Lecture 2.pdf

We are interested in how plants regulate their size. To figure out which genes in plants are involved in regulating size, we performed random mutagenesis and identified a mutant (shorty). Problem is, we don't know the gene that is mutated. Today, we will look at how to figure out which gene is mutated in the mutant Biology 301 You got a mutant that has a defect in growth!! now what? Molecular Biology Lect 2: Gene Identification ➢  Gene mapping ➢  based on gene linkage (recombinant frequency) ➢  use of molecular DNA markers as mapping landmarks Wiypety shorty Extra materials ➢  Extra reference material on mapping with molecular markers in Lab Manual Appendix A2.5 ➢  For more info on PCR as a technique, take advantage of “Current Protocols in Molecular Biology” (Ch 15 intro & 15.1) – available at McGill Life Sciences Library under eBooks – Current Protocols Online You need to identify which gene is mutated in shorty. 2 1 Method of how you identify the gene affected by mutagenesis depends on how you mutagenized it. In general, there are two types of mutagens 1) insertional mutagens: a piece of DNA that can integrate in random places in genome. In arabdopsis, it is called T-DNA. By introducing this DNA to a host and inserting itself into random place, you are hoping it goes into middle of a particular gene or into a regulatory sequence required for expression of a gene and thus, interferes with its function. THe advantage is that you can use the DNA you inserted as a template to figure out where in the host gene this DNA got inserted into; one technique you can use is called TAIL-PCR. In TAIL-PCR, we can use part of T-DNA as primer, but we don't know the sequence of gene next to T-DNA --> so we add a bunch of random oligos which can bind many places --> we're hoping one of them bind near where insertion site is and PCR extends towards mutagen --> this will allow us to amplify the region where T-DNA got integrated --> once you get amplification, you send it for sequencing (we know sequence of genome of most model organisms) Identification & cloning of a gene depends on Whhere s ts picureiiiiiciiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiis mutagen taken? ➢  insertional mutagens ➢ mutagenize genes by sticking a large chunk of DNA in the middle of it or its regulatory sequences ➢ inserted DNA acts as a “tag” to identify the gene involved ➢ can use “TAIL-PCR” to amplify flanking genomic DNA – sequence and compare to genome to identify gene T-DNGene ➢  chemical mutagens – e.g. ethylmethane sulfonate (EMS) ➢ point mutations -- affect proteins through truncation or “loss” of key amino acids (CAG: glutamine " TAG: stop codon) ➢ There is no “tag” to identify the gene involved ➢ clone with positional or map-based cloning 2) chemical mutagenesis. EMS commonly used. It introduces point mutations and you want it to affect coding 4 sequences and affect proteins. Because it is point mutations, we can't use tail-pcr. This is where you need positional or map-based cloning Gene mapping: When genes are linked, they are physically linked -- they are on -this is to locate the gene of interest by identifying the nearby same chromosome and probably very close to each other. The way landmarks. We know the location of the landmarks in the genome. you distinguish whether genes are linked or not is to perform a -landmarks can be morphological or molecular markers bunch of crosses. Slide shows two extreme examples -how do you determine which landmark is close to gene of -you first cross WT with homozygous recessive to create interest? that is determined by linkage b/w gene and landmark. heterozygous. Then, you cross heterozygous with tester (homozygous recessive) --> you have equal chance of getting phenotypes shown in slide Gene Mapping Gene linkage ➢  Locating a GOI in the genome by identifying the nearby “landmarks”. Genes on the same chromosome Independent assortment ➢ Locations of “landmarks” are “mapped out” in the genome, ➢ Landmarks could be either morphological or molecular markers Nearby landmarks are identified by determining linkage ➢  Recombinant = the production of new combinations of alleles of different between the GOI between the landmarks genes during meiosis (gametogenesis) 6 5 (cont from above) Half are parental (same as parents) and half are recombinants (new combo of traits). We get equal frequency of the progeny is b/c of independent assortment (genes segregate independently) If two genes are linked (left), you will end up with parental type progeny. But there can be recombination. Good thing about recombination is that the closer the genes are to each other, the less likely they will cross over. Recombination randomly and it's physical reasons why if genes closer have less chance of crossing over. Frequency of recombination can be used as indication of how close genes are (lower frequency = closer) Thus, even if two genes are on same chromosome, you can have cross over during meiosis creating a recombinant. Gene linkage & Gene linkage & recombination map distance ➢  The closer genes are together, less ➢  Linked genes " recombination due to crossing over crossing over can occur between them for physical reasons All Parental ➢  The frequency of recombination is Recombinant determined by the Recombinant physical distance Parental between two genes 7 8 How close genes are to each other can be calculated by determining the recombinant frequency (RF) -RF expressed as percentage; 1% RF = m.u = cM -if RF is less than 50%, two genes are linked. 50% is magic In scenario where genes are on same chromosome, you can number b/c if two genes aren't linked (if they are on diff have recombinants but in general, you will have more chromosomes), there is 50% chance of getting recombinant progeny parental types. Frequency of recombinants depend on how -population size can make diff in accuracy close genes are to each other. Determining gene linkage by recombinant Gene linkage frequency Recombinant frequency (RF) = # recombinants # parentals + recombinants ➢  If genes are linked ➢ % Parental type > ➢ RF is expressed as percentage recombinant type ➢ 1% RF is also known as a map unit (m.u.) or centiMorgan (cM) ➢  Closely linked genes ➢  If RF < 50%, two genes are linked ➢  lower RF, closer the genes are together (DNA elements) segregate together ➢  If RF 50% or greater, genes are unlinked ➢  Population size can make a difference on the accuracy of gene mapping 9 10 Example: -you do mutagenesis in fly and identify mutant which you name "m" (gives small wings). Otherwise, this fly has WT background -flies have 4 chromosomes. Which chromosome is gene m on? You need to take mapping line that has 4 phenotypes associated with the 4 chromosomes. Thus, this mapping line carries 4 mutations, each corresponding to a particular chromosome. Gene M will be WT in mapping line (haven't done mutagenesis there) -You create F1 progeny --> this will be heterozygous for all genes -then, you can try to get a tester fly which is homozygous recessive for all genes (ie mm ww bb hh vv). But this is not practical b/c it is difficult to get and fly gets really sick if it has this many mutations. Using the this is map (morpholoigal markers) in drosophila mapping line is also no good b/c it is WT for gene of interest. So what we -these are genes where we know where they are in genome do is cross F1 with F1 and you focus on one marker at a time and we know the phenotypes that are associated with this gene -in F2 progeny, you don't care about flies without mini wing phenotype (ie -eg there is a gene that will cause fly to have yellow body don't care about flies that are mm) (cont below) - 1 2 3 4 Gene mapping with visible (morphological) Gene mapping by w v markers recombinant frequency h b X Mutant Mapping line P mm WW BB HH VV MM ww bb hh vv Tester (mm ww bb hh vv) Looks like wild type F1 mM Ww Bb Hh Vv X MM ww bb hh vv mM Ww Bb Hh Vv mm bb or mm Bb or mm BB F2 Gene mapping: Locating a GOI in the genome by identifying the nearby ➢  Analyze RF between mm and each markers “landmarks” (by determining RF) 11 12 -you only care about mm phenotype b/c this means that this gene element cae from mutant (see P generation) -let's assume m is not on the second chromosome (where "b" is). In this case, ratio of F2 would be mmbb:mmBb:mmBB = 1:2:1 -But if gene m is on the second chromosome, you'll have less chance of getting mmbb (b/c to get bb, that must have come from mapping line and recombined with mutation)