The least you need to know:
- eukaryotic genes: their elements and how they are regulated (think about everything that needs to happen in order for a eukaryotic gene to be transcribed in a given cell)
- the conept of "reporter gene" and how to use a reporter gene to test the activity of a cis-regulatory element (or to define it)
- how to clone a gene based on positional information
- how to clone a gene based on sequence homology
- when and how to use a Southern blot, a northern blot, chromosome walking, RT-PCR, footprint analysis (and how to interpret results)
- how to sequence an entire genome (steps involved)
- how to assemble a contig using molecular markers
- what a microarray is
- transposable elements: generalities, as well as P element's hybrid dysgenesis (in general) and how to use P elements to add a piece of DNA to a fly's genome, and how to exploit them to clone a gene
- transposable elements in a cross (like the question we did with corn and the Ac and Ds elements)
- types of transposable elements (very general-class I and class II)
Happy studying!
29 comments:
When prepping the DNA for footprint analysis...why is it denatured to be ssDNA?
Question about "gene tagging" Ch 14 slide 36.
The first step in this process tells us to make 2 dysgenic strains:
- one with intacted IRs but a defective transposase
- and the other with defective IRs but a working transposase.
I see how you can make the first strain using embryonic transformation with a clipped wing construct. But, how do you "make" a strain of flies that have an immobile source of transposase? Aren't the IRs required for the insertion process?
FOOTPRINTING:
you don't NEED it to be ss (technically), but you'll have to get rid of one of the 2 strands because remember how they are both labelled at one end? If you kept both, you would get 2 different fragments of each size, and you'd never know how to align them...
example:
*------XXXXX----------
------XXXXX----------*
imagine the XXX are where the protein binds. You'll get labelled fragments that are -, --, ---, ----, -----, ------, and also -------, -------- and --------- (from the other stand. Hence, it does not look like the protein is bound....because by keeping both strands we are mixing the fragments that go in "opposite directions"! Try to draw it out carefully, for example with the protein binding 10 bp away from one end and 50 bp away from the other end.
You'll see that you'll obtain fragments of all lengths, from 1 to 50 bp if you keep both strands!
SOURCE OF THE TRANSPOSASE:
You can't really build it in a very controlled manner, however, if you have a strain where you introduce a P element that makes the transposase. Then you get the P element to jump around, and at some point, through imperfect excision, you'll probably get one strain where the P element "loses" (or gets a mutation in) one of its IS.
it's a great question, by the way. You definitely have your P elements down!
Follow up on Footprint question:
That makes sense except that in step 2 of the course note description of Footprint analysis we cut off the "distal" P32 before we add the endonulclease.
Are these 2 steps redundent or are both necessary for footprint analysis to work?
Footprinting:
sorry about misinterpreting your question.
For our purposes, the two steps are redundant, as you say.
Thanks!!!
It's all coming together now...
I want to clarify what is meant by the following. Am I right in describing:
Cloning from Positional info
- combining info on a particular pedigree (genetic mapping) with molecular marks from individuals in that pedigree to narrow down the physical location of a particular allele. Once that location is narrowed down, the implicated DNA segment is sequenced???
Cloning by Sequence Homology
- starts with a gene of know function in one organism. Using that gene to make a probe and probing the genome of a different organism???
Sorry to be so vague, but I want to make sure I know what’s expected.
Yes, that's the idea. If you know and understand how to proceed to do these, you are going to be fine.
Keep up the goood work!
If anyone is still confused about DNA footprinting there is a pretty good article about it on wikipedia.
http://en.wikipedia.org/wiki/DNA_footprinting
I am confused about how p-element can insert cloned construct into somatic cells if it is only mobilized in germline cells? As showned in the chap 14 lectures slides 33-35? What is the difference between this example and the one showned in the textbook?
Thank you very much!
P-elements:
imagine a modified P element construct inserts itself into a germ cell of an embryo. When that germ cell becomes an egg or a sperm, and it is fertilized (or fertilizes an egg), the fly resulting from that fertilization event will have the p element construct in all its cells.
ALSO: remember that when we do P element transformation we don't rely on the p element's transposase (in fact, that part of the element is removed from it). We do add a separate source of transposase (the helper plasmid), that can be expressed everywhere.
Cheers
I do understand that, the affected germline will cause the F2 flies to display alternative phenotype in their soma, I am just confused as to how the F1 flies (supposed to have normal phenotype and only "intected germline) will have alternative phenotype as shown in the lecture slides? It's just that the lecture example and the textbook example sort of contradict each other.
I understand that the regular p-element only mobilizes in germline cells because it requires spilicing event of its mRNA which only occurs in the germline cells. So is the "helper p-element" able to mobilize in both germline and soma cells of the embryo unlike the normal p-element because its transposase mRNA does not require the splicing?
Sorry if my wording is somewhat confusing!
If you add a natural P element to a strain that does not have P elements, and you do this by crossing a female that does not have P elements to a male that does, then the F1 will have "messed up germline". This will cause any or all of the following:
no F2; very mutated F2; recombination in males, etc.
If, on the other hand, you insert a modified P element into an embryo by injecting plasmids, things are different. The transposase gene made by the helper plasmid is expressed everywhere (no splicing required, you are correct!) in the embryo. The modified P element in the other plasmid can jump from the plasmid to anywhere in the genome, and this can happen in any nucleus (i.e. a nucleus that will become part of the cells that will make wings, legs, intestine, etc). Hence, as these embryos develop to adulthood, they may have mutant phenotypes.
Keep the questions coming!
Pam
Cloning from Gene tagging:
According to chapter 14 slide 36, we can use colony lift to identify plasmids carrying the "P-tagged" gene; however, how do we know that the plasmid will contain the ENTIRE gene and not just a fragment (in which case it can still be picked up by the probe)? Do we ensure that we get the ENTIRE gene of interest by digesting the genome with enzyme that is known to NOT have an RE site within the gene of interest? But then if we do not know the sequence of the gene of interest how can we know it doesnt contain a certain RE site?
Would it be more efficient to clone the tagged gene using inverse PCR instead of colony lift?
(again, with inverse PCR, how do we know we've captured the entire sequence of interest since only a internal sequence is used as probe. How do we know if we've "extended" long enough in the flanking regions?"
Would this be a trial and error process then?
Thanks! And sorry if the wording sounds confusing!
There are some questions about imprinting in Tom G's extra problems from chapter 10. I kinda know what imprinting is but I'm almost positive we NEVER went over it in class. Is imprinting a testable topic?!?
i had a question about contigs sts notation
say a sequence is PCQ....but you don't know if it's PCQ or QCP...do you have to write..it could be either..or it is assumed that PCQ can also be QCP....
and how would we write that in bracket...(PCQ)?...i dont' really get it...because then it's saying C isn't known either?
Q: about P element
is IS the same as IR?
soo does a P element excise out...then jump into another gene in a location w/ an IR/IS to make a staggered cut?
...and does the excision from the orignal gene get mutated in any way?
IMPRINTING:
if tested, they will remind you what it is. You just need to remember that it exists.
STS notation:
if you don't know the order of a cluster of clones, (e.g. your PCQ) but you know that they are, as a cluster, in a certain position with respect to another clone, you would say for example:
CDE[PCQ]; where P, C and Q could be in any order.
Transposons:
sometimes the IR is the same as the IS. All we need to know is that when we have a precise excision, the elemt jumps out and does not leave anything behind.
In reality, we don't really know how the excision/insertion occurs-we can only make inferences from what we see that's sometimes left behind.
Don't need to know the mechanisms.
follow up on transpson
soo does the P element excision that jump have an IR/IS...
or does the insertion have the IR/IS for the P element to jump into...
because it seems like in transformation...the insertion has the IR/IS...but in dysgenic flies..that P element excision has the IR/IS? i'm just a bit confused because i don't understand if the IR is needed to jump out...or for place for P element to up in?
Hi Pam,
I think you missed my question on colony lift and reverse PCR, thanks!
You question is basically about how do we know that we have "grabbed" the whole gene in the clone that we picked up by colony-lift hyb or by doing inverse PCR.
It's a great question-in fact most of the time we DO NOT get the entire gene. But it does not matter, at this point: all we need is a little piece of it, and this little piece will allow us to make a probe corresponding to it and with this probe we will chromosome walk one step...in other words we will use this little piece of the gene of interest as a probe to screen a wild-type library, and this will allow us to to pick up a clone that contains, hopefully, the whole gene of interest!
Same idea for inverse PCR: it will at least allow us to find a littl bit of the gene of interest, which is really all we need!
I would like to clarify:
Is it true that P elements can duplicate themselves without utilizing RNA Pol, by the model of transposition where it comes from a plasmid and cointegrates into another plasmid/genome? Thus mutations occur without the cell ever replicating.
But it can also make a perfect or imperfect excision. In that case, there is no duplication of the P element?
Thanks!
No, not P elements. P elements don't duplicate themselves-retrotransposons do.
In eukaryote, class I transposon has retrovirus like activity, after its mRNA is transcripbed, part o it translates inrto a reverse transcriptase, which rtranscribes rest of the mRNA into cDNA, this cDNA goes back to the nucleus and is incorporated into the genome in another location (what is the exact mechanism of this? Is transposase still involved?? How does the cDNA gets back to the nucleus??)
Class II transposon is DNA transposon, and P element is an example of this. If does not make a copy of itself. Rather, with the help of transposase, which recognize the IR sequenc flanking it, it is excised (perfectly OR inferfectly), then inserted into another part of the genome. Location of inseartion is characterized by flanking of two identical sequence (due to the staggered cut made during insertion)
Hope this clear things up a bit for you.
However I am still a but confused about the mechanism of class I transposon.
Spatial pattern of early embryogenesis is established by combination of various morphogen (maternal + zygotic). What about temporal patterns? Are the timing determined by cascade and/or external stimuli?
Hey transposons people!
You are doing an excellent job at clarifying things for eachother. You won't need to know the exact mechanisms for retrotransposons-in fact, few people in the world understand what is really going on (or at least they think they understand).
Cheers
Pam
Temporal patterns of development:
what a great question. As far as I know, much, much less is known about temporal patterns of expression. There are signalling cascades that are involved (like, one thing has to happen first, then the next, and the next, etc), but why it is that each event always happens at a given point in development is not completely clear.
In vertebrates, for example, development is 'staggered'-the anterior part of the embryo starts developing first, so it's always "ahead" in the process. WHY does it start in the anterior part first? In response to some signals, but how that exactly works, we don't know.
Cheers
Pam
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