Last updated:

08/18/98

Designed by:

Davis's Ent.

Read the DISCLAIMER before proceeding.

Genes and Heredity

Garrod - This dude identified the first genetically based human disease, sometime in the early 1900’s.

It’s symptoms included black urine (uhhh… that’s pretty disgusting), which is caused when alkaptons are oxidized when exposed to air. The alkaptons got there because who ever had the disease lacked an enzyme that was needed to break down alkaptons. He also hypothesized that the disease was inherited. Of course, everyone thought he was nuts.

Decades later, they figured out that he was actually right. This disease is called alkaptonuria.

Scientists traced it down to a gene that dictates the production of the specific enzyme that breaks down alkaptons. It is an autosomal recessive disorder that followed Mendel’s laws of inheritance. Descendents got the disease

Beadle and Tatum (1941)

These dudes, working again, after finding their mutants in Drosophila started looking for mold in bread. (Pretty fun job, eh?) Just like the white eyed male mutant flies before, they looked in Neurospora, hoping to find other mutants that would back up their work.

Normal "wild-type" Neurospora survives only when it’s got enough food to eat… without these essential nutrients, it’ll die. So, they tried to grow the stuff on MINIMAL medium, stuff that had the absolute minimum of stuff, hypothesizing that any mutants wouldn’t be able to survive because they wouldn’t have certain key enzymes to live off of such small stuff. SO, any mutants that didn’t survive, they took to another table and tried to give them back each nutrient that the medium lacked from the COMPLETE medium one by one, until they found what ever it was they could live on. (e.g. argine, tryptophane or uracil)

Any mutants, etc. were called auxotrophs (auxo means "to increase", troph means "nutrients")

As a result, the one gene à one enzyme theory developed. Over the years, this was revised to one gene à one polypeptide, because not all proteins are enzymes. This tied changes in the DNA to changes in specific enzymatic activities, which in the end, changes genotype or phenotype.

Sanger (he da MAN!)

This guy determined the amino acid sequence of proteins. (Don’t have to know how he did it… he just did… and got the Nobel Prize for it too. If you actually wanna read about it, go to page 378)

He used this method to show that sickle cell anemia is actually caused by a change in one amino acid. In the protein sequence (polypeptide chain), one amino had been changed and was different than what was SUPPOSED to be there.

Later, it was shown to be one change in the DNA’s nucleotide sequence (as predicted by the genetic code)

Central Dogma of Molecular Biology (KNOW THIS!!!)

DNA à RNA à protein

The whole point is that the DNA controls ALL! It makes the RNA that in the end makes the proteins that control what and who YOU are.

now DNA ß RNA also known (reverse transcription by viral transcriptase)

Note that there are also RETROVIRUSES that are RNA that can actually RE-ENGINEER your DNA via a viral "reverse transcriptase". This is how HIV works, and it really fucks everything up!!!

RNA Molecules

differences between DNA and RNA

The big difference between deoxyribose and ribose is the deoxy- part of it. DEOXYRIBOSE is missing an oxygen atom from on part of the pentose, so it’s just a lone H instead of a complete OH group.

Another difference is that Uracil replaces Thymine in RNA. These two nitrogenous bases are also very similar in structure, except that one carbon on Thymine has a methyl (CH₃) group and Uracil has a single hydrogen.

types of RNAs

mRNA - messenger RNA, has coding sequence for protein(s) (for example: UUGCTAA)

tRNA - transfer RNA, carry activated amino acids to ribosome (The tRNA carries specific amino acids on one end and an anti-codon that latches on to the corresponding codon on the mRNA)

rRNA - ribosomal RNA, part of the ribosome (Remember this from Masaracchia? These come out of the nucleus, etc. Right? These sites are where the mRNA and tRNA stick together)

snRNA - small nuclear RNA, involved in splicing out introns (more on the intron / exon thing later… don’t u know?)

Transcription (RNA Synthesis)

RNA polymerase, transcription bubble

A big thingy type of enzyme called RNA polymerase swoops in and starts to open up the dual stranded DNA. It’s a lot like DNA replication, and uses the rNTPs (remember? UTP, CTP, ATP, and GTP). There’s an ori (or transcription bubble) that opens up, and the RNA polymerase comes in and starts chuggin’.

What’s cool is that the mRNA just kind of peels of as it’s made. In the wake of it, the DNA winds back together automatically

What’s even COOLER is that multiple mRNA polymerases can work at the same time on the same strand of DNA (I think…)

promoter (where the mRNA binds to DNA and start transcription)

Certain sequences of DNA act as promoters, where the RNA polymerase can hook up with the DNA and start making all that fun RNA. TRANSCRIPTION occurs (that’s when the message is just kinda copied… just a different handwriting… see?)

5’ à 3’ direction of synthesis à (This is the way we make RNA… make RNA… make RNA… all through the morning…)

uses ribonucleotide triphosphates, does not proofread, no primer required

That’s right. CTP, ATP, GTP, and UTP (these are the rNTPs like I said before!)

It doesn’t proofread (the polymerase doesn’t give jack if it screws up…)

Plus, you don’t need a primer. (it’s in the 5’ to 3’ anyway… no OKAZAKI frags…)

complementary pairing with DNA template (anti-parallel)

In other words, the DNA is the RNA’s template and they are ANTIPARALLEL (RNA is made in the OPPOSITE direction. The RNA is 3’ à 5’)

primary RNA transcript (generally processed later; exception - bacterial mRNAs not processed)

OK… so you’ve got an ugly little pre-mRNA type thing after transcription. You still gotta process it! It’s got a lot of blank stuff that doesn’t even code for anything. You want to take out unneeded material called INTRONS and keep the EXONS. So, the genes are spliced, and you get the REAL mRNA. COOL, huh?

Translation (protein synthesis) (kinda like translating English to a foreign language… let’s say, Filipino! J )

ribosomes are RAD

rRNAs and proteins

ribosomal RNA or rRNA is actually 60% RNA and 40% protein (believe it or not)

small and large subunits

made up of large and small, they come together as soon as they see mRNA and tRNA on the way

mRNA (the messenger RNA)

codon - don’t ya know? It’s an mRNA base triplet. Each triplet codes for one particular amino acid. The sequences of these determine the sequence of the polypeptide chain.

AUG - called the "start" codon, it ALWAYS begins mRNA molecules and codes for a methionine to come in and start everything up.

ribosome binding site (RBS) - the ribosome actually has TWO sites where tRNA (the ones that bring in the amino acids) come in and match up with the mRNA (the sequence from the DNA with codons)

There’s the P Site or Peptidyl-tRNA binding site, which is where the current tRNA is letting go of its protein for the protein to hook up to the ever growing polypeptide chain.

Then there’s the A Site, or Aminoacyl-tRNA binding site, where the next tRNA, amino acid, and anti-codon, is just itching to go get ‘em.

UAA, UAG, UGA à stop translation -- These three codons don’t actually code for a tRNA and amino acid as others normally do, but instead, they code for a protein release factor. This comes in and tells the growing to stop, so there’s no more stuff to add on, etc. SO STOP THAT ALREADY!!!

read mRNA 5’ à 3’ --- uhhh… yeah… mRNA is read in the 5’ to 3’ direction. OK?

reading frame (significance, how set, what if changed accidentally)

Oh yeah… this is a REALLY big deal… Let’s say you had the sentence: "The Red, mad Filipino killed the BOOM-BAY". If you jumbled all the letters together, you’d get "theredmadfilipinokilledtheBOOM-BAY". You might think that the first word was THERE, but it’s NOT! That’s what happens when you have a FRAMESHIFT. Normally, codons are read in "3 lettered words", like UGA, TTT, or CAC. If something happens to alter the reading frame, the sequence UUU-GGG-AAA could be read UUG-GGA-AA? (missing the first U) Now that would screw EVERYTHING up, wouldn’t it?

tRNAs or transfer RNAs… JUST WHAT ARE THEY? They’re adapters, "converting the nucleotide sequence of codons to amino acid sequences"

Everyone knows this concept by now, right? These tRNA molecules play a key role in creating the polypeptide sequence.

They’re actually made of real RNA sequences with the loop type things that keep the whole structure together in the 3 dimensional form

The lowest loop has three nucleotide bases that are collectively the ANTICODON that pairs up with the CODON on mRNA.

The amino acid hooks up on the 3’ end, while the 5’ end… well...is just kinda there I guess… (there’s a picture on page 307… go stare at it if you’re bored - the simplified structure (letter "c" on the diagram) actually looks like a stick of dynamite. GO LOOK if you don’t believe me!!!)

Each tRNA brings in a different amino acid (kinda… if you don’t understand what I’m talking about, don’t worry about it)

The tRNA picks up amino acids from the cytosol (the cell is just jam packed FULL of them!!!). But they just don’t instantly latch on. That’s what "Aminoacyl-tRNA Synthetases are for!!!

activating or tRNA charging enzymes, Aminoacyl-tRNA Synthetases (20 different, 1 for each amino acid, use ATP for NRG)

Well, this is actually a whole phreakin’ FAMILY of enzymes - 20 different ones (one for each amino acid hookin’ up to a tRNA)

FIVE STEP PROCESS:

1. The active site of the enzyme binds the amino acid and an ATP molecule (it needs NRG!!!)

The ATP loses two phosphate groups and joins to the amino acid as AMP (adenosine MONOphosphate)

The appropriate tRNA covalently bonds to the amino,

Displacing the AMP from the enzyme’s active site.

The enzyme the releases the Aminoacyl-tRNA, the "activated" amino acid.

2. anticodon (tRNA region which pairs w/ codon)

Yeah… whatever. You know this, right? The tRNA anticodon pairs with the mRNA codon, bringing in the amino acid that it’s supposed to have… OK?

Decrypting the GENETIC CODE

triplet (4³ or 64 possible codons), non-overlapping code -

since there are 20 amino acids and 4 different bases, there had to be 64 possible codons (4 to the third). Why? 4 ² is not enough to cover it all… Plus, none of them overlap – meaning that one codon codes for only ONE amino acid (not multiple amino acids per codon)

degeneracy (define/explain), more than one codon / amino acid

Degeneracy or "wobble" as the book calls it (funny name there, eh?) is the fact that you’ve got extra codes that you can use up (You only needed 20, and you’ve got 64!!!) So, multiple codons can stand for one amino acid. That way, if there’s any accidental mutation, etc. most of the time it won’t do jack, because it’ll still code for the same amino acid (check the chart on page 303 of you still don’t get it…)

61 codons call for amino acids, but the AUG codon is used for methionine and a "Start" signal - so proteins start with methionine as first amino acid

There are 3 stop or "nonsense" codons for stops (there are no tRNAs for these, just a protein release factor). They are UAA, UAG, UGA

universal code - What’s pretty cool is that all this codon stuff is actually more or less universal for all organisms - they use nearly identical codons for the same amino acids. THAT ROX!!!! (kinda… I guess… well… maybe not…)

mitochondrial / chloroplast changes in genetic code usage (also, note prokaryotic nature of organelle gene expression and theory to explain), antibiotics can harm organelle gene expression – why?

This one’s a little tough, so listen up. It’s theorized that mitochondria (as well as chloroplasts, were actually bacteria from like along time ago that got trapped within our own cells (can you say SYMBIOSIS?) Well, they’ve got their very own DNA and do a lot of stuff on their own.

reading frame - like I said before, know the meaning of the term , and that it’s just set by the start AUG codon

mechanism of protein synthesis (don’t YOU feel like making proteins TOO?)

Initiation (ribosome, mRNA, initiator met-tRNA, protein factors, AUG, ribosome binding site – identifies start AUG codon and interacts with small sub-unit rRNA)

Ok.. this is where it all begins. You first have to get everything together à the mRNA comes in and binds with a small ribosomal sub-unit and a special tRNA initiator. Everything starts at the 5’ end of the mRNA sequence, at a particular sequence of mRNA.

Down stream, there’s the awaiting AUG codon that says, "Come on!!! Let’s make some proteins already!!!" So, when the timing’s right, the special tRNA initiator brings in the methionine amino acid. The large ribosomal sub-unit then hooks up and you’ve got a functional ribosome. Some tRNAs are constantly being fed into the whole thing. GTP provides NRG for the initiation process.

Elongation - basically, the polypeptide sequence gets longer as more stuff is processed.

Codon Recognition - The mRNA codon waiting in the A site of the ribosome forms H-bonds with the incoming tRNAs anti-codon, carrying the appropriate amino acid. An elongation factor (a protein) pushes the tRNA into the A site. This also requires the hydrolysis of a phosphate bond from GTP.

Peptide Bond Formation - A little thing in the big ribosomal sub-unit rushes the two amino acids along and says, "Hey… I don’t got all day! MAKE A PEPTIDE BOND ALREADY!!!" So, they do. The POLYPEPTIDE SEQUENCE that is on the tRNA in the P site moves itself to hook up with the one amino acid on the tRNA in the A site. So, it just got longer.

Translocation - The tRNA in the P site just kinda floats away. It doesn’t have any business in there any more. The tRNA in the A site is translocated to the P site where the other one WAS. As the tRNA moves around, it is still hydrogen bonded to the mRNA, so they two move forward as a unit. This movement is fueled by yet another GTP molecule. The movement is in the 5’ to 3’ direction. Then, it starts all over again.

Termination - "It’s all over… THEY IN TROUBLE…"

By now, it’s all over. Elongation goes on until it hits a stop (UAA, UAG, or UGA).

A release factor protein binds directly to the termination codon in the A site. This causes the ribosome to add a water molecule to the growing polypeptide chain. This reaction hydrolyzes the completed polypeptide chain from the tRNA in the P site, freeing everything up, and letting everything go. THE END!

poly(ribo)somes - This just means that you can have multiple ribosomes on a single mRNA. This occurs in both eukaryotes and prokaryotes. This allows proteins to be made faster.

coupled transcription / translation - This occurs in prokaryotes only. Their DNA doesn’t have all the intron / exon crap that we eukaryotes do. (There’s no room for it in them anyway!) Since there is nothing to splice out, you can go straight from transcription to translation. The mRNA peeling off of the DNA in prokaryotes can go immediately hook up with their ribosome friends (polyribosomes, if you want to!)

Diphtheria toxin – inactivates translocase in eukaryotes – DO WHAT???

RNA processing in eukaryotes - now THIS is complicated stuff…

introns and exons - when pre-mRNA peels off of the DNA, there’s a bunch of extra crap in there that doesn’t really code for anything. So, you gotta take them out. You WANT to keep IN the EXONS, but take OUT the INTRONS. (introns removed to make mRNA from primary transcript)

capping of eukaryotic mRNA on the 5’ end - when mRNA is first formed, a modified version of a guanine (G) nucleotide is attached. This seems to serve two important functions:

It helps protect the mRNA from hydrolytic enzymes that will destroy it instantly.

It is the signal sequence I was talking about earlier when I was talking about the small ribosomal sub-unit "attach-to-me" sign.

poly A tail on 3’ ends of eukaryotic mRNAs - On the OTHER end, when it’s all done, anywhere from 30 to 200 adenine nucleotides are added on, again to protect the mRNA and (scientists are still trying to figure out) maybe regulate protein synthesis in facilitating the export of mRNA from the nucleus to the cytoplasm.

snRNPs - pronounced "snurps"! - taking out them INTRONZ during RNA splicing

snRNPs are short for small nuclear ribonucleoproteins.

They play a key role in taking out the introns out of the pre-mRNA just after it is transcribed from the DNA. (the pre-mRNA is technically called hnRNA, which stands for heterogeneous nuclear RNA, because the it is a mix of intron and exons kept within the nucleus. (REMEMBER: IT TAKES PLACE IN THE NUCLEUS)

snRNPs contain snRNA, small nuclear RNA. A bunch of these snRNPs join together to help form a spliceosome. It cuts at specific points in introns and immediately joins the surrounding exons. A clearer picture can be found on figure 16.21 on page 316.

Ribozymes - these are RNA molecules that function as catalysts to help splice other types of RNA transcripts like tRNA and rRNA.

Differences between eukaryotic and prokaryotic gene expression

Mutation

Mutation - a change in the nucleotide sequence of DNA (it really screws everything up because the proteins get read wrong, etc.)

The TYPES of mutations:

Point mutation - it just happens at a certain point in the DNA à chemical changes in just one nucleotide in a single gene

Insertion - an extra nucleotide is added in somewhere along the line. No big deal, right? WRONG!!! There’s a reading frame shift that occurs, and it will seriously mess EVERYTHING up.

Deletion - here, instead, a nucleotide is missing from the sequence. Just about the same result as insertion.

Translocation - These are just TOTAL screwups that occur during gametogenesis and gene regulation. Entire portions of the DNA are moved around, so not only are wrong amino acids made due to a reading frame shift, but they’re also in the wrong order in the polypeptide chain to begin with! AHHHH!!!!

Ames test for mutagenic compounds

The man w/ the plan. His name is Bruce Ames, and he created a test that measured the mutagenic strength of various chemicals (ability of these chemicals to mess up your DNA badly)

You take the suspected mutagen and put it in some Salmonella. This is special Salmonella though, because it needs histidine to survive. Well, place it in some stuff that DOESN’T have it. Then add rat liver extract for good measure (for biological activation and to simulate cool stuff).

If there are tons of colonies present in the end, then that means that something in your "suspected mutagen" reengineered the DNA of the Salmonella so that they could make histidine to survive. (Dr. Benjamin said that they tried this w/ Marcchino cherries which were coated with Red Dye #2 à it failed… It doesn’t matter because I HATE CHERRIES ANY WAY!!!)

If there aren’t many / any colonies present, then it’s alright. (I guess…)

Sources of mutation

X-rays

X-rays are a really high energy form of electromagnetic radiation (this is where chemistry starts seeping back into biology… scary, isn’t it?) The X-rays can break apart DNA, cause deletions, and create translocations. It basically has enough power to blow the DNA to smithereens! (It can take apart H-bonds REALLY easily.)

UV light

UV light attacks your pyrimidines (remember? T and C) When harmful UV light hits them, it can rearrange the bonds in two adjacent pyrimidines so that they bond to each other, and not to the DNA’s complementary strand. These are called pyrimidine dimers, and the fuck everything up.

Chemicals

Basically, this is what the Ames test tested for. Certain chemicals can alter your DNA through reaction to modify bases (like add on bromo- group to every other nucleotide) or even miss-pair bases (so now a T will fit with a G, etc.)

Spontaneous

Not much on this. (I don’t think he really talked about it.) I guess it’s just when things just don’t go right and everything is spontaneous. (Hmm… who knows?)

Consequences of mutations

germ line cells, genetic defects - When mutations mess up gametes, genetic defects can be produced in the offspring. (Chances are that the offspring won’t live. Too bad… L )

somatic cells, cancer, tumors - When mutations mess up body cells that you already have, you get cancers and tumors. It really sucks. Then you die… L .

Cancer

Epidemiology - studying the relationship between a disease and its cause.

Epidemiologists graph and study all cancers across the US, graphing how many cases per a given year, and then correlating that with causes that might have caused them.

For example, 20 or 30 years after the rise of smoking and looking "cool", everyone came down with lung cancer. Before that time (when no one smoked) lung cancer didn’t even exist. There are also similar correlations between:

Meat & intestinal (colon) cancer,

Fat intake & breast cancer, and even

Cancer vs. Age (This will make a lot of sense as you keep reading - It’s the whole "Check a box" idea.)

CANCER, CANCER, CANCER (words to know: )

virus - what the hell is THIS doing here?

oncogenes - cancer causing genes (things that you DON’T want - these code for the continuous, uncontrolled growth of whatever cell it’s in)

tumor suppressors - genes that code for proteins that normally help prevent uncontrolled cell growth (cancer). Anything that messes up these genes could help the onset of cancer.

cancer - DUHHH… you should know this by now… "Uncontrolled growth of cells… they just keep going, and going, and going…"

RSV & Peyton Rous (1910)

RSV stands for the Rous Sarcoma Virus, a retrovirus containing RNA that actually has the ability to change the sequence of DNA in a cell. It contains oncogenes, which will kill a chicken (injected with it) almost immediately. (It is an example of an acute transforming virus.)

It was also found that if you injected extract from the dead chicken, with out any cells at all in it, into another chicken, it would die to. (It was an instant killer.)

transforming retroviruses - they carry oncogenes (those cancer causing ones) that can reengineer your DNA so that they’ll have them (and eventually, you’ll get cancer!)

proto-oncogenes - genes found normally in animal cells that normally regulate cell growth, cell division, and cell adhesion. These are cousins of oncogenes, and can readily be changed into one via gene amplification, chromosome translocation, gene transposition, and possible point mutations. (You probably won’t need to know all that - I just put it in for the hell of it).

Regularly, the proteins coded by the proto-oncogenes directly alter the division of the cell. If the proto-oncogene is turned into an oncogene, then it can directly alter anything. In this way, there is no stopping an oncogene, so it acts in a dominant fashion.

oncogene products found in nucleus (DNA binding proteins), in the cytoplasm and in / around plasma membrane

The loss of tumor suppressor genes is responsible for the genetically inherited increased risks of cancer. In this way, since you have to take over two of them, they are recessive?

Plasmids

Plasmids are small replicons found in primarily in bacterial cells

They carry valuable, but generally non-essential genes

antibiotic resistance

some catabolic pathways

Restriction enzymes - Enzyme that recognize a specific sequence of DNA and cut it up (remember BIOLAB? J )

It is used as bacterial defense against invading DNAs (bacteriophage) (it chews up foreign DNA)

It can be used to cleave DNA at specific sequences (e.g. G/AATTC for Eco R 1)

It often creates "sticky ends" on cleaved DNA

Cloning

pSC101 - first chimeric DNA (cloned segment in plasmid) by Cohen and Boyer - 1973 (don’t need to know names or plasmid here)

cloning procedure

cleave target DNA and plasmid vector with restriction enzyme

use DNA ligase to tie ends of plasmid to target DNA fragment (insert DNA into plasmid)

transform recombinant plasmid into E. Coli cells

screen for cells containing recombinant plasmid (antibiotic resistance, lacZ activity screening - blue/white)

applications of recombinant DNA technology

production of pharmaceuticals (especially proteins such as insulin, interferon, human growth factor, TPA, etc.)

herbicide resistant plants (using Ti plasmid and Agrobacterium)

virus resistant plants

"insecticidal plants"

transgenic farm animals (particularly those making pharmaceuticals)

piggyback vaccines (harmless viruses which code for surface antigens from disease causing viruses or other organisms)