New User Special Price Expires in

Let's log you in.

Sign in with Facebook


Don't have a StudySoup account? Create one here!


Create a StudySoup account

Be part of our community, it's free to join!

Sign up with Facebook


Create your account
By creating an account you agree to StudySoup's terms and conditions and privacy policy

Already have a StudySoup account? Login here


by: Ceasar Fritsch


Marketplace > Texas A&M University > Biochemistry > BICH 631 > BIOCHEMICAL GENETICS
Ceasar Fritsch
Texas A&M
GPA 3.92


Almost Ready


These notes were just uploaded, and will be ready to view shortly.

Purchase these notes here, or revisit this page.

Either way, we'll remind you when they're ready :)

Preview These Notes for FREE

Get a free preview of these Notes, just enter your email below.

Unlock Preview
Unlock Preview

Preview these materials now for free

Why put in your email? Get access to more of this material and other relevant free materials for your school

View Preview

About this Document

Class Notes
25 ?




Popular in Course

Popular in Biochemistry

This 6 page Class Notes was uploaded by Ceasar Fritsch on Wednesday October 21, 2015. The Class Notes belongs to BICH 631 at Texas A&M University taught by Staff in Fall. Since its upload, it has received 10 views. For similar materials see /class/225854/bich-631-texas-a-m-university in Biochemistry at Texas A&M University.

Similar to BICH 631 at Texas A&M

Popular in Biochemistry




Report this Material


What is Karma?


Karma is the currency of StudySoup.

You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!

Date Created: 10/21/15
BICH 631 Isolation of Mutants Selections and Screens Paper Szybalski W 1992 BioEssays 14495 500 A Screens Screens involve examination or analysis of a mixture of mutants and nonmutants either naturally occurring collections or those created by a directed mutagenesis for differences between wild type and putative mutants 1 General Issues 11 Background variation of ph enotypes in isogenic lines Phenotypic variation in normal wild type backgrounds in inbred stocks needs to be established prior to screening for mutant phenotypes WT control screen Variation in phenotype can occur due to environmental perturbations spatial growth effects stochastic variation in rate of development eg sometimes caused by differences in seed development that cause differences in seedling vigor that do not have a genetic basis This baseline information is required for an effective screen The better you understand a phenotype better you can design a screen Example Starting with 96 seed from an inbred rice plant grow seedlings in a controlled environment room and assay height time to owering or biomass accumulation in the vegetative phase Graph of plants vs variation in any of these traits Why is there variation This variation does not have a genetic basis all plants had the same genotype This background variation in inbred individuals must be quantified and taken into consideration when assessing the significance of a phenotype in a mutant line and when screening for mutant phenotypes false positives Philosophy Know your phenotype 12 Methods for dealing with phenotypic variation not caused by genetic variation characterize sources of variation biological samples environment assays replication replication replication pool individuals to average phenotypes conduct replicated studies in randomized fashion to average environments spatial 13 Genesmutations in interacting gene networks that generate pleiotropic ph enotypes Consider whether differences in the phenotype you are screening for arise from a biochemical pathway or regulon that affects many phenotypes or a pathway or branch of the pathway that has amore limited scope of action and thus a limited set of phenotypes Example Stay green drought tolerance phenotype in sorghum Example Flowering pathway 14 Variation in genetic background Uniform and nonuniform genetic backgrounds can be screened effectively for mutations Variation in nonisogenic backgrounds provide much greater potential range and frequency of allelic variation for discovery variation in numerous different alleles epistatic interactions but also much more complexity to resolve downstream Consider whether mutants may be lethal ie embryo lethals that need to be maintained in the heterozygous state do you screen M2 RILs NILs populations Q Yeast contain 6000 genes and are one of the mostly highly studied genetic model systems known Nearly all pathways phenotypes have been searched for mutants When the yeast genome was sequenced it was found that of the 60000 genes 2000 had been directly identified in various mutantbiochemical studies 2000 could be identified as homologs of genes with known function in other organisms and 2000 had unknown function Why did yeast geneticists miss these 2000 genes in prior studies gene families genome duplications ie trace ancestral duplications in Os ploidy dosage scope of screening vs genes needed for yeast function in vivo 7 very small differences in genotypephenotype are selected over time 7 these differences are often below the detection limit of our screens 15 The screen is usually the most important element in downstream snccess high throughput or low labor intensive or automated will the screen identify a wide range of mutations or narrow range scope will there be background that leads to false positives false negatives how will false positives be identified what cut off will be used to distinguish between mutant and wild type do you want a broad range of discovery open ended or narrowly focused search space ie developmental mutants in zebraf1sh was open ended initially because it was not clear what would be found this was very successful when to assay especially important in organisms with multistage development example GSl GSZ G83 and seed development in plants regulatory framework changes at each stage DNA methylation etc single cells assays or whole organism phenotypes multicellular organisms 16 How many cells lines should be screened depends on the complexity of the phenotypes genes that modulate the trait depends on the mutation frequency of different genes in the pathway depends on time and funding 2 Examples of Screens 21 Carlson et al screened N22000 colonies to nd 31 mutants four in a new locus SNFl 22 Somerville et al did GC assays on 3000 Arabidopsis EMS mutagenized plants to nd mutants in fatty acidlipid biosynthesis Leaf punches were taken from each line lipids extracted pro les obtained using a GC and compared to WT 23 MENDEL 7 generated 1500 Arabidopsis transgenic plants overexpressing every transcription factor in the genome and then screened the resulting mutants for variation in response to water limitation lowhigh temperature nutrient uptake etc Q What was the rationale for this approach Q What are potential confounding factors 24 Mullet lab screened for differences in chloroplast numbersize characterize wild type 40 ct cell average size microscope screened EMST DNA tag mutant populations using the same assay attempted to enrich using chlorophyll content prescreens explain why this did now work well found 8 mutants that vary in chloroplast number l2 cell vs 40 mutants in FtsZ genes involved in plastid division many unknown 25 Screens based on bluewhite color differences on indicator plate assays or any difference in spectral quality NF pH sensitive dyes are sometimes used to identify differences in enzyme activity chromogenic indicators such as Xgal 5Bromo4 chloro3indolylBDgalactoside Xgal is colorless until hydrolyzed by B galactosidase releasing a dark blue dye basis of the whiteblue screen for plasmids that encode lacZ encodes B galactosidase Xgluc XP are similar indicators negative control background activity in target organism or chromophores very efficient colonies are localized on plates and large numbers can be screened automated screening and colony picking Qbot colony picking FACs cell sorters still need to set cut off parameters ie how blue is blue Fusion proteins can produce light blue colonies Note lacZ is also used as a rep01ter gene a gene that substitutes for a native gene because it is easy to assay ie in promoterreporter gene constructs Note Luciferase and GFP green uorescent protein are often used as reporter genes because their activityabundance can be detected using noninvasive spectroscopic techniques photon counters such as a CCD camera or uorescence imaging for GFP B Selection and Enrichment Selection An experimental design that targets recovery of only a selected phenotypic class the experimenter only examines individuals that express that phenotype 1 Selection vs screening Example selection for ampicillin resistance as a means to isolate bacteria with recombinant plasmids mix plasmid DNA with competent cells treat cells to increase uptake of DNA add rich media plate 108 cells on a agar plate ampicillin next day 71000 amp resistant colonies are observed 99999000 cells selected against if you had to screen colonies instead need to screen 1000 plates of 1000 colonies to get one transformed colony selection allows much higher level of sensitivity selections are less important when mutants are not rare or if populations are small selections are very important to nd rare events ie rare recombinants in suc2 alleles 2 Types of selection bacteriocidal or fungicidal etc 7 a treatment that kills bacteria or fungi etc bacteriostatic or fungistatic 7 a treatment that prevents cell growth Example growth of bacteria on sucrose as the only carbon source allows suc cells to take over the population Suc cells are selected against but they are not dead just waiting for a carbon source physical selections remove a subset of a population based on a physical difference Example 1 remove or select bacteria that display a surface antigen using antibodies Example 2 phage display 7proteins are expressed as fusions such that they are located on the surface of a phage particle The subset of the population displaying the desired protein can be selected by differential binding to plates that contain target ligands biopanning Overhead1 Phage display cycles of selection for a speci c protein behavioral selections See Seymore Benzer s selection for Drosophila that respond to an ordorant Selection for insect s ability to learn to avoid pain associated with odor Overhead2 Selection for Drosophila that learn to avoid ordorpaz39n 3 Common selections 32 Drag resistances Antibiotics 7 act by binding to a specific target enzyme inhibiting it or subverting its normal activity Overhead3 Table of common antibiotics and their targets Resistance to antibiotics can be acquired by several di erent mechanisms modifying the target ie mutations in RNA polymerase to change rifampicin binding overexpressing the target effectiveness depends on drug concentration bypassing the need for the target alternative pathways modifying or inactivating the drug ie amp resistance conferred by B lactamase which modifies the drug by cleaving it thus inactivating the drug very useful for selection of plasmids on amp plates ie chloramphenicol acetyl transferase that inactivates CAP by acetylation preventing the drug from reaching the target transporter KOOE etc tetracycline resistance based on expression of a membrane pump that exports tet many tumor cells become resistant to chemotherapy via activation of pumps Resistance provides insight into mechanism of action Example rifampicin resistance was traced to mutations in rpoB the gene that encodes the B subunit of E coli RNA polymerase 32 Selections for and against the same function gain and loss of function Szybalski 1982 Describes protocols selecting for and against the function of HGPRT or HPRT hypoxanthine guanine phosphoribosyl transferase Review of nucleotide metabolism Overhead4 Biochemical pathways to purines i Two pathways feed purine and pyrimidine nucleotides into the NTP pool de novo synthesis pathway nts are synthesized by steps leading to IMP purine or UMP pyrimidine salvage pathway preferred if possible to save energy ii Salvage pathway converts preformed bases or nucleosides into nucleosides or nucleotides Overhead5 Salvage pathway key enzyme is HGPRT couples free guanine G or hypoxanthine H to PRPP 5phosphoribosyl l pyrophosphate PPi release iffeed G HGPRT makes GMP if feed H HGPRT makes IMP inosine monophosphate then converted to GMPAMP APRT converts free adenine to AMP T 0 select against H GPRT activity Szybalski fed cells 8azaguanine 8AzaG cells that take up 8AzaG die not sure of mechanism just must be incorporated via HPRT into 8 azaGMP used to select for HPRT loss of function cells with only de novo pathway survive T 0 select for H GPRT activity Szybalski needed a condition where only cells with HGPRT activity can grow needed to block de novo purine biosynthesis but HGPRT minus cells would not grow This was satisfied by use of HAT medium hypoxanthine aminopterin thymidine hypoxanthine provides the substrate for DHFR via the salvage pathway aminopterin was used to inhibit dihydrofolate reductase DHFR DHFR converts dihydrofolate to tetrahydrofolate lcarbon units carried by tetrahyrofolate are required in two steps in purine synthesis therefore inhibition of DHFR blocks de novo purine biosynthesis thymidine was added to overcome DHFR block in synthesis of TMP tetrahydrofolate is required for methylation of uracil by thymidylate synthase thymidine can also be made de novo or salvaged via TK HAT medium selects for TK active salvage pathway Szybalski used these cells and techniques to accomplish several things 1 Measured spontaneous mutation rates to azaH azaG 10394cell generation frequent probably involved in permeability 10396cellgeneration HGPRT loss of function 2 Worked out methods for transforming human cells with DNA transformed HPRT cells to HPRT cells with DNA from HRPT cells on HAT media optimized DNA transformation methods DNA spermine phosphate formed a precipitate on the cells later found that Cal F was critical to form Ca phosphate ppts linear relationship between concentration of DNA and transformation efficiency sensitive to DNase but not RNase 3 Used to select for cell hybrids Kohler and Millstein later used the hybridoma technique for production of monoclonal antibodies immortalized antibody genes in spleen cells 4 Basic technology required for gene therapy transgenics


Buy Material

Are you sure you want to buy this material for

25 Karma

Buy Material

BOOM! Enjoy Your Free Notes!

We've added these Notes to your profile, click here to view them now.


You're already Subscribed!

Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'

Why people love StudySoup

Steve Martinelli UC Los Angeles

"There's no way I would have passed my Organic Chemistry class this semester without the notes and study guides I got from StudySoup."

Allison Fischer University of Alabama

"I signed up to be an Elite Notetaker with 2 of my sorority sisters this semester. We just posted our notes weekly and were each making over $600 per month. I LOVE StudySoup!"

Jim McGreen Ohio University

"Knowing I can count on the Elite Notetaker in my class allows me to focus on what the professor is saying instead of just scribbling notes the whole time and falling behind."


"Their 'Elite Notetakers' are making over $1,200/month in sales by creating high quality content that helps their classmates in a time of need."

Become an Elite Notetaker and start selling your notes online!

Refund Policy


All subscriptions to StudySoup are paid in full at the time of subscribing. To change your credit card information or to cancel your subscription, go to "Edit Settings". All credit card information will be available there. If you should decide to cancel your subscription, it will continue to be valid until the next payment period, as all payments for the current period were made in advance. For special circumstances, please email


StudySoup has more than 1 million course-specific study resources to help students study smarter. If you’re having trouble finding what you’re looking for, our customer support team can help you find what you need! Feel free to contact them here:

Recurring Subscriptions: If you have canceled your recurring subscription on the day of renewal and have not downloaded any documents, you may request a refund by submitting an email to

Satisfaction Guarantee: If you’re not satisfied with your subscription, you can contact us for further help. Contact must be made within 3 business days of your subscription purchase and your refund request will be subject for review.

Please Note: Refunds can never be provided more than 30 days after the initial purchase date regardless of your activity on the site.