BISC 219/F10: Lab 5

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[[BISC 219/F10:Gene Mapping  | Lab 2: Gene Mapping]]<br>
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== Lab 5: Continue Mapping the Mutation ==
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[[BISC 219/F10: Lab 3  | Lab 3: Linkage Test Part 1]]<br>
+
'''Mapping''' Because Fall Break is coming and we don't want you to have to come in to do work during break, please place 2 young adult double mutant hermaphrodites from a true breeding plate onto new each of 2 new plates. Make sure there are no males on the plate from which you select your worms! Label these plates with your <font color="purple"> PURPLE </font color= "purple">Sharpie.<BR><BR>
-
[[BISC 219/F10: Lab 4  | Lab 4: Linkage Test Part 2 and Mapping]]<br>
+
We will incubate your worms at lower temperature to slow them down until Lab 6. <br>
-
[[BISC 219/F10: Lab 6  | Lab 6: Score]]<br>
+
-
 
+
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== Lab 5: Mapping ==
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'''Mapping:''' Cross wild type males (+ +/+ +) X your double mutant (d u/d u). Remember to follow the normal precautions to insure that all progeny are either self progeny (d u/d u mutants) or double heterozygous cross progeny (d u/+ +). Set up duplicate crosses to be sure you get enough worms.<br>
+
<br>
<br>
-
Label both plates with your initials, date and the cross set up + +/+ + (M) X d u/d u (H) with your <font color= purple> '''PURPLE''' </font color= purple> Sharpie.<br>
 
<br>
<br>
-
Incubate your worms at 23°C for 3 days<br>
+
 
 +
== Complete Complementation Testing==
 +
Examine your complementation crosses. '''The main consideration is whether or not there are any '''MALE progeny of Dpy phenotype''' present in the progeny of cross #2''' <br>
 +
WHY male?  Think about the hallmark of a sucessful cross!  What else can hermaphrodites do other than mate with males - might be why you have Dpys on more than one plate.<br>
<br>
<br>
-
'''3 days after lab:'''<br>
+
If you find a cross with Dpy male progeny pick one or two to a fresh plate and take their picture being sure to get their tails in the picture. This is your "proof" of failure to complement.<br>
-
To allow us to '''FINALLY''' determine the map distance between our two mutations, by determining the number of recombinant gametes which is related to the number of recombination events between the two genes, pick 3-4 wild type males (double heterozygous-d u/+ +) to each of two new mating plates and mate them to L4 hermaphrodite double mutants (homozygotes d u/d u) - this is called a test cross.  <br>
+
-
<br> 
+
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Label both plates with your initials, date and the cross  d u/+ + males X  d u/d u (H) with your <font color= purple> '''PURPLE''' </font color= purple> Sharpie.<br>
+
-
<br>
+
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Incubate your worms at 23°C for until next lab<br>
+
<br>
<br>
 +
For each plate consider that: <br>
<br>
<br>
-
== Complementation ==
+
'''A) If your unknown mutation and your known mutation under study are in different genes (not allelic):''' the heterozygote male that you incorporated into Cross #2 will produce two types of gametes: type 1) with a ''dpy-u'' mutation and a wild type copy of the ''dpy-k'' gene and type 2) with wild type ''dpy-u'' gene and wild type ''dpy-k'' gene. One the other hand, the hermaphrodite you added will produce only one type of gamete-- we'll call it gamete type 3) with wild type ''dpy-u'' gene and a mutation in ''dpy-k''. <br>
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'''You will now set up Cross #2:''' Pick 3-5 males from the cross plate initiated last week onto three new mating plates (remember these are heterozygous for your dpy mutation so they are phenotypically wild type but they carry dpy (dpy/+). Again, it is essential that only males be transferred onto these plates.  Add three L4’s from each of the 3 known dpy reference strains to the mating plates with the males.<br>
+
<br>
<br>
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'''Available Dpy strains:'''
+
These gametes can be combined so that you will have two possible genotypes. Combining gametes type 1 and 3 you get ''dpy-u'' from the male and wild type for the hermaphrodite so the progeny are heterozygous at the ''dpy-u'' locus (''dpy-u''/+) and for the dpy-k locus you get wild type from the male and ''dpy-k'' from the hermaphrodite (+/''dpy-k''). These individuals are phenotypically wild type since they have one wild type allele for each of the genes. Combining gametes 2 and 3 for the ''dpy-u'' locus you get wild type alleles from the male and the hermaphrodite (+/+) and for dpy-k you get wild type from the male and ''dpy-k'' from the hermaphrodite (+/''dpy-k''). These individuals are phenotypically wild type since they have at least one wild type allele for each of the genes. In conclusion if your unknown mutation (''dpy-u'') and your known mutation are in different genes (not allelic) will you observe any mutants in the progeny of cross #2?<br>
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{| border="1"
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! Chromosome 1 !! Chromosome 2 !! Chromosome 3 !! Chromsome 4
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|-
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| <center>''dpy-5'' </center> || <center>''dpy-2''</center> || <center>''dpy-1'' </center>|| <center>''dpy-4''</center>
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|-
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| <center>''dpy-14'' </center>|| <center>''dpy-10'' </center> || <center>''dpy-17''</center> || <center>''dpy-13''</center>
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|-
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| <center>''dpy-24''</center> || <center>''dpy-25'' </center> || <center>''dpy-18''</center> || <center>''dpy-20''</center>
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|-
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|}
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<br>
<br>
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Label your plates, using your <font color= orange> '''Orange''' </font color= orange>  Sharpie, with your initials and the date and the cross dpy/+ (M) X dpy-k (replacing k with the gene number of each of the reference strains)/dpy-k.
+
[[Image:Complementation test cross 2 dif genes.jpg]]<br><br>
-
Incubate the worms at 23°C until next lab period.<br>
+
'''B) If your unknown mutation (''dpy-u'') and one of the previously identified mutations under study (''dpy-k'') are in the same gene (allelic): ''' the heterozygote male that you incorporated into Cross #2 will produce two types of gametes: type 1) with a ''dpy-u'' mutation and type 2) with wild type gene. One the other hand, the hermaphrodite you added will produce only one type of gamete we'll call it gamete type 3) with the ''dpy-k'' mutation. <br>
-
<br>  
+
-
 
+
-
'''3 days after lab:'''<br>
+
-
Examine each of the 3 plates initiated last laboratory period. '''The main question is whether there are any '''MALE progeny of Dpy phenotype''' present in the progeny of cross #2''' <br>
+
-
WHY male?  Think about the hallmark of a sucessful cross!  What else can hermaphrodites do other than mate with males - might be why you have dpys on more than one plate.<br>
+
<br>
<br>
-
For each plate consider that: <br>
+
These gametes can be combined so that you will have two possible genotypes. Combining gametes type 1 and 3 you get ''dpy-u'' from the male and ''dpy-k'' from the hermaphrodite so the genotype is (''dpy-u/dpy-k''). These individuals are phenotypically mutant. Combining gametes 2 and 3 you get wild type from the male and ''dpy-k'' from the hermaphrodite so the genotype is (+/''dpy-k''). These individuals are phenotypically wild type since they have one wild type allele. In conclusion if your unknown mutation (''dpy-u'') and your known mutation are in the same gene (allelic) will you observe mutants in the progeny of cross #2<br>
 +
[[Image:Complementation test cross 2 same gene.jpg]]
<br>
<br>
-
'''A) If your unknown mutation and your known mutation under study are in different genes (not allelic)''' we will call the two genes responsible for the mutations A and B, respectively. The heterozygote male that you incorporated into Cross #2 will produce two types of gametes: type 1) with a defective gene A (carrying the dpy mutation) and a wild type gene B and type 2) with wild type gene A and wild type gene B. One the other hand, the hermaphrodite you added will produce only one type of gamete-- we'll call it gamete type 3) with wild type gene A and mutant gene B (carrying the dpy-k mutation). <br>
+
Can you now id the ''dpy'' gene responsible for your Dpy phenotype?
 +
If you discovered that one of the reference '''dpy'' strains was allelic to your dpy mutation, congratulations! You now know the name of your mutated gene and are ready to start the next phase of our Forward Genetics study: characterization of the nature of the mutation and, perhaps, discovery of a functional area of the protein product. DNA sequence analysis and bioinformatics are next. <BR>
 +
 
 +
If none of your reference strains failed to complement (meaning that everything tested was non-allelic), you may not have learned much about the identity of the gene associated with your mutation but that is not, necessarily, a bad thing. Does that make it more likely that the gene you are characterizing has not been previously identified as associated with a Dpy phenotype and may be a newly characterized mutation? It will be a lot easier to publish this work if so:)
 +
<BR>
 +
 
 +
== Complementation Testing Analysis ==
 +
From your complementation analysis, you may have discovered the name of your dpy gene of interest. To learn more, you can enter the ''dpy''gene name into the ''C. elegans'' database: [http://www.wormbase.org | Wormbase at http://www.wormbase.org Wormbase]<BR>
 +
 
 +
Click on the link to Wormbase above and enter the gene name into the box at the top of the page and click Search.  It will either bring you directly to that page or it will bring you to a page with mutiple hits - click on the link that provides a definition for what the gene does.<br>
<br>
<br>
-
These gametes can be combined so that you will have two possible genotypes. Combining gametes type 1 and 3 for gene A you get dpy-MB? from the male and wild type for the female so gene A is (dyp-MB?/+) and for gene B you get wild type from the male and dpy-k from the female so gene B is (+/dpy-k). These individuals are phenotypically wild type since they have one wild type allele for each of the genes. Combining gametes 2 and 3 for gene A you get wild type alleles from the male and the female so gene A is (+/+) and for gene B you get wild type from the male and dpy-k from the female so gene B is (+/dpy-k). These individuals are phenotypically wild type since they have at least one wild type allele for each of the genes. In conclusion if your unknown mutation (dpy-MB?) and your known mutation are on different genes (not allelic) will you observe any mutants in the progeny of cross #2?<br>
+
On this new page you should find all the known information about this particular gene.  Its name, who named it, what the gene encodes - if that is known, and much more. At the bottom will be a list of references - or a link to a list of references. Does it appear that you are working with a well characterized gene?<br><br>
-
<br>
+
-
'''B) If your unknown mutation (dpy-u) and your known mutation under study (one of four) are on the same gene (allelic): '''we will call that gene A. The heterozygote male that you incorporated into Cross #2 will produce two types of gametes: type 1) with a mutant gene A (carrying the dpy-u mutation) and type 2) with wild type gene A. One the other hand, the hermaphrodite you added will produce only one type of gamete we'll call it gamete type 3) with mutant gene A (carrying the dpy-k mutation). <br>
+
-
<br>
+
-
These gametes can be combined so that you will have two possible genotypes. Combining gametes type 1 and 3 for gene A you get dpy-MB? from the male and dpy-k from the female so gene A is (dpy-MB?/dpy-k). These individuals are phenotypically mutant. Combining gametes 2 and 3 for gene A you get wild type from the male and dpy-k from the female so gene A is (+/dpy-k). These individuals are phenotypically wild type since they have one wild type allele. In conclusion if your unknown mutation (dpy-MB?) and your known mutation are on the same gene (allelic) will you observe mutants in the progeny of cross #2<br>
+
 +
Spend some time with Wormbase and marvel at all the hard work and years of research that went into discovering all this information about this tiny little nematode that causes us no harm (non-parasitic). Why do you think so many smart people have devoted so much of their time and energy to working out the genetics of "appearance or movement challenged" little worms? We will talk more about model organisms and the power of functional and comparative genomics in our next series.
-
<br>
+
== Assignment ==
 +
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab.
 +
 
 +
<div class=noprint>
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==Links to Labs& Project Info==
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Series1:<BR>
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[[BISC 219/F10: Worm Info| Worm Info]] <br>
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[[BISC 219/F10: Gene Linkage| Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start]]<BR>
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[[BISC 219/F10: Lab 2  | Lab 2: Sex-Linked or Autosomal Finale]]<br>
 +
Series2:<BR>
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[[BISC 219/F10: Gene Mapping Info | Background: Classical Forward Genetics and Gene Mapping]]<br>
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[[BISC 219/F10: Lab 2 Mutant Hunt | Lab 2: Mutant Hunt]]<br>
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[[BISC 219/F10: Lab 3  | Lab 3: Linkage Test Part 1]]<br>
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[[BISC 219/F10: Lab 4  | Lab 4: Linkage Test Part 2, Mapping and Complementation]]<br>
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[[BISC 219/F10: Lab 5  | Lab 5: Finish Complementation; Mapping Con't]]<br>
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[[BISC 219/F10: Lab 6 | Lab 6: DNA sequence analysis; Mapping Con't]]<BR>
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[[BISC 219/F10: Lab 7  | Lab 7: Complete Mapping: Score]]<br>
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Series3:<BR>
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[[BISC 219/F10:RNA interference | Schedule of Reverse Genetics Project]]<BR>
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[[BISC 219/F10:RNAi General Information| RNAi General Information]] <br>
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[[BISC 219/F10:Media Recipes | Media Recipes]]<br>
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[[BISC 219/F10: RNAi Lab 5  | Lab 5: Picking your gene to RNAi]]<br>
 +
[[BISC 219/F10: RNAi Lab 6  | Lab 6: Cloning your gene of interest]]<br>
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[[BISC 219/F10: RNAi Lab 7  | Lab 7: Picking your transformant]]<br>
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[[BISC 219/F10: RNAi Lab 8  | Lab 8: Plasmid purification and transformation]]<br>
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[[BISC 219/F10: RNAi Lab 9  | Lab 9: Induction of bacteria for RNAi]]<br>
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[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br>
 +
[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br>
 +
</div>

Current revision

Contents

Lab 5: Continue Mapping the Mutation

Mapping Because Fall Break is coming and we don't want you to have to come in to do work during break, please place 2 young adult double mutant hermaphrodites from a true breeding plate onto new each of 2 new plates. Make sure there are no males on the plate from which you select your worms! Label these plates with your PURPLE Sharpie.

We will incubate your worms at lower temperature to slow them down until Lab 6.


Complete Complementation Testing

Examine your complementation crosses. The main consideration is whether or not there are any MALE progeny of Dpy phenotype present in the progeny of cross #2
WHY male? Think about the hallmark of a sucessful cross! What else can hermaphrodites do other than mate with males - might be why you have Dpys on more than one plate.

If you find a cross with Dpy male progeny pick one or two to a fresh plate and take their picture being sure to get their tails in the picture. This is your "proof" of failure to complement.

For each plate consider that:

A) If your unknown mutation and your known mutation under study are in different genes (not allelic): the heterozygote male that you incorporated into Cross #2 will produce two types of gametes: type 1) with a dpy-u mutation and a wild type copy of the dpy-k gene and type 2) with wild type dpy-u gene and wild type dpy-k gene. One the other hand, the hermaphrodite you added will produce only one type of gamete-- we'll call it gamete type 3) with wild type dpy-u gene and a mutation in dpy-k.

These gametes can be combined so that you will have two possible genotypes. Combining gametes type 1 and 3 you get dpy-u from the male and wild type for the hermaphrodite so the progeny are heterozygous at the dpy-u locus (dpy-u/+) and for the dpy-k locus you get wild type from the male and dpy-k from the hermaphrodite (+/dpy-k). These individuals are phenotypically wild type since they have one wild type allele for each of the genes. Combining gametes 2 and 3 for the dpy-u locus you get wild type alleles from the male and the hermaphrodite (+/+) and for dpy-k you get wild type from the male and dpy-k from the hermaphrodite (+/dpy-k). These individuals are phenotypically wild type since they have at least one wild type allele for each of the genes. In conclusion if your unknown mutation (dpy-u) and your known mutation are in different genes (not allelic) will you observe any mutants in the progeny of cross #2?

Image:Complementation test cross 2 dif genes.jpg

B) If your unknown mutation (dpy-u) and one of the previously identified mutations under study (dpy-k) are in the same gene (allelic): the heterozygote male that you incorporated into Cross #2 will produce two types of gametes: type 1) with a dpy-u mutation and type 2) with wild type gene. One the other hand, the hermaphrodite you added will produce only one type of gamete we'll call it gamete type 3) with the dpy-k mutation.

These gametes can be combined so that you will have two possible genotypes. Combining gametes type 1 and 3 you get dpy-u from the male and dpy-k from the hermaphrodite so the genotype is (dpy-u/dpy-k). These individuals are phenotypically mutant. Combining gametes 2 and 3 you get wild type from the male and dpy-k from the hermaphrodite so the genotype is (+/dpy-k). These individuals are phenotypically wild type since they have one wild type allele. In conclusion if your unknown mutation (dpy-u) and your known mutation are in the same gene (allelic) will you observe mutants in the progeny of cross #2
Image:Complementation test cross 2 same gene.jpg

Can you now id the dpy gene responsible for your Dpy phenotype? If you discovered that one of the reference 'dpy strains was allelic to your dpy mutation, congratulations! You now know the name of your mutated gene and are ready to start the next phase of our Forward Genetics study: characterization of the nature of the mutation and, perhaps, discovery of a functional area of the protein product. DNA sequence analysis and bioinformatics are next.

If none of your reference strains failed to complement (meaning that everything tested was non-allelic), you may not have learned much about the identity of the gene associated with your mutation but that is not, necessarily, a bad thing. Does that make it more likely that the gene you are characterizing has not been previously identified as associated with a Dpy phenotype and may be a newly characterized mutation? It will be a lot easier to publish this work if so:)

Complementation Testing Analysis

From your complementation analysis, you may have discovered the name of your dpy gene of interest. To learn more, you can enter the dpygene name into the C. elegans database: | Wormbase at http://www.wormbase.org Wormbase

Click on the link to Wormbase above and enter the gene name into the box at the top of the page and click Search. It will either bring you directly to that page or it will bring you to a page with mutiple hits - click on the link that provides a definition for what the gene does.

On this new page you should find all the known information about this particular gene. Its name, who named it, what the gene encodes - if that is known, and much more. At the bottom will be a list of references - or a link to a list of references. Does it appear that you are working with a well characterized gene?

Spend some time with Wormbase and marvel at all the hard work and years of research that went into discovering all this information about this tiny little nematode that causes us no harm (non-parasitic). Why do you think so many smart people have devoted so much of their time and energy to working out the genetics of "appearance or movement challenged" little worms? We will talk more about model organisms and the power of functional and comparative genomics in our next series.

Assignment

Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab.

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