred in one direction into the other strain  

• F+ bacteria = donor of genetic material using an F-factor • F- = recipient

- become recombinants

• Physical contact between the cells needs to take place

- donor cell forms an extension that attaches to recipient (F pilus)  - Recombination and sharing of material to take place  

F-factor Transmission

1) Conjugation between the cells  

2) Endonucleases nick one strand off of the F-factor and it is transferred  from that cell to the recipient cell through the pilus  

- F-factor aids the transfer of DNA from F+ cell to F- cell.  - Outside strand that was nicked is unwinding a single strand of the DNA and moving into recipient cell  

3) The DNA complement is synthesized for both single strand in each cell  - Occurs concurrently in donor and recipient cells  

Following conjugation:  

• F-factor copy is always passed on and one always stays in the original F+ • Recipient (F-) cells become donors (F+) because they have gained that F  factor

The F-factor is a Plasmid

• Plasmid: an autonomous unit of inheritance of circular double stranded  DNA  

- Usually involved in resistance  

• Name follows function:

- F-factor –> ________________

- R plasmid –> ________________

- Col plasmid –> ________________protein

Becoming a Hfr Strain…

• F-factor enters the cell and integrates its DNA into the cell’s, converting the host cell into an Hfr cell

- Hfr (high frequency recombinant cell)  

• The new Hfr cell conjugates with the original cell  

• RE cuts the F-factor and the cut site creates origin

- Origin – location where RE nicks DNA (binds to DNA then gets cut  - Cut out internal portion and clean the rest

• Transfer the single strand through the pilus  

• DNA replication occurs in both cells

- Conjugation did not happen lnon

- F- cell

• Conjugation interrupted before complete transfer

• A & B genes make it in to recombine with homologous region of host  chromosome

• F-factor did not fully make it so the cell remains an F-

- Changes in allelic composition of recombinant  

- Tells us ordering of genes as well as physical distance btetween them  o Distance –> amt of time it takes to get new allelic variant  

Lecture 18

10/05/16

Ch. 8 –> Genetic Analysis and Mapping in Bacteria and Phage

Conjugation: Recap

• Mutant (Hfr) strain integrated the F-factor blasmid into main bacterial  chromosome

- once have strains, allow them to come in contact with F- cells and  conjugation takes place

- FIG 8-8

Transformation

• alternative approach to getting bacteria with recombinant chromsomes - haploid bacterial cell with main bacterial chromsome

- complement bacterial cell has receptor on surface  

• integrate foreign DNA into a bacterial chromsome

• double stranded DNA from outside of the cell enters the bacterial cell  through a receptor site

- receptor site: where DNA from bacteria can be taken up  • One strand enters the cell, the other strand is digested

• Foreign DNA pairs with its homolog in the bacterial host chromosome  through excision and replacement  

- ligase “glues” new into place

• ________________forms from the two different DNA strands  - does not last long

• Lots of mutations from transformed DNA  

• after replication there will be 1 recombinant chromosome and 1 original  due to the one single strand replacing part of the plasmid

• After cell division:  

- 1 transformed cell

o TGC with ACG

 Recombinant cell

- 1 untransformed cell

o TAC with ATG

Transformation and Mapping

• short fragments of transformed DNA causes neighboring genes to be  transformed together if they are close enough  

- Only relatively short pieces can be taken up and the length of the  heteroduplex is quite constrained

- Can start to identify genes that are close together on a chromosome  • Two independent and simultaneous transformation events are rare (product law)

• Compare transformed v. untransformed cells to map linkage groups  First…azi –> ton –> lac –> gal… (last

Those close to each other co-transform a lot (ex: azi –> ton) Those further apart rarely do (ex: azi & lac)

Those furthest apart NEVER do (ex: azi & gal)

** time is our measure for how far apart genes are in Conjugation ** - No heteroduplex

- Single stranded DNA moves into recipient cell

** in transformation, frequency with which they co-transform is our measure  distance**

- Heteroduplex

- Double stranded DNA moves through receptor site  

T4 Bacteriophage (Transduction)

• A bacteriophage is a virus that infects bacteria with it’s genetic makeup by  binding to host cell to insert its DNA into

T4 Lytic Cycle

• bacteriophage attaches via a receptor-protein binding site  - Recognizes its appropriate host… likes to attack E. coli bacteria • DNA crosses membrane and replicates inside host cell

- Squats and injects DNA into cytoplasm of bacteria cell - Cascade of changes in bacterial cell

o Bacteriophage chops up main bacterial chromosome

o Uses cellular machinery to replicate its own DNA within the cell  

• virus molecules recombined

• Lysozyme breaks open host cell and new bacteriophages and viruses exit - Must reassemble the newly formed bacteriophages within cells - These new bacteriophages lyse and break out of host cell

Transduction

• Genetic recombination in bacteria is mediated by the phage that infected  them because some recombined genes leave and others with original  bacteriophage DNA also leave

• During the lytic cycle, defective phage package bacterial DNA in their  head, then exit via lysis  

- Defective phage: head capsules packaged with fragmented bacteria  chromosomes instead of DNA from virus

• The phages find another host to infect with bacterial DNA and the host  gains new genes  

- Small proportion of defective viruses are now able to find new host  cells

Transduction and Mapping

• a few genes contain phage and bacterial DNA

• several genes contain only bacterial DNA inside the phage head  

Standard principles apply:  

• genes that are located together are co-transduced

• Distant genes don’t  

Ch. 9 –> DNA Structure and Analysis

Genetic Material

Storage and Replication 

• The whole genome is contained within a haploid set of cells which can be  copied during the cell cycle  

Expression 

• on and off switches help certain genes to be turned on at any given time  

Evidence for DNA (not protein)

• Avery et al. experimented with 2 strains of staphylococcus) bacteria - IIR (non virulent)  

- IIIS (virulent)

• Noticed that IIR became virulent when mixed with heat-killed IIIS

- Suggests transformation occurs  

FIG 9-2

More Evidence

• Hershey and Chase experiment:

- Bacteria (E. coli) and phage (T2)

• Knew that phage components would enter a host cell and reproduce  • Used radioisotopes:

-32P: E coli DNA

-35S: protein  

Q: Why not stop after eliminating protein?

A: Could have been something other than DNA that the experimenters did  not think about

- Experimental procedure also could have been messed up and allowed  transformation in all of them

Lecture 19

10/7/16

Ch. 9 –> Structure and Analysis

More Evidence (DNA, Not Protein)

• Protein contains sulfur but not phosphorus

- DNA contains phosphorus but not sulfur

OVERVIEW: experiment involved a focus on viruses on whether they had  protein or DNA for genetic material

- Start off with 2 experimental lines  

o Viruses placed in jar of E. coli host cells living in medium of  radioactive phosphorus

o Viruses placed in jar of E. coli host cells living in medium of  radioactive sulfur

- Progeny phages become labeled and infect unlabeled bacteria  - Separate viruses from bacteria and see what it was that made it into  the bacterial host cell

o Whatever the genetic material is, is the stuff injected into the  bacterial host cell

- Now have differentially labeled viruses that will interact with host cells  that are unlabeled

- Labeled phages infect unlabeled bacteria  

- Centrifuge to get rid of virus “ghosts” still stuck to outside of cell (like a cicada lol)

o Infected bacteria are labeled with 32P

o Phage “ghosts” are labeled with s5S

o Infected bacteria are unlabeled  

Indirect Evidence in Eukaryotes

• Prediction 1 suggests that DNA should be doubled in diploid cells compared to haploid

• Prediction 2 suggests that DNA should be absorbed as UV (the mutation  inducer) is activated

- Both predictions were validated  

• Also observed…

- Know that haploid gametes produced from organisms must have half  the genetic material than parent cells

o No difference between amount of protein, but amount of DNA is  halved  

 Consistent with idea that it is DNA is the genetic material - Also know that genetic material can be mutated with exposed to UV  radiation

o Whichever of the two is the true genetic material should absorb  the most UV light  

 DNA absorbed UV light most efficiently with more  

mutations produced  

Direct Evidence in Eukaryotes

• Recombinant techniques:  

- After DNA insertion, eukaryotic genes were produced by bacteria - Can also occur with transgenic animals

RNA (NOT DNA) in Some Viruses

• Exception to the Rule  

- RNA injected into host cell is then converted to DNA via Reverse  Transcription  

- HIV is a retrovirus so virus itself uses RNA not DNA  

• reverse transcription helps retroviruses convert their RNA back into DNA  once they are in the host cell.  

• viral RNA can be made now through the host cell’s genome  

DNA Molecules

• Nucleotides – basic unit of DNA

- Nitrogenous base

- Sugar

- phosphate

• Bases: purines include A and G; pyrimidines include C and T/U - RNA replaces T with U (uracil)

- One other difference: DNA tends to be double stranded whereas RNA is single stranded  

DNA Structure

• Chargaff et al. found repeated patterns of base pairs:  

- Amount of A = amount of T  

- C + G ≠ A + T

o AKA not necessarily equal proportion between C&G and A&T  C goes with G and A goes with T  

• Franklin’s x-ray diffraction – dna double helix the lead to observation of  overall arrangement (even though she didn’t recognize it at the time)

The DNA Model

• Watson and Crick formulated a model to account for earlier observations  (published and some unpublished)

• Proposed that base complementary & pairing provides a mechanism for  DNA replication  

- Double hydrogen bond holding A and T together

- Triple hydrogen bond holding C and G together

• Recognized that this could help us realize how DNA is replicated

RNA Structure and Function

• Usually, single-stranded, except when folded back on itself, or in some  animal viruses

- Loops back on itself and hydrogen bonds between complementary  bases can occur causing it to look doubled (stems) with single parts  (loops)  

o Stems and loops  

DNA codes for main 3 types of RNA:  

- rRNA –> ribosomal RNA

- mRNA –> messenger RNA that helps expression

- tRNA –> transcription RNA that carries amino acids to the ribosome to  make proteins  

Ch. 10 –> DNA Replication and Recombination  

DNA Replication

• Main function of DNA is storage and replication  

• replication is important for keeping genetic continuity  

- when replication happens, either of the 2 strands both become a  template

- synthesis of a complementary strand happens with lots of accuracy via DNA polymerase

• high accuracy is necessary so that not many errors occur  - DNA polymerase

Mode of DNA Replication  

• Semiconservative: each single strand from the double stranded helix  serves as a template  

- New helix = 50/50 mix of new and old DNA

Other Possible Replication Modes  

• Conservative: after replicating the two original strands will pair back up to  their original structure.  

- 2 new strands separate and associate with one another o original template goes back together  

• Dispersive: helix chopped up and reorganized with every strand being a  mixture of the old and new DNA

- each of the 2 DNA strands serve as a template for a new  complementary strand for each

- chopped up and arranged into mosaic like structure helix - along the length of a new single strand you have old + new mosaic like double helix

Meselson-Stahl Experiment

• 3 different modes lead to 3 different helix compositions

- realized that the 3 alternatives make different predictions  • DNA tests utilize the nitrogen component with different isotopes;  -15N (heavy)  

-14N (light)  

- centrifugation  

o incorporates nitrogen and these versions were available to them  o after centrifuging, you can distinguish between the two  • Grew the E. coli on 15N media

• Transferred it to 14N media  

• Isolated DNA from cells each generation

- each generation of a bacteria, they looked at proportion of heavy vs.  light that had been incorporated into the chromosome  

o all of DNA incorporates heavy nitrogen and falls into bottom of  tube  

if semiconservative – all bacteria would have DNA that were one light  nitrogen and one heavy nitrogen together  

- found that it had a different weight and formed a band

- repeated cycle of 1 round of replication of bacteria and centrifuging

Predictions

• After 1 generation – 1 intermediate density DNA band

- semi-conservative

- 2 helixes that are both light and heavy 50/50

• After 2 generations – 2 intermediate and light DNA bands:

• After 1 generation – 2 heavy and light DNA bands

- conservative

o one helix that is heavy and one helix that is light  

• After 2 generations – 2 heavy and light DNA bands

• After 1 generation – intermediate density DNA band

- dispersive  

o mosaic like structure

o 50/50 heavy and light  

• After 2 generations – 1 intermediate DNA band

Lecture 20

10/10/16

Replicaiton Origin and Direction

• _______________________– separates the double-stranded DNA into single  strands at the origin  

• After one initiation even the DNA immediately replicated is called a replicon • In E. coli, replication is bi-directional with a single origin and replicon

For DNA replication to take place

- first separate the strands

o each single strand serves as a template

- complimentary strand attaches  

- “unzip” DNA

DNA Synthesis in Bacteria

________________

- nucleotide base pairs for elongation  

- maintains accuracy/fidelity by an auto-corrective system  - to function it needs dNTPs, a template, and magnesium  

Unwinding the Helix

• Origin

- _________

- repetitive DNA sequence (TATA etc.)

• ________________

- a helicase

- breaks H bonds

- denatures strands

- opens helix

• _______________________

- helicases  

- destabilize strands

What keeps the helix open?  

- ________________________________proteins

• the enzyme DNA gyrase flattens out eh helix that form ahead of the  replication fork via

- Cut and re-paste type of enzyme  

Elongation from a Primer

• DNA polymerase during the extension/elongation step will always bind to  RNA at the origin  

• RNA primers are only about 10-12 base-pairs long  

• RNA primers are made by the enzyme primase

Leading v. Lagging Strands

• One strand is 5’ –> 3’ and its complement is 3’ –> 5’

- Antiparallel

• DNA polymerase III synthesizes from the 5’ end to the 3’ end  • ________________– long and continuous fragments

• ________________– shorter and discontinuous fragments

For the lagging strand:  

• DNA polymerase I

- Cuts out primers

- Replaces with complement bases  

• DNA ligase  

- Glues the fragments together  

For both strands:  

• DNA polymerase III

- Pairs all the missing bases constantly and simultaneously  

3’ ––> 5’ Exonuclease Activity  

• Polymerases can pause what they’re doing during synthesis to reverse and  correct any errors

- Proofreading to increase accuracy  

- checking for polymerases error bc every now and then a mutation will  occur  

DNA Replication in Eukaryotes

Several similarities to Prokaryotes:  

• replication fork forms at the origin as the helix unwinds

• there is a bi-directional synthesis with leading and lagging single strands of the DNA  

• Elongation –> DNA Polymerase  

Also some important differences:

• There is more DNA to be copied and it is packaged within the nucleosome • linear chromosomes; not circular  

- centromere

- telomere  

Multiple Replication Origin  

• yeasts have lots of origins

• mammals have around 1000s  

• Replication “bubbles” each make 2 replication forks  

Initiation of Replicatoin  

• timing of replication needs to be controlled so no more copies are  replicated than needed

- origins control this

• origin recognition complex (ORC) – these proteins tag the origin locations in the G1 stage  

• pre-replication complex – when present, replication occurs - when replication does occur these proteins leave and reassemble at  the next G1

Lecture 10

9/16

Recombinant DNA

- Combining DNA that is from different organisms  

o Do not occur naturally

o Not a product of crossing over

 Ex: gene from fruit fly in bacteria  

• Gene from two different species that would not normally mate to create  mosaic-like chromosomes  

- Can also be used to isolate and copy specific DNA

Recombinant DNA

• ________________: use recognition sites to cut DNA into fragments • ________________: takes up the cut DNA and incorporates it into its own by  sealing the sticky ends

• ________________________________: have the ability to take up the  vectors/plasmids

- Receptor sites – take plasmids up and bring them into cell so that DNA  replication can proceed inside of the competent cell  

Restriction Enzymes (REs)

- Original source from bacteria as a defense mechanism against  ________________.  

• ______ and _______DNA at both strands

- Cuts in zigzag fashion

• The fragments are now called ________________

- Single stranded DNA overhang creates “sticky ends”

Vectors (= plasmids)

• Transports the small DNA fragment into a competent cell  - Helps replicate inserted DNA  

Good vectors… (specific properties for easier cloning) - ________________– region that would be cut by enzymes so that it opens  up to insert DNA fragment of target gene in it

o it is versatile and can be cut by many different enzymes o the region is a functional gene called Lac Z gene and is involved  in breaking down food and changes color of cell when it is  function ing properly

 only works when NO inserted DNA in genes  

 bacterial cell with this plasmid will break down food and  turn blue  

- _________________________– bacteria takes this up and will be able to  grow on a medium  

o allows bacteria cells to survive on a growth medium

• Replicate

• Are versatile because they have many cut sites for RE

• Transformed hosts can survive on the growth medium/go on to make more  copies

Sticky ends and DNA ligase

FIG. 17-2

• Start with cleavage with ________________from eukaryotic gene and one  from bacterial plasmid (vector)

• Fragments with complementary Tails

• Annealing allows recombinant DNA molecules to form by complementary  base pairing  

• DNA ligase seals the gap  

A recombinant DNA molecule

• If the DNA molecule took up the DNA successfully, blue colonies will form  - if unsuccessful or broken, white colonies will form  

• host cell needs the ampicillin resistant gene to survive ampicillin treated  growth medium and divide so that colonies will form  

What would happen if one of this vector failed to take up the plasmids at all?  - They would die before they’d even be able to be seen because they  would not be resistant to ampicillin and would die from the growth  medium  

Transforming the Host Cell

• E. coli is a good bacterial host/competent cell  

• competent cells will take up the DNA when electrically or heat shocked  - Heat shocked in lab?  

• yeast cells can be used as host cells to study eukaryotic genes

Polymerase Chain Reaction (PCR)

• ________________________________is the DNA polymerase used - Needs high temps  

- Very accurate  

• 3 steps – ________________________________________________ - Makes lots of copies of the DNA

• primers help set boundaries  

PCR: first cycle

• stress the dsDNA with high temperatures to ________________and split the  strands into 2 ssDNA

- 95°C

- each strand acts as template strand

• Primers ________________ to each ssDNA strand at around 50°C

- Primers are fairly short and polar

o 3’ and 5’ ends

o what TAQ polymerase adds base pairs to  

• Taq adds base pairs to the 3’ of the primer (5’ of DNA complementary  strand) (________________)  

• Each cycle doubles the number of templates for the next cycle  - start with 2 strands and go through 8 cycles  

o 28 = 256 strands

 exponential increase

Advantages (v. Cloning)

• host cells are not needed

• takes way less time  

• More versatile due to primers because it creates more cut sites for DNA  polymerase

• Not a lot of DNA is necessary  

Limitations (v. cloning)  

• primers need some baseline info about the organism’s DNA  • Contamination is easy  

- Can accidentally clone DNA from a different source; need to be  cautious you are not amplifying your own DNA

Lecture 11

9/19

Cloning: recap

• cloning isn’t always perfect, sometimes the bacteria fails to take up the  inserted target DNA

- If something goes horribly wrong – all those bacterial cells will fail to  grow because they lack the gene that makes them resistant to  ampicillin  

Steps are involved in PCR and in correct order:  

- Denature –> annealing –> extension

More applications…  

• organism identification for illegal activities such as whale meat sales to  food industry  

• Chromosome walking  

- Essentially start at a particular place on a chromosome and extend  outward; generating an entire chromosome sequence  

• finding DNA mutations like when you use PCR

Following cloning or PCR

• ________________________________

- Many different restriction enzymes to cut specific DNA into desired  fragments

- separate fragments using electrophoresis and other methods • Now can find out information from the cut sites

- how many cut sites

- order of cutting

- distance between the cutting

• restriction fragment length polymorphism (RFLPs) helps us identify DNA  variation

Electrophoresis

• Technique for separating DNA fragments according to differences in size  using a gel to act as a viscous matrix as well as an electric charge to pass  through this gel

- Send electric current from negative –> positive end; DNA migrates  towards positive end (Anode)

- Bands with longer fragments will take more time to move through the  gel

- Bands with shorter fragments will take less time  

Medical Diagnosis  

• RFLP analysis can help find disease-associated alleles in a genome  • Ex: ________________l

- A substitution mutation causes a different amino acid sequence which  leads to a ß-globin protein and  

- Homozygous: detrimental; heterozygous: effects show later  • A change in restriction enzyme recognition sites cause a new banding  pattern which can be used for diagnosing the disease the pattern represents  

Pharmacogenomics

• A lot of medications have common and/or fatal side-effects because  medication is variable to work for everyone…usually on works for about 60%  of the population

• Pharmacogenomics: selecting drugs and dosage based on the individual’s  genetic makeup  

- Individuals with same disease metabolize 6 MP differently  - More dependable than trial and error

- Differences due to genetic make-up (TPMT genotype)

- Genetic assay allows customized treatment

Complimentary DNA (cDNA)

• cDNA: only the DNA that codes for some specific protein  1. Extract mRNA

2. Convert mRNA back into its complimentary DNA using Reverse  transcription PCR

Reverse Transcription PCR

• In Reverse Transcription, mature mRNA is what we use to make templates  for coding  

• Oligo-DT primer will anneal to the 3’ end of the double stranded molecule • Reverse transcriptase enzyme makes complementary DNA by extended  from the Oligo-DT primer

• a hybrid molecule of both mRNA and DNA is created so RNAse H enzyme  will digest that mRNA strand

• DNA polymerase will be used by any of the remaining mRNA  • the now double-strand of DNA acts as a snapshot of all the coding DNA in  the genome

DNA Sequencing

• How to determine how genes are organized by nucleotides  - Help determine genetic differences between organisms • Used to only be done by chain termination sequencing, or the Sanger  method.  

Chain Termination Sequencer  

• lots of colored peaks, one for each nucleotide, from a chromatogram  

• reads roughly 800 bp at a time

• a slow and expensive method compared to those newer methods

Lecture 12

9/21

“Next-generation” Sequencers

• ________________________________

- Faster and cheaper

o Reads around 150 bp

• ________________–

- Also faster and cheaper and reads around 400 bp per read.  ** relevant to align and assemble nucleotide sequences**

Ch. 18

Genomics and Bioinformatics

New-ish fields

** Review genomics, proteomics, and bioinformatics  

Genomics

• The study of the complete genetic information of small creatures that live  in the depths of the earth, who guard buried treasure

Karyotype is highly variable

Organism

Diploid number (2n)

African Wild Dog 

78

Badger

32

Carp

104

Adders-Tongue

1262 (!)

Cotton

52

Organismal “complexity”

• Organsimal complexity is not proportional to the  

________________________________________________ or to ________________size  (absolute number of base pairs)

- Genome size – total amount of DNA (base pairs) within a haploid  genome  

Genome size also variable  

Organism

Billion bp (1000 million)

African Wild Dog 

2.66

Snapping Shrimp 

15.45

Carp

1.61

Poison Dart frog

8.70

Hugh-man

(human)

3.03

relate to absolute size of genome?

How does number of chromosomes  

- Size of African wild dog genome is quite larger than carps’ - Chromosomes are of different size (African Wild Dog’s are larger than  carps)  

- Frogs have a very large genome size compared to humans

Sequencing a Genome

• ________________– complete __________ set of all the DNA in a cell - Not consistent size across organsism  

- Viruses have the smallest genomes, prokaryotes have median size,  and eukaryotes usually have the largest genomes  

• DNA sequencing  

- “shotgun” sequencing

o great for whole genome sequencing

 Fragment the genome using restriction enzymes

 Sequence the fragments

 Assemble/order the pieces using some sort of  

electrophoresis  

Genomic Libraries

• Basically a whole genome that has been fragmented that can be inserted  and stored inside of vectors

• Does not involve PCR  

• Just get one copy of each sequence using the fewest number of fragments - BAC or YAC clones

Shotgun Sequencing Technique

• Fragment the genome

- Use specific REs one at a time  

• Get combinations of fragments  

• Computationally sequence the fragments

• End up assembling a whole chromosome sequence

Assembling “contigs”

• ________________– stretch of DNA sequences within one gene and across  adjacent genes that overlap

HP Computing (align, assemble)

• de novo alignment and assembly

- Not a lot of baseline data  

• Resequencing  

- Some baseline information  

Diploid Genomes

• heterozygous genotypes are possible so it may be necessary to align the  gene variants  

- Allele 1 v. Allele 2

DNA Sequence Alignment

• Scenario 1 – the reads do not start in same place

- Align sequences to see a match-up

- Mismatch between 2 nucleotides – point mutation/DNA sequence  substitution

- They are not identical  

Gene Pools

• ________________– a gene pool can contain many allelic variants  • Might have to align the different variants of a gene

- Look out for different kinds of polymorphisms  

DNA Sequence Alignment

• Scenario 2 – many individuals and probably many alleles - Align sequences to see a match-up

o At least 5 different alleles  

 Take multiple sequence alignment and count the number of different sequences that exist

• Bioinformatics allows us to figure all of this quickly  

Annotating a Genome

• Need to identify genes with names and locations to interpret

Protein-coding genes have some hallmarks:

- Start codons for mRNA include AUG and for DNA = TAC o T pairs with A, A pairs with U, and C pairs with G <– in RNA - No stop codons until necessary so that a whole reading frame is  created

- Regulatory region upstream from primer  

o ________-box

o Binding sites – often have conserved genetic DNA sequences

Can also compare to annotated sequences in databases:  

- ________________searches can help identify sequences using unknown  stretch of DNA or amino acid sequences using the NCBI database to  find the closest match

o Query data base with stretch of DNA or amino acid sequence and find the “best” match that exists in data base… can infer the  likely chromosome or origin or function of the portion of DNA that is included in the search

Public databases

• National Center for Biotechnology Information (NCBI)

- Contains DNA sequences, annotated DNA sequences from whole  organisms and model organisms  

• Human chromosome maps, including known polymorphisms - Blue text – protein coding genes  

• Role of bioinformatics is not just to align and assemble existing short reads  of DNA sequences to generate whole genomes, but also in comparison of  whole genomes across individuals within same species or across different  species

BLAST Searches

• Query sequence –> stretch of sequence in which we generated and don’t  know its chromosomal origin or its function; going to compare it to its closest match in the blast search

- Best match highlighted in blue

- FIG 18-3

• Mouse sequence already annotated

- look for the matching portion

 an insulin receptor gene on chromosome 8

- Mouse = subject

• Rat (query) sequence was from an unknown genomic location • Now those identity and function can now be inferred based off of mouse  sequencing  

- Rat = query  

- Can infer that the 280 base pair sequence from the rat probably  originated from the same genomic location in the rat genome  - Can also infer that the 280 rat sequence does not contain stop codons  where they don’t belong if we expect this to be a protein coding  sequence…

o or if we were able to identify an AUG start codon or even an  upstream regulatory region  

Comparative Genomics

• covered so far

- how a whole genome sequence is produced

o shotgun sequencing – fragmenting, sequencing, and aligning and assembling

o annotate genome by identifying through inference what the  identity and function of DNA sequence is  

o once fully genome’d, compare it to the whole genome of a  second, third, or fourth species

• Compare different organisms and their genomes whether they are closely  related or not

- considerable similarity among organisms you would not have thought  • Helps scientists understand structure and function and expression of  human mutant diseases  

o comparative genomics provides a way to identify useful model  organisms that can help in experimental crosses that may inform us how to treat human genetic diseases

• Important evolutionary insights from comparative genomics - direct comparison between genome sizes between different classes of  organisms  

o prokaryotes, eukaryotes, viruses

- Viruses – genome sizes small relatively; moderately strong correlation  between genome size and number of protein coding genes - Prokaryotes – strong linear relationship between genome size and  number of protein coding genes in the genome

- Eukaryotes – diffuse relationship (but still correlated) but not that tight; some large genomes with relatively few protein coding genes (large  portion of genome is comprised of non-coding DNA)

Lecture 13

9/23

From Article:  

• Adaptive change evolved rapidly (w/in 6,000 years)

• Predation rate increases for white mice in dark backgrounds and dark mice  in lighter backgrounds  

- Suggests that phenotype is adaptive and depends on the context of  the environment in which the mice lives

• Single DNA sequence change in protein coding portion – amino acid  replacement

- Different protein variant that functions differently

- How they figured out allelic variant with white coat colored is derived –  A SNP is 1 nucleotide along a stretch of protein coding region… this is  the only variant of focus that exists in natural populations…derived or  inherited?

o Used phylogenic approach  

o Derived because most mice in lineage is dark, only every now  and then a white phenotype will arise

o Tells us what the gene does

• Describes details to controlled cross and gives indication of what the  results mean

- Dominance/recessiveness

- 1 gene or more than 1

- gene is pleiotropic and RR is dark and CC is light so RC is intermediate  - copy of normal allele and copy of mutant allele

o look at figure 4

- proportion of chromosomes that carry the allelic variants - mosaic chromosomes – different phenotypes  

o 75 dominant phenotype and 25 recessive – standard Mendelian  assumption

 does not hold in this case (varying phenotypes)

 not always equal fitness

- Table 1 – shows results of the controlled cross focusing on the F2  generation and their phenotypes; genotypes have been sequenced o PVE is main column of interest because it shows the percentage  of variancts explained

• Figure 2 – basically what we get is 2 cell culture lines identical in every way except for which allelic variants of MC2R gene they have

- Alleles were expressed

- Figure shows how the 2 different cell lines perform with respect to  generating melanin

- MC1R has an activator which shows dark pigment and an antagonist  which produces a lighter color  

o Lots of activating protein – normal allele produces lots of melanin while mutant allele does not produce as much  

 Shows that DNA sequence mutation that alters amino acid  is encoded to the protein produced –> respond differently  in cell  

• The light coloration on the Gulf Coast is due to the MC1R allele while the  light coloration of the mice on Atlantic coast is not due to the MC1R allele.  This suggests that there are different ways to obtain that phenotype  depending on the environment of the population  

- White population on Atlantic coast evolved differently – nothing to do  with MC1R allele  

• Does evolutionary change proceed gradually through many small  mutational steps or can adaptation occur via a few large leaps? • Does adaptation generally proceed through dominant or recessive  mutations? Any of the above

• Do beneficial mutations tend ot affect protein function, or ists spatial or  temporal expression?  

• Are same genes and mutations responsible for similar traits in different  poulations or species  

Lecture 14

9/26

** will be exam questions about the Research Experiment we read and studied **

Ch. 19 –> Applications and Ethics of Genetic Engineering and  Biotechnology

Modified Organisms

• ________________: Recombinant technology to add and remove genes in an  organism  

- Ex: making crops such as cotton insect resistant by inserting a bacteria gene into their genome

• ________________: using living organisms to make some product better - Genetically engineered species  

- Number of jobs in this field is constantly increasing  

- Allows rapid growth of a product

Insulin-Producing Bacteria

• ________________: proteins used by one organism, that probably has  recombinant DNA, to create pharmaceutics for another

- Ex: _________: hormone that regulates carbs and fat metabolism - Can make large quantities of insulin in a pure way due to it being the  first human protein that can be made by a genetically engineered  organism  

• Polypeptide chains A and B are bonded together in insulin  - Originally produced A and B separately and then fused them together  • Human genes encode A and B separately and those genes are put next to  the bacterial lacZ gene

- After transcription and translation, a fusion protein is formed o Promotor region: where polymerase is binded  

o lacZ is bacterial gene not relevant to producing insulin

o insulin gene is important and of eukaryotic origin  

o white colonies are the ones that have taken up the plasmid  o fusion protein – string of amino acids encoded by insulin gene A

• Extract the proteins

• chains separated from lacZ

- first step  

• Subunits bond creating a fully functioning insulin molecule  - first widespread application of genetic engineering for human health  - no risk of passing on diseases  

Transgenic Animals

• Bacteria is useful but does not consistently work well

- can’t have successful translation of mRNA transcript generated by  electrolytic gene when happening with prokaryotic machinery  o bacterial machinery for transcription and translation doesn’t  always work well when dealing with mRNA transcripts from  eukaryotic DNA  

• Cannot modify eukaryotic genes directly

- May have reduced activity or be completely inactive

• So eukaryotes are more often used as a “biofactory”

Vaccines from GE organisms  

• Cause our immune systems to make antibodies that will make us resistant  to the disease  

• Normal vaccines are injections of weak or dead pathogen - weak but still living: ________________

- dead: ________________

- Heat kill a virus – virus still has all its surface proteins  

• Can make subunit vaccines with genetically modified organisms

- Contain only surface proteins of the pathogen

- Still stimulates immune response  

- This approach can slow the rate at which a virus evolves  

Subunit Vaccines

• __________: protects against HPV, help prevent cervical cancer • immune protection against HPV as long as the infection has not been  caught yet

• FDA recommends to get this vaccination before adolescence - Political resistence in sense that in some states that STDs aren’t a  problem for young kids

- FDA argues it’s better safe than sorry

- Argument of possible side effects – are birth defects possible?  

GE and Agriculture

• ________________________________– bringing gene from different species into  an organism  

• phenotypic variants  

• Selective breeding: cross organisms that could create a desirable  phenotype in offspring

- Corn –> thousands of generations of selective breeding causes shift of  phenotype  

- Has been going on forever

• Recombinate technology can speed up process of selective breeding  • GE is good to create organisms for insect resistance, herbicide resistance,  and gaining nutritional enhancements  

Herbicide-resistant crops

• Roundup seems like itd be good buuuut…  

- Inhibits photosynthetic pathways so must be put directly on weeds not  the crop  

• Roundup inhibits EPSP synthase production

- Shuts down a gene crucial for photosynthesis  

• Create Roundup-resistant gene from a virus

- Agrobacterium from soil goes into roots of plants and spreads  throughout; genes that reside on chromosome in bacteria are  translocated into the main chromosome of the plant’s nucleus o How EPSP is reintroduced into the plant cell

• Recombinant plasmid to be transported into the crop plant through the soil - Increase amount of EPSP in plants so that when they are sprayed with  Roundup the crops don’t die because they have more of those EPSP

- Problem: Roundup is not as harmless as originally thought – female  frogs turned into male frogs in fields sprayed with roundup; lots of  human health problems as well linked to Roundup  

- Aka should not be spraying lots of Roundup anyways

- GE did exactly what it needed to do but the consequences of the  herbicide caused problems

Plant Resistant Crops

• Bacillus thuringiensis (Bt) produces a protein that kills some insect  herbivores when they eat the crop and it’ll form crystals in their stomach - Cotton growers  

- Cause of declines in Monarch butterfly (non-target)?

o Pollen plants produced travel far and wide but the protein is still  on the pollen, killing butterflies due to the GE cotton

o Accidental non-target kill

Lecture 15

9/28/16

Learning Goals

• Describe applications of genetic engineering to agricultural crop plants and livestock  

• Recall how crossing-over frequency relates to physical distance between a  pair of genes  

Ch. 19 –> Applications and Ethics of Genetic Engineering and  Biotechnology

Recap

- Extra copies of EPSP gene introduced into genome of crop plants  - Negative consequences of overuse of Roundup NOT crop GE - Protected 24/7 because they contain a bacterial gene to protect it from insect attack

- Non-target kill of insects that do not try to feed on crop o Monarch butterflies killed off due to pollen that also had the  bacterial gene

Nutritional Enhancements

•In certain parts of the world such as Asia and Africa, people are commonly  vitamin A deficient  

- Malnutrition is a worldwide problem  

• Rice is a commonly eaten food in these countries  

- 3rd world countries have lots of vitamin A deficiencies  

• Golden rice can now contain ß-carotene which is a vitamin A precursor  - ethical consideration: companies that put a lot of money in research  and development want to recoup their expenditures

o farmers need to buy this golden rice specifically from one  company every time  

o paten on genes unethical?  

o Using humans as research subjects for effects of golden rice is  not good  

Transgenic Cows

• Utters easily can obtain the Staphylococcus aureus infection - block ducts

- contaminates milk  

- Also leads to death of cow itself  

• introduce lysostaphin gene from S. simulana, expressed in milk  - Natural enemy of staphylococcus aureus because it produces a protein  detrimental to the growth and production of protein  

• This enzyme breaks down the cell wall, acting as a natural antibiotic in milk to wards off infection  

- Helps in longevity of dairy cows  

Genetic Engineering in the News

• Species – homo sapiens

• Habitat – everywhere

• editing genes in human embryos: using CRISPR/Cas for introducing  mutations that are resistant to HIV

- Shouldn’t really be messing with genomes of humans

- Global leaders got together and agreed to not use this CRISPR  technology for modification of humans  

- Some Chinese researchers defy the ethics and experimented by editing human embryos; adding a mutation that damages a gene linked to  natural HIV resistance  

o Moderate success in the possibility of producing fetuses immune  to HIV

CRISPR-Cas System

• Bacteria create a “database” of viral infections and use it as a form of  prokaryotic immunization  

• database: a set of spacer DNA regions on the bacterial chromosome,  matching parts of viruses genome  

• immunization: targets and modifies those parts of the virus DNA sequences (editing), renders it inviable  

- Releases enzymes that edit DNA of virus that is attacking bacteria,  making that virus ineffective  

- Can specify what kind of editing  

Ch. 7 –> Linkage and Mapping in Eukaryotes

Physical Linkage

• Genes on the same chromosome, if they are close together, may not be  separated during Meiosis I

Basic Expectations of No Linkage

FIG 7-1a

• Genes on different chromsomes may be independently assorted without  crossing over due to random combination of alleles in the gametes  

Basic Expectation: Strong Linkage

FIG 7-1b

• The two genes on the same chromosome are close together so they will be  ________________________________________________

• same combination of alleles as parent

Basic Expectation: Weak Linkage

FIG 7-1c

• if two genes are on the same chromosome and are far apart, there is a  ___________linkage

• will probably be separated during crossing over  

• combination of alleles not seen in the parent

- outside chromatids ________________

Recombination and Mapping

• The further apart two genes are together on a chromosome, the __________  probability they have for crossing over *

** need consider multiple meiotic events; not just 1!**

• measured by how many recombinant gametes are formed - helps determine distance between the two genes as well  

Hypothetical example:

- Pair of homologous chromosomes; same size, same genes,  heterozygous

- Physical distance  

- 8 separate meiotic events – 8 different points of crossing over per  chromosome  

- crossing over effects top portion of chromosome  

• Probability a cross-over point lands over certain genes is based on distance between those genes

- the further apart the genes are, the probability of cross-over points  increases  

Lecture 16

9/30/16

Ch. 7 –> Linkage and Mapping in Eukaryotes

Linkage and Mapping Recap

• physical linkage alters phenotypic ratio

- not 9:3:3:1

• Gametes can be parental, with unchanged chromosomes or recombinant,  when crossing-over occurs

• About a 50/50 ratio between the two  

- unmodified chromosomes

- proportion increases the further apart they are and decreases the  closer they are

** Need consider multiple meiotic events, not just one**

Linkage and Recombination

Following Meiosis, several outcomes possible:

• Only parental gametes

- occur 2 genes are very close together

- a cross-over point in between is unlikely  

• Mostly parental gametes

- 2 genes are fairly close together

- some crossing over occurs, but rare

• 1:1 parental vs. recombinant gametes:

- 2 genes are far apart, always decoupled by crossing-over

Mapping

• How far apart two genes are determines how often they’ll be affected by  crossing over

- can measure the map distance in map units to predict frequency of  crossing over  

o 1 mu = 1% recombination between a pair of genes

Single Crossovers

During prophase I:

• crossing over is limited to one pair of non-sister chromatids and locations  are random  

- When A and B X a and b are close to each other, segments of two non sister chromatids are exchanged, but the linkage between the A and B  alleles and a and b alleles is unchanged  

- Could cross over at any point along the chromosomes

Multiple Crossovers

During prophase I:

• multiple crossovers are possible

- Can occur concurrently during same round of meiosis  

o Aka simultaneously at 2 locations at the same time

• must follow inheritance of minimum 3 different genes and their alleles • 2 independent and simultaneous crossing over events

- Takes probability of each event and puts it together –> smaller  probability  

Mapping Accuracy

• crossing over frequency is positively correlated to crossing over location  • So many deviations can arise and look like original state even if its  recombinant

- AB X ab –> non-detectable recombinant bc it essentially restored  original state

- Assume they are close together but in reality they are quite far apart  because double cross-over did occur

o Leads to decrease in accuracy  

• This can cause error because they don’t look like a recombinant but they  are

• Solution: focus on genes that are fairly close instead of further apart  - When distances are small, the theoretical v. actual relationship are  pretty spot on, but when distances are larger, the relationship can be  skewed  

Lecture 17

10/03/16

Ch. 8 –> Genetic Analysis and Mapping in Bacteria and Phage

Bacteria and Phage

Bacteria = Prokaryotes 

• unicellular

• 1 usually circular haploid chromosome

- May or may not have a plasmid  

Bacteriophages = Viruses 

• infect prokaryotic and eukaryotic cells

- Original source of Restriction Enzymes (used as a defense mechanism)  - Genetics in the news – CRSPR gene editing system isolated from  bacteria – natural mechanism for protecting from viruses  

Bacteria, Mutations, and Growth

•DNA mutation characteristics:

- Spontaneous  

- Constantly arise

o High rates of mutation in human genomes in places like  Chernobyl due to high radiation, etc.  

• How populations get new genetic variances  

- Why mutations important in evolutionary process – what natural  selection acts on

- Ex: beach mice  

- Usually detrimental to the individual  

• because the organisms are haploid, phenotype clearly expresses the  genotype

- No dominant allele overriding recessive

• __________________________– where nutrients can be manipulated for  experiments  

- Important: minimal medium has just bare essentials

- Enriched media – has lots of nutrients  

Synthesizing Compounds

Different strains of same species of bacteria:

• ________________

- Can biosynthesize its necessary amino acids  

- Just needs carbon and ions

• ________________

- Have sustained these mutations

- Growth medium must be spiked so that they get the right nutrients  they need

• Both prototrophs and auxotrophs can grow on enriched media • prototrophs can grow on minimal media

- Enriched media spiked with whatever amino acids are needed

Recombination via Conjugation

All inform us about gene order

• ________________– one bacterial cell passes on its genetic makeup to  another bacterial cell to allow genetic diversity.  

• Followed by ________________________________

- One chromosomes cuts out a set of its genetic material and puts in a  new set  

- One remains unmodified whereas in crossing-over, both homologous  chromosomes are modified  

• altered genotypes and phenotypes

- Changing genetic makeup of chromosome in recipient cell  - The giving cell shares only one of the two complimentary strands so it  does not lose any information when sharing with recipient cell

Lederberg and Tatum’s Experiment

• Strains A & B = auxotrophs 

• + functions like it should  

• - non functional  

• Two different strains, both auxotrophic but in their own ways • Mixture of strain A and B  

- Minimal media  

- Strain A or B alone –> no growth which means no prototrophs - No growth in Strain A or B

o Gain of function mutation –> ruled out

o Assume it must be something about the mixture of the 2  autotrophic strain to create the prototrophic strain  

 Did they need to come into contact with each other?

Q: Why are gain-of-function mutations unlikely?

- Mutations strike randomly, should also be seen in controls - Need a particular and complex combo of gains (product law)

Davis’s Experiment

• Release of DNA directly into the media does not occur because bacterial  cells must come in contact with each other

- Not enough for the two strains to be in close proximation –> must have contact

Conjugation, Donors, and Recipients

• DNA is transferred in one direction into the other strain  

• F+ bacteria = donor of genetic material using an F-factor • F- = recipient

- become recombinants

• Physical contact between the cells needs to take place

- donor cell forms an extension that attaches to recipient (F pilus)  - Recombination and sharing of material to take place  

F-factor Transmission

1) Conjugation between the cells  

2) Endonucleases nick one strand off of the F-factor and it is transferred  from that cell to the recipient cell through the pilus  

- F-factor aids the transfer of DNA from F+ cell to F- cell.  - Outside strand that was nicked is unwinding a single strand of the DNA and moving into recipient cell  

3) The DNA complement is synthesized for both single strand in each cell  - Occurs concurrently in donor and recipient cells  

Following conjugation:  

• F-factor copy is always passed on and one always stays in the original F+ • Recipient (F-) cells become donors (F+) because they have gained that F  factor

The F-factor is a Plasmid

• Plasmid: an autonomous unit of inheritance of circular double stranded  DNA  

- Usually involved in resistance  

• Name follows function:

- F-factor –> ________________

- R plasmid –> ________________

- Col plasmid –> ________________protein

Becoming a Hfr Strain…

• F-factor enters the cell and integrates its DNA into the cell’s, converting the host cell into an Hfr cell

- Hfr (high frequency recombinant cell)  

• The new Hfr cell conjugates with the original cell  

• RE cuts the F-factor and the cut site creates origin

- Origin – location where RE nicks DNA (binds to DNA then gets cut  - Cut out internal portion and clean the rest

• Transfer the single strand through the pilus  

• DNA replication occurs in both cells

- Conjugation did not happen lnon

- F- cell

• Conjugation interrupted before complete transfer

• A & B genes make it in to recombine with homologous region of host  chromosome

• F-factor did not fully make it so the cell remains an F-

- Changes in allelic composition of recombinant  

- Tells us ordering of genes as well as physical distance btetween them  o Distance –> amt of time it takes to get new allelic variant  

Lecture 18

10/05/16

Ch. 8 –> Genetic Analysis and Mapping in Bacteria and Phage

Conjugation: Recap

• Mutant (Hfr) strain integrated the F-factor blasmid into main bacterial  chromosome

- once have strains, allow them to come in contact with F- cells and  conjugation takes place

- FIG 8-8

Transformation

• alternative approach to getting bacteria with recombinant chromsomes - haploid bacterial cell with main bacterial chromsome

- complement bacterial cell has receptor on surface  

• integrate foreign DNA into a bacterial chromsome

• double stranded DNA from outside of the cell enters the bacterial cell  through a receptor site

- receptor site: where DNA from bacteria can be taken up  • One strand enters the cell, the other strand is digested

• Foreign DNA pairs with its homolog in the bacterial host chromosome  through excision and replacement  

- ligase “glues” new into place

• ________________forms from the two different DNA strands  - does not last long

• Lots of mutations from transformed DNA  

• after replication there will be 1 recombinant chromosome and 1 original  due to the one single strand replacing part of the plasmid

• After cell division:  

- 1 transformed cell

o TGC with ACG

 Recombinant cell

- 1 untransformed cell

o TAC with ATG

Transformation and Mapping

• short fragments of transformed DNA causes neighboring genes to be  transformed together if they are close enough  

- Only relatively short pieces can be taken up and the length of the  heteroduplex is quite constrained

- Can start to identify genes that are close together on a chromosome  • Two independent and simultaneous transformation events are rare (product law)

• Compare transformed v. untransformed cells to map linkage groups  First…azi –> ton –> lac –> gal… (last

Those close to each other co-transform a lot (ex: azi –> ton) Those further apart rarely do (ex: azi & lac)

Those furthest apart NEVER do (ex: azi & gal)

** time is our measure for how far apart genes are in Conjugation ** - No heteroduplex

- Single stranded DNA moves into recipient cell

** in transformation, frequency with which they co-transform is our measure  distance**

- Heteroduplex

- Double stranded DNA moves through receptor site  

T4 Bacteriophage (Transduction)

• A bacteriophage is a virus that infects bacteria with it’s genetic makeup by  binding to host cell to insert its DNA into

T4 Lytic Cycle

• bacteriophage attaches via a receptor-protein binding site  - Recognizes its appropriate host… likes to attack E. coli bacteria • DNA crosses membrane and replicates inside host cell

- Squats and injects DNA into cytoplasm of bacteria cell - Cascade of changes in bacterial cell

o Bacteriophage chops up main bacterial chromosome

o Uses cellular machinery to replicate its own DNA within the cell  

• virus molecules recombined

• Lysozyme breaks open host cell and new bacteriophages and viruses exit - Must reassemble the newly formed bacteriophages within cells - These new bacteriophages lyse and break out of host cell

Transduction

• Genetic recombination in bacteria is mediated by the phage that infected  them because some recombined genes leave and others with original  bacteriophage DNA also leave

• During the lytic cycle, defective phage package bacterial DNA in their  head, then exit via lysis  

- Defective phage: head capsules packaged with fragmented bacteria  chromosomes instead of DNA from virus

• The phages find another host to infect with bacterial DNA and the host  gains new genes  

- Small proportion of defective viruses are now able to find new host  cells

Transduction and Mapping

• a few genes contain phage and bacterial DNA

• several genes contain only bacterial DNA inside the phage head  

Standard principles apply:  

• genes that are located together are co-transduced

• Distant genes don’t  

Ch. 9 –> DNA Structure and Analysis

Genetic Material

Storage and Replication 

• The whole genome is contained within a haploid set of cells which can be  copied during the cell cycle  

Expression 

• on and off switches help certain genes to be turned on at any given time  

Evidence for DNA (not protein)

• Avery et al. experimented with 2 strains of staphylococcus) bacteria - IIR (non virulent)  

- IIIS (virulent)

• Noticed that IIR became virulent when mixed with heat-killed IIIS

- Suggests transformation occurs  

FIG 9-2

More Evidence

• Hershey and Chase experiment:

- Bacteria (E. coli) and phage (T2)

• Knew that phage components would enter a host cell and reproduce  • Used radioisotopes:

-32P: E coli DNA

-35S: protein  

Q: Why not stop after eliminating protein?

A: Could have been something other than DNA that the experimenters did  not think about

- Experimental procedure also could have been messed up and allowed  transformation in all of them

Lecture 19

10/7/16

Ch. 9 –> Structure and Analysis

More Evidence (DNA, Not Protein)

• Protein contains sulfur but not phosphorus

- DNA contains phosphorus but not sulfur

OVERVIEW: experiment involved a focus on viruses on whether they had  protein or DNA for genetic material

- Start off with 2 experimental lines  

o Viruses placed in jar of E. coli host cells living in medium of  radioactive phosphorus

o Viruses placed in jar of E. coli host cells living in medium of  radioactive sulfur

- Progeny phages become labeled and infect unlabeled bacteria  - Separate viruses from bacteria and see what it was that made it into  the bacterial host cell

o Whatever the genetic material is, is the stuff injected into the  bacterial host cell

- Now have differentially labeled viruses that will interact with host cells  that are unlabeled

- Labeled phages infect unlabeled bacteria  

- Centrifuge to get rid of virus “ghosts” still stuck to outside of cell (like a cicada lol)

o Infected bacteria are labeled with 32P

o Phage “ghosts” are labeled with s5S

o Infected bacteria are unlabeled  

Indirect Evidence in Eukaryotes

• Prediction 1 suggests that DNA should be doubled in diploid cells compared to haploid

• Prediction 2 suggests that DNA should be absorbed as UV (the mutation  inducer) is activated

- Both predictions were validated  

• Also observed…

- Know that haploid gametes produced from organisms must have half  the genetic material than parent cells

o No difference between amount of protein, but amount of DNA is  halved  

 Consistent with idea that it is DNA is the genetic material - Also know that genetic material can be mutated with exposed to UV  radiation

o Whichever of the two is the true genetic material should absorb  the most UV light  

 DNA absorbed UV light most efficiently with more  

mutations produced  

Direct Evidence in Eukaryotes

• Recombinant techniques:  

- After DNA insertion, eukaryotic genes were produced by bacteria - Can also occur with transgenic animals

RNA (NOT DNA) in Some Viruses

• Exception to the Rule  

- RNA injected into host cell is then converted to DNA via Reverse  Transcription  

- HIV is a retrovirus so virus itself uses RNA not DNA  

• reverse transcription helps retroviruses convert their RNA back into DNA  once they are in the host cell.  

• viral RNA can be made now through the host cell’s genome  

DNA Molecules

• Nucleotides – basic unit of DNA

- Nitrogenous base

- Sugar

- phosphate

• Bases: purines include A and G; pyrimidines include C and T/U - RNA replaces T with U (uracil)

- One other difference: DNA tends to be double stranded whereas RNA is single stranded  

DNA Structure

• Chargaff et al. found repeated patterns of base pairs:  

- Amount of A = amount of T  

- C + G ≠ A + T

o AKA not necessarily equal proportion between C&G and A&T  C goes with G and A goes with T  

• Franklin’s x-ray diffraction – dna double helix the lead to observation of  overall arrangement (even though she didn’t recognize it at the time)

The DNA Model

• Watson and Crick formulated a model to account for earlier observations  (published and some unpublished)

• Proposed that base complementary & pairing provides a mechanism for  DNA replication  

- Double hydrogen bond holding A and T together

- Triple hydrogen bond holding C and G together

• Recognized that this could help us realize how DNA is replicated

RNA Structure and Function

• Usually, single-stranded, except when folded back on itself, or in some  animal viruses

- Loops back on itself and hydrogen bonds between complementary  bases can occur causing it to look doubled (stems) with single parts  (loops)  

o Stems and loops  

DNA codes for main 3 types of RNA:  

- rRNA –> ribosomal RNA

- mRNA –> messenger RNA that helps expression

- tRNA –> transcription RNA that carries amino acids to the ribosome to  make proteins  

Ch. 10 –> DNA Replication and Recombination  

DNA Replication

• Main function of DNA is storage and replication  

• replication is important for keeping genetic continuity  

- when replication happens, either of the 2 strands both become a  template

- synthesis of a complementary strand happens with lots of accuracy via DNA polymerase

• high accuracy is necessary so that not many errors occur  - DNA polymerase

Mode of DNA Replication  

• Semiconservative: each single strand from the double stranded helix  serves as a template  

- New helix = 50/50 mix of new and old DNA

Other Possible Replication Modes  

• Conservative: after replicating the two original strands will pair back up to  their original structure.  

- 2 new strands separate and associate with one another o original template goes back together  

• Dispersive: helix chopped up and reorganized with every strand being a  mixture of the old and new DNA

- each of the 2 DNA strands serve as a template for a new  complementary strand for each

- chopped up and arranged into mosaic like structure helix - along the length of a new single strand you have old + new mosaic like double helix

Meselson-Stahl Experiment

• 3 different modes lead to 3 different helix compositions

- realized that the 3 alternatives make different predictions  • DNA tests utilize the nitrogen component with different isotopes;  -15N (heavy)  

-14N (light)  

- centrifugation  

o incorporates nitrogen and these versions were available to them  o after centrifuging, you can distinguish between the two  • Grew the E. coli on 15N media

• Transferred it to 14N media  

• Isolated DNA from cells each generation

- each generation of a bacteria, they looked at proportion of heavy vs.  light that had been incorporated into the chromosome  

o all of DNA incorporates heavy nitrogen and falls into bottom of  tube  

if semiconservative – all bacteria would have DNA that were one light  nitrogen and one heavy nitrogen together  

- found that it had a different weight and formed a band

- repeated cycle of 1 round of replication of bacteria and centrifuging

Predictions

• After 1 generation – 1 intermediate density DNA band

- semi-conservative

- 2 helixes that are both light and heavy 50/50

• After 2 generations – 2 intermediate and light DNA bands:

• After 1 generation – 2 heavy and light DNA bands

- conservative

o one helix that is heavy and one helix that is light  

• After 2 generations – 2 heavy and light DNA bands

• After 1 generation – intermediate density DNA band

- dispersive  

o mosaic like structure

o 50/50 heavy and light  

• After 2 generations – 1 intermediate DNA band

Lecture 20

10/10/16

Replicaiton Origin and Direction

• _______________________– separates the double-stranded DNA into single  strands at the origin  

• After one initiation even the DNA immediately replicated is called a replicon • In E. coli, replication is bi-directional with a single origin and replicon

For DNA replication to take place

- first separate the strands

o each single strand serves as a template

- complimentary strand attaches  

- “unzip” DNA

DNA Synthesis in Bacteria

________________

- nucleotide base pairs for elongation  

- maintains accuracy/fidelity by an auto-corrective system  - to function it needs dNTPs, a template, and magnesium  

Unwinding the Helix

• Origin

- _________

- repetitive DNA sequence (TATA etc.)

• ________________

- a helicase

- breaks H bonds

- denatures strands

- opens helix

• _______________________

- helicases  

- destabilize strands

What keeps the helix open?  

- ________________________________proteins

• the enzyme DNA gyrase flattens out eh helix that form ahead of the  replication fork via

- Cut and re-paste type of enzyme  

Elongation from a Primer

• DNA polymerase during the extension/elongation step will always bind to  RNA at the origin  

• RNA primers are only about 10-12 base-pairs long  

• RNA primers are made by the enzyme primase

Leading v. Lagging Strands

• One strand is 5’ –> 3’ and its complement is 3’ –> 5’

- Antiparallel

• DNA polymerase III synthesizes from the 5’ end to the 3’ end  • ________________– long and continuous fragments

• ________________– shorter and discontinuous fragments

For the lagging strand:  

• DNA polymerase I

- Cuts out primers

- Replaces with complement bases  

• DNA ligase  

- Glues the fragments together  

For both strands:  

• DNA polymerase III

- Pairs all the missing bases constantly and simultaneously  

3’ ––> 5’ Exonuclease Activity  

• Polymerases can pause what they’re doing during synthesis to reverse and  correct any errors

- Proofreading to increase accuracy  

- checking for polymerases error bc every now and then a mutation will  occur  

DNA Replication in Eukaryotes

Several similarities to Prokaryotes:  

• replication fork forms at the origin as the helix unwinds

• there is a bi-directional synthesis with leading and lagging single strands of the DNA  

• Elongation –> DNA Polymerase  

Also some important differences:

• There is more DNA to be copied and it is packaged within the nucleosome • linear chromosomes; not circular  

- centromere

- telomere  

Multiple Replication Origin  

• yeasts have lots of origins

• mammals have around 1000s  

• Replication “bubbles” each make 2 replication forks  

Initiation of Replicatoin  

• timing of replication needs to be controlled so no more copies are  replicated than needed

- origins control this

• origin recognition complex (ORC) – these proteins tag the origin locations in the G1 stage  

• pre-replication complex – when present, replication occurs - when replication does occur these proteins leave and reassemble at  the next G1

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