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Genetics review

Link to genetics review. May be updated later.

Hardy-Weinberg equilibrium

Here is a link to the assignment.

Link to Pedigree and Karyotype exercise

Here is a link to a site at Stanford that helps you understand pedigree. If the link downloads a file instead of opening a page, you have to do the following: right-click or control-click the link and select "copy link." Then paste it into an address line and change the ending from "swf" to "html." Then it should work.
There is a short quiz you can take. I won’t see the results.
and here is the Karyotype activity
Below is a blog I wrote on the topics. You can use it as a reference if it helps.

Karyotyping

:
(as usual, images from wikipedia).
Karyotyping is a technique for looking at all the chromosomes in a cell. Briefly, a pro-metaphase cell is is stained with a dye that binds chromosomes. There the traditional dye, giemsa, stains the chromosomes in a banding pattern that is different for each one. So, the banding pattern of chromosome 1 is different from that of chromosome 2, etc. These days, there are specific color-coded probes that make it easier. The cell is then photographed and the picture is enlarged. Then, traditionally by literally cutting up the picture, but now digitally, the chromosomes are cut out and aligned so that we can see that the individual has 2 copies (one from mom and one from dad) of each of the 22
autosomes. The largest is chromosome 1 and the smallest is chromosome 22.
Karyotype
Then look at the last pair: The really tiny one, smaller even than chromosome 22, is the “Y” chromosome (so this is a male). The larger one next to it is the X.
Look how much cooler it is with the modern technique:
ColorKaryotype

As you can see, this individual is a female.
Using techniques such as this, we can look for large changes to the DNA, rearrangements of the chromosomes we call “inversions,” “deletions” (both self explanatory), translocations (where a piece of one chromosome is spliced to another), extra or missing copies of one chromosome (“trisomy” or “monosomy”) or, even whole extra sets of chromosomes.
Some common disease states result from this. Trisomy 21 causes Downs syndrome.
This can occur when the sister chromosomes of 21 do not separate at meiosis I (this is called “nondisjunction”). That results in a 2 gametes getting no chromosome 21 and 2 others getting 2 copies of it. If one of that latter group fuses with a normal gamete, the resulting zygote has 3 copies.
Check out this karyotype:
XXY
Do you see what’s wrong? This person has Kleinfelter’s syndrome, where he has 2 X chromosome and a Y.

Pedigree Analysis:


Here is the pedigree example given in the Campbell Book.
Widows
Females are circles, males are squares. People with the trait being followed usually are filled in with a color, people without the trait are not. This trait, the appearance of the “widow’s peak,” is a dominant allele on one of the Autosomes (not sex linked). How can I tell? Remember that the recessive allele can be covered up by a dominant allele. So, two people with the trait, if it is dominant, can produce a child without it. Notice that this happens in the mating in the second generation. Two “widow’s peak” people had a non widow’s peak child. You never see enough children to invoke statistical predictions in a generation. You should see only ¼ homozygous recessive in the 3rd generation…but there are only two kids. Even if there were 4, the numbers are not good enough to make predictions.
I know it is not on the X because an affected mother in generation 1 married an unaffected man and had an affected daughter. That would be impossible for a recessive allele on the X (the daughter would have to have 2 copies, one from each parent. But Dad cannot have it since he does not show the trait). I know it is not on the Y because it appears in women.

Here is the pedigree for hemophilia (a severe bleeding disease) in the royal families of Europe. The mutation seems to have occurred in Queen Victoria.
  1. Is this trait sex-linked?
  2. How do you know?
  3. How do we know that Irene (number “3”) is a carrier?
  4. Is it possible that Queen Elizabeth is a carrier?
  5. Could the disease show up in any of the children Prince William and Kate Middleton might have?
HemopheliaPedigree

Classes of Punnett squares

Here you will find the examples of the "classes" of Punnett squares. That is, how the alleles will assort depending on the parents genotypes.

More genetics stuff

Some variants in inheritance.


Multi-gene trait. There's really not much to this. Some traits…really most interesting traits…may be inherited, but be based on more than one gene. So, there is no single gene for being tall. There is no allele for being 6' tall. But, that doesn't mean that height is not inherited. Many different loci may contribute to it. Here is a hypothetical plot of what you might expect if there are three loci that contribute to height, each with two alleles where the dominant of each allele contributes positively to height (note, there is no reason any of those assumptions should be true). You might see a distribution that looks something like this:
Multigenedist

So, you can get a distribution of heights based on some combination of alleles at different loci. In this case, we are assuming simple additive interactions among the genes. It an be more complex (See below(.

Pleiotropy
This is sort of the opposite of multi-gene trait. Here, one gene can affect many traits. We've discussed this in the context of cytoskeletal proteins before. For example, mutations to a microtubule-associated motor protein could affect male fertility (sperm flagellum), airway function (cilia on airway epithelium) and vesicle transport and secretion of proteins. Everywhere that protein is needed, you would see some effect.

Epistasis
Finally, there is epistasis. Proteins interact with other proteins, so variations in one gene can affect how you see the phenotype caused by another. Here is a classic example I stole from another website at the university of Georgia. It covers coat color in.
Labrador retrievers. One locus, the B locus, controls the color of the pigment eumelanin. Eumelanin can be either brown in color (bb) or black BB or Bb.
Another gene, known as the "E" locus (for extensor…never mind) is needed to deposit eumelanin in the fur of the dog. It encodes a protein called MC1R and where it is expressed determine whether the eumelanin gets into the fur. The ee homozygote does not deposit eumelanin at all in the fur while Ee or EE do.
Thus, if the dog is ee, it will be yellow no matter whether it makes black or brown melanin. All the possibilities on the 4x4 matrix are shown below.

Epistasis



Problems for Genetics

There are several problems we will work on together. This first one is pretty straightforward.

Here is the
first one I want you to use.

The second one.

Aa third one
And one more.

Intro Genetics

This focuses mostly on terms we will use. Read More...

Answers to worksheet

I wrote brief answers (no diagrams) to answer the worksheet. You can find that worksheet here.

Types of mutations

Types of small mutations.


I discovered that I for genetic code lecture, I forgot to talk about the three types of small changes, mutations, to DNA. There are also large rearrangements like deletions, insertions (such as the type in exon shuffling that can lead to new protein functions). But, also there are small, single-nucleotide changes, such as the one cited in the sickle cell disease question.
Mis-sense mutation. This is where a nucleotide is substituted and results in the wrong amino acid being encoded in the protein. An example of this is the glutamic acid (GAG) to Valine (GUG). In the "THECATWASBADTHEDAYSHEBITTHEDOG" case, it might be "THE
BATWASBADTHEDAYSHEBITTHEDOG"
You can still read it, but the sense of it has changed.
Frame-shift mutation: this inserts or deletes one or two bases so that you are no longer reading in the correct frame. So, "THE
TCATWASBADTHEDAYSHEBITTHEDOG" after THE, you read TCA TWA….everything is messed up after that.

Finally, there is something called a nonsense mutation, which is when a stop codon is created. So, if the UUG codon for Tryptophan was changed to UUA, it result in the protein being terminated early. Note that the frame shift mutations usually result in encountering a stop in the new frame pretty quickly.

You can also have "synonymous" mutations: a change to the DNA that does not change the protein. For example since GUA and GUU both encode valine, that switch would not change the protein.

One more thing, I forgot to mention signal peptides. These are sequences of amino acids at the amino terminus of the newly made protein that tell the ribosome that the protein is destined to be secreted or made in the membrane. It shouldn't surprise you that, again, we need both the structural data and signals to tell the machinery what to do.

RNA Processing

General processing and splicing of mRNA
Read More...

Gene-to-Protein 1

NOTE: Reading change from what I said today: Read pages 325-334.


We are just beginning to look at how transcription and translation are performed. Step 1: how does the cell know where, exactly, does a gene begin.
Answer, there are proteins binding mainly in the major groove that can recognize (“read”) sequence. Read More...

RNA Processing

RNA Processing

Below is my presentation slide from the day:
processing1All Most of what I’ll be talking about here occurs specifically in eukaryotes.
As usual, most images are from Wikicommons.
Following transcription, the “pre-mRNA” must be processed on its way out of the nucleus to the cytoplasm.
First, notice that the sequence that made up the promotor does not end up in the pre-mRNA. This is a general theme: With each step in the DNA→RNA→Protein pathway, the information needed to specify regulation of each step generally is lost as we go to the next step. This makes sense, since it is no longer needed.

Poly-A tail and GTP cap.


When the RNA polymerase reaches the end of the sequence that’s supposed to be transcribed, it hits a signal called the “polyadenylation signal.” The sequence in the DNA (coding strand) is 5'-AATAAA. Of course, the polymerase is reading the template strand, so you could say the “signal” really is 3' TTTATT. The sequence varies a fair bit. This signals the RNA polymerase to leave the DNA. The sequence now in the pre mRNA (AAUAAA, or something similar) recruits a protein complex that will cleave the mRNA near the 3' end and an enzyme called PAP, for Poly Adenyl Polymerase, uses ATP to add a long series of A’s to the end of the mRNA. These A’s, and there may be hundreds of them, are NOT encoded anywhere.
The utility of the tail seems to be in recruiting several poly-A binding proteins that protect the mRNA from nucleases that degrade it, facilitate the next steps in processing, the transport of the mRNA out of the nucleus to the cytoplasm, and regulation of translation.
Some prokaryotes do a version of poly-A tail also.
The other initial change is the addition of the GTP cap. Actually, it’s a modified G with a methyl group on position 7 of the base, cleverly called 7-methyl GTP. Wiki has a
short discussion of the mechanism of transfer. There is a specific enzyme that does the capping and the cap looks something like this:
GTPCap

Note the “inverted” 5'-5' link. The cap structure also protects the end of the mRNA from degradation.
RNA splicing:
We will only touch on this here. It’s sort of a favorite of mine so we will have a lecture on it next. The pre-mRNA has regions called “introns” (for “intervening sequences” with a cool “tron” ending), and Exons. The exons encode the protein and are what end up in the final mRNA. The introns are spliced out. We will talk about the mechanism and the implications later.
The final processed mRNA might look something like this:

processed Transcript
It comprises the coding sequence, the cap, the 5' and 3' “untranslated region” (UTR) and the poly-A tail. I think all the names are pretty self-explanatory. The 5' UTR, in particular, will contain sequences that contribute to regulating translation. Only the green stretch above will make it into the protein.



RNA Splicing part 1


Take a look at
this video which is presented by Cold Spring Harbor Lab, where I used to work years ago and where some of the work I discussed today was done.

Two benefits of splicing


While no one believes that RNA splicing evolved
because of these benefits, these are real benefits organisms now enjoy because of it.
  1. Alternative splicing: Not all the exons are included when splicing of specific mRNAs takes place. Different versions of the mRNA may be formed. In the example I gave earlier, exons 15-19 could all be joined up in sequence, or, exon 15 could be spliced directly to 19, leaving out 16, 17 and 18. This results in different versions of the protein, with different functions. The domain structure of proteins makes this possible.
  2. Exon shuffling: Also because of the domain structure of proteins, it is possible to add exons to genes via recombination and create different versions of the protein. Any chunk of DNA that has portions of introns on its ends that then is spliced into another intron will do no damage to an existing gene (It can be spliced out at the mRNA level). But, it also provides the possibility, through alternative splicing, to evolve proteins with new functions.
Key words:
Spliceosome: (there’s a good descriptive name) It’s the assembly of proteins and RNA that carry out splicing.
snRNPs (pronounced “snurps”). These are “small nuclear ribonuclear proteins.” They are the components of the spliceosome.
snRNAs: small nuclear RNAs are the main catalytic components. It is the RNA that carries out the reaction. They have names like “U1,” and “U2.” etc.


Self Splicing:
The story about how RNAs got spliced became more obvious when self-splicing RNAs were found. I mentioned these today.
The general reaction looks like this:
Splicing
There are two, successive trans esterification reactions that occur in a concerted way and use energy from an additional GTP that is brought in by the complex. The first takes a branch point sequence near the 3' end of the intron. The 2' OH of that branch point attacks the 5' exon/intron boundary. This splices the intron into a lariat and frees the 3' end of the exon, which then attack the 5' end of the next exon, resulting in the spliced exons and a lariat structure of the intron.
In the self-splicing form, “internal guide sequences” form base-paired structures that hold the players together and facilitate the attacks (which usually involve that 2' OH in an intermediate.
You can see below how the stem-loop structures formed using base pairs, then can fold into three-dimensional structures, facilitating catalysis. Yes, I know, it should have been obvious.

Self3

You can see how the base pairing can be used to bring the active sites together in more detail here. By the way, these last two images are used without permission. The one below is from the Molecule of the Month blog/discussion and the one above is from Trends in Ecology and Evolution.
Closeup

Normal mRNA splicing (non self-splicing)


The story is not all that different in the splicing of mRNA. There are guid sequences and a branch-point lariat. The chemistry varies a little as to who attacks whom when and where. But, the main difference is that the sequence of the intron is not that important for the structure to work. Instead of the intron folding into a complex structure, the separate RNAs in the snRNPs form those structures and then bind the specific sequences in the intron and at the intron-exon borders.