Genetics (Ch.10-#)

Molecules responsible for heritability must:

1. Be able to STORE complex information

2. REPLICATE faithfully/copy with fidelity (from parent to offspring)

3. ENCODE phenotype (through effects on RNA and Protein)

4. Must allow for VARIABILITY between individuals (due to sexual reproduction)

Levene (guy)

He proposed that DNA consisted of repeating units containing all four bases in equal proportions, theory incorrect because fixed repeating arrangement would not allow enough variation to encode diverse phenotypes of different organisms

• DNA (polymer) made of nucleotides (monomer)

• Each nucleotide is composed of a phosphate, base (purine, pyrimidine), and sugar (deoxyribose, ribose)

• Adenine, Thymine, Guanine, Cytosine

• Tetranucleotide Theory (WRONG)—> Doesn’t allow for variation, cannot encode different phenotypes

Chargaffs Rules:

1. Distribution of the bases varies from species to species

2. A=T

3. G=C

Chargaff’s Findings

Compared DNA from organisms (E.coli, Yeast, Sea Urchins, Rats, humans)

Comparing multiple species matters because a single sample such as E.Coli would not establish how base composition varies across organisms

• observed that base composition varies among species, A approx=T, G approx=C

• Ratio >1 numerator more abundant A/T , G/C

• Purines=A,G Pyrimidines=C,T

Griffith

Bacterial Transformation

• studied bacterial transformation

• living bacteria can take up material released from dead bacteria and acquire new traits

• When bacterial cells break open (dead, weak membrane), DNA fragments and plasmids can enter the environment

• Fragment integration (one of the ways bacteria can acquire variation)???

Avery, MacLeod, and McCarty

Identifying the transforming substance

• they investigated the chemical nature of Griffith’s transforming substance

• Prepared material from heat-killed virulent bacteria and treated separate samples with:

RNase, degrades RNA

DNase, degrades DNA

Protease, degrades protein

Substance that doesn’t KILL lacks the transforming substance

• Tested whether the treated material could transform nonvirulent bacteria

• Transformation continued after RNA or Protein degrades, but not after DNA degrades (it is transforming substance)

NOTE: transforming substance also absorbed UV light like DNA and was not degraded by protease and rnase

Hershey and Chase

Testing whether DNA or protein enters bacterial cells

• Used bacteriophages (viruses that infect bacteria)

• T2 phage contained DNA and a protein coat

• Used radioactive isotopes to track the two components (35s, 32p)

After phages infected E. Coli, the samples were blended (to shear protein coats) and centrifuged (to separate protein from cells)

• Results supported the conclusion that DNA, not protein, entered bacterial cells

Wilkins and Franklin

Used X-ray crystallography to investigate DNA structure

• X-ray passes through crystallized DNA to produce a diffraction pattern, pattern provides evidence about the structure. Diffraction pattern is analyzed

Franklin and her student Gosling produced image photo 51, provided evidence of DNA helical structure

Reflection

Light bounces back

Refraction

Light bends as it passes between media

Watson and Crick

1953, proposed a model for DNA structure based on:

1. Levene

2. Chargraff’s Rules

3. Franklin and Wilkins’ DNA structure X-ray data

DNA

A polymer made of nucleotides held together via phosphodiester linkages (bond between the phosphate group of one nucleotide and the 3’ hydroxyl group of another nucleotide’s sugar)

• This forms the backbone

• Deoxyribose is more stable due to missing Oxygen

Phosphodiester Linkage

The bond between the phosphate group of one nucleotide and the 3’ hydroxyl group of another nucleotide’s sugar

• Holds nucleotides together

• Forms the backbone of nucleic acids (DNA, RNA)

Primary Structure of DNA

The linear sequence of nucleotides, joined together by phosphodiester linkages.

Nucleotide Structure

Contains:

1. Phosphate

2. Ribose sugar (Ribose or Deoxyribose)

3. Nitrogenous base (Purine/Pyrimidine bases; ATGCU)

Nitrogenous Base binds to the 1’ Carbon of the sugar (purine corner N of house)(pyrimidine bottom N/N1)

Negative charge of Phosphates (binds to 5’ carbon of sugar) makes DNA Acidic

Purine Nitrogenous Bases

Adenine (A6), Guanine (O6A2)

Pyrimidine Nitrogenous Bases

Cytosine (O2,A4), Thymine (U+5Methyl), Uracil (O2,4)

DNA Secondary Structure

Double helix formed by complementary base pairing

• Antiparallel strands (double stranded)

• Bases are H-bonded to each other

• Sugars & Phosphates are on the outside (Backbone; deoxyribose sugars linked by phosphate))

DNA tertiary and quaternary structure

The 3D folding and coiling of double helix into complex structures (e.g. chromosome)

B Form DNA (watson and crick form)

(aqueous cellular environment)

• 10 base pairs per turn

• Bases spaced 36 degrees apart

• Complete turn 3.4 nm

• Helix has major and minor grooves

• RIGHT HANDED Helix

A-DNA

Exists when water is limited

• shorter and wider than B form

• DNA-Protein complexes (found in)

• RIGHT HANDED Helix

Z Form DNA

LEFT HANDED Helix

• Rich in CG repeats

• Sites of Active Transcription

RNA Secondary Structure

Single stranded

• Base pairing can exist between the bases on SAME strand

• Hairpin Structure (consists of paired stems and loops)

• Stems

• May contain numerous hairpins allowing them to fold up into complex structure

Special Structure (can form in DNA & RNA): H-DNA

Three-stranded (triplex)

• long sequence of only purines or only pyrimidines

• Common in mammalian genomes, more prone to breakage/breaks more often

Methyl Groups (DNA Methylation)

Some enzymes add Methyl groups to DNA

• Bacterial DNA is frequently methylated (allows for the cell to detect foreign DNA (A and C))

• Eukaryotic cells use methylation to regulate gene expression (5-methylcytosine) think Epi genetis

Naming system for bases

Nucleotide:

dAMP (deoxyadenosine 5’ monophosphate)

Nucleoside:

dA (deoxyadenosine)

Eukaryotes

Have a nucleus

Linear Chromosomes

Multicellular

Membrane-bound organelles (ex: mitochondria)

EX: Fungi, plants, animals, diatoms, malaria

Prokaryotes

No nucleus

lack membrane-bound organelles

DNA is long and CIRCULAR supercoiled chromosome in nucleoid region

single celluar

EX: Archaea and Bacteria

Endosymbiotic Theory

Ancestral prokaryote with nucleoid—> Infolding of plasma membrane forming protonucleus—>Engulfs aerobic bacterium—>Engulfs cyanobacterium—>Ancestral Eukaryote (with mitochondria and chloroplasts)

Supercoiling

Makes DNA structure more compact and is stabilized using proteins

Occurs in both prokaryotic and eukaryotic cells

Topoisomerases

(+) Over wound

(-) Under Wound

Topoisomerases

Enzymes that add or remove rotations of DNA (to manage its supercoiling)

(+) Supercoil (Overwound)

Closes down DNA, makes it tighter and more compact

(-) Supercoil (underwound)

Allows 2 strands to move or twist off of one another

(During processes such as transcription)

Type IA (Topoisomers)

(+) Supercoiling

5’ ssDNA (single-stranded) cleavage

Cleaves One side

Does NOT require ATP

EX: Reverse gyrase with helicase

• Only enzyme that introduces (+) supercoil in DNA

• also found in Archaea

Type IB Type IC (Topoisomerases)

(+) Supercoiling

3’ ssDNA (Single-stranded) Cleavage

Cleaves one side

does NOT require ATP

Controlled rotation

Chromatin

Consists of DNA and packing proteins (histones)

Eukaryotic DNA remains packed during interphase

• Epigenetic changes effect chromatin structure

Euchromatin

Less condensed structure of DNA where transcription occurs

Located on Chromosome arms

Unique Sequences

Many genes

Replicated throughout S Phase

Often transcribed

common in crossing over

Heterochromatin

More condensed (“true” chromatin) structure

Located at centromeres, telomeres

Repeated sequences

Few genes

Replicated in Late S phase

uncommon in crossing over

Types:

• Constitutive

• Facultative

Constitutive Heterochromatin

Remains condensed (ALWAYS heterochromatin)

like at centromeres

Facultative

Is condensed (heterochromatic) only at particular developmental stages

How does the cell package long amounts of DNA into the nucleus?

Through the tertiary structure of DNA (chromatin) wrap around Histone proteins and Nucleosomes

Nucleosome (NCP)

The basic repeating unit of chromatin

• 147 base pairs of DNA

• Protein octamer of 4 histone proteins (2 of each; H2A, H2B, H3, H4)

• Separated by Linker DNA (a stretch of DNA whose length varies in different cell types)

Histone Core Particle (HCP)

Octamer

• High in Lys & Arg (positively charged amino acids)

• The positively charged amino acids attract DNA’s negatively charged sugar phosphate backbone (167 base pairs)

• Excludes H1

Histone tails

• 11-37 Amino acids long

• can be modified, affecting transcription and replication

H1

Not apart of HCP octamer

Binds to 20 base pairs of DNA (and linker dna?) to lock the nucleosome components in place.

Epigenetic effect on chromatin

Compaction prevents transcription of DNA into RNA

even euchromatin (that is reactive/actice) has nucleosomes which block transcription of DNA

Chromatin remodeling is required for DNA transcription (to unblock it)

Telomeres

The caps/tips of LINEAR chromosomes

• usually consists of repeating A and T followed by several G

• There is a G rich 3’ overhang of 50-500 bases

The overhang can form a T-loop

• Helps protect chromosome ends from degradation

Shelterin protects the ends from repair

• multiprotein complex

• binds to telomeres and protects end

Telomere shortening

DNA polymerase cannot replicate the ends of chromosomes, so telomeres get Shorter with every Cell Division

• Germ cells and stem cells have telomerase enzyme to lengthen telomeres and prevent shortening

• Cancer cells sometimes have increased telomerase activity

Too Short Telomeres…

• Leads to Senescence

Senescence

Process of deterioration with age