1) Gene structure, organization and regulation (in prokaryotes and in eukaryotes)
2) The techniques, tools and strategies to study gene structure, organization and regulation.
So far, you have learned about:
1) Gene structure, organization and regulation in prokaryotes
--> examples of the lac operon, example of the operon seen in problem set 2
2) A number of techniques to study gene structure and regulation, including:
--> how te see if a gene of interest is expressed (if it produces an enzyme, we can use enzyme assays, like for the B-Gal; in all cases we can also look for presence of RNA corresponding to our gene of interest, e.g. with northern blots). We'll see more methods later.
--> how to clone a gene of interest (you have seen two strategies so far: the first one is to clone a gene through functional complementation, the second is to clone it based on sequence homology). Again, you will learn about other strategies to clone genes later on in the course.
Keep these points in mind!
Cheers
Pam
PS: in response to popular request, step-by-step answers to problem sets 2 and 4, and to the questions on the handouts, will be posted on the 335 resource page (by Wednesday afternoon).
pam i couldnt attend tutorial today :(
ReplyDeletecould u give a brief summary of what was covered?
I am unclear on how restriction mapping tells you that your presumptive clone supposedly containing the gene of interest, actually contain the gene of interest. Also the textbook mentioned something about genetic landmarks that I do not understand. One last thing, the text says that Southern Blotting requires the genetic material to be blotted on the membrane to be single stranded. If this is the genomic DNA, do the restriction enzymes make the DNA single stranded? thanks!
ReplyDeleteToday (Tuesday) we worked on a series of practice questions (for those who did not get a copy of them, I'll have more copies tomorrow). The main topics were restriction mapping (data analysis and making restriction maps of plasmids and linear molecules) as well as probing/Southern blots.
ReplyDeleteRESTRICTION DIGESTIONS TELL US a lot of stuff... for example, they tell us whether a plasmid contains an insert, and how big this insert is. If we know what restriction sites are present in a fragment/gene of interest, and how far apart they are, a restriction digestion of a clone will also tell us whether the insert contained in the plasmid likely includes our gene of interest or not. For instance, if we know that the gene of interest has 3 EcoRI sites, and that the distance between the 1st and 2nd site is 300bp, and between the 2nd and 3rd it's 1200bp, and it also has 2 PstI sites, with the 1st one 400 bp downstream of the 1st EcoRI site, and the 2nd one 30 bp upstream of the 3rd EcoRI site, etc... then we can perform a series of restriction digestions on our putative clone of interest and, if the results that we obtain are consistent with the data we have about the positions anddistances of all the restriction sites, then we are quite confident that what we have cloned is indeed the gene of interest. If the results don't match the expected data, then we know that our clone is not worth using for future applications, because it contains some other sequence that we are not interested in at the moment.
SOUTHERN BLOTTING.... uses double stranded DNA. the DNA that you run on the gel, and that you blot on the membrane, is double stranded. However, in order to allow the probe to hybridize to its complementary sequences, you will treat the membrane (with the ds DNA bound to it) so that the 2 strands of DNA will come apart and stay apart during the treatment. So, the DNA will be single stranded, but only tempararily. After the treatment, as we want the probe to hybridize and 'stick', we put the membrane under conditions that allow the DNA to go back to double stranded.
THE RESTRICTION ENZYMES do not make the DNA single stranded. They just cut the DNA at specific sites.
I realize that this is a lot of information, and it is particularly hard to digest and assimilate if you have not had molecular biology labs before....you guys are doing a great job!!
Cheers
Pam
Hi Pam,
ReplyDeleteI attempted the midterm problems (posted on the 335 website), but have some questions about the answers. If you have time in tutorial tomorrow, could you help me? However, I am unable to bring a paper copy of the questions, as when I attempted to print them out, it did not work!
Jasmine
Hi, I was wondering is Craig going to cover everything from chapter 20 for the midterm?
ReplyDeleteHi Pam! What exactly is the function of X-gal? I know if lactose is present it turns blue, but what does it do molecularly?
ReplyDeletethanks
Hello everyone!
ReplyDeleteI talked to Craig today and he said that everything until (and including) what you'll be covering in class today is testable on the MT. He will go over parts of the practice MT tomorrow in lecture, and we can go over parts of it today in the tutorial.
The function of XGal....? That's a good question. I don't know. It's an artificially made chemical... I'll try to find out.
Cheers
Pam
Hello Pam, you said the answers will be posted by wednesday after noon, but I can't find them.
ReplyDeleteHi Pam,
ReplyDeleteI was confused with terms in previous mt.
1. What does it mean by " homologous recombination (Q2-201)?"
2. Also when we are asked to draw coding sequence (Q2-201), does it mean sth like start/stop codon??
3. Last, what is Q2 -202(Identify clones that contained the desired fragments) asking us?? Is it asking us how to purify the gene of interest (e.g. colony-life hybridization technique)??
thanks!!!
Hello everyone!
ReplyDelete1) ANSWERS TO PROBLEMS: still trying to post them (for some reason they don't seem to be going up).
2)PREVIOUS MTs:
-homologous recombination" means "crossovers"
- coding sequence is the part of the gene that encodes the amino acids of the protein
- identifying the clones that contain the desired fragments means, you'll have a bunch of colonies, each colony will contain a plamid, each plasmid will (or won't) contain an insert/fragment, and only a subset of the fragments are our desired fragments. How are we going to know which colonies contain a plasmid that contains the fragment(s) that we're interested in?
(Yes, colony-lift hyb would work if you have a probe!)
Cheers for now
Pam
hi Pam
ReplyDeleteI was reading up on functional complementation and I was wondering how would we check if there was functional complementation versus my mutant reverting back to wildtype.
i.e. bacterial wt is orange, mutant is white
orange> white
through functional complementation I would transform wt orange plasmid into the mutant white bacteria, grow the bacteria on a medium and see all orange so yay!!
but what happens if some of the orange colonies we see actually don't have the orange plasmid but instead have a reverted white mutant gene that reverted back to orange?
Can I test for this reverted gene? I was thinking that I would cross it with a WT but orange > white so I would always see orange.
can I test to see if the white gene reverted back to orange or is it something I will never know?
This is a very good question indeed.
ReplyDeleteThe first thing to keep in mind is that reversion is relatively rare (more so than successful complementation with a plasmid). Also, whenever doing these kinds of experiments, we always do a control where we don't transform the mutant bacteria, which will give us an idea of the rate of reversion (we'll know that all the orange colonies obtained in the control are NOT due to complementation).
Your strategy of crossing the revertant would work...except remember it's a bacterium...how are you going to do it?
Having said this, a revertant (from the experiment, not from the control) may contain no plasmid at all, and in this case you'll find out very quickly that it's a revertant because, when you'll try to extract and purify the plasmid out of it, you'll obtain nothing, no plasmid at all. A revertant may also contain a plasmid, but a 'random' plasmid, not one with the WT orange gene. In this case, you'll be able to extract and purify such plasmid but, if you'll do a restriction digestion, you'll notice that its restriction map does not look like the plasmids obtained from your other orange colonies. Moreover (and this is the 'ultimate' test), if you take such plasmid, and transform it into mutant (white) bacteria, it won't be able to complement the mutation--> no orange colonies.
I hope this helps.
Cheers
Pam
Hi Pam, I have a question about RFLP. Why would restriction enzyme sites disappear in one allele and the other doesn't in the first place?
ReplyDeleteThe technique works, but there's an assumption here that the gene of interest will contain restriction enzyme sites pattern that differs between the 2 alleles.
Is that true for all genes, or is it limited to certain genes only? If we can have one site disappear, couldn't there then be 2 sites, making it 3 fragments instead of always 2? Or is this site mutated so that the RE site disappeared in the first place?
Hi!
ReplyDeleteRestriction sites don't 'disappear'. The thing is, genomes are polymorphic, there is variability among individuals. Some regions of the genome have very small variability, so basically everybody has the same sequence there--same restriction sites in every individuals. In other spots, there is high variability...For instance, if we looked at a particular position on a chromosome, you may have a A and I may have a G. This has absolutely no consequences, except if we looked at the sequence around this site we may found that it is a recognition sequence for a restriction enzyme... for example, it may be GAATTC in your genome, but GGATTC for me. See that you'll have the A, that forms a cut site, but I have the G, so the enzyme won't recognize that site and won't cut.
The gene of interest typically DOES NOT contain RE patterns that differ between two alleles (although it can be the case sometimes, like for the globin gene, as you saw in lecture). Any RFLP that is close to a gene of interest can be used as a useful molecular marker. Potentially we could find useful RFLPs linked to any gene we want.
Also, the restriction pattern does not always need to differ between the 2 alleles of the gene.
Restriction sites are quite far apart, so it's less frequent that we have pattterns where 2 sites are polymorphic within the span of the same probe (remember, we are using Southern blots here...so we need probes).
I hope this helps. Please ask again in class tomorrow if you have more questions!
Pam
Hi Pam, I'm still confused about promoter-bashing and enhancer-bashing. Can you explain what are the differences between these two techniques? I tried googling and one of the websites says "Promoter bashing involves fusing a series of truncated promoter fragments to a reporter gene" What's a reporter gene? Also, when you do digestion in enhancer-bashing, how would you know that the part you are cutting up is the enhancer and not some other regulatory sequences?
ReplyDeleteThanks!
Well before you can start 'bashing' you need to define your enhancer or promoter. The 2 techniques are virtuallly identical...
ReplyDeleteA reporter gene is a gene that we can easily see if it's expressed (like the lacZ, or the gfp, or the luciferase gene).
We'll talk more about this topic today!
Cheers
Pam