genetics 3200 exam 1

5 characteristics of genetic material

1. Capacity for replication (replication) 2. Stability over time (stability) 3. Ability to undergo mutation (mutability) 4. Ability to express phenotypes (expression) 5. ability to be passed down to offspring (heritability)

3 parts of DNA

Sugar (deoxyribose), phosphate, nitrogenous base (a, t, g, c)

3 parts of RNA

Ribose sugar, phosphate, nitrogenous base (a, u, g, c)

pyrimidines

single ring structure → cytosine, thymine, uracil

purines

double ring structure → adenine and guanine

Makes 2 H-bonds

A and T base pairs

Makes 3 H-bonds

G-C base pairs

secondary structures of DNA

double helix structure, hairpin (stem-loop), and loop (caused by intramolecular hydrogen bonding)

Prokaryotes genetic content

no nucleus, cell is relatively small, one circular DNA molecule, no histones, small amount of DNA, no membrane bound organelles 

eukaryotes genetic content

nucleus, cell is relatively large, multiple linear DNA molecules, histones, large amount of DNA, membrane bound organelles

Tertiary structure of DNA

higher folding that takes place in cells (allows huge amounts of DNA to fit into prokaryotic cells and eukaryotic nuclei) 

Supercoiling

additionally winded/twisted double helix DNA; supercoiled DNA takes up less space than relaxed DNA

positive supercoiling

clockwise direction of winding; overrotated

negative supercoiling

• counterclockwise direction of winding, underrotated

• MOST DNA IS NEGATIVELY SUPERCOILED

Topoisomerases

add or remove rotations in DNA by breaking and then rejoining DNA strands 

euchromatin

usually transcriptionally active, relaxed chromatin, lightly stained regions of chromosomes

constitutive heterochromatin

usually transcriptionally inactive (silenced), tightly condensed, darkly stained regions of chromosomes, highly compacted even during interphase 

facultative heterochromatin

condensed or relaxed under specific conditions

restriction enzymes

• recognizes specific sequence of bases anywhere within the DNA

• endonuclease cuts phosphodiester bonds of both strands

cohesive ends

• type of dna cleavage

• look at top strand and see what side (5’ or 3’) arrow is closer to; either 5’ or 3’ overhang

blunt ends

• type of dna cleavage

• even cut through both strands

gel electrophoresis

• Separates DNA, RNA, or proteins by size using an electric field, with smaller molecules moving farther through the gel than larger ones.

• Used to visualize, compare, and estimate the size and amount of biological molecules in different samples.

gel electrophoresis - size

• smaller molecules move towards bottom

gel electrophoresis - charge

• negatively charged molecules move towards positive electrode at bottom of gel

gel electrophoresis - shape

• tightly condensed molecules move towards bottom

how are gel electrophoresis and blotting used to examine gene expression and interpret the results of such experiments?

• Gel electrophoresis separates DNA, RNA, or proteins by size

• blotting transfers those molecules to a membrane where specific DNA sequences, RNAs, or proteins can be detected using probes or antibodies

• Gene expression is interpreted by the presence, size, and intensity of bands on Northern (RNA) or Western (protein) blots, with darker bands indicating higher expression and absence of bands indicating little or no expression.

3 models for DNA replication

semiconservative, conservative, dispersive

semiconservative replication

each daughter duplex contains one parental and one daughter strand

conservative replication

one daughter duplex contains both parental strands and the other contains both daughter strands 

dispersive replication

each daughter duplex contains both interspersed parental and daughter segments

how does experimental evidence show that DNA replication is “semi-conservative”?

• After one generation of the meselson and stahl experiment, they found that the DNA had one intermediate band (one strand parent and one strand daughter)

• After two generations, there were 2 bands, one in the middle and one light band

• Rules out conservative bc one heavy band and one light band (none in the middle)

• Rules out dispersive bc all the bands would be in the middle (mix of both parental and daughter segments)

Theta replication

• occurs in e. Coli and other bacteria;

• replication intermediate looks like the Greek letter;

• single origin (Ori) of replication;

• single replication bubble and 2 replication forks;

• bidirectional replication from the origin

Linear replication

• occurs in all eukaryotes;

• each chromosome has multiple origins;

• each replication bubble has 2 replication forks;

• bidirectional replication from each origin

replication steps

initiation, unwinding, elongation, and termination

Initiation

• single replication origin (oriC)

• 245 bp minimal sequence, A-T rich

• Initiator protein binds to oriC and causes a short section of DNA to unwind; 

Unwinding

• Helicase binds to the lagging strand, moves in the 5’ -> 3’ direction and breaks hydrogen bonds

• SSBs (single-strand-binding proteins) stabilizes single-stranded regions

• DNA gyrase (a topoisomerase) relieves torsional strain in front of each fork

Elongation

• DNA synthesis is initiated at RNA primers;

• synthesized by primase, a specialized RNA polymerase;

• the RNA primers are complementary and antiparallel to each template strand;

• DNA polymerase cannot initiate DNA strand synthesis without a primer; ALWAYS MADE 5’ -> 3’;

• a single RNA primer needed for each leading strand;

• new primers needed for each Okazaki fragment on the lagging strands

DNA gyrase

• topoisomerase

• relieves torsional strain in front of each fork

Helicase

• binds to the lagging strand, moves in the 5’ to 3’ direction and breaks hydrogen bonds

Primase

• synthesizes RNA primers that are complementary to OriC sequences

SSB (single strand binding proteins)

• stabilizes single-stranded regions

DNA polymerase III

• synthesizes DNA in the 5’ to 3’ direction

DNA polymerase I

• uses 2 activities to complete replication;

  • 5’ to 3’ exonuclease activity removes the RNA primers;

  • 5’ to 3’ polymerase activity adds DNA nucleotides to the 3’ end of the DNA segment preceding the primer

DNA Ligase

• catalyzes formation of a phosphodiester bond between adjacent DNA segments (3’ OH on one segment, 5’ PO4 on the other segment)

RNA primers

Primase synthesizes RNA primers that are complementary to OriC sequences

Leading Strand

• synthesis of one daughter strand occurs continuously in the same direction as fork progression 

Lagging strand

• synthesis of the daughter strand occurs discontinuously, in the opposing direction to fork progression (via Okazaki fragments)