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113 Cards in this Set

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proteome
proteins expressed by genome
Explain using lactate and NAD+ how an assay works
assay is biochemical test which searches for unique property of a protein.

measures the enzyme activity of lactate dehydrogenase

lactate + NAD+--ladehy--->pyruvate + NADH + H+

Test looks for absorbtion of light that NADH has that NAD does not. (340 nm in one minute))
What are the two things you need for purifying protein?

Once you have them, what can you measure?
protein assay to measure enzyme activity

protein concentration

with these two measure specific activity
specific activity
ratio of enzyme activity to amount of protein in enzyme

the idea is to maximize and increase the specific activity through purification
homogenate
source of protein from cells and tissues
differential centrifugation
homogenizer disrupts cells

homogenate is centrifuged in step by step process yeilding:

500 x g for 10 min = nuclear fraction

10,000 g's for 20 min = mitochondrial fraction

100,000 gs for 1 hour = microsomal fraction
Describe salting out
increased salt decreases a protein's solubility

there are different concentrations for different proteins i.e.
0.8 M ammonium sulfate for fibrinogen
2.4 M ammonium sulfate for serum albumin

concentrates dilute protein solutions
Dialysis
separates proteins from small molecules and salts, but not other proteins

can remove ammonium sulfate from protein preparation

cellulose membrane is semipermeable
what are nucleic acids comprised of?
DNA and RNA

individual monomers are a sugar, base and phosphate

DNA uses 2' deoxyribose for sugar

RNA uses ribose

the linear polymer is linked via phosphodiester bond from 3' end of one monomer to 5' start of next monomer
Base pairings?
Adenine and Thymine

Adenine and Uracil (RNA)

Guanine and Cytosine
Which bases are from purine?
Adenine and Guanine
Which bases are from pyrimidine?
Thymine and Cytosine

Uricil
What is a nucleoside?

What are the names of the nucleosides for RNA?

DNA?
base and a sugar with a B-glycosidic linkage

RNA--adenosine, guanosine, cytidine, uridine

DNA--deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine
where does the b-glyocsidic linkage occur with A & G (purine base)?
N--9
where does the B glycosidic linkage occur in C, T and U (pyrimidine bases)?
N--1

all bases attach to C-1 of sugar.
why is a nucleotide different from a nucleoside?
nucleotides include the Phosphodiester linkage

Pi attaches to 5' of sugar
what is ATP?
Adenosine Tri Phosphate

nucleotide
What is 3' dGMP?
deoxyguanosine 3' monophosphate
What's the nucleotide name for Adenine for RNA and DNA?
RNA- Adenylate
DNA - deoxyadenylate
what's the nucleotide name for DNA and RNA for Guanine?
RNA -- guanylate

DNA -- deoxyguanylate
What's the nucleotide name for Thymine/ uracil for RNA and DNA?
RNA - uridylate
DNA- thymidylate
WHat's the name for nucleotide for cytosine RNA and DNA?
RNA-- cytidylate

DNA--deoxycytidylate
Describe the composition of DNA
comprised of the 4 nucleotides
deoxyguanylate, deoxyadenylate,
deoxycytidylate
thymidylate

5'-pApCpGpT-3' chain polarity
and only one direction.
E coli DNA
single strand 4.6 mil nucleotides long
Describe a double helix
2 oppositely oriented helical polynucleotide chains coiled around common axis

bases are inside, sugars are outside

bases perpindicular to axis

rise of 3.4 A

structures repeat every 34 A so 10 bases / turn and rotation of 36 degrees / base

diameter 20A
How are bases paired (DNA)?
G=C
A=T

held together by weak H bonds
1-5 kcal/mol

van der waals forces = 0.5-2.0 kcal / mol

bases are hydrophobic and clustered inside while polar sugars (- charge) are exposed to water
What is semiconservative replication?
basically shows that the double helix splits open and replicates.
In the daughter generation one strand on new helix is from parent and one is nacent
Explain the Meselson and Stahl experiment
E Coli parent DNA labeled with 15N.
Then they grew Ecoli in 14N environment. Showed the incorporation of 14N during replication

They could detect the difference due to the density difference (1.66 vs. 1.76 g/mol)

after one replication, density was half for 14N and half for 15N

after 2 generations equal amounts of hybrid and 14N DNA
What is DNA melting?
DNA helix can be reversibly melted
heat to Tm (temp at which 50% of structure is lost)~70 degrees C

or

add alkali or acid to ionize bases
What is hypochromism?
a way to determine if bases are stacked or not

Stacked bases absorb less light than unstacked

so at Tm single strands will abosorb more uv light at 260 nm
What are some characteristics of circular DNA?
continuous

may be supercoiled to superhelix for compactibility

may be relaxed
Describe a stem loop structure
made by ssDNA or ssRNA

may form watson-crick base pairs with other mismatched base pairs

can have non-standard base pairing
What is DNA polymerase?
Kornberg discovered it catalyzes DNA synthesis

adds deoxyribonucleotides to preexisting DNA template

directed by the existing DNA template

some remove mismatched pairings
What does DNA replication require to take place?
DNA n

dNTP (of all 4 bases)

5' deoxynucleoside triphosphates

Mg2+

free 3'OH primer
How does the template direct DNA replication?
goes in a 5' -----> 3' direction

3' OH mounts a Nucleophilic attack on the alpha triphosphate chain attached to the 5' of the previous nucleotide

PPi (pyrophosphate) is given off
describe the order in which a protein is made.
DNA transcription to mRNA

mRNA takes it to ribosome

RNA translates with help from tRNA the codons into proteins
Describe mRNA (E.coli)
template with codons which serves as the master for translation.

1.2 kb long average for E coli

5% of total RNA (ribosomal RNA, tRNA make up rest)
Describe tRNA
one for each of 20 amino acids

~75 nucleotides
~25 kDa

carries the activated amino acid and with its corresponding anticodon aids in translation in ribosome

makes up about 15% of RNA
Describe rRNA
ribosomal RNA

makes up 80% in E. coli
This 80% is comprised of 23S, 16S and 5S species
Describe snRNA
only in Eukaryotes

works in splicing out the inons
Describe miRNA
micro RNA

class of small non-coding RNA's (~21 nucleotides

These guys bind complementary mRNAs and therefore inhibit translation
describe small interfering RNA
siRNA
small non-coding RNA
bind up mRNA and degrade it
Describe RNA polymerases
catalyzes transcription

uses dsDNA or ssDNA as template

makes and elongates RNA chains
What does an RNA polymerase need to start elongation?
ATP, GTP, UTP, CTP

Mg2+ or Mn2+

template but NO primer needed
Describe transcription
RNA polymerase aids for the free 3'OH to mount nucleophilic attack on the incoming alpha phosphate

reaction is aided by hydrolysis of PPi

No proofreading here.

The new mRNA strand is identical to the coding DNA strand. (not identical to template, but complementary to template)
How do mRNA in Eukaryotes differ from those in Prokaryotes?
There is a 5' cap and a 3' poly AAAAAAAA structure added after transcription
Describe the role and mechanism of tRNA
brings amino acids to ribosome for making proteins

aminoacyl tRNA synthetase uses ATP to make aminoacyl-tRNA which is a unique tRNA for each amino acid

each one of these aminoacyl-tRNA's have a unique anticoding site which will recognize the codon on mRNA
what is and what is the significance of a degenerate genetic code?
amino acids can have more than one codon.

64 possible codons for 20 amino acids

3 triplets are stop codons so the leftover 61 code for the 20 amino acids

degenerate code decreases harmful mutations and having too many chain terminators
Where is the amino acid attached to tRNA?

What's on the other end
3' end of tRNA attaches to the carboxyl carbon of the amino acid

5' end is phosphorolated
Which two amino acids only have one codon?
Trp

Met
Which three amino acids have 6 codons?
Leu

Ser

Arg
What are the stop codons?
UAA

UAG

UGA
What is the codon for Trp
UGG

Take a Trp to australia to get some uggs
What is the codon for Met?
AUG

Mett's birthday is in AUG
XY___ and XY____ ALWAYS encode for the same amino acid
C and U
XY____ and XY_____ USUALLY code for same amino acid
G and A
For E. coli what starts translation?
On the mRNA chain a 5' shine dalgano (purine rich) marker occurs at -10, and then an AUG or GUG is +1 which tells the fMet to bind there and begin
For eukaryotes what starts translation?
At the 5' cap, Met-tRNA binds to the first AUG it sees and goes from there.
How does translation know when to stop?
UAA, UAG and UGA are stop codons

This binds protein which is not tRNA and it uses release factors to release the new protein
Describe how universal the genetic code is.
Nearly universal but some deviations

in humans UGA is a code for Trp and not a stop codon
instead we have AGA and AGG as stop codons and we don't code for Arg

mitochondrial DNA is different because they make their own tRNA's

16 organisms deviate from the standard genetic code
what are introns and exons
introns are unused code and exons are expressed genes which means we must splice these guys.

Humans have 8 introns which can be 50-10000 nucleotides long
Describe human hemoglobin B chain with regard to introns and exons
3 exons and 2 introns
Describe how we know about introns and exons?
pre m RNA is heavier--has a 15S rate as opposed to the actual mRNA which is 9S rate.
What is a spliceosome?
assemblies of proteins and small RNA molecules

recognizes that introns begin with GU and end with AG preceeded by essential pyrimidine tract
What would be the benefit of exon shuffling or alternative splicing?
shuffling allows for recombined genes which may provide diversity of function

alternative splicing may lead to new proteins being formed
How do restriction enzymes provide the bases for gene exploration?
restriction endonucleases cut DNA in certain spots where they recognize specific sequences and cut both strands
Where do restriction enzymes know where to cut?
the cleavage site is 4-8 bp at a palindromic symmetry site.

They see this symmetry and hydrolyze the phosphodiester bonds here

can leave staggered (sticky) or even (blunt) cuts.
EcoRI
5' G*AATTC 3'
3' CTTAA*G 3'

derived from E. coli
What are the endogenous and laboratory uses for restriction endonucleases
In cells, these guys cleave foriegn DNA without harming host DNA

In labs, we use it to cleave DNA into fingerprints so we can work with it better
Describe SV40
5.1 kb circular DNA

has 1 EcoRI cleavage sit

has 4 HpaI

has 11 HindIII
Once you have DNA fragments what do you do with them to manipulate them?
Gel electrophoresis will separate up to 1,000bp

(more porous agarose up to 20 kb)

Pulse Field Gel Elect. separates chromosomes
what does ethidium bromide do?
stains dsDNA fragments after separated by gel elect. and has sensitivity to show 50ng with orange fluorescence
Explain how to identify restriction fragments using 32P probe

which technique is this?
Separate different fragments in gel

denature them to make ssDNA

use a nitrocellulose sheet

a strand, matching the one you are looking for has 32P incorporated. when it finds its ppair it links up and audioradiography will show you which one it is on paper

southern blotting
What other things can southern blots reveal about a restriction fragment?
mutations on restriction fragment

grounds for polymorphism RFLP restriction fragment length polymorphism
What is northern blotting?
same technique as southern but for RNA
Describe dideoxy method for DNA sequencing
The method of incorporating a small amount of dideoxynucleoside triphosphatees which serve to effectively terminate replication at various points

primed with synthesized fragment

use DNA polymerase

add all the deoxyribonucleoside triphosphates and 2' 3' dideoxy

gel and audioradiogram to sequence
what's a more modern less radioactive sanger method?
fluorescence tagging

can run all four bases in one reaction

up to 500 bases can be determined

automated. can generate more than 1 million bases / day
What method is used for making DNA or DNA probes?
solid phase phosphite triester method

use glass bead

add excess reagents to drive rxn to completion

sequential addition of protonated (proteceted) deoxyribonucleoside 3' phosphoramidites

wash soluable protectors away
what are the reagents for phosphite triester method?
deoxynucleoside 3' phosphoramidite

DMT dimethoxytrityl

BCE B cyanoethyl

I2 (oxidizer)

dicholoroacetic acid

NH3
What is the role of NH3 in phosphite triester method?
remove all protecting groups and remove the deoxyribonucleoside 3' phosphoramidite from the glass bead
what is role of dichloroacetic acid in phosphite triester method?
removes DMT from 5' end of newly added deoxyribonucleoside.
where does the triester form in phosphite diester method on activated monomer and chain?
phosphit on 3' of activated monomer attaches to 5 ' OH of chain.
What's the efficiency of the phosphite triester method?
99%

10 min per cycle

elongation is never 100% so the longest one is the golden one. You can find this using PAGE or HPLC
Once you makes some DNA using the phosphite triester method, what can you do with it?
make probes (32P or fluorescence tag and then set it loose in fragments to find the needles in the haystack)

use it as a primer for new neighboring DNA

make new designer genes which can make new funcitons or new proteins
What is PCR?
Polymerase Chain Reaction
amplifies your sample
What are the 3 steps in PCR>
strand separation (heat)

hybridize primers (cool with a ton of primers in there --20-30 nucleotides long)

heat to 72 so Taq polymerase can start making the strand 5' -3'

repeat 25-30 times

n cycles = 2tothe n results
Describe recombinant DNA technology
DNA fragment + a vector (plasmids or lambda phage)

host (E. coli)
Describe how Eco RI works with plasmid pSC101
cuts pSC101 at a specific site leaving a sticky end.

If you use Eco RI to cut any piece of inserting DNA those same sticky ends will line up and voila.
What is DNA ligase?
DNA ligase catalyzes the formation of phosphodiester bonds

needs a free 3'OH and 5' phosphate as well as ATP or NAD+
Describe the cohesive ends (Linkers) method.
If you have a DNA segment you can add cohesive ends to put the tape on the ends.

polynucleotide kinase phosphorylates the 5' end of a linker

T4phage ligase seals them (can be blunt ended).

then use EcoRI to make the sticky ends and insert into your vector
what is a polylinker?
a synthetic DNA fragment programed with several sequences that many restriction endonucleases recognize. Can custom make your sticky ends.
why are plasmids and lambda phages good vectors?
they get the recombinant DNA into bacteria easily which you can then select for.

Plasmids carry genes for inactivation of antibiotics and can replicate independently of host genome
What is it about pBR322 that makes it good for cloning?
has genes for resistance to tetracycline and ampicilin

the EcoRI site isn't in these genes for the ab resistance
but it does have other restriction enzyme sites which will interrupt the resistance
Describe selection using pBR322
pBR322 uses the enzyme cut in amp. resistant element and foreign DNA ligated in.

Now these guys are going to be amp sensitive. Still resistant to tetra.

grow E coli on plates of tetra.

transferred in matching postions to new plates . One plate has tetra and another has amp and tetra. Now you will know which ones are amp sensitive
What size is a plasmid?
2-100+ kb
What is the size of lambda phage?
48 kb
What are the differences between a lytic and lysogenic pathway?
lytic kills host cell quickly and produces ~100 virons

lysogenic inserts DNA into host cell genome and it is replicated for many generations

Upon activation it expresses dormant viral DNA
What is lambda gt-lambdaB
mutant lambda phage with only 2 EcoRI sites as opposed to the normal 5

enter bacteria way easier than plasmid
Describe lambda gt-lambdaB and how it gets packaged.
EcoRI cleavage gives two arms, and foreign DNA inserted to make it 93% (fits in the 75-105% size requirements
what is cosmid lambda?
hybrid of lambda phage and a plasmid

accomodates large DNA inserts of 45 kb!!
What is a BAC
Bacterial artificial chromosome

engineered version of ecoli fertility F factor.

accomodates inserts of DNA in 100,000-300,000 bp

selectable with chloramphenicol resistance

put in bacteria by electroporation
What is a YAC
Yeast Artificial Chromosome

accomodates inserts as big as 2,000,000 bp

lots of stuff in em that we didn't really cover but that makes them ideal for big genomes
What is a genomic digest?
mechanically sheared fragments of a 3,000,000 kb genome

makes random overlapping fragments which can be PAGEd

isolate 15 kb long molecules and add linkers and then insert into lambda phages and then grown in E coli.

gives you genomic library
Why do we need DNA hybridization?
Allows us to screen the genomic library
What is DNA hybridization?
the recombinant phages are plated on a lawn of bacteria where they like guys grow a plaque.

Nitrocellulose is then applied and we add NaOH to lyse and denature the DNA. 32P probe DNA is introduced and the target sequence is radioaudiographed.
How do we know how to make the probe for human hybridization screening?
take the mRNA from an enriched source of what you are looking for. Make a cDNA from this mRNA and use as probe

use amino acid sequence from target (use sequences with a lot of Trp and Met

Synthesize the probes with 32P

Use all the probes and isolate and sequence positive clones
Why do we need a probe with minimal degeneracy?
lots of peptides are encoded by several oligonucleotides. Sequences where there is alot of Met or Trp are best because they only have one codon
Why isn't Ecoli. good for mammalian mRNA?
E. coli doesn't have the ability to splice, so we have to make cDNA from mRNA (which doesn't have introns)
What is the difference between SINES and LINES?
Short intersperced elements (DNA that doesn't code for proteins)
1,000,000 Alu sequences of 300bp

Long intersperced elements
nearly 1 million LINES up to 10 kbp
how do we make cDNAs
mRNA + oligo T primer with 3'OH + 4 dNTP's

oligo T pairs with poly A at 3' end of mRNA

reverse transcriptase makes a DNA complementary to RNA template

increase the pH and dissolve mRNA

add terminal transferase which adds oligo GGG to 3' end of the ssDNA

then add oligo CCCC to make cDNA
What antibody do we use in immunochemical screening of cloned cDNA?
125I antibody
Describe DNA microinjection method/
0.1 mm micropipet

yeilds 2% viability

foster mother mouse
what is GH 21-kd protein?
rat growth hormone

in rats it is promoted by metallothionein promoter

have an activator