Genetics Exam 1

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Transfer

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replication, or reproduction (vertical transfer), or pass genetic material to non-related organisms (horizontal transfer) via:
-transformation,
-conjugation,
-transduction, or
-transfection

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Avery, Macleod & McCarty

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They took isolated components from lysed type IIIS bacteria and mixed that w/living IIR bacteria & asked which transformed IIR to IIIS.
Result: Nucleic Acid transforms IIR & protein part doesn't transform.

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Hershey & Chase Results

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Most of 35S (radioactive isotope of sulfur) protein (80%) in the supernatant is going to be radioactive & result in proteins tagged. DNA will not be tagged b/c it has no sulfur in it.

Most of 32P DNA (65%) in the pellet.

If there is a transfer from E. coli to bacteriophage then it must be DNA b/c of the above results.

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Nucleic Acid (DNA & RNA) Structure:

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polymer of nucleotide subunits that are covalently attached to each other via phosphodiester linkages.

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Chargaff

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Determined base composition of DNA from different organisms

Exp: Hydrolized DNA into unlinked nucleotides and measured concentration of each.

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Rosalind Franklin (Maurice Wilkins)

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x-ray crystal structure of DNA molecule = rough 3D placement of atoms

Properties: DNA formed a double strand,
- purine joined w/pyrimidine,
-double strand is helical,
-there are 10 base pairs per turn of the helix

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Watson & Crick Double Helix Characteristics

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-sugar phosphate backbone on the outside b/c they are hyrdrophilic
-nitrogenous base on the inside b/c they are hydrophobic
-A's form 2 hydrogen bonds w/T's
-G's form 2 hydrogen bonds w/C's
-bases stack up on top of each other
-2 strands interact w/each other in an anti-parallel fashion(5'--3', 3'--5')

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Energetics of helix formation

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For any biological molecule, the forces that drive attainment of the final 3D conformation depend on interactions of atoms of the molecule with atoms of itself and between atoms of the molecule and atoms of its environment.

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RNA:

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5' - 3'

RNA can form double stranded helix's when complementary strands are available.

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Short regions of complementary sequences form

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local regions of double helix within an RNA single strand

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Unpaired nature of most of RNA single strand leaves

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hydrophobic bases exposed to acqueous environment of the cell.

As a result, RNA molecules are able to do a lot more than DNA., i.e.: splicing, transcription, expression.

RNA is more structurally and functionally diverse than DNA.

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DNA in the cell exists primarily

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as chromosomes

Most DNA molecules in a cell are long linear or circular chromosomes rougly 1-200 per cell depending on the organism.

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gene

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is defined as the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide.

This includes: coding region
transcriptional control regions
(enhancers)
splice sites
polyA sites

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Francis Crick: Experimental evidence for the triplet code

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T4 wild type forms small fuzzy plaques on E. coli.

Mutagenize T4 w/proflavin(mutagen) causing single BP insertion or single BP deletion.

Must have occurred in a critical place such as a gene.

1 class of mutants produced large clear plaques (rII mutants).

RESULTS: a wild-type plaque(normal) morphology was obained only when three (-) mutations were combined in the same phage. The three (-) mutations in the same phage restored the normal reading frame. These results were consistent with the hypthesis that the genetic code is read in multiples of three nucleotides.

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wild type =

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r+
-these are non-mutant types, typically found in the wild.

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rII

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single bp insertion in the r gene
-gene product is non-functional
-cell of r protein results in a clear plaque

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rII mutant =

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mutanegized w/ proflavin
-most plaques were clear but a few were fuzzy which is the wild type phenotype
-some fuzzy plaques were "true revertants" (wild type sequence was restored)
-some were "pseudo reverts" which is,
-----------2nd mutation (up or down)
----------restores the wild-type phenotype(2nd side repression)
----------2nd side repressor: second mutation at a DNA site other than the site of the initial mutation that "supresses" the effect of the initial suppression.

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For Crick the 2nd site repressor

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was a single bp deletion near the single bp insertion of the original insertion.

-He then collected 3 different 2nd site suppressors & then separated the 2nd site mutation from the original insertion.
(+)(-)a = (+) insertion, phenotype clear
fuzzy(wild type) = (-)a deletion, phenotype clear

Conclusion: genetic code is read 3 bp's at a time & removal of 3 seperate bp's is equivalent of removing a single codon.
Each codon codes for one amino acid.

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single isolated suppressor (-)a

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Phenotype clear

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(-)a(-)b or (-)a(-)c or (-)b(-)c

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phenotype clear

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(-)a(-)b(-)c

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fuzzy

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Nirenberg & Ochoa

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Need an assay to identify and quantify incorporation of amino acids into polypeptide using pool of synthetic RNAs.

Established patterns between the specific base sequences of codons and the amino acids they encode.

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Polypeptide(protein) =

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Cell extract + synthetic RNA pool + 20 amino acids

RNA = ribonucleotide diphosphate + polynucleotide phosphorelase(II)

19 AA's will be non-radioactive (cold)

1 AA will be radioactive (hot)

to learn about codons we use 2 nucleotides at a time.

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Gobind Khorina

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Could synthesize short RNAs, 2-4 nucleotide polymers of RNA with a known sequence..

Short RNAs could then be ligated together to form a long chain (copolymers).

e.g. 5' AUC 3'

-now could make a copolymer by connecting short polymers

5' AUC AUC AUC AUC AUC.... 3'
AUC = Ile 33% of radioactivity are these amino acids
UCA = Ser 33% of radiactivity are these amino acids
CAU = His 33% ....

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Eukaryotes Genes

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protein encoding genes almost always consist of a single cistron

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Prokaryotes Genes

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many polypetides are encoded on an mRNA with other polypeptides = polycistronic

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Chromosomes:

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double-stranded DNA

-promoter dictates what strand is used as template strand b/c it determines what direction transcription goes due to which direction the RNA polymerace is facing.

-RNA is always complementary to template strand.

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How are genes defined:

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1) Forward genetics: isolate a mutation that produces a phenotype & then find where mutation is in DNA & examine surrounding region to define a gene.

2) Reverse genetics: identify potential gene in the DNA & then make a mutation in the "gene" & then ask if it has a phenotype.

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ORF's are used

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to identify/facilitate reverse genetics.

Applies to protein coding genes, not RNA genes.

Means a stretch of DNA that potentially encodes a protein (from start codon to stop codon). Only applies to a protein-coding gene.

ORF is any region of DNA where Start codon to Stop codon is greater than 150 nt's.

The longer the sequence, the more likely it is a real gene.

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AUG to Stop

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is approx 60 nt, equaling 20 AA

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Genome-entirety of an organism's hereditary material

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-chromosome
-plasmids (if present)
-mitochondrial DNA (if present)
-chloroplast (if present)

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Virus Characteristics

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-DNA or RNA
-ss or ds
-small: a few to a few hundred genes often overlapping ORF's, meaning efficient use of sequence

-viruses are a nucleic acid inside the protein coat

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Bacteria Characteristics

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-usually have 1 chromosome that are often circular
-much bigger than viral chromosome
-always DS DNA
-less overlapping of ORFs
-intergonic regions are relatively small

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How to compact DNA to fit inside bacterial cell:

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1) Looping: occurs through random binding of DNA-binding proteins

2) Supercoiling: double-helix is further coiled causing chromosome to compact. Two types:

a) Positive Supercoiling: goes in same direction as helical coil(clockwise)
b) Negative Supercoiling: opposite (CC)

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What Causes Supercoils:

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1) Topoisomerase II (aka DNA syrase) creates negative supercoils

2) Topoisomerase I relaxes supercoils

Most DNA in bacteria is negatively supercoiled

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How Supercoiling Occurs:

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1) Cut backbone of 1 strand
2) Pass the other strand through the break
3) reseal the break

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Eukaryote Chromosome Characteristics

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-all chrom. are ds DNA
-often linear
-usually more than 1 chromosome per cell
-much larger than bacterial chromosomes
-intergonic regions are large
-require add. levels of compaction & organization
-DNA is negatively supercoiled, ds

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Levels of Organization

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1) DNA wraps around histone proteins = DNA wrapped around histones = nucleosome

2) 30 nm fiber; requires H1 protein

3) 30 nm fiber interacts with the nuclear matrix; forming of radial loop domains

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Nucleosome

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H2A, H2B, H3, H4 comprise a nucleosome.

H1 coats DNA b/t nucleosomes.

Nucleosomes are spread along the DNA like "beads on a string."

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Markus Noll: Experimental Evidence for "beads on a string" model:

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-rat cell nuclei + disrupted H1 - DNA interaction

-treated isolated DNA w/DNase I(enzyme that is non-specific endonuclease)

-low DNase 1: i.e. result is 5 random cuts.
medium DNase 1:you'll get more random cuts per distribution & each piece will be generally smaller
-high DNase 1: even more cuts & size will be even more smaller

Result:

1) single nucleosome comprises 200 bp of DNA
2) nucleosome are distributed evenly across entire chromosome

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Endonuclease

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-cuts within ds DNA to break phosphodiester BB of both strands
-large molecule that can't cut in a nucleosome

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2 Types of Heterochromatin

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1) Constitutive Heterochromatin: always heterchromatic

ex's: telomere and centromeres; contain no genes

2)Facultative Heterochromatin: can be converted to euchromatin & vice versa

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C value

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amount of DNA sequence per cell of organism

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Human genome project

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-3 total billion base pairs on 23 chromosomes

-approx 20,000 - 25,000 protein-coding genes equals 1.5% of the genome

-other 98.5% comprises:

1) RNA genes
2) Regulatory sequences
3) Introns
4) Junk (repetitive DNA): organisms w/ a higher C value than humans have more junk

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Explanation for C value paradox

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some organisms have higher C value w/o additional complexity

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3 Classes of Human DNA based on sequence Complexity(repetitiveness)

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1) Unique sequences: DNA that is not repeated or only repeated a few times (most genes)

2) Moderately Repetitive: DNA sequence that are repeated a few hundred to a few thousand times; includes junk & non-junk DNA (i.e. ribosomal, histone genes are non-junk)

3) Highly Repetitive: DNA sequences repeated 10's of millions of times.

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Transposable elements:

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can be divided into:
1) SINES: short interspersed nuclear elements. These are small, don't have any genes. E.g. Alu suquence, approx. 300 bp long, 11% of human genome.

2) LINES: Long intersperssed nuclear element. Much bigger than SINES. Contain genes that are responsible for their transposition, include endonuclease and reverse transcriptase. Genes are also responsible for transposition of SINES. Account for approx 21% of human genome.

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Single BP mutation

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1) BP substitution:
a) transition: purine <-> purine or pyrimidine <-> pyrimidine
b) transversion: purine <-> pyrimidine

2) single BP insertion or deletion

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Multiple BP insertion or deletion

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1) Insertions & deletions greater than 1 BP

2) Inversions, i.e. (prob. deleterious)

3) Translocation: likely to lead to non-functional gene; prob. deleterious

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What causes aneuploides?

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Caused by chromosome non-disjunction during gamete formation.

1) Meiotic Non-Disjunction: break occurs causing homologs to stay together. Every cell in organism will have the aneuploidy b/c it originates from zygote.

2)Mitotic Non-Disjunction: same thing as meiotic but every cell will not have aneuploidy. Depends on type of tissues and how early the non-disjunction occurs.

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Barr Bodies

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-Highly condensed version of X chromosome
-they start to occur relatively early in development
-entirely heterochromatimized
-led to proposal that Barr body is an inactive X chromosome

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Lyon Hypothesis:

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-Mechanism of X chromosome inactivation.
-Hypothesized that in mammalian females early in development in each cell an X chromosome is randomly converted into a Barr body & once converted all succeeding cells in that line have the same X homolog inactivated.

-this explains calicoism

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Testing the Lyon Hypothesis

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-use a female mouse w/2 different alleles of a gene encoding G-6-P DH

-this gene is on the X chrom
-2 allels can be differentiated from each other b/c 1 is a fast migrating form and the other is a slow migrating form

-take one of those cellls and put a single cell into each of a # of cell culture flashes & grow a tissue culture of each

Result:
-50% of cultures showed only fast-migrating form & other 50% showed only slow migrating form
-once an X is inactivated all the succeeding X cells of that type are inactivated

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Mutations on protein-coding sequence

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1) Single BP Mutation (substitution)
a) silent: no affect on AA sequence
b) missense: single AA substitution
c) nonsense: change an AA coding codon into a stop codon

2) Single BP insertion/deletion
a) frameshift mutation: normally deletions if closer to beginning but not to end

3) Multiple BP Mutations
a) insertion/delection of a non-multiple of 3 will cause a frameshift (closer to beginning, less likely to be deletions, but, closer to end will not be deletions)
b) multiples of 3: not a frameshift