Lecture 1 Flashcards
What is the difference between a prokaryotic and eukaryotic cell?
- prokaryotic: single celled, lacks a membrane bound nucleus, genetic material is stored in a form of a nucleoid, outer membrane/cell wall/inner membrane
- eukaryotic: has other membrane bound organelles; mostly in multicellular organisms but not all the time
What is the Central Dogma of Molecular Biology?
DNA transcription RNA translation PROTEIN
Gene expression
In 1941, who did experiments on red bread mold Neurospora and what did they find?
Edward Tatum and George Beadle: linked genes and enzymes
- Neurospora grows well on minimal medium. Minimal midium consists of a few simple sugars, inorganic salts, and biotin
- But in order for them to grow, they must be able to synthesize other essential nutrients such as amino acids and vitamins
- Are the enzymes that are a part of these essential biosynthetic pathways needed to make these essential nutrients controlled by genes?
Exp:
1. Irradiated Neurospora spores with x-rays to induce genetic mutations. If these enzymes were really controlled by genes, then the X-ray exposure should stop their growth.
2. They selected for mutants. They placed samples on both complete medium and minimal medium. They found growth on complete medium only. So nutritional mutants were not able to grow in MM.
3. Then they had to figure out what kind of nutrition was need for these mutants to grow. Was it vitamins or aa? So they placed samples on MM+vitamins and MM+aa. They found that mutant strains grew on MM+aa.
4. Now, which aa was necessary for mutant growth? They tested the sample in multiple tubes of MM+ (an aa). The tube with MM+arginine was the only one where mutants grew. So, Arginine was being blocked from synthesis.
Conclusions:
They isolated many mutants each with a single mutated metabolic pathway enzyme and proposed a direct link between genes and enzymatic reactions.
ONE GENE- ONE ENZYME HYPOTHESIS: GENES WORK BY CONTROLLING THE SYNTHESIS OF SPECIFIC ENZYMES
Who discovered DNA and how?
1869
Friedrich Miescher
-found NUCLEIN in the nuclei of white blood cells isolated from pus on old bandages
What was nuclein found to be made up of?
H O N P
(no S)
-had a unique ratio of P:N
Who discovered chromosomes and how?
1879
Walther Flemming
-used aniline dyes to stain cells of salamander embryos and saw threadlike material as the cells divided. This is where he started to describe chromosomal behavior during mitosis.
The study of chromosomes is called_____.
cytogenetics
What was the chromosomal theory of heredity?
Early 1900s
Thomas Hunt Morgan
-used drosophila to demonstrate that chromosomes carry the genetic material.
-but he didn’t know if it was DNA or protein?
-at the time, proteins were widely believed to be the genetic material because
1. DNA was though to lack the chemical diversity needed
2. DNA was thought to only provided a structural framework to the chromosome.
Briefly name the 3 classic experiments that demonstrate that DNA is the genetic material and proteins are not.
- Frederick Griffith
1920s - Avery, MacLeod, McCarty
1940s - Hershey and Chase
1950s
Describe the Frederick Griffith Experiment.
- english microbiologist
- worked with pneumonia-causing bacteria
- observed that nonpathogenic bacterial strains could be transformed to pathogenic strains when mixed with heat-killed pathogenic bacterial cells
Experiment:
- Living S-cells injected to mouse: S-colonies found in dead mouse tissue
- Living R-cells injected to mouse: R-colonies found
- Heat killed S-cells injected to mouse: mouse is healthy and no colonies found
- Living R-cells & Heat killed S-cells: both R and S colonies found
Concluded that something in the dead S cells “transformed” the live R cells to pathogenic S cells
What is pathogenicity of bacteria determined by?
The capsule gene
- R colonies: rough (this is the gene in its nonfunctional form
- S colonies: smooth**pathogenic cells have a carbohydrate layer around the capsule
Describe the Frederick Griffith Experiment.
- english microbiologist
- worked with pneumonia-causing bacteria
- observed that nonpathogenic bacterial strains could be transformed to pathogenic strains when mixed with heat-killed pathogenic bacterial cells
Experiment:
- Living S-cells injected to mouse: S-colonies found in dead mouse tissue
- Living R-cells injected to mouse: R-colonies found
- Heat killed S-cells injected to mouse: mouse is healthy and no colonies found
- Living R-cells & Heat killed S-cells: both R and S colonies found
Concluded that something in the dead S cells “transformed” the live R cells to pathogenic S cells.
Griffith did not know the chemical identity of this transforming principle in 1928.
What was nuclein of Friedrich Mieschers pus samples, found to be made up of?
H O N P
(no S)
-had a unique ratio of P:N
What is pathogenicity of bacteria determined by?
The capsule gene
- R colonies: rough (this is the gene in its nonfunctional form
- S colonies: smooth**pathogenic cells have a carbohydrate layer around the capsule
Describe the Avery, Macleod, and McCarty Experiment.
Extended Griffith’s experiment to determine whether the chemical identity of the “transforming principle” was protein, DNA or RNA.
-Made highly purified cell fractions from heat-killed S cells and found that the active fraction that had transforming capability had a C H N P composition consistent with that of DNA.
Describe the Avery, Macleod, and McCarty Experiment.
Extended Griffith’s experiment to determine whether the chemical identity of the “transforming principle” was protein, DNA or RNA.
-Made highly purified cell fractions from heat-killed S cells and found that the active fraction that had transforming capability had a C H N P composition consistent with that of DNA.
Exp: Took S-cell extract, treated with with enzymes and cultured it with R-cells.
- Treated with protease (found R and S colonies)
- Treated with RNase (found R and S colonies)
- Treated with DNase (found only R colonies)
Concluded that DNA was the transforming principle. Because only when the destroyed DNA, the transformation did not occur.
Describe the Avery, Macleod, and McCarty Experiment.
Extended Griffith’s experiment to determine whether the chemical identity of the “transforming principle” was protein, DNA or RNA.
-Made highly purified cell fractions from heat-killed S cells and found that the active fraction that had transforming capability had a C H N P composition consistent with that of DNA.
Exp: Took S-cell extract, treated with with enzymes and cultured it with R-cells.
- Treated with protease (found R and S colonies)
- Treated with RNase (found R and S colonies)
- Treated with DNase (found only R colonies)
Concluded that DNA was the transforming principle. Because only when the destroyed DNA, the transformation did not occur.
Describe the Hershey and Chase experiment.
- used T2 virus that infects E. coli bacteria by injecting it with its genetic material. This was composed of DNA and protein in approximately equal amounts. **The DNA contains P but not S
- *The Proteins contain S but not P
-Labeled using radioactive isotopes:
DNA with 32P
Proteins with 35S
What are isotopes?
same proton
different neutrons
different atomic mass
What are radioisotopes?
unstable
Their atomic nuclei spontaneously decompose to form nuclei with higher stability. The energy and particles released during the decomposition process are called radiation.
Describe the Hershey and Chase experiment.
- used T2 virus that infects E. coli bacteria by injecting it with its genetic material. This was composed of DNA and protein in approximately equal amounts. **The DNA contains P but not S
- *The Proteins contain S but not P
Exp: Labeled using radioactive isotopes
- Labeled phage DNA with 32P and let it infect E.coli. Saw that phage progeny contained 32P labeled DNA.
- Labeled phage proteins with 35S and let it infect E.coli. Saw that phage progeny did not contain any 35S labeled protein.
Concluded: Mainly DNA, not protein is inherited from parental phage.
What are isotopes?
same proton
different neutrons
different atomic mass
What are radioisotopes?
unstable
Their atomic nuclei spontaneously decompose to form nuclei with higher stability. The energy and particles released during the decomposition process are called radiation.
What are radioisotopes?
unstable
Their atomic nuclei spontaneously decompose to form nuclei with higher stability. The energy and particles released during the decomposition process are called radiation.
Who discovered DNA structure and composition?
1953
Watson and Crick
-proposed a 3D structure: double helix with paired subunits called nucleotides
monomer: nucleotides
polymer: DNA or RNA
What is a nucleotide composed of?
pentose sugar
phosphate group
nitrogenous base
Describe the structure of a pentose sugar.
1’ connects a base
2’ in DNA connected to H and in RNA connected to OH
3’ phosphodiester bonds
4’ H
5’ phosphates connects; also used to form phosphodiester bonds
How does the pentose sugar in a nucleotide differ in DNA and RNA?
DNA 2’ H
RNA 2’OH (less stable)
Purines
2 ringed structure
aromatic, hydrophobic
A and G
Pyrimidines
1 ring structure
C and T
Describe the structure of a pentose sugar.
1’ OH connects a base
2’ in DNA connected to H and in RNA connected to OH
3’ OH; phosphodiester bonds
4’ H
5’ phosphates connects; also used to form phosphodiester bonds
Pyrimidines
1 ring structure
C and T
What is the difference between a nucleoside and a nucleotide?
nucleoside: ribose sugar and base
nucleotide: ribose sugar, base, and phosphate
* When a ribose sugar connects its 1’C to a base and its 5’C to a phosphate group, it loses H20 through a dehydration reaction
What kind of bonds like nucleotides into a chain?
phosphodiester bonds
- covalent
- between 3’OH of one nucleotide and the 5’ Phosphate of another nucleotide
1. creates an alternating sugar-phosphate backbone
2. creates polarity of the chains (3’ end and a 5’ end)
What are the 2 Chargaff’s Rules?
- total purines= total pyrimidines
This means that the total number of A and G = the total number of T and C - Base composition ratios of A:T and G:C vary between species
Who helped solve the structure of DNA, in which Watson and Crick took credit for?
Rosalind Franklin
X-ray diffraction pattern
Explain the Model for DNA structure proposed by Watson and Crick.
- Double stranded
- sugar-phosphate backbone on the outside and bases in the middle
- 2 strands of the helix are held together by H-bonds
- A always found with T and G always found with C
- strands are antiparallel
- most DNA is R-handed
Explain the Model for DNA structure proposed by Watson and Crick.
- Double stranded
- sugar-phosphate backbone on the outside and bases in the middle
- 2 strands of the helix are held together by H-bonds
- A always found with T and G always found with C
- strands are antiparallel
- most DNA is R-handed
A-T
How many H-bonds?
2 H-bonds
G-C
How many H-bonds?
3 H-bonds
How is it possible that each base can exist in two alternative tautomeric states?
spontaneous flipping occurs at a very low rate between H-bond donors and acceptors on the base. If this happens at the wrong time, then it causes replication errors. If this happens when being copied, then a wrong base can come across it
What are 2 ways in which the double helix is stabilized?
- Base pairing: H-bonding
2. Base stacking: van der Waals/ hydrophobic effect