MBS 344: Exam 1 Vocab

process of evolution by natural selection

1. DNA mutation

2. genetic diversity

3. phenotypic variation

4. different survival through fitness and competition

5. change in population characteristics over time→ evolution

Lamarckian evolution is based are ____________.

spontaneous generation

(individual evolution through use)

phenotype vs geneotype

observable characteristics vs genetic information

DNA vs RNA

deoxy→ missing an OH at 2C

nucleotide vs nucleoside

phosphate gp vs no phosphate gp

why does double helix exist?

energetically favorable→ hydrophobic interior and hydrophilic exterior

(binding of two strands)

three stabilizing chemical bonds in double helix

1. covalent bond→ phosphodiester backbone

2. H-bond→ between bases

3. hydrophobic effects→ base stacking due to aromatic rings

all these bonds make the structure very stable

hyperchromic effect

temperature increase→ strand separation (H-bond and base stacking altered)

absorbance increase

hypochromic effect

temperature decrease→ strand unite (H-bond and base stacking altered)

absorbance decrease

identify the peptide termini

N-termini: first amino acid

C-termini: last amino acid

significance of R-gp in amino acids

contain chemical properties that allow interactions w/ one another

levels of protein structure

primary: linear peptide bond (covalent)

secondary: 2D folding and H-bonding → Beta sheat and Alpha helix → independent of side chain

tertiary: collection of secondary and 3D folding→ hydrophobic + side chain interaction + disulfide

quaternary: collection of tertiary→ disulfide + side chain+ hydrophobic interaction

disulfide bonds in proteins

tertiary→ stabilize

quaternary→ combine chains

proteins that live outside the cell need s-s (secreted and transmembrane proteins)

cytosol proteins do not need s-s

three critical components of plasmid in DNA cloning

1. restriction sites

2. origin of replication

3. antibiotic resistance

DNA cloning (definition)

creating a recombinant DNA

restriction enzymes

cut DNA @ specific sites

DNA cloning process

1. gene and plasmid cut the the same RE (digested)

2. sticky ends bind w/ corresponding BP

3. DNA ligase closes gaps in phosophodiester backbone→ recombinant plasmid

product of directional cloning

expression vectors

use two RE, one for each end

purpose of bacterial transformation?

amplify recombinant plasmid by inserting in bacteria

how is DNA cloning used in lab?

1. DNA sequencing

2. gene editing

3. probes

PCR and primer function

amplify a region of DNA w/ a primer

2 primers→ forward and reverse → allow specific region of amplification and replication

PCR process

1. denature: ds→ ss

2. anneal: primer added

3. extend: DNA polymerase, ss→ds

gel electrophoresis in PCR

separates PCR product by size using charge of backbone→ determine success of PCR, one band

PCR + DNA cloning

1. isolate DNA from cell

2. perform PCR using primers with restriction sites

3. analyze PCR products on agarose gel

4. digest PCR product and plasmid with same RE

5. ligase to produce recombinant plasmid

6. transform plasmid into bacteria

7. plate cells on media w/ antibiotic

8. sequence DNA in cloned plasmid

hybridization

two complementary ssDNA or ssRNA sequences bind to form a ds hybrid

probe

allow DNA analysis by detecting presence of specific sequence

southern blot (definition)

determine if correct DNA segment identified

only DNA→ dead cell

southern blot process

1. isolate DNA and cut with RE

2. gel electrophoresis to separate DNA by size

3. denature and transfer DNA to gel

4. incubate DNA in solution w/ probe→ hybridization

5. autoradiography used to detect radioactive probe

fluorescence in situ hybridization (FISH) purpose

visualize chromosome to detect disease with fluorescent probes

dead and alive cells

DNA and RNA

FISH process

1. collect cells, arrest @ M-phase (chromosome is the thickest), fix to slide

2. denature→ ssDNA

3. add fluorescent probe and allow hybridize

4. analyze fixed cells w/ fluorescent microscope → look for probe

poly-dT primer

primer used in cDNA conversion, allows reverse transcriptase to bind

not specific

RT-PCR

measure expression of specific gene w/ cDNA

band size=expression

RT-PCR vs PCR

same: use primer to amplify and gel electroph.

difference: DNA vs cDNA template

qPCR

quantitative way of measuring amplification w/ fluorescent detection→ more precise them images

SYBR green vs probe in qPCR

SYBR→ cheaper, less accurate, detects all fluorescent (not gene specific)

probe→expensive, more accurate, detect fluorescent w/ DNA polymerase (gene specific)

Cq/Ct value

establish the threshold cycles needed to reach significant amplification by comparing samples

Cq~ (1/expression)

northern blot process

1. isolate RNA

2. gel electrophoresis to separate RNA by size

3. transfer RNA to gel

4. incubate RNA in solution w/ probe→ hybridization

5. autoradiography used to detect radioactive probe

no RE nor denaturation

northern blot

band size ~ expression of RNA

fusion protein

normal protein gene combined with another gene→ one protein w/ two part

→ used as signaling molecule, isolate proetin

GST tag

used in fusion protein to isolate protein

high affinity for GSH

GFT

used in fusion protein to indicate protein location w/ in cell

highly fluorescent

type of reporter gene

reporter gene

used in fusion protein to study the functions of regulatory DNA elements like promoter and enhancer

SDS-PAGE

gel electrophoresis for proteins→ separate by size→ use SDS and BME to denature proetin into a linear sequence

SDS→ detergent denatures protein and adds (-)

BME→ reducing agent eliminates disulfide bonds

western blot definition

antibodies used to detect the amount of a specific protein on a membrane

thicker band~more protein

western blot process

1. denature protein (SDS-PAGE)

2. transfer to membrane

3. incubate w/ primary antibody

4. add secondary antibody that binds primary, used for visualization of product

Coomassie stain vs western blot

Coomassie→ nonspecific detection of protein (used as a loading control in SDS-PAGE)

western blot→ specific detection of proteins

Prokaryotic vs Eukaryotic genome

Pro: no nucleus, one circular DNA, DNA naked, one origin of rep, gene rich

Euk: nucleus, many linear DNAs, nucleosome (protein), many origins of rep, gene desert, has centromere and telomere

Both: chromosomal DNA, DNA & RNA polymerase

three DNA sequences in prok

1. protein coding region

2. origin of replication

3. repetitive sequence

also present in euk chromosomes

two stragetgies to compact prok chromosome

1. loop domain

2. supercoiling

DNA underwinding

remove a turn on B DNA→ create structural strain→ energetically unfavorable→ promote supercoil

two functions of supercoiling

1. compaction

2. strand separation (transcription+replication)

what casues “laddering” in euk DNA?

DNA protected by protein in a uniform manner

core histones→ octamer

H2A, H2B, H2, H4

two of each to make octamer

H1 histone

alters exit and entrance of strand

how does histone bind DNA?

histone→ basic amino acids (+)

DNA→ phosphate backbone (-)

Euk DNA compaction levels

1. supercoiled DNA

2. 10 nm→ histone octamer + DNA

3. 30 nm→ histone octamer + DNA + H1

4. chromosome scaffold

5. further loops and coils

chromatin

DNA + protein

chromatin remodeling complex

alter nucleosome arrangement on chromosome

use ATP to eject/replace nucleosome

histone variants

alternative substitutes for histones

variants→ H3 and H2A

no variants→ H2B and H4

significance of N-terminus tail

phosphorylated or acetylated→ intermolecular context to loosen/tighten nucleosome

histone modifications of H3K4, H3K9, H3K27

acetylation→ of all three residues causes increased transcription (euchromatin)

methylation→

• H3K4me: increased transcription

• H3K9 & H3K27: decrease transcription

histone modifications H3.3, CENPA, H2AX

H3.3→ stabilize euchromatin w/ pole= permanently open

CENPA→ adds hooks on centromere for kinetichore binding

H2AX→ throughout genome, phosphorylated when double strand break occurs

purine vs pyrimidine

purine→ 2 rings (G, A)

pyrimidine→ 1 ring (C, U, T)