Some Genetic Stuff....

[sps27]

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I was reading the chapter of Expression of Genetic Info (TBR), sub section on Genetic engg and cloning and had some doubts that I wanted to ask....

1) are we likely to get a DNA sequence and asked where the RI enzyme will make its cut? From what I've read it usually looks at a palindromic sequence and makes its cuts there. The book mentions enzymes like BgI 2, Hae 2, Hae 3, Hind 3 etc., enzymes (all for Ecoli) which look for specific palindromic sequences and make their cuts. How imp is all of this? I mean, it gets confusing after awhile to retain.

2) also, it seems like there is not 1 enzyme but many that clean up and check for errors after DNA replication. DNA polymerase 1 and 3 is mentioned in case of slippage or deletion of single bases, mismatch repair enzyme (looks for a GATC box sequence), and AP endonuclease is mentioned which hacks an AP site (meaning a site where a Uracil was introduced when Cytosine deaminated) in the sequence. Once again, it gets confusing. I wonder how much I need to know in all of this.

3) seems to me that exo-nuclease and endo-nuclease are generic terms used for a family of enzymes. Those that do repair work of DNA are termed exo-nuclease and those that digest DNA make cuts etc., are termed endo-nuclease. Is that understanding correct? Does RNA Polymerase 1 have exo-nuclease activity?

4) there is also mention of transcription + translation happening together esp., in relation to Trp Operon. So I think something like that can only happen in Bacteria where a ribosome is right behind an RNA Polymerase and tRNA molecules are available because there is no defined nucleus. Because I doubt tRNA molecules are lurking around in the nucleus of a Eukaryote. Right? I can see mRNA but not tRNA there? But then again, I could be wrong. Let me know. Thanks!

 

[mcloaf]

I can help I think with some of the info, but I took the MCAT two years ago so it's a little bit harder for me to say with certainty what you will and won't need to know.

1. You should definitely know the general mechanism of restriction enzymes, but I'd be pretty shocked if they expected you to know the sites where each one cuts. I guess you can if you have extra brain space, but IMO that's a waste of time.

2. You're right in that there are numerous mechanisms of DNA repair, but really there aren't that many. DNA polymerase has intrinsic repair activity which helps take care of errors as they occur during DNA replication. For single strand problems you have base-excision repair, nucleotide-excision repair, and mismatch repair. For double strand breaks you have non-homologous end joining and homologous recombination (and microhomology mediated end joining, if you want to get really specific). You'd be super well prepared if you knew a little bit about each of those, but I'm not sure if that's necessary. At the very least I would know that polymerases have intrinsic repair abilities, and that you have a couple sets of different mechanisms to repair single strand mutations and double strand breaks.

3. Exo- and endonuclease refers to where in the DNA the enzyme works. Exo (out) nucleases work by chewing away at the end of DNA strands, while endo (in) nucleases work by cutting within strands of DNA.

4. You're right. Transcription and translation can occur simultaneously in prokaryotes because the genetic material is not separated from the cytosol by a nuclear membrane. In eukaryotes the transcribed information must exit the nucleus before it can be translated because ribosomes are located in the cytosol.

 

[sps27]

I can help I think with some of the info, but I took the MCAT two years ago so it's a little bit harder for me to say with certainty what you will and won't need to know. .

I was unclear on exo and endo nucleases but reading your response has made me research that a bit more. Thanks mcloaf for your answers. I appreciate it.