1
Q
genetic material must be able to..
A
- contain the information to construct an organisms - a cookbook with ALL the recipes
- pass from parent to offspring and from cell to cell during cell division (transmission)
- be accurately copied (replication)
- account for the known variation between organisms
2
Q
what is the genetic material?
A
-how is material passed from generation to generation?
3
Q
what is the genetic material?
A
- late 1800’s scientist postulated a biochemical basis
- researchers convinced chromosomes- carry genetic information
- chromosomes= proteins + nucleotides
- protein= more complex
- expected to be the genetic material
4
Q
Griffiths bacterial transformations
A
- late 1920’s Frederick griffith work with Streptococcus pneumoniae
- strains that secrete a capsule look smooth and are fatal in mice
- strains that do not secrete capsules look rough; not fatal
5
Q
Griffiths experiment
A
- injected living type S bacteria into mouse- mouse died. Type S cells are virulent
- inject living type R bacteria into mouse. Type R cells are benign
- inject heat-killed S into mouse- benign
- inject heat-killed S bacteria into mouse and living R bacteria- virulent type S strain in dead mouse’s blood. Living R cells transformed into virulent S cells by a substance from the heat-killed type S cells
6
Q
Griffiths bacterial transformations results
A
- genetic material from heat-killed type S bacteria was transferred to living type R bacteria
- R-bacteria were transformed
- Griffith did not know the biochemical basis of his “transforming principle”
7
Q
Griffith’s bacterial transformation; “transforming principle”
A
- met requirements for genetic material
- variation- some bacteria make a capsule, some do not
- the R strain acquired the information to make capsule (a trait)
- the transformed R cells replicated this information and transmitted it to new cells during cell division to cause an infection
8
Q
Avery, Macleod and McCarty
A
- what substance is being transferred from the dead type S bacteria to the live type R?
- used purification methods to purify different macromolecules (DNA,RNA and proteins)
- only purified DNA from type S could transform type R bacteria
9
Q
What happens just before a cell divides?
A
cells exactly double their amount of DNA
10
Q
Chargaff; DNA position
A
- he looked at the composition of DNA
- 3 components- a pentose sugar, phosphate group and a nitrogenous base
11
Q
Chargaffs Rule
A
- amounts of the 4 bases were not equal
- however the amount of A=T and amount of G=C
- Chargaffs Rule
- double helix counts for this
12
Q
Structure of DNA
A
- too small to see through microscope at the time
- portions of DNA’s structure could be inferred through a technique called X-ray diffraction
- purified DNA bombarded with X rays
- takes years to obtain a well made X-ray crystallograph
- Rosalind Franklin did this
13
Q
Watson and Crick; DNA structure
A
- Wilkins gave x-ray graph to Watson and Crick
- associated the graph to the double helix
- found ball and stick model consistent with data
- double helix
- a purine w/ a pyrimidine
- correct width of helix
- fits with Chargaff’s A=T and G=C
14
Q
5 levels of DNA structure
A
- nucleotides
- strand
- double helix
- chromsomes
- genome
15
Q
Nucleotides
A
- DNA structure
- the building blocks of DNA and RNA
16
Q
Strand
A
- DNA structure
- a linear polymer strand of DNA or RNA
17
Q
double helix
A
- DNA structure
- the two strands of DNA
18
Q
chromosomes
A
- DNA structure
- DNA associated with an array of different proteins into a complex structure
19
Q
genome
A
- DNA structure
- the complete complement of genetic material in an organism
20
Q
nucleotides: DNA
A
- Phosphate grouo
- Pentose sugar- deoxyribose
- Nitrogenous base
- Purines (A, G)
- Pyrimidines (C,T)
21
Q
nucleotides: RNA
A
- Phosphate group
- Pentose sugar
- oxyribose - Nitrogenous base
- Purines (A,G)
- Pyrimidines (C, U)
22
Q
Conventional numbering system
A
- sugar carbons 1’ to 5’
- base attached to 1’
- phosphate attached to 5’
23
Q
strand structure
A
- sugar-phosphate backbone
- bases on the inside
- stabilized by H bonding
- specific base pairing
- major and minor grooves
24
Q
Chargaff’s rule; strand structure
A
- A pairs with T via 2 H bonds
- G pairs with C via 3 H bonds
- keeps width consistent
- 10 base pairs per turn
25
Phosphodiester bond
-nucleotides covalently bonded via phosphodiester bond- phosphate group links 2 sugars
26
complementary; antiparallel
- 2 DNA strands are complementary
- 5'- GCGGATTT-3'
- 3'- CGCCTAAA- 5'
-2 strands are antiparallel
one stand 5' to 3' others 3' to 5'
27
1. conservative (DNA replication)
- the parental double helix stays together, the new double helix is completely new
28
2. Dispersive (DNA replication)
each daughter double helix is a random mosaic of parental DNA and newly synthesized DNA
29
3. semiconservative (DNA replication)
-each daughter double helix contains one parental strand and one new strand
30
how does DNA replicate?
- the double helix must open
- strands separate
- weak H bonds allow this
- sequence on one strand is a template for the other strand
- new nucleotides must obey the AT/GC rule
- the new strand is the "complementary sequence"
- end up with 2 identical DNA molecules
31
Origin of replication
- starting point
- opens up to a replication bubble
- produces 2 replication forks
- bidirectional replication
- bacteria have a signal origin
- eukaryotes have multiple points of origin- speeds up process
32
DNA replication in the cell (in vivo)
- chromosomes are BIG, millions of bases
- many proteins needed to copy a chromosome
- copying occurs at many sites
- or cell will take too long to divide
33
DNA helicase (protein in replication)
-binds DNA and travels 5' to 3' using ATP to separate strand and move fork forward
34
DNA topoisomerase (protein in replication)
relieves additional coiling ahead of replication fork
35
Single-strand binding proteins (replication)
keep parental strands open to act as templates
36
DNA polymerase (protein in replication)
-synthesizes new DNA strand- requires an RNA primer (short stretch of nucleotides to "grow off" of)
37
DNA polymerase uses dNTPS
- free nucleotides with 3 phosphate groups
| - breaking covalent bond to release 2 phosphate groups provides energy to connect adjacent nucleotides
38
DNA polymerase- 2 enzymatic features
1. can't begin DNA synthesis on a bare template strand
- DNA primase must make a short RNA primer
- primer later removed and replaced with DNA
2. DNA polymerase can only work 5' to 3'
39
leading strand
- DNA synthesized as one long continuous moelcule
- DNA primase makes one RNA primer
- DNA polymerase attaches in 5' to 3' direction
40
lagging strand
-DNA synthesized 5' to 3' as "Okazaki fragments"
41
leading and lagging strands
- DNA polymerase "hopes" back to replicate DNA 5' to 3'
| - DNA ligase fills gaps
42
DNA replication and errors
- error rate about 1/billion bases- really good!
- approx. 3 errors every time a human cell copies its DNA
- error rate of nucleotide addition by DNA polymerase about 1/100,000
- H-bonds between A&T/ G&C more stable than mismatches
43
DNA replication & Errors (mutations)
- DNA polymerase proofreads the sequence as it adds bases
| - other DNA repair enzymes remove/fix errors as well
44
Telomeres
- no place for upstream primer, so DNA polymerase cannot copy the tip of the DNA strand with a 3' end
- if this replication problem were not solved, linear chromosomes would become progressively shorter
45
Telomerase
- enzyme attaches many copies of DNA repeat sequence to the ends of chromosomes known as telomeres
- E.g GGGTTA(humans)
- telomere at 3' does not have a complementary strand and is called a 3' overhang
- complementary strand is embedded in telomerase
- prevents chromosome shortening
- provides upstream site for RNA primer
46
Telomeres and aging
- body cells have a predetermined life span
- skin cell sample grown in a dish will double a finite number of times
- infants, about 80 times
- older person, 10to20 times
- inserting a highly active telomerase gene into cells causes them to continue to divide
47
Werner Syndrome
- telomeres and aging
- DNA helicase gene mutation
- leads to lack of DNA repair and shortening of telomeres
- DNA replication impaired
- significant premature aging
48
telomeres and cancer
- in 99% of all types of human cancers. telomerase is found at high levels
- prevents telomere shortening and may play a role in continued growth of cancer cells
- mechanism is unknown
49
eukaryotic chromosome structure
- typical eukaryotic chromosome may be hundreds of millions of base pairs long
- length would be 2 meters
- must fit in cell 10-100um
- chromosomes composed of chromatin
- DNA + protein
50
Level 1 of DNA packaging; DNA wrapping
- DNA wrapped around histone protein complexes to form nucleosome
- "beads on a string"
- shortens length of DNA moelcule 7-fold
51
Level 2 of DNA packaging; 30nm-fiber
current model suggests asymmetric, 3D zigzag of nucleosomes
| -shortens length another 7-fold (49-fold..)
52
Level 3 of DNA packaging; Radial loop domains
-interaction between 30nm fibers and nuclear matrix
53
3 levels of DNA packaging
- each chromosome located in discrete territory
- level of compaction of chromosomes not uniform
- Euchromatin vs. Heterechromatin
54
Cell Division
-when cells prepare to divide, chromosomes become even more compacted
BIO 111
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