To Purify a gene
- 1) Method of isolating cell components
- 2) an assay for genetic materialness
He found a way to isolate the nuclei from cells in pus and found out the main consistituent of the neuclei is a componet called nuclein which turned out to be DNA
Inserted S(smooth) and R (rough) bugs in rats and the Sbug killed the mice and the R bugs were killed by the mice. When he put in both the mice died, therfore the R bug transformed into Sbugs. The R bug was called the transforming principle.
Avery, Macleaod and McCarty
Found that the transforming material was DNA
Al Hershey and Martha Chase
Took a virus and labelled its DNA with radioactive Phosphorus and protein with radioactive Sulfur. They allowed the virus to infect the bacteria and then threw the mix in the blender. There was only radioactive phosphorus left, therefore the 32P containning DNA must be the viral genetic material.
Watson and Crick
Found the structure of DNA
Rosalind Franklin and Maurice Wilkins
Use X-ray diffraction to show that the DNA was double stranded and helical
The OH group is on which carbon? and where is the phosphate group?
The OH group is on the 3' carbon and the phosphate group is on the the 5' carbon
He found the relationship between A-T and C-G, there are 3 hydrogen bond between C-G and 2hydrogen bond between A-T
- The bases are hydrophobic planar molecles
- A-T and C-G.. important because the hydrogen bonds are more stable that way and because by pairing purine and pirimidines the distance between the two strands remain constant
- The strand are antiparrallel to each other..reverse compliments of each other.
Types of replications
Concervative Replication- they each make new strands and the two old strands and the two old strands re-anneal
Semiconcervative: each old strand made and remained annealed to a new strand
Dispersive replication- DNA would break apart and rejoin to produce four strands, each with a mixture of old and new DNA.
Melselson and Stahl
Showed that DNA does semiconcervative replication. They labelled DNA by growing bacteria with a heavier isotope of Nitrogen. After one duplication half was heavy and the 2nd half the DNA was half as the half as heavy.
DNA polymerase, grabs a nucleotide that is complementary to the next nucleiotide in line on the template stand....it takes the nucleuotide and breaks the bond between the alpha phosphate(attached to sugar) and beta phosphate. It the attaches the alpha phospate to the 3' hydroxyl group of the last nucleiotide
- 5' to 3' direction (3'direction)
- Helicase unwinds the DNA
- Primase adds primers(complementary RNA) to strands because Polymerase III needs 3' hydroxyl group
- DNA polymerase III does work on lagging and leading strand
- Origin of replication is infront of replication fork
- Okazaki Fragments are the segments of primers
- DNA plymerase I chews up RNA primers and uses the newly synthesised DNA as a primer to fill in gaps.
- DNA ligase joins the ends of the newly synthesized strand
How does the cell deal with mistakes made my DNA Polymerases
- Polymerase has proofreading
- Mechanism thaat works during recombination called mismatch repair
- Mechanism thats works the rest of time called excision repair
- Excision repair also works on skin during exposure the UV light
Studied ppl with alkapturia..cause arthritis and black urine. The cause is a genetic error that results in the loss of a one specific enzyme
Beadle and Tatum
- They reckoned that i genes code for protein and you gave a metabolic pathway where each step in the pathway catalyzed by a different enzyme, then it would be possible to identify mutants that are arrested at each step in the pathway
- They isolated the mutants called auxotrophs as opposed to the wild type prototrophs
- Each gene specifies a particular enzyme
- One gene- one protein
Determined the first peptide sequence of a protein of a protein insulin.
Crick, Syndney Brenner and Francois Jacob
- Formulation of Central Dogma
- The idea that information flows from DNA-RNA-Protein..never goes the reverse way
RNA has a ___ hydroxyl group
RNA has a 2' hydroxyl that DNA does not have
- RNA polymerase (does not need a 3' hydroxyl) sits down on DNA at the beginning of the gene and starts creating a strand of RNA complimentary to the DNA.
- RNA polymerase extends the RNA strand until it gets to the end of the gene
- mRNA now has all the information necessary
Marshall Nirenberg, JH Matthaei and Gobind Khorana
Showed that if you add a polyU RNA to ribosime you could produce a protein of all phenyalanine...they basically found codons(3 base pair)
The code is said to be......
- mRNA goes to the cytoplasm and gets decoded by tRNA ( there is no particular affinity between amino acids and the corresponding codon)
- tRNA has an anticodon that will bind according to the base pairing rules to codon on mRNA
- On the other end theres the amino acid the encodes for that codon. Theres atleast one tRNA for every amino acid.
- The ribosome, by making tRNA bind to the codon then catalyses the polymerization of the amino acids, creates a sting of amino acids.
What enzynme determined what amino acid gets attached to each tRNA
aminoacyl tRNA synthase. The code is determined by synthase. The tRNA are not very smart.
What is said about the genetic code
- it is arbitrary
- the code is universal
The strand which is not transcribed and the ont that is called?
the one that is not transcribed is called the sense strand/coding strand and the one that is transcribed is the template strand/ antisense
What is the beginning of the gene called, type of leway
its called the promoter. The promoter sequence itself is not transcribed. Here is a case where DNA transmits information directly to a protein. Similary there is a termination sequence in the DNA telling the RNa polymerase when to stop [TATA] box is a transcription complex for the promoter region and its cause theres only double bonds there so its easier to breaak.
Describe the translation with tRNA and ribosoms
This small complex of small subunit, tRNA and mRNA recruit the large ribosomal subunit. The large subunit has two tRNA binding sites, the Asite and the P site. When a ribosomal subunit is recruited it bind so that the met-tRNA is in the Psite and then it lines up in the A site.
Proteins have a carboxylic side and an amino side, side they add on is the hydroxyl side because its 5' to 3'
What realease everything for termination to happen
The release factor binds to the A site causing the last tRNA to release the peptide chain and the ribosomal complex to dissassemble. Then the peptide folds into a functional protein.
Why can tRNA use more than one codon?
The 5' residue of the anticodon( which corresponsds to the 3' residue of the codon) has some wobble. That is it doesnt always have to match perfectly.
Polysome is when a ribosome binds to the same transcript starting t the start codon, on after the other.
Linus Pauling with help Vernan Ingram did what?
Found that sickle cell was cause but a very small change in the the base pair GAG...instead GTG
Types of Mutations
- Point mutation: small change in single gene
- Silence mutations: changing and A to T or C to G
- Missense: single nucleiotide change
- Nonsense: change in an amino acid to stop codon
- Deletions: causes frame shift if not full codon
- Insertion: causes frame shift if not full codon
- Chromosomal mutations: changes that affect a large portion of a chromosome, often affecting many genes
***These can be reciprocal translocations, were two chromosomes trade pieces***
- Deletion: remove a large piece of the chromosome including many genes
- Duplication: duplicate large chunks of the chromosome
- Inversion: when a piece of the DNA flips around, re-enters the chromosome in reverse direction
- Translocation; result when a piece of DNA jumps from one chromosome to another chromosome
What happens if your somatic cells or your germ cells get a mutation?
If its a somatic cell is will have more mutated somatic cells but not your your progeny and it might die or kill you,
If its you germ cells it it will affect your prgeny and then the progeny will have somatic cells with the mutation
Virus particles: simple protein coat or capsid and nucleic acid
What cycle do different phages undergo
- Vilrulent phage undergo a lytic cylce reproductive cycle: the phage binds to the bacteria and injects its DNA
- causes the bacterica to immediately produce more phage proteins and more phage DNA(instead of its own)
- and when the cell is used up the cell lyses and the newly assembled viruses are released.
- temperate phage under go lysogenic cycle. The virus takes a wait and see approach. It inserts its DNA (now called prophage) into the bacterial chromosome and wait while the bacteria divide (replicating th prophage alonf with their own DNA). When the moment is right the prophage pops out the chromoso,e and resumes, temporaly, the lyctic reproductive cycle.
Can phage combine?
Yes and they share DNA...yay!
Does something close to lysogenic phage. The go of the reverse of the central dogma. they make DNa copy from an RNA genom. The DNA copy of a retrovirus can be intergrated into the host chromosome and remain there, inactive, for long time before making virions.
GOOGLE AUXOTROPHIC PHENOTYPE!!!
When prophage excises itself from the chromosme. I can pick up a gene from the bacterial chromosome.The bacterial gene becomes part of the virus genome ad will be inserted into the chromosome of another bacterial when infected by that phage
Sometimes the phage do a poor job of chopping up the bacterial DNA and instead of packaging phage DNA into the virions they package chunkcs of bacterial chromosome. When those virions infect another bacterium, the chunk of bacterial DNA gets injected ito the infected bacterium and can be incorporated into th chromosome. Generelized transduction can be used to map genes.
Bacteria can sometimes take up naked DNA floating around in their enviroment as shown by Griffith. The genomic DNA can then be integrated by recombinatio. However, there is a type of non-genomic DNA called plasmid
What are plasmids that carry antibiotic resistance genes?
- Two strians of DNA..F+(has and F- plasmid..kinda like the male) and F-
- F+ donate a strand of their Fplasmid to F- to make a F+ plamsmid
- what makes this if the F- gets intergrated into the chromosome
- 1)conjugation occurs much more frequently
- 2) F DNA takes some of the chromosomal DNA and conjugates it too
- 3) the transffered DNA can be incorporated into the chromosome of the F- cell by recombination.
- These integrate F strains are called Hfr (high frequency of recombination)
inducible enzymes are enzymes that functions only when needed, constitutive enzymes are always on
- In the absense of lactose
- The lac repressor binds to the lac operator that blocks transcription
- When lactose enters the lactose binds to the repressor and it changes shape and releases the operator and transcription is now allowed the start
- B-galactosidase, Bgalactoside permease an B-galactoside trransacetylase
The presence of tryptophane blocks the transcription. CPR protein acts this way....look at video
Part on gene that codes for proteis
Part of gene that doesnt code for anything. to cut them out you use splicing. To take the introns out and put exons together you use snRNPs that catalyze the whole rxn
How do you stabolize mRNA
- A guanosine cap is added to the 5' end
- 3' untranslated region (3' to the stop codon) is snipped off and a string of~100 adenosine nucleotides, a poly A tail added to the 3' end
What is added to the chromosome that doesnt code for anything?
Telomere is added so that the DNA doesnt keep getting shorter. Its the enzyme telomase that adds telomeres. Creates a buffer for chromosome for everytime it shortens
What are other sources of non coding DNA?
- Centromeres: attachment of kinetochore
- Theres also transposal elements: these are DNA elements that can hop around the genome.
- Make copies of themselves by being transcribed then using reverse transcriptase to make DNA copy if the transcript that then reinserts in the genome.
- LTR retrotransposons look like retro viral proteis , but cant leave cytoplasm b/c doesnt code for any proteins that is functional. nonLTR retrotransposons(LINES) dont look like retrovirus but do encode for protein that can be inserted in genonome,.
- Alu elements dont code for any protein but rely on reverse transcriptase produce by other elements to allow hem to jump
splice themselve out of the genome then jump back by encoding a special enzyme called transposase
Ordinary mRNA gets copied into DNA and inserted into the genome producing processed pseudogenes. A chromosomal duplication can produce multiple copies of a gene some o which eventually are inactivated by mutation to produce a non funtional pseudogene
Found self splicing introns, neither DNA nor RNA came first. Ribozymes are ribozomes almost made entirely of RNA
House keeping genes?
Cells that express different genes....
The amount of given protein might control the rate of transcription, transport out of the nucleaus and mRNA stability translation protein function
- Transcription control
- Process control
- Transport Control
- mRNA stability control
- Translational control of protein synthesis
- Posttranslation control of protein function
Transcription Factors for Pol II
TFIID is a complex of proteins includuin TBP..the TATA binding protein that binds the TATA box DNA. TFIID recruits more TF's that eventually recruit RNA Polymerase II
Silencer and enhancer
- 1) Can act at a distance upstram or downstream
- 2) They act in either orientation
- Enchencers bind to enhancers and silencers bind o repressors (as a rule it is the enhancer and silencer binding transcriptio factors that determine what proteins are synthesised)
- The important thing is that they bind specific sequences by having protein domains that probe the major groove of the DNA(the wide groove)
heterochromatin vs euchromatin
heterochromatin stains darkly and does not transcrb in contrast to euchromatin which is light and transcribes
curled up X chromosome that form this ball thing
- seen in Calico cats
- all females and have a sex linked on X chromosomes
Not very well understood but cancer cells do it...amplify there rRNA gene so they gave millions
somtimes when splicing an intro and exon will get spliced which will cause a missing chunck. the presnence of absence of a chance can affect the way the protein functions. Its seen on a sex determining trait of these rando flies
if RNA was stable there would be no point to in regulating transcription
- changing the amount of capping
- factors binding to the RNA to prevent ribosome attachment
- RNA interference occurs when the cell makes small interfering RNA's that do not encode a protein but are complementary to the mRNA of a gene. With the help of protein called Dicer, theses siRNA's bing to the complementary RNA and blcok translation
- enzymes that phosphylate other proteins called kinases.
- Selective protein degradation is also an important way of controlling protein activity....marked a ubiquitimatiom(protein destined for degradation)
Why do ppl do genetic engineering?
- 1) To make an organism express a new phenotype
- 2) understand how the gene itself works
Frederick Banting and Charles Best
discovered that if you inject insulin in a dog it can survive
Formation of insulin
- Could use pig insulin b/c single amino acid differemce
- Make DNA copy of the mRNA from which the introns have already been spliced out. Use reverse transcriptase enzyme from a retrovirus. This DNA copy of th mRNA is called cDNA
- Clone ALL cDNAs then try to figure out which bacterium has the insulin cDNA.
- Must put the cDNA into plasmid. these are called vectors
- Cut plassmid so its linear. To cut use restriction endonucleases or a restriction enzymees. DNA recognises the restriction enzyme called restriction cite.
- Ragged, sticky end is left by the restviction enzyme cDNA need DNA ligase to create phosphodiester bonds between the cDNA and plasmid vector
- The phenotype that is allowed to grow is called the transformation marker.
- Now created a cDNA library Take good gene and move to plasmid good at making protein- expression vector
- Put in DNA probe to identify complement by heating and re annealing.
- Then do agorose gel electrophoresis (DNA is negatively charged)
- Then take what you need use lac or trp operon to multiply