Anonymous said: What would happen if you overdigest your probe with DNase I? What would you see on the gel? Would you see multiple small bands since the molecule is not cut only once now?

There would be so many small bands that it all becomes kind of blurry. 


Image from here. I think its MNase but I think that the same thing will happen.

Anonymous said: Pol I and Pol III complexes are much more stable than those for Pol II-why?

Disclaimer: I did not learn this in class.

That said, RNA polymerase II differs from the other two in that it has a CTD, or C terminal domain. The CTD prevents degradation and it consists of many repeats of the same protein pattern. If the cell lacks those repeats and only has a few, well, stuff isn’t going to work out for the cell. In order for elongation to occur, this CTD has to be phosphorylated. However, when the Pol II is hyperphosphorylated, or all its binding sites have been phosphorylated, this protein called the von Hippel-Lindau protein comes along and marks it for ubiquitination. 

If you have any other questions, feel free to ask, or check out these links.


von Hippel-Landau protein

importance of CTD

CTD repeats

this is a nice overview (with pictures!) of the different types of RNA polymerase

Anonymous said: What is the difference between DNase and MNase? What is the difference in how they cut around nucleosomes?

Disclaimer: I learned this all from the Internet and have never covered this in class.

That said, 

DNase cuts DNA and MNase cuts linker DNA between two nucleosomes. MNase is a type of DNase. MNase cuts mostly AT base pairs. It can induce double-strand breaks within nucleosome linker regions, but within the nucleosome, it can only induce single-strand breaks.

DNase cuts its sites in 10 nucleotide intervals, starting from the 5’ end. 

If you have anymore questions, hit me up!  

Here are links I used (or further information):


The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

DNase-sensitive sites in Nucleosomes: 

Precise location of DNase I cutting sites in the nucleosome core:

(Reblogged from starchythoughts)
The MEK pathway and osteosarcoma.

The MEK pathway and osteosarcoma.

Radiation is a well-documented aetiologic factor, being implicated in approximately 3% of osteosarcomas. An increased incidence is likely to be seen, as more patients survive long enough after primary irradiation to develop this complication. The interval between irradiation and appearance of osteosarcoma ranges from 4 to more than 40 years (median: 12-16 years). Osteosarcomas have also been associated with the use of intravenous radium 224 and Thorotrast (diagnostic radiocontrast agent). Exposure to alkylating agents may also contribute to its development. Approximately 2% of patients with Paget’s disease develop osteosarcoma and cases of osteosarcoma in patients older than 40 years are associated almost exclusively with this premalignant condition. Other conditions associated with an increased risk of development of osteosarcoma, are solitary or multiple osteochondroma, solitary enchondroma or enchondromatosis (Ollier’s disease), multiple hereditary exostoses, fibrous dysplasia, chronic osteomyelitis, sites of bone infarcts and sites of metallic implants for benign conditions. The incidence of osteosarcoma is increased in several well-defined hereditary disorders associated with germ-line alterations of tumour suppressor genes. By far the strongest genetic predisposition to this disease is found in patients with hereditary retinoblastoma (germ-line mutation of the retinoblastoma gene RB1 on chromosome 13q14), where osteosarcoma occurs 2000 times more frequently in the skull after irradiation and 500 times more frequently in the extremities than would be expected in the general population. The Li-Fraumeni syndrome (germ-line mutations in the p53 gene) is associated with a 15-fold increase. Rothmund-Thomson, Bloom and Werner (adult progyria) syndromes are also associated with an increase in osteosarcomas.


  • Table 1: Osteosarcoma subtypes within central and surface tumours CENTRAL (MEDULLARY)
  • a. Conventional high-grade central osteosarcoma
  • b. Telangiectatic osteosarcoma
  • c. Intraosseous well-differentiated (low-grade) osteosarcoma
  • d. Small cell osteosarcoma
  • a. Parosteal (juxtacortical) well-differentiated (low-grade) osteosarcoma
  • b. Periosteal osteosarcoma - low- to intermediate-grade osteosarcoma
  • c. High-grade surface osteosarcoma
(Reblogged from stratum-lucidum)

This song thing might be a trend??? Song is relevant since p53 and ubiquitin get all close? Sais pas. Here goes.

p53 and mdm2

Break it down. I have three major ideas here: p53, mdm2, ubiquitination.

Let’s start with ubiquitination. More like u-be-quitting-nation amirite? Yes, thank you, I am aware that my jokes are terrible and I should stop using party metaphors for everything.

Ubiquitin is everywhere. Like actually, it used to be called ubiquitous immunopoeietic polypeptide. A mere 76 amino acids, this highly conserved protein does ALL the things. One thing it does is ubiquitination, which is a type of proteolysis, which is breaking down proteins into amino acids, which is done by enzymes. See, when a cell makes proteins, it knows they’re going to go out of fashion or get degraded some time. How does it tell the proteins this in a nice way? It sends the fashion police. Not really. It attaches ubiquitin to the protein. 

This force tag teams it out. The E1 enzyme activates ubiquitin to start (it uses ATP to attach ubiquitin’s tail to its own cysteine amino acid). Then, it hands off the metaphorical baton to E2, which works with E3 to recognize obsolete proteins and attach ubiquitin to them. 1 ubiquitin works as signaling, 4 or more mean imminent degradation. The 4 or more ubiquitins drag the protein and drop it in the 26S proteasome. Don’t be fooled by the pro-tea-some. Its name looks like it is all for drinking warm beverages under a blanket while cuddling, but it will rip you apart. Like, literally, into chains of about 8 amino acids. 

It works like a paper shredder. The ubiquitin is a ‘key’ that attaches to the regulatory particles to open the proteasome and dump in the protein, where active sites on the inside will chop it up. 

Where does p53 come in to this? It’s one of the proteins that gets dumped in. Don’t worry, this one is important. 

p53 is a tumor suppressor gene. The majority of cancer cells have mutated copies of p53. However, this mutation is recessive, so both copies have to be faulty. When there is DNA damage after G1, the cell halts and the cell tried to repair itself with BRCA2, or if the damage is too extensive, goes through apoptosis. When there is no cellular stress, mdm2 ubiquitinates p53. Mdm2 is an E3 ligase. This works in a negative feedback loop: p53 allows for the transcription of mdm2 during normal cell function (low p53 levels), and mdm2 ubiquitinates p53 to keep the levels low. When the cell is stressed, mdm2 doesn’t ubiquitinate the p53 and the cell cycle stops. What what prevents mdm2 from binding? Here’s a few ways that p53 is activated:

  • phosphorylation (word on the street is this might not actually activate shit) - kinase phosphorylate in response to radiation, DNA virus infection, and the like. Once activated, p53 doesn’t care about any phosphates, so that’s why the phosphorylation might not be crucial to degradation, but rather, regulation. The phosphate groups bind to at Ser 15 and Ser 20, which repeals interaction.

  • acetylation - unacetylated p53 can induce the feedback loop, buut the acetylation stops the mdm2-mediated repression. The acetyl groups are attached at the same lysine residues as ubiquitin, so p53 cannot be degraded by mdm2.

  • tumor suppressor p14ARF binds to p53 to prevent mdm2 interactions and is all like swiper no swiping . It stabilizes the p53 and possibly degrades the mdm2.

  • and then mdm2 can self -ubiquitinate to keep levels low. yup.

k, final picture

Recap picture time!

Activating the p53 causes cell cycle arrest; degradation, on the other hand, allows for the continuation of the cell cycle. Several post-translational mods affect p53, such as acetylation, ubiquitination, phosphorylation, and enzymatic processes. 

(Source: Spotify)

Got our tests back today… uh yeah nowhere to go but up, I guess.

This weekend, I WILL finish typing up all the notes. I swear. I have to. I’ll also start a big picture post, and therefore, my poster. 

Wrapped up some presentations, learned about the APC, PLKs and other stuff. I’ll use pictures from the presentations in my class later on.

The audio is relevant, I swear. I mean, S phase and initiation is all about the Macklemore x Ryan Lewis. But first, in late G1, mitogenic signals lead to E2F transcription.

Then, the party starts! That was a cue to start playing the audio file. The PRC (pre replication complex) gets down on the floor. But, see, it’s like that gaggle of girls who move as one, like those fish in finding nemo. 

There’s like no difference there, I swear. Let’s start. 

S phase promoting factor accumulates to signal this start, then breaks down at the end. The ORCA (origin replication complex [this is an E2F target gene]) binds to the origin on the DNA. Then the cdc6 binds to the ORC. This picture is kind of misleading; the cdc6 makes a sandwich. Here’s my notes (I tried). And, yes, I did indeed photoshop my notes.

The cdc6 the recruits cdt1, a replication factor, to get them all beverages or something. Then cdc6 also gets MCM proteins(mini chromosome maintenance), all 6 of them, to bind to the DNA. Since MCM is a helicase, they start unzipping DNA’s genes. (You seen what I did there? I had to work that line in there.) Cdc45 then piles on top of this whole thing. Lastly, cdc7, alias cyclin A because cdc7 is just too famous to go partying, turns out to be a kinase and - gasp - phosphorylates MCM, which activates it. This GINS/SING/INGS/GNIS protein thing then comes along and binds. RNA polymerase also shows up since it ain’t DNA rep without an RNAP. 

Time for the DNA replication! Cyclin A aka cdk2 aka cdc7 phosphorylates cdc6 and cdt1, then kicks them off the ORC. Wait why is cyclin A being a killjoy? See, DNA replication only happens once a cycle or things get whack. Disassembling the PRC is the way to go. This the part where the parts of the PRC start singing the chorus of the above song (ORC can’t hold them fer sure). 

There’s two ways of making sure the PRC is gone for good: nuclear exclusion (ban them from the club/nucleus) or degradation (no metaphor here since cyclin A ubiquitinates them). Cyclin A phosphorylates the ORC to prevent cdc6 and cdt1 from binding again, then ubiquitinates them, too, for good measure. MCM is no exception - it get kicked out of the nucleus. 

Also: cyclin A is an E2F target gene. At this rate, you’d think E2F just does everything.

(Source: Spotify)