DEVELOPMENTAL GENETICS AND ENHANCER TRAPS

What do you need to remember about developmental genetics?

1) Different cells, and cells in different positions, are different because they express different combinations of genes.

2) How do cells end up expressing different combinations of genes? There are 2 main reasons: 

a) they inherit different cytoplasmic determinants (for example, if in a cell a particular protein is not evenly distributed throughout the cytoplasm, the 2 daughter cells will inherit different concentrations of this protein). In the special cases of embryos that at some point are like the Drosophila one, with many nuclei in a big communal cytoplasm, if a morphogen is distributed unevenly  in the communal cytoplasm, then nuclei in different regions will be exposed to different concentrations of the morphogen. The net result of inheriting, or being exposed to, different concentrations of morphogens is that different genes will be expressed at different levels;

b) cells communicate with each other and with their environment; cell-to-cell communication, and cell-to-environment communication is transduced into signalling cascades, resulting in expression of particular subsets of genes.

3) How can we have uneven distribution of an RNA or of a protein in the cytoplasm? Again, there are a number of ways. We can anchor the RNA to a particular spot, so that when it is translated, the protein product will diffuse from this source and thus form a concentration gradient. We can also constantly, actively transport the RNA or the protein product to one region of the cytoplasm. We can let the RNA get evenly distributed, but have a protein, localized in one specific region and forming a bit of a concentration gradient, that prevents its translation. This will create a 'sink' and therefore a concentration gradient of the protein product of our RNA.

4) Remember maternal effect genes! Remember that at the earliest stages, a zygote does not transcribe/translate its own genes. It 'lives off' the products that the mother dumped in the egg's cytoplasm. 

FOR ENHANCER TRAPS

Here are a few links that, are better than any explanations I gould give, and also give lots of example of how people use these great tools:

http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/Def-E/enhancer_trap.html

Example of uses (just read the abstracts to get an idea!):

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-3YXJ34N-H&_user=1022551&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=965372155&_rerunOrigin=google&_acct=C000050484&_version=1&_urlVersion=0&_userid=1022551&md5=61b5e2743be61fd22e71815ae982073f

http://www.ncbi.nlm.nih.gov/pubmed/8049476

Promoter traps (have a look at the abstract):

http://www.pnas.org/content/103/47/17813.full

Transposable elements

What do you need to know about transposons?

Transposons may insert into a gene, thus disrupting it/interrupting it. Effectively, they can therefore be mutagenic.

Transposons do indeed 'jump', but they can typically do so only in a short window of time.

'Jumping' is less frequent than 'not jumping'.

When we deal with transposons, we introduce a significant amount of randomness...

You need to remember that a completely functioning transposon must have at least intact insertion sequences (inverted repeats) and a functioning transposase gene.

Examples of transposons are the corn Ac element, Drosophila P element, and many others. Although they all work based on the same principles, don't mix them up. (Flies also have a whole bunch of transposons with interesting names: hobo, gypsy, nomad, tyrant, and so forth).

There are mutated transposons that can't produce transposase anymore. In order for them to transpose (i.e. 'move'), another source of transposase is required.

Other mutated transposons can't move anymore because their IRs are not intact. However, they can still produce transposase.

You should be able to predict phenotypics frequencies in the offsprings of crosses between individuals that carry different versions of a transposable element (like in your Ac/Ds question).

You should also be able to diagnose the presence of a transposable element... remeber:

mosaicism ('patches'), a high mutation rate, and a high reversion rate are all indications that there might be a transposable element involved.

You will also need to know about hybrid dysgenesis and how to use P elements to make transgenic Drosophila and to clone a Drosophila gene: there is a handout on the 'parallel' 335 page.

PLEASE POST HERE ALL YOUR QUESTIONS ON TRANSPOSABLE ELEMENTS!

ASSEMBLING CONTIGS AND SEQUENCING GENOMES

Post all your questions about genome sequencing and about contigs under this post!

Also note that, on the 335 resource page (link on the right) there are now a couple fo additional questions to practice assembling contigs, and an explanation on how to go about solving the RFLP question with the FCD.

Cheers

Pam

PS: remember that Friday (tomorrow) it's 'spring break'!

PROMOTER AND ENHANCER BASHING

If you have any questions regarding promoter bashing or enhancer bashing (or, how to find an enhancer) please post them here!

A 335 LOPPIN_ADAPTED QUESTION

This is a modified version of a question that was in the previous editions of the book.

The loppin 'ant' gene has been cloned. We know that it is only expressed in the antennae of female loppins.

Propose a strategy/experimental procedure to identify and define the regulatory regions that confer this expression pattern to the gene (that is, the regulatory regions that are necessary and sufficient to make the gene only expressed in females' antennae).

Have fun!

Pam

UP TO THIS POINT...

... remember that there are 2 big parts to 335:

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).

FOR THE WEEKEND...

Make sure you enjoy the sunshine!

If you feel like working on BIOL335, you can start with probelm set #4. You'll have to look up some of the techniques in your book. You may also find some help on the techniques page and on jove. On jove, you'll have to type in the technique you're looking for.

IMPORTANT: MONDAY WE'LL MEET AT 1.45 IN ROOM 2449

How do you get a plasmid into E. coli? Here is a video!

And here is how you get the plasmids out of the cells.

FRIDAY JULY 3RD

On Friday our tutorial will still be at 2.30pm. I'll have two sets of lac operon practice quesitons for you, and we'll briefly go over biosynthetic operons. Also, bring your problem set 4! You'll have enough background to start working on it.

Cheers

Pam

PS: we won't move the tutorial to the morning.

THINGS TO KNOW FOREVER...

Here are a few things that we touched on last Tuesday, and that you should always remember:

- genes have promoters

- transcription does not start at the AUG, and is not stopped by stop codons! (The RNApolymerase does not know the genetic code, it just transcribes until it hits a "transcription termination" signal)

- plasmids can exist as independent "mini-chromosomes" inside bacteria, or they may be integrated into a bacterial chromosome

- plasmids may carry, in addition to all the plasmid genes, some bacterial genes.

FOR THURSDAY AND FRIDAY:

we'll work on prokaryotic gene regulation (two main examples), bring your textbook and problem set#2!

Much more info on prokaryotic gene regulation will be posted in the next couple of days. For more info on the lac operon ...there are links on the right!

Cheers

Pam