Required Properties of Genetic Material
1. Reliable storage of all information
2. Controlled expression of stored information
3. Accurate replication
4. Capable of some change
Proved by Griffith's mouse experiment where some of the R strain bacteria took in a fragment of the S strain DNA that encoded for synthesis of the polysaccharide coat. Further experimentation was done by Avery and his colleagues.
Did experiment with phage. Used radioactivity to label either phage DNA or protein. The conclusion was that only the DNA was transfered to the host and that it must be direct synthesis of new phage.
=building bock of nucleic acid
is made up of 3 parts: pentose sugar, nitrogenous base, phosphate.
If an H is attached to the base then it is deoxyribose (DNA), but if an OH is attached to the base then it Ribose (RNA)
Base: double ring
adenine and guanine
Base: single ring
thymine (DNA) and uracil (RNA)
How the nucleotides attach to each other. Number of phosophates may vary. 1=monophosphate, 2=diphosphate, 3=triphosphate. Has to be triphosophate if it is going to be added to nucleic acid
Chargoff base composition
found that A=T & G=C by using sensitive techniques to quantify amount of each base in DNA from different species
Technique used to discover the double helix nature of DNA (photo 51). Several scientist who used this message were Rosalind Franklin, Linus Pauling, and William Astbury.
6 Characteristics of Watson/Crick Model
-Right handed helix
-Sugar phosphate backbone outside; bases extend inward
-Complete turn ever 10 (really 10.4) bases
-Alternating major and minor grooves
-Arrange in complementary pairs bonded via H-bonds
Analytical Techniques for DNA
-Absorbance of UV light: directly proportional the amount of light is absorbed and the amount of nucleic acid
-Ratio Abs260: abs 260 indicates protein contamination (want low ratio)
-Melting Temp: measured when 50% of the strands are melted, absorbance is higher when it has melted, tells us the length of of the DNA (longer pieces have higher melting temp, and when there are more G/C)
-Molecular hybridization is used to find specific sequences and are often probed using radioactivity (souther spotting, in situ hybridization, FISH)
The separation of nucleic acids or proteins, on the basis of their size and electrical charge, by measuring their rate of movement through an electrical field in a gel.
DNA is negatively charged
Possible Models of DNA Replication
-Semi-conservative (actual way): each DNA replication strand is made up of one "new" and one "old" strand
-Conservative: the parent strand is used as a template and then returns to its original structure and the new strand is completed
-Dispersive: most complex theory. Each strand is made up of partial parental and of partial new strand.
studied replication of e. coli. Used different isotopes (14-N or 15-N) to track parent and daughter DNA strands. This experiment showed that the semiconservative model of DNA replication was correct because only one intermediate band was created after one generation. After two generations two bands were formed (one intermediate and one light band.
the enzyme that assembles nucleotides into a new DNA strand which was discovered by Kornberg using "cell-free in vitro DNA synthesis system" by systematically separates the e. coli lysate in to fractional pieces. There are three types polymerase 2 is the important one.
Pol 1 - Repair and some replication (high concentration) Kornberg discovered this DNA pol
Pol 2 - All repair
Pol 3 - Majority of replication (low concentration)
DNA Polymerase proofreads during replication. It has 3' to 5' exonuclease activity. This allows it to repair by using the energy from the previous nucleotide. It catches approx. 99% of the errors
Helps starts replication and is removed by DNA pol 1. Makes up part of the Okazaki fragment
Synthesizes "primer" and is made of RNA.
Separates the strands and uses ATP
makes final phosphodiester bond
The part on the lagging strand that contains the primer and the piece that is replicated to the next primer. Contains 1000 to 2000 nucleotides
Origin of Replication
occurs during the initiation phase and is where the strands "open." Contains a sequence of DNA recognized by the enzymes that start it
less polymerase used then during eukaryotic replication and single replication of origin per chromosome.
Similar to prokaryotic replication except for three main differences. There are many more polymerases, there are multiple origins per chromosome (still make bubbles), and has to replicate chromosome ends.
enzyme used in embryonic cells to maintain chromosome length. Is not active in adults just embryos, however it is active in cancer cells.
Different Chromosome Structures
+ supercoiling= too many helical turns
- supercoiling= too few helical turns (can help open up the strands of DNA)
center part of the chromosome that attaches the two chromatids together
a fibrous structure with a size of about 400nm, located within the centromere. It is the site of microtubules attachement during cell division.