biochem exam 3 Flashcards
aldoses
ketoses
trioses
pentoses
hexoses
aldoses: aldehyde, sugar
ketoses: ketone, sugar
trioses: 3 carbon sugar
pentoses: 5 carbon sugar
hexoses: 6 carbon sugar
Where the chiral centers are in
aldoses
ketoses
trioses
pentoses
hexoses
aldoses
ketoses
trioses
pentoses
hexoses
need help
Number of possible stereoisomers as a function of number of chiral centers
2^n
if 4 chiral
2^4 = 16 stereoisomers
“D” and “L” are defined in reference to glyceraldehyde
D: OH on right
L: OH on left
In nature, almost all monosaccharides are “D” isomers (though there are some “L” sugars). Know what this means.
means that the OH furthest from the carbonyl is to the right
epimers
epimeric pairs
enantiomers
diastereomers
epimer: only 1 chiral center is different
epimeric pair: molecules with only 1 chiral carbon that is different
enantiomer: all chiral centers are different
diastereomer: all but 1chiral center is different
The standard abbreviations for:
fructose
galactose
glucose
mannose
ribose
fructose (Fru)
galactose (Gal)
glucose (Glc)
mannose (Man)
ribose (Rib)
D-Aldoses that you have to know
do you know the structures yet?
what are the number of carbons and which are ketoses and which are aldoses
D-ribose
D-glyceraldehyde
Dihydroxyacetone
D-fructose
D-glucose
D-Galactose
D-Mannose
D- Aldoses:
D-glyceraldehyde - 3
D-ribose - 5
D-glucose - 6
D-Mannose - 6
D-Galactose - 6
D-ketoses:
Dihydroxyacetone - 3
D-fructose - 6
YES, I KNOW THEM. GOD IS GOOD
NO, I DON’T AND GOD IS STILL GOOD
- there are only two D-ketoses and fructose is 6 and Dihydroxyacetone is 3, the rest of the molecules are D-aldoses*
- do linear sugars spontaneously cyclize in solution if so, what molecules can they form
how are the two molecules formed?
can anything else be done to the molecules so that they turn to another molecule
cyclized sugars with hemiacetals or hemiketals form from linear sugars
hemiacetal:
aldehyde + OH = hemiacetal
hemiacteal + OH = acetal
hemiketal:
ketone + OH = hemiketal
hemiketal + OH = ketal
- What pyranoses and furanoses are
pyranose: 6-membered oxygen-containing ring
furanose: 5-membered oxygen-containing ring
- What anomers (anomeric pairs) are (α and β), and what mutarotation is (did he talk about this?- he actually skipped it…)
anomer: The carbonyl carbon becomes a new chiral center called the anomeric carbon. Which is where we determine if there is a hemiacetal or not :)
forms if the OH attacks from the top or the bottom
- What Haworth perspectives are
the ring form of the sugar where the OH is either up (beta) or down (alpha)
- There are many derivatives of hexose. Remember N-acetyl-β-D-glucosamine, but don’t memorize structures.
N-acetyl-β-D-glucosamine has
- acetyl group
- OH & CH2OH up
- is a glucose
- amine group
it is the subunit of chitin!
- Most monosaccharides are reducing sugars, what does this mean
what makes a disaccharide a reducing sugar
what disaccharides are reducing sugars and which are not
reducing sugars: carbonyl group can be oxidized
When a disaccharide is formed, if the anomeric carbon of at least one of the monomers remains ‘free,’ and has a hemiacetal the disaccharide is a reducing sugar; but if both anomeric carbons are part of the O-glycosidic bond, the disaccharide is not a reducing sugar.
Maltose and lactose are reducing sugars, while sucrose and trehalose are not.
how are blood glucose levels measured
how can average blood glucose over time be measured
Blood glucose levels are measured using glucose oxidase (more specific than Fehling’s Reaction).
Average blood glucose level over a period of time (2-3 months) can be measured by measuring the concentration of glycated (glycolsylated) hemoglobin (GHB; a.k.a. hemoglobin A1c).
The shorthand for indicating the structures of disaccharides, such as lactose and sucrose
lactose = Gal(β1→4)Glc (a reducing sugar) or sucrose = Fru(β2↔1α)Glc (non-reducing)
- The disaccharides in Maltose & Trehalose
Sucrose
Lactose
Maltose & Trehalose (Glc, Glc), Sucrose (Fru, Glc), and Lactose (Gal, Glc)
which of the following is an aldopentose
D- glyceraldehyde
D-ribose
D-glucose
Dihydroxyacetone
D-fructose
D-ribose
Sugars form Rings: The Cyclization of Sugar
An aldehyde and an alcohol can react to form a _____.
If another alcohol is available, the reaction can happen again to form an _____.
This same theme can happen with _____.
This is important because ______ have ____ groups (aldoses), or _____ groups (ketoses), and alcohols!
These groups can react with themselves, in the same molecule, and _____!
An aldehyde and an alcohol can react to form a hemiacetal.
If another alcohol is available, the reaction can happen again to form an acetal.
This is important because monosaccharides have aldehyde groups (aldoses), or ketone groups (ketoses), and alcohols!
These groups can react with themselves, in the same molecule, and cyclize!
Cyclization takes place as a result of interaction between the functional groups of distant _____
Between ____ and ____ to form a cyclic hemiacetal in aldohexoses
Between ____ and _____ To form a cyclic hemiketal in ketohexoses
what is the structure of a hemiacetal
what is the structure of an acetal
Cyclization takes place as a result of interaction between the functional groups of distant carbons
Between C-1 and C-5To form a cyclic hemiacetal in aldohexoses
Between C-2 and C-5 To form a cyclic hemiketal in ketohexoses
hemiacetal: on carbon there is:
OR
OH
R
H
acetal:
OR
OR
R
H
For D-sugars: CH2OH is ___
alpha OH is ___
beta OH is ___
For L-sugars: CH2OH is ___
alpha OH is ___
beta OH is ___
For all:
alpha OH is ___ CH2OH
beta OH is ____ CH2OH
For D-sugars: CH2OH is up
alpha OH is down
beta OH is up
For L-sugars: CH2OH is down
alpha OH is up
beta OH is down
alpha OH is opposite CH2OH
beta OH is same side CH2OH
what is the hexose derivative that you are supposed to know
what is it used for
N-acetyl-β-D-glucosamine
(bacterial cell walls, and other things)
Many sugars (including glucose) are _____!
The carbonyl (aldehyde or ketone) can be ____
For example, the linear form of D-glucose can participate in ____ reactions.
An example of this is an older way of testing blood glucose. _____ Reaction
any sugar with a hemiacetal can ‘open up’ to form a _____
Many sugars (including glucose) are reducing agents!
The carbonyl (aldehyde or ketone) can be oxidized
For example, the linear form of D-glucose can participate in REDOX reactions.
any sugar with a hemiacetal can ‘open up’ to form a reducing sugar
as it is reducing, it is itself being oxidized
what happens to a reducing agent as it reduces another compound
what must a reducing sugar have an oxidizable aldehyde group. Ketoses are reducing sugar because they can be converted to aldoses
a reducing agent is oxidized as it reduces another compound
a reducing sugar must have an oxidizabe aldehyde group. Ketoses are reducing sugar because they can be converted to aldoses
A hemiacetal, when opened up, forms an ____, which can be ____
If hemiacetal: “____ _____”
If not, not ____
is it reducing?
A hemiacetal, when opened up, forms an aldehyde, which can be oxidized because it is a carbonyl!
If hemiacetal: “reducing end”
If not, not reducing
is it reducing? Look for hemiacetal
Measuring [Blood glucose]- old fashion way
what do you need to monitor for diabetes
what is the rxn that is involved
what does the brown color mean
diabetes need to monitor the concebtration of glucose in their blood, in order to adjust theur insulin dosage
the rxn used is
D-glucose + O2 + glucose oxidase (which oxidizes glucose) forms D-glucono-delta-lactone + H2O2
this turns blood “brown” and we meausre the color change
the more brown, the more glucose in blood
what is another way to measure glucose
what does hemoglobin react with and what does it form
what is this molecule also called
what does this molecule tell you about the person
measure A1C
Glucose reacts with Hemoglobin to form “glycated hemoglobin (GHB)”
GHB is A1C
gives broader picture of your “glucose habits” because
[GHB] is proportional to average G1C over the preceding 2-3 months
monosaccharides to disaccharides
how can monosaccharides form disaccharides
through what bond can two sugar molecules be joined and between what type of carbons
what atoms form the O-glycosidic bond:
is maltose a reducing sugar, why or why not
Monosaccharides can form di- and poly-saccharides through glycosidic bonds!
Two sugar molecules can be joined via a glycosidic bond between an anomeric carbon and a hydroxyl carbon
O-glycosidic bond:
between the anomeric (hemiacetal) carbon of one sugar and an -OH group of another sugar
maltose is a reducing sugar because it has an available hemiacetal
Disaccharides: Glycosidic Bonds
what is numbered
what do the numbers refer to
what is possible between the same monosaccharides
what are enzyme specific for
what type of bond can humans not digest and what polysaccharide is it in
for all Disaccharides,
α: OH ____. CH2OH
β: OH ____ CH2OH
These glycosidic bonds are numbered
The numbers (ex: 14) refer to the carbons involved in the bond. 1 is the anomeric carbon. 5/6 is the CH2OH carbon.
Multiple linkages are possible between the same monosaccharides.
Enzymes are specific for these linkages.
Humans cannot digest most β-glycosidic bonds which is in cellulose and lactose
For all:
α: OH opp. CH2OH
β: OH same side CH2OH
what you need to know about disaccharides
maltose
lactose
surcose
trehalose
what monosaccharides make it up and which are reducing and which are non-reducing
what do you have to be reducing and non-reducing
maltose: Glc (a 1 - 4) Glc - reducing
lactose: Gal (b 1 - 4) Glc - reducing
sucrose: Fru (b 2 - 1a) Glc or Glc (a 1 - 2b) Fru - non-reducing
trehalose: Glc ( a 1 - 1a)Glc - non-reducing
reducing has a hemiacetal (OR, OH, R, H) and non-reducing has an acetal (OR, OR, R, H)
Polysaccharides!
what does poly mean
what does saccharide mean
what are 3 major functions
what kind of molecules are storage and what are they stored for
what kind of molecules are structural
cell surface polysaccharides are what kind of molecule
what is the nomenclature
what are they also known as
“Poly” (many) “saccharide” (sugar)
Polysaccharides are carbohydrates made up of many sugars bound together
Major Functions: Storage, Structure, Recognition (cell-cell recognition)
Starch and Glycogen are storage (energy) molecules
Chitin and Cellulose are structural molecules
Cell surface polysaccharides are recognition molecules
Nomenclature:
Homopolysaccharide vs. Heteropolysaccharide
These are also known as “glycans”
Polysaccharides!
In nature, most carbohydrates exist as polysaccharides
Homo/Hetero
Linear/Branched
Glucose is usually tied up in a large polysaccharide
Homoepolysaccharide
unbranched: same monomers made into a polymer but not branched
branched: same monomers made into a polymer but branched
heteropolysaccharides
two monomer types. unbranched
- made of two different monomers into an unbranched polymer
multiple monomer types, branched
- - made of two different monomers into a branched polymer
Polysaccharides
why store carbohydrates as polysaccharides rather than as monosaccharides
Osmolarity
1 - storing glucose in glycogen = lower [free glucose]
2 - glycogen is largely insoluble and therefore does not contribute to osmotic pressure
3 - without glycogen, glc conc. would be ~0.4M leading to cell explosion because water would rush into cells (?)
Polysaccharides
why store carbohydrates as polysaccharides rather than as monosaccharides
concentration gradient and thermodynamics
[Glc] inside the cell is less than outside.
Glc tends to move into the cell.
If Glc was free…
[Glc] inside the cell is greater than outside.
Glc cannot move into the cell.
Storage Homopolysaccharides - Starch
Starch (in plants) is composed of two polysaccharides, what are they
amylose & amylopectin
Amylose: unbranched glucose polymer (α1 - 4 linkage)
Amylopectin (also glucose): linear parts (α1 - 4 linkage)
- Branches out every 24-30 residues (glucose) using α1 - 6 linkages
Starch granules are thought to be clusters of amylose and amylopectin
Double helical structures can form
Non-reducing end glucose sugars are removed sequentially.
amylose and amylopectin both have glucose monomers
Storage Homopolysaccharides – Glycogen
which organism is it the main storage for
why is it more compact
Main storage polysaccharide in animals
Greatest abundance in liver and muscle cells
Similar in structure to amylopectin, but with more branch points
Unbranched glucose polymer (α1 - 4 linkage)
Branches out every 8-12 residues (glucose) using α1 - 6 linkages - glycogen has more frequent branching than amylopectin which branches every 24-30 glucose
It is more compact. Why? - less surface area and also more space to chop off glucose
Why Branching?
to use polysaccharides as sources of energy, degradative enzymes must degrade polymers into monomers
these degradative enzymes act only on non-reducing ends (has acetal)
each branch ends with a non-reducing sugar
branching makes possible more rapid degradation
Structural Homopolysaccharides - Cellulose
Cellulose is the primary structural component of plant cells
Most abundant polysaccharide in nature
Unbranched: 10,000-15,000 glucose monomers
β-(1-4)-linked glucose (previous polymers were α glucoses); water-insoluble, tough
Starch was a tightly coiled helix, cellulose is a straight chain
Wood (50% cellulose), Paper (90%), Cotton (90%)
How is Cellulose so Strong?
what does the bond allow for
there are hydrogen bonds:
- between the sheets to strengthen the structure
- intrachain hydrogen bonds
- interchain hydrogen bonds
(also seen in the base pairing of DNA/RNA)
β-(1-4)-linked glucose allow for the formation of hydrogen bonds
most animals cannot use cellulose as a fuel source because they lack the enzyme to hydrolyze β-(1-4)-linkages
Most animals do have the α-amylase enzyme to digest amylose (starch)
so basically because it has the β-(1-4)-linked glucose which allows for hydrogen bonds
Structural Homopolysaccharides - Chitin
is it branched
what is the monomer
is it abundant
what is it the main part of
where is it also found
straight-chain (unbranched), like cellulose
monomer is N-acetylglycosamine
the second-most abundant polysaccharide
the main part of exoskeletons in lobsters and insects
chitin is also a component of the cell walls of some fungi to inhibit the synthesis of chitin
Structural Heteropolysaccharides - Peptidoglycan
where are they found and are the flexible or rigid
what is the pattern of their subunits
what are its subunits and how are they arranged
what drug blocks its synthesis
Peptidoglycans: rigid component of bacterial cell walls
Alternating monosaccharides (repeating disaccharides)
Peptide-cross-linked linear polysaccharides
Penicillin and related antibiotics block synthesis
Structural Heteropolysaccharides – Glycosaminoglycans (GAGs)
what is the pattern of its subunits
are there cross links
what is it a component of
are they polar, what do they do
what are examples
Repeating disaccharides (…ABABAB…) with an amino side-group
No cross-links
Components of the gel-like extracellular matrix (ground substance – connective)
Highly polar molecules attract water lubricant/shock absorbers
Examples: hyaluronan, chondroitin sulfate, keratan sulfate, heparin
Structural Heteropolysaccharides – Glycosaminoglycans (GAGs) examples
hyaluronan
chondroitin sulfate
keratan sulfate
heparin
Glycoconjugates
what are they
what are they made of
what are the 3 types
Information carriers,
“anchors” embedded in the cell membrane, plus oligo- or polysaccharides (floating outside of the cell membrane – into the ECM)
proteoglycans = proteinn + glycosamineglycans
glycoproteins. = protein + carbohydrate
glycospingolipids = membrane lipid + oligosaccharide and lipopolysaccharides
Glycoconjugates - Proteoglycans
what are they composed of
where is it found
what is the main site of biological activity
what is its function
Core protein + glycosaminoglycans
Found on the cell surface or in extracellular matrix
1 or more glycosaminoglycan chain(s) covalently bound to a core protein
The glycosaminoglycan is often the main site of biological activity
Proteoglycan Aggregates
ECM component
Absorbs/release large amounts of water (polarity)
Functions as “shock absorber”
Glycoconjugates - Glycoproteins
how are they linked
what are they composed of
where can you find this
what are examples
what is the O and N linkage
Glycoproteins: proteins covalently linked to carbohydrates through N- or O-linkages
Protein + 1 or more oligosaccharide(s)
Carb components are smaller/more structurally diverse than in proteoglycans
Cell surface/ECM
Hormones, Immunoglobulins (IGs)
O-Linkage:
Between hydroxyl groups of serine or threonine and N-acetylgalactosamine
N-Linkage:
Between asparagine and N-acetylglucosamine
Glycoconjugates - Glycolipids and Lipopolysaccharides
what is another name for this
what are the composed of
where does this happen
what are they binding sites for
LPS
where are they most dominant
is it a toxin and if so what kind
what are they targets for
Glyco(sphingo)lipids:
Oligosaccharides covalently bound to lipids via a glycosidic linkage
On the outer surface of plasma membranes
Often binding sites for proteins (antigens, blood group determinants)
Lipopolysaccharides:
Dominant surface feature of gram-negative bacteria (E. coli, Salmonella enterica)
Endotoxin (released when bacteria dies inflammatory/immune response)
Prime targets of antibiotics
what are the main groups of glycoconjugates?
(1) proteoglycans
(2) glycoproteins
(3a) glycolipids
(3b) lipopolysaccharides.
Some lectins
what is the function of each and where does it attach onto
human selectin: mediate inflammatory response
viral lectins (esp. influenza): attach onto oligosacahrides on human cell membrane
H. pylori lectin: attach onto the oligosaccharides of gastric cells
cholera & pertussis toxins: attach to host cell oligosaccahrides
- it may be possible to prevent disease by blocking the lectin
human selectins
Human selectins mediate the inflammatory response (RA, asthma, psoriasis, MS, transplant rejection) – and are therefore potential drug targets
Lectins
what are they
where are they found?
what do they serve
what do human selectins do
what are P-lectins on
Proteins that specifically and strongly bind carbohydrates (usually attached)
Found in all organisms.
these proteins in viruses and bateria attach onto the oligosaccharides/other carbohydrates of other proteins and can use NA to chop off and release viral contents from the cell
Serve a wide variety of cell-cell recognition, signaling, and adhesion processes (CAMs)
Human selectins mediate the inflammatory response (RA, asthma, psoriasis, MS, transplant rejection) – and are therefore potential drug targets
P-selectins on endothelial cells and oligosaccharides on leukocytes
Lectins and Viral Infections
what is the cell membrane covered with
what do some proteins have
what is influenza and what is it called
what does it bind
what is going on in simple terms
what does neuraminidase do
what drugs is used for the flu
what do neuraminidase inhibtors do
The cell membrane is covered in glycoproteins.
Some proteins are “decorated” with a carbohydrate called neuraminic acid (sialic acid). Neu5Ac is typical.
Influenza has a receptor-binding membrane fusion glycoprotein (lectin) called Hemagglutinin (HA)
it binds to sialic acid-attached glycoproteins
( so the flu virus has a glycoprotein or lectin called Hemagglutinin or HA that can bind onto the carbohydrate neuraminic acid or sialic acid)
AND a neuraminidase (NA) which clips sialic acid off of the glycoprotein
neuraminidase inhibitors - Oseltamivir (Tamiflu) - competitive inhibitor, so this drug binds onto the enzyme instead of the substrate
Neuraminidase inhibitors are drugs that block the function of the viral neuraminidase protein. By blocking this protein enzyme it stops the release of viruses from the infected host cell and prevents new host cells from being infected.
Lectins and Human Health
H.pylori
-where does it attach onto
what can block adhesion
cholera and pertussis
- where does it bind
- what can prevent disease
what diseases is it linked to
what are some Pharmacological tools
H.pylori attaches to gastric cells surface oligosacchariddes
- analogs of those oligos may block adhesion
cholera toxin and pertussis toxin are lectins that attach to host cell oligosaccharides
- it may be possible to prevent disease by blocking the lectin
Linked to all kinds of cancer, ulcers, inflammation, etc.
Pharmacological tools: PTx blocks Gi so can’t turn off AC (cAMP)
CTx leaves AC on (cAMP)
Sodium and water out diarrhea (death?)
Starch: Amylose
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Storage
Homopolysaccharide
Glc
α1→4
unbranched
Starch: Amylopectin
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Storage
Homopolysaccharide
Glc
α1→4 unbranched
branched (α1→6)
Glycogen
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Storage
Homopolysaccharide
Glc
α1→4
branched (α1→6)
Cellulose
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Structural
Homopolysaccharide
Glc
β1→4
unbranched
Chitin
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Structural
Homopolysaccharide
GlcNAc
β1→4
unbranched
Peptidoglycans
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Structural
Heteropolysaccharide
Repeating disaccharide
peptide cross-links
Glycosaminoglycan
function
type of polysaccharide
core subunit/monomer
linkage
is it branched/unbranched/crosslinked
Structural
Heteropolysaccharide
Repeating disaccharide
Proteoglycans
Composition
Location (example)
Protein + 1 or more glycosaminoglycan(s)
Cell surface
Glycoproteins
Composition
Location (example)
Protein + oligosacch. (smaller, more diverse)
Cell surface (e.g., glycophorin)
Glycolipids
Composition
Location (example)
Lipid + oligosacch.
Outer memb. surf. (e.g., blood group determinants)
Lipopolysaccharides
Composition
Location (example)
Lipid + oligosacch.
Outer memb. surf. of gram-neg. bact. (target of antibodies)
Human selectins (on human cell surfaces)
Bind to oligos on:
Function or examples
(Potential) drug action
Other human cells
Inflammatory response
May block interaction, response
Bacterial lectins (on cell surfaces)
Bind to oligos on:
Function or examples
(Potential) drug action
Human cell surfaces
H. pylori
Oligo analogs to block adhesion
Viral surface lectins
Bind to oligos on:
Function or examples
(Potential) drug action
Human cell surfaces
Influenza
Anit-virals prevent release, prolif.
Toxins (released by microorganisms)
Bind to oligos on:
Function or examples
(Potential) drug action
Human cell surfaces
Cholera, pertussis
Attach to (block) toxins
what are the two bases of Nucleotides and Nucleic Acids?
how do you remember them
what is a nucleoside/how do you remember it
what is a nucleotide?
what makes ribose different than deoxyribose
what are the 4 bases of DNA and RNA?
Bases:
purine (smaller word, bigger molecule)
pyrimidine (bigger word, smaller molecule)
nucleoside
- a nitrogenous base (purine or pyrimidine) with a pentose sugar on the side
nucleotide:
– a nitrogenous base (purine or pyrimidine) with a pentose sugar on the side and a phosphate
ribose vs deoxyribose
- ribose: OH on 2’ carbon
- deoxyribose: H on 2; carbon
bases on DNA vs RNA
- DNA: AGCT
- RNA: AGCU
uracil instead of thymine is the main difference
differences in Nucleotides and Nucleic Acids: Bases
for purines, how many rings & what are the two types
what is different between the 2 bases
for pyrimidines, how many rings & what are the 3 types
what is different between the 3 bases
what are the nucleotides for DNA & RNA
for purines: 2 rings
- adenine and guanine are similar
- adenine has just an N while guanine has a NH
- adenine has just a CH while guanine has an NH2
for pyrimidines: 1 ring
- cytosine has N
- thymine has NH & CH3
- uracil has NH
difference between thymine and uracil: thymine has a CH3 and uracil does not
DNA: AGCT
RNA: ACGU
Sugars: Ribose v Deoxyribose
What are the 2 types of furanoses?
What is the sugar residue in nucleotides called? and what is the name of the specific sugar?
how does RNA compare with DNA
how does the difference affect DNA or RNA
what are ribo/deoxyribonucleic acids?
DNA & RNA are both beta-furanose rings (B-furanose rings) XD GREAT JOB!; both are furanose rings
the sugar residue in nucleotides is a pentose (sugar with 5 carbon atoms). The specific sugar is ribose
RNA = ribose (2’ -OH)
DNA = 2-deoxyribose (2’ –H)
each sugar in DNA has 1 less -OH group than each sugar in RNA
this small difference affects secondary structure, function, and stability
ribonucleic acids = polymers of ribose
deoxyribonucleic acids = polymers of deoxyribose
We can move that phosphate group around the sugar…
where is the standard position
can be :
standard 5’
2’
3’
2’, 3’ cyclic
for standard 5’
- we can move the P around
- can even be cyclic!
- 3’, 5’ - cyclic adenosine monophosphate (cAMP)
what are the two types of bases and what kinds of bases do they fall under
are these the only types of bases
purine
- adenine
- guanine
pyrimidine
- cytosine
- thymine
- uracil
- There are minor bases in addition to the above-mentioned common bases
DNA cloning
preface
cut
-what must you cleave
what must you cut
what must you cut
paste
what must you select
what must you ligate
insert
what must you insert
how is this done
grow
what will the bacteria do
what will occur
what must you do to the product
now what must you do
preface: figure out your cloning vector. Figure out what gene/protein product you want to clone
cut
- cleave a site on your vector for your gene
- cut your gene of interest out of a chromosome (or wherever you get your genes)
- cut DNA with restriction endonuclease(s) ( I think the vector’s DNA)
paste
- select an appropriate cloning vector
- ligate/join your gene fragments of interest into your cloning vector using DNA ligase
insert
- insert DNA (in the cloning vector) into the host cell. Usually a series of heating and cooling steps.
- done by transformation
grow
- the bacteria will propagate, and with it, the plasmid (vector) with your gene of interest.
- Gene expression will occur, and your product will be produced.
- Extract and purify the product
- so basically, grow the cells and identify those with the desired recombinant DNA.
select your cloning vector
what is the first step
what must you now select
how many types are there and arrange them from biggest to smallest
cut:
- gene of interest
- vector to fit
1 - other plasmids (limit: 15,000-bp DNA)
2 - bacteriophages (notably, bacteriophage lambda)
- lambda: 48,502 bp - much larger than most plasmids; can accommodate larger DNA (total length 40,000-53,000bp)
- these are viruses for bacteria
3 - bacterial artificial chromosomes (BACs)
- DNA of 100,000-300,000 bp
- no longer talking about a plasmid but now a whole chromosome!
4 - yeast artificial chromosomes (YACs)
- advantage: yeast is a eukaryote and these genomes tend to be larger, so they can fit a much larger piece of DNA in there
- DNA up to 2,000,000 bp
rank the cloning vectors from smallest to largest
other plasmids
bacteriophage lambda
bacterial artificial chromosomes
yeast artificial chromosomes (YACs)
of the following types of cloning vectors, which are the largest
bacterial artificial chromosomes
bacteriophage lambda
other plasmids
yeast artificial chromosomes (YACs)
yeast artificial chromosomes (YACs)
cutting DNA, specifically
how do we cut DNA?
what does it recognize
is it specific or just cuts everywhere
what do some make
where do they cut DNA
what do they recognize and bind to
get a restriction endonuclease (or enzyme)
all kinds of enzymes, each recognizing, a different sequence, but only palindromes (a sequence that is the same forward and backward)
super specific
some make “sticky ends” where there are overhangs
some make “blunt ends” where there are no overhangs
what to know:
- restriction endonuclease (or enzyme) cut DNA only at palindromic sequences
- they recognize specific palindromic DNA sequences, bind to DNA at those sites, and cut the DNA
The enzymes that are used to cut DNA at specific nucleotide sequences are…
A - Kinases
B - Phosphatases
C - DNA Ligases
D - DNA Polymerases
E - Restriction Endonucleases
Restriction Endonucleases
cut at palindromes!!!!!!
Collections of Genetic Information
what is a cDNA library
what enzyme can you use to go from RNA to DNA
cDNA library
- collection of mRNA-derived DNA fragments (complementary DNA) cloned into vectors
Can go from RNA to DNA using reverse transcriptase from retrovirus which HIV is one
don’t think you have to know!
to do that:
- isolate and collect mRNA (which is a direct result of gene expression)
- treat with reverse transcriptase which turns RNA into DNA
- insert DNA into plasmid and insert plasmids into bacteria
- grow bacteria
- purify DNA and put it into the collection
CRISPR-Cas components
for gene editing
the CRISPR-associated protein (Cas) - a non-specific endonuclease
single guide RNA (sgRNA) - a piece of RNA that includes a sequence complementary to the target DNA and a sequence that can bind to the Cas
sgRNA with the Cas bound is a ribonucleoprotein (RNP)
CRISPR: what you need to know
what can it do to DNA
modify/change (ex: inactivate or activate)
remove or replace (remove a defective gene)
or add a gene
Nucleotides and Nucleic Acids – Let’s Go Crazy
what do these words mean in regards to nucleotides
monophosphate
diphosphate
triphosphate
how many phosphates can adenine and guanine have and what do those molecules do
how much energy does breaking 2 or 3 phosphate bonds release
mono - 1 phosphate
di - 2 phosphates
tri - 3 phosphates
adenine
- AMP, ADP, ATP
guanine
- GMP, GDP, GTP
- these are energy-storage molecules
breaking of bond
- 2: release 2 amounts of energy
- 3: release 3 amounts of energy
Functions of Nucleotides - Cofactors
what do cofactors do
what are some energy carriers?
what happens each time a phosphate is removed
examples of Enzyme Cofactors are what
what are the two parts of coenzyme A
cofactors help enzymes function
energy carriers: ATP, ADP, AMP –> adenosine
Each time a phosphate is removed, energy is released.
examples of Enzyme Cofactors
- Coenzyme A, NAD, FAD
coenzyme a: lipid + nucleotide (ADP)
Functions of Nucleotides - Cofactors
Building blocks of nucleic acids (obvs)
Energy Carriers: ATP ADP AMP Adenosine
Each time a phosphate is removed, energy is released.
Components of Enzyme Cofactors
- Coenzyme A, NAD, FAD
- Electron carriers in metabolic redox rxns
a way to spot reduction: more hydrogens!
What are the 4 stand-alone functions of nucleotides?
energy carriers
- ATP, ADP, AMP
- GTP, GDP, GMP
Enzyme Cofactors
- coenzyme A, NAD, FAD
Regulatory
- cAMP, cGMP, ppGpp
information
- DNA, RNA
Functions of Nucleotides - Regulatory
what are the 3 types
what are the 2 secondary messengers
what does alarmone do
Regulatory Molecules:
cAMP, cGMP, ppGpp
All of these are involved in the regulation of various enzymes
cAMP, cGMP: second messengers
ppGpp: “Alarmone” Inhibits the growth of bacteria when there is a shortage of resources.
- so bacteria will not grow if there are not a lot of resources
Functions of Nucleotides – Polymers! (DNA and RNA)
DNA and RNA, are the monomers or polymers
how are the phosphates connected to the sugars?
how are the sugars linked?
which sugar are phosphates attached to
what is the directionality
is the backbone hydrophilic or phobic, why?
where is the rest of the molecule besides the backbone
what is the charge at neutral pH
they are polymers!
connected through Phosphodiester bond:
C−O−PO2-O−C
Sugar-P-sugar-P
Links the 3’C of one sugar and the 5’C of another sugar.
5’ -> 3’
Phosphates are attached to the 5’C of one sugar and the 3’C of the next. Polymers start with 5’, and end with 3’ (no phosphate at the end)
5’-3’ directionality
backbone is hydrophilic:
a - phosphate -PO4-
b- sugar -OH (RNA)- OH makes it more hydrophilic
the rest of the molecule is buried inside
charge at neutral pH is negative
Hydrolysis
how is the phosphodiester bond formed and between what groups
because of this, what must break this bond, is it harder or easier to break RNA vs DNA, why or why not
what conditions does this happen at
what is subject to hydrolysis and is it a fast process
what enzyme breaks phosphodiester bonds
what are examples of them
The phosphodiester bond is formed as a result of the condensation reaction between phosphate groups and hydroxyl groups of two sugars…
So Hydrolysis must break them…RNA – easy (due to the extra OH on the 2’). DNA – hard.
this happens in alkaline conditions (high pH)
The covalent backbone is subject to hydrolysis, though this is a slow process
Phosphodiesterases (PDEs)!
For now, DNAase, RNAase
DNA & RNA are subject to hydrolysis but this is usually a very slow process
Nucleic Acids – Polymers of Nucleotides
Oligonucleotides
Polynucleotides
Customarily written as:
Functions:
how many pi does ACGTA have
how many pi does ACGT have
Oligonucleotides (fewer than ~ 50 nucleotides)
Polynucleotides (DNA, RNA)
Customarily written as 5’-to-3’, left-to-right
Functions:
Storage of genetic info (DNA)
Transmission of genetic info (mRNA)
Protein synthesis (tRNA and rRNA)
Processing of pre-mRNA (snRNA)
Catalysis (Ribozymes – RNA) RNA can act as an enzyme
In terms of genetic information, this corresponds to “N-to-C” in proteins
there is no phosphate at the last base
example:
ACGTA has 5 phosphate
ACGT has 4 phosphates the last A does not
Nucleic Acids – Structure and Properties
Bases in the nucleic acids are generally ___ , or close to it.
Maximum light absorbance is ___ nm
The bases are ____ at near-neutral pH.
“Base-____” is very important for their structure
- What kind of interactions do you think we have?
“Base-pairing” is also very important
- Hydrogen bonding
- Inter-strand
- Intra-strand
Watson-crick base pairing, how many hydrogen bonds between AT & AU & GC
is DNA parallel or anti-parallel
Bases in the nucleic acids are generally flat, or close to it.
Maximum light absorbance is 260 nm
The bases are hydrophobic at near-neutral pH (leading to [intra-strand] base-stacking)
“Base-stacking” is very important for their structure
What kind of interactions do you think we have?
“Base-pairing” is also very important via:
Hydrogen bonding
Inter-strand
Intra-strand- sometimes in RNA
Watson-Crick basepairs: Hydrogen bonding
A & T have 2
A & U have 2
G & C have 3 - stronger
DNA strands run antiparallel
Nucleic Acids – Structure Types
What are the levels oof structure
Primary: sequence (easy)
Secondary: local interactions and patterns (helix)
Tertiary: 3-dimensional, longer-range, possibly involving other molecules
Secondary Structure: 3-D Watson-Crick Model of DNA
Main features of the Watson-Crick model of DNA
- is left or right handed
- is the backbone hydrophillic or phobic
- where are the bases and through what kind of interactions
- what kind of grooves does it have
- are the strands parallel or antiparallel
what charge is the exterior surface and why does it have that charge
what kinds of bonds do the base pairs make with each other
how many bonds does each base pair make with its complementary base pair
Two polynucleotide strands wind together to form a long
- right-handed double helix
- hydrophilic backbone outside
- bases inside (stacked, base-paired [purine: pyrimidine], perpendicular to the helical axis), “Base-stacking” planar bases stack in the interior through hydrophobic interactions and van der Waals forces
- Major and minor grooves
- anti-parallel strands
Exterior surface: negatively charged phosphate groups
H-bonds between the bases
base composition:
A + G (purines) =C + T (pyrimidines)
A = T
C = G
Secondary Structure: 3-D Watson-Crick Model of DNA
how many strands and are they parrallel
is it left or right handed
what is on the inside, how are they arranged, how are they to the axis, what is paired and how many bonds
which bases are equal in #
2 antiparallel strands
right handed double helix
outside: hydrophilic backbone
inside: bases
- stacked
- perpendicular to the axis
- paired: G 3 bonds to C, A 2 bonds to T
overall base content
- # of G = # of C
- # of A = # of T
varies from species to species
3 forms of DNA
a long segment of DNA often has sub-segments if different conforms
B- form = watson-crick, most common
A- form:
- in lab,
- Favored in the absence of H20
- right-handed like B-form
- wider than B
- more compact than B
- not yet found in vivo
Z-form :
- naturally occurring
- left-handed
- found in stretches of DNA
Main features of DNA and RNA polymers:
what forms the backbone, what is the pattern and what is the bond that forms it
what are phosphates attached to, what are the ends of the polymers, and is there usually a phosphate at the 3’ end
is the backbone hydrophilic or phobic, what are the two reasons why
1
- alternating sugar and phosphate groups form the backbone
- phosphodiester bond linkage
2
- phosphates are attached to the 5’ C of one sugar and the 3’ C of the next
- the polymers have 5’ and 3’ ends; there is usually a phosphate at the 5’ end, but usually not at the 3’ end
3 - the backbone is hydrophilic
(a) because at physiological pH, each phosphate group is negatively charged and
(b) because of the free –OH groups on the sugars in RNA
What is the normal way of writing DNA/RNA sequences?
which part is phosphorylated and which part is not
write DNA/RNA sequences with the 5’ end (phosphorylated) on the left, and the 3’ end on the right (not phosphorylated).
Be familiar with schematic representations such as on page 276, or the shorthand … ACGTA …
which of the following are enzyme cofactors
ATP, GTP, CMP
coenzyme A, NAD, FAD
cAMP, cGMP, ppGpp
DNA, RNA
coenzyme A, NAD, FAD is the right answer!!!!!!!!
ATP, GTP, CMP - energy carriers
cAMP, cGMP, ppGpp - regulatory
DNA, RNA - information
which of the following are regulatory molecules
ATP, GTP, CMP - energy carriers
coenzyme A, NAD, FAD
cAMP, cGMP, ppGpp
DNA, RNA
cAMP, cGMP, ppGpp is the right answer!!!!!!!!!
ATP, GTP, CMP - energy carriers
DNA, RNA - information
coenzyme A, NAD, FAD - enzyme cofactors
why does the reduction of the carbonyl oxygen to a hydroxyl group in D-glyceraldehyde result in glycerol that cannot be given a D or L configuration?
because glycerol does not have a chiral carbon and so no matter which way it points, it will look the same: it is a mirror image
and so it cannot be labeled as D or L because D or L is based on chirality
storage molecules
what are the two types
where are they found
are they branched or unbranched
what linkage are they branched or unbranched
starch and glycogen
starch
- composed of two polysaccharides: amylopectin and amylose
- amylopectin: unbranched at (a 1 - 4) branched at (a 1 - 6) at every 24-30 residues
- amylose: unbranched at (a 1 - 4)
- in plants
- glucose subunits
- homopolysaccahride
glycogen
- branched at (a 1 - 6) at every 8-12 residues
- unbranched at (a 1 - 4)
- in animals
- in liver and muscle cells
these are homopolysaccarides!!!!
structure molecules
what are the two types
where are they found
are they branched or unbranched
what linkage are they branched or unbranched
cellulose
- in plant cells
- unbranched (B 1 - 4) linked D-glucose
- the anomeric carbon is in the β configuration - as a result of the β configuration, these polymers are strong, straight chains; compare structure of amylose (α configuration, resulting in tightly-coiled, compact helices;)
- cannot be digested by humans :(
- straight chain
- most abundant polysaccharide
chitin
- in insect exoskeleton and fungi cell wall (anti-fungal agents inhibit the synthesis of chitin)
- unbranched N-acetylglucosamine
- straight chain
- second most abundant polysaccharide
- The advantage of branching
(quicker degradation by enzymes that attack only non-reducing sugars)
what are the two types of homopolysaccharides
structural: cellulose and chitin
storage: starch and glycogen
what kind of molecules are blood groups
glycolipids
what are the two types of heteropolysaccharides
both are structural!
peptidoglycan (have cross-links, in cell wall of bacteria)
glycosaminoglycans (no cross-links, in extracellular matrix, a part of proteoglycans)
what are the 3 types of glycoconjugates
these are anchors in the cell membrane
keeps membrane intact
- Information carriers, consisting of “anchors” embedded in the cell membrane, plus oligo- or polysaccharides (floating outside of the cell membrane – into the ECM)
3 types:
- proteoglycans (glycosaminoglycans + protein)
- glycoproteins (protein + carbohydrates)
- glycosphingolipids (membrane lipid + oligosaccharides + lipopolysaccharide)
maltose
what two monosaccharides is it made of
what is the linkage?
is it reducing
2 glucose molecules!
Glc(a 1 -> 4)Glc linkage
reducing!
lactose
what two monosaccharides is it made of
what is the linkage?
is it reducing
galactose & glucose
Gal(b 1 -> 4)Glc linkage (like cellulose!!!!), lactose intolerant
reducing
sucrose
what two monosaccharides is it made of
what is the linkage?
is it reducing
fructose & glucose
fru (B2 - 1a) Glc or Glc (a1 - 2B)Fru
non-reducing
trehalose
what two monosaccharides is it made of
what is the linkage?
is it reducing
2 glucose (like maltose!!!!!)
Glc (a1 - 1a)Glc
non-reducing
why store carbohydrates as polysaccharides rather than as monosaccharides
osmolarity
concentration gradient
Inside of a cell: Osmolarity: water follows solute, water goes from [high osmolarity] to [lower osmolarity. Osmolarity = how much solute. More glucose, means higher osmolarity because there’s less water/water is taken up by glucose. So less water in the cells will cause water outside of the cells (because water goes from high to low) to move into the cell and cause swelling
High glucose inside, could cause glucose to go outside. Will cause no glucose to move inside because there is already a [high] inside. Putting glucose inside would push against the concentration
reducing/oxidizing sugars
what does it mean to be oxidized
what does it mean to be reduced
sugars are ___
Form more bonds with oxygen = most oxidized (lose electrons)
Form less bonds with oxygen = most reduced. This is a reducing agent, reducing agent means oxidized. By reducing other molecules, it can be oxidized
sugars are reducing agents
“a reducing agent is a chemical species that “donates” an electron to an electron recipient”
D and L stereoisomers of a given monosaccharide are…
A. Epimers
B. Mirror images of each other
C. Diastereomers
D. None of the above
B. Mirror images of each other
Which of the following is an aldopentose?
A. D-glyceraldehyde
B. D-ribose
C. D-glucose
D. Dihydroxyacetone E. D-fructose
B. D-ribose
Which of the following is a ketohexose?
A. D-glyceraldehyde
B. D-ribose
C. D-glucose
D. Dihydroxyacetone
E. D-fructose
E. D-fructose
Lactose consists of…
A. 2 Glucose monomers
B. Glucose + Galactose
C. Glucose + Fructose
D. Galactose + Fructose
B. Glucose + Galactose
Which of the following is not a reducing sugar?
A. Fructose
B. Galactose
C. Lactose
D. Maltose
E. Sucrose
E. Sucrose
Which of the following is a correct representation of the structure of sucrose?
A. Fru(β2→1α)Glc
B. Gal(β1→4)Glc
C. Glc(α1→1α)Glc
D. Glc(α1→4)Glc
E. Glc(β1→4)Glc
A. Fru(β2→1α)Glc
In what way is cellulose different from any starch molecule?
A. It is unbranched/linear
B. It is branched
C. The repeating monomer is N-acetylglucosamine
D. Glucose is in the β configuration
D. Glucose is in the β configuration
N-acetylglucosamine is the repeating monomer in…
A. Amylose
B. Amylopectin C. Cellulose
D. Chitin
E. Glycogen
D. Chitin
Which of the following is a type of structural heteropolymer?
A. Starch
B. Glycogen
C. Cellulose
D. Chitin
E. Peptidoglycans
E. Peptidoglycans
Hyaluronate, chondroitin sulfate, and heparin are types of…
A. Peptidoglycans
B. Glycosaminoglycans
C. Proteoglycans
D. Glycoproteins
E. Glycolipids
B. Glycosaminoglycans
Proteoglycans contain…
A. Oligosaccharides
B. Oligonucleotides
C. Glycosaminoglycans
D. Lipids
E. Lectins
C. Glycosaminoglycans
Lipopolysaccharides are found…
A. In exoskeletons
B. In the extracellular matrix
C. On the surface of viruses
D. On the surface of bacterial cells
E. On the surface of parasites
D. On the surface of bacterial cells
The blood group determinants are…
A. Proteoglycans
B. Glycoproteins
C. Glycolipids
D. Lipopolysaccharides
C. Glycolipids
Human selectins…
A. Bind to oligosaccharides on the influenza virus
B. Bind to oligosaccharides on influenza H. pylori cells
C. Mediate the inflammatory response
D. Block viral proliferation
C. Mediate the inflammatory response
Palindromes (and Mirror Repeats)
What can they form
What recognizes this sequence and cuts there
What is a palindrome, what is it complimentary with, how many base pairs do you need to qualify
Palindromic sequences can form loops!
Also, restriction endonucleases (enzymes) recognize these sequences and cut there = useful for molecular biology!
palindrome: inverted repeat; self-complementary within each strand)
need at least 4 base pairs to qualify
which of the following contains a palindrome
ATCAATTAGTTA
TACCATATGGTA
GCTTTACGAAAT
TGATCGACTAGC
TGATCGACTAGC
Palindromes (and Mirror Repeats): Structures
what do single stranded palindromes form
what do double stranded palindromes form
are cruciform areas in the genome stable and what is it prone
what are the proteins BRCA1 and p53 a part of and what do they bind
. What happens if BRCA1 or p53 goes wrong?
Single-stranded palindromes form hairpins [stem-loops]
Double-stranded palindromes form cruciform structures
Cruciform areas of the genome tend to be unstable and prone to mutations, deletions.
BRCA1 and p53 proteins, functioning in DNA repair, bind preferentially to cruciform structures.
. What happens if BRCA1 or p53 goes wrong?- cancer
RNA
does it always have a single or double strand
what is mRNA, rRNA, tRNA
which one is a good example of tertiary structue and what is an example of one/can it be an enzyme
what can viruses use as genetic material
what does reverse transcriptase do
Always composed of a single strand.
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
- A good example of a tertiary structure
- Ribozymes RNA Enzymes
Other RNA
- Viruses can use RNA as their genetic material
- Reverse transcriptase: RNA to DNA
mRNA!
how is it produced
where is it in the central dogma
is it right handed or left handed and how is its base pairs arranged
Product of transcription from DNAvia RNA polymerase (Ch. 26)
DNA to mRNA to Protein
Right-handed helixwith base-stacking
Pre-mRNA prior toprocessing.
RNA – Structure: Hodgepodge-y (mix)
how many strands does it always have
what directionality is the primary structure
can it form base pairs with itself
what is the usual base pairing via hydrogen bonds
what happen occasionally in RNA
are secondary and tertiary structures possible
Always single-stranded
The primary structure is the 5’ 3’ sequence
Can form base pairs with “self” secondary structures or with other RNA or DNA molecules
The usual base-pairing via hydrogen bonds:
A = U
G ≡ C
Occasionally G = U (non-Watson-Crick base pair usually due to folding)
Secondary and Tertiary Structures are possible!
RNA – Structure: Helical
what is it formed by
what is the typical form
what the form observed in lab
what is the form never observed
what can it form with itself
Formed from anti-parallel complementary segments of a strand
Typically A form (Right-handed w/base stacking)
Z form observed in the lab
B form never observed; RNA has no B form but DNA does (typical Watson-crick)
can form a double helix with self
- In its double-stranded regions, RNA exists mostly commonly in A form (RH helix, with base stacking); B form not observed; Z form made in lab
Nucleic Acid P-Chem: Denaturation and Annealing
what can happen to nuclei acids
what can denature nucleic acids
during this process what does dsDNA become
do this also happent to RNA too
how can you determine the state of the DNA
Nucleic acids can be denatured and renatured (annealed)
Remember proteins?
This occurs via disruption of hydrogen bonds through changes in temperature, pH, and ionic strength, like protein denaturing cause some forces are around
dsDNA becomes ssDNA
RNA secondary and tertiary structures, too!
Can determine the state of the DNA via the absorption values (native v denatured)
- Denaturation (unfolding) and re-naturation (annealing): can be monitored by measuring UV absorption; no covalent bonds break, only H-bonds
Denaturation and Annealing
what would make the Tm higher and why
does RNA or DNA have a higher melting point, why or why not
what does Tm mean
The higher the G ≡ C content, the higher the Tm Why?- stronger DNA has more GC (which is 3 bonds)
RNA has a higher melting point than DNA
due to # of hydrogen bonds -due to OH on 2’ of RNA it is stronger and has a higher melting point
need more heat to get 50% saturation
tm = melting point: mid-point (50%) of melting curve (native denatured)
Nucleic Acid Hybridization
what do complementary segments of DNA or RNA do with one another, what is an example
what determines if the binding is strong or not
where can this be applied to
complementary segments of DNA or RNA CAN ANNEAL TO EACH OTHER. for example
- a small “probe” sequence
Complementary parts of pieces of DNA even if surrounded by non-complementary regions: the greater the complementarity, the stronger the binding
- application: research, comparing relatedness of species, based on DNA similarily/diversity
which of the following pieces of double-stranded DNA, all with equal numbers of base-pairs, would have the highest Tm
15% G
20% A
80% [A + T]
25% C
30% T
20% A
Chemical Transformation of Nucleotides and Nucleic Acids
are non-enzymatic rxn fast or slow
what can it cause
nonenzymatic; usually very slow/rare
cause of mutations
3 important types of spontaneous (though rare) rxns
which of the following deoxyoligonucleotides would hybridize with DNA containing the sequence
5’ GAGGCTACAT 3’
A - 5’ TACATCGGAG 3’
B- 5’ CTCTAGCGTA 3’
C. (5’)-CTCCGATGTA-(3’)
D. (5’)-ATGATCGCTC-(3’)
E. (5’)-ATGTAGCCTC-(3’)
E. (5’)-ATGTAGCCTC-(3’)
Spontaneous Deamination
what does cytosine to uracil release
what does bisulfite change
what would you see when you sequence the DNA
what is BS-seq used for
what is DNA methylation important for
what does deamination involve
what happen uner typical cellular conditions
what happens spontaneously
Cytosine to uracil releases ammonia.
Bisulfite changes all non-methylated cytosines to uracil. Methylated cytosines remain intact. —oh so bisulfite really is a deaminating agent! (alonbg with nitrates, nitrites and nitrosamines)
Sequence this treated DNA, where you still see cytosines is where you have a methylation.
BS-Seq - detection of DNA methylation
DNA methylation is very important in Epigenetics!
removal of exocyclic amine (ohhh NH2 outside of the pyrimidien ring of cytosine; deamination)
under typical cellular conditions, 1 in every 10^7 cytidines are lost every 24 hours
spontaneous removal of amines from the bases
Breaking the Sugar-Base Bond (Depurination)
which bond is broken
every 24 hours, how many purine are lost
Spontaneous removal of bases leads to what
break the N-B-glycosyl bond
under typical cellular conditions, 1 in every 10^5 purines are lost every 24 hours
Spontaneous removal of bases leads to
- Degraded
- Can cause a mutation or be repaired
UV Irradiation Thymine Dimer Formation!
where is UV light from and how does it effect thymine
what happens to the backbone as a result of this
unless what
what will happen to polymerase if it is bad
what can this cause
what is this the primary cause for
radiation causes how much DNA damage by environmental agents
UV light (the sun) “glues” thymines together.
Puts a bump in the backbone. Can be fixed!
Unless it’s bad…
Then polymerase will make mistakes during replication/transcription
Can cause cancer via mutations.
Primary cause of melanomas.
radiation causes ~10% of all DNA damage by environmental agents
Increasing the Frequency of These Reactions
what are deaminating agents
what do alkylating agents do and what is an example of it
what do some cells have
deaminating agents (in some foods)
- nitrites, nitrates, nitrosamines = nitrous acids
- bisulfite
- (balance: risk-benefit)
alkylating agents (disrupts base-pairing
- ex: dimethylsulfate
oxidizing agents (most important agent)
- cells have defense mechanism, but not system is 100% perfect
Which nucleotide in the oligonucleotide CAGAT contains a free 5’ phosphoryl group?
A. A
B. C
C. G
D. T
B. C
How many phosphoryl groups does the oligonucleotide CAGTA contain
A. 1
B. 2
C. 3
D. 4
E. 5
F. 0
E. 5
Which of the following contains a palindrome?
A. ATCAAT TAGTTA
B. TACCAT ATGGTA
C. GCTTTA CGAAAT
D. TGATCG ACTAGC
D. TGATCG ACTAGC
Which of the following is true of RNA?
A. Single-stranded, no base-pairing
B. Base-pairing and B-form helices
C. Base-pairing and A-form helices
C. Base-pairing and A-form helices
Which of the following pieces of double-stranded DNA, all with equal numbers of base pairs, would have the highest Tm?
A. 15% G
B. 20% A
C. 80% [A + T]
D. 25% C
E. 30% T
B. 20% A
Which of the following deoxyoligonucleotides would hybridize with DNA containing the sequence:
(5’)-GAGGCTACAT-(3’)
A. (5’)-TACATCGGAG-(3’)
B. (5’)-CTCTAGCGTA-(3’)
C. (5’)-CTCCGATGTA-(3’)
D. (5’)-ATGATCGCTC-(3’)
E. (5’)-ATGTAGCCTC-(3’)
E. (5’)-ATGTAGCCTC-(3’)
Agents such as nitrites, nitrates, and nitrosamines may lead to…
A. Nucleic acid deamination
B. Cleavage of the sugar-base bond in DNA
C. Formation of T-T dimers in DNA.
A. Nucleic acid deamination
Hoogsteen pairs are often found in…
A. A-DNA
B. B-DNA
C. Z-DNA
D. Triple-helix DNA
E. Double-helix DNA
D. Triple-helix DNA
- Know what the Polymerase Chain Reaction (PCR) is – what it does.
A method that amplifies (makes many copies of) specific DNA sequences
DNA cloning steps simplified
(1) cut DNA with restriction endonuclease(s)
(2) select an appropriate cloning vector
(3) join the fragments using DNA ligase
(4) insert the DNA in a host cell (transformation),
(5) grow the cells and identify those with the desired recombinant DNA.
cut
paste
insert
grow
competitive inhibitor for lectins
q.26
will outcompete for the active site on neuraminidase or can add more substrate
- Antidote to prevent or reverse ricin-mediated entry of the toxin - competitive inhibition or add more substrate! Ricin will bind either the oligosaccharide (competitive inhibitor) or more of the substrate instead of the cell surface target which will prevent the entry of the toxin
hairpin/stem loops and cruciforms
What are hairpins and stem loops for
What are cruciforms for -
- Are they stable
- what proteins are a part of DNA repair and what do they do to cruciforms
- what happens if those proteins are not working properly
What do hairpins and cruciforms need to form
What is a palindrome
What conformation are RNA hairpin & DNA cruciforms in
Hairpin and stem loops for RNA
Cruciforms for DNA - unstable and prone to mutation. BRCA1 & p53 are a part of DNA repair which can bind onto cruciforms to fix it but if the BRCA1 & p53 proteins are not working properly or have a mutation. And this causes BRCA1 breast cancer and p53 cancer
Both hairpin and cruciform need palindromes to form!
Palindromes: read the same forwards and backward
RNA hairpin vs DNA cruciform: RNA - hairpin is in A conformation, DNA - cruciform is in B form
Adenine vs guanine
look at the way they look and you can see how many bonds they make
A makes 2 bonds
G makes 3 bonds
cytosine vs thymine vs uracil
Cytosine has NH2
Thymine and uracil have 2 carbonyls and thymine has CH3 and uracil does not
what is the difference between a hemiacetal and a glycoside
hemiacetal: when an aldose or ketose condenses with an alcohol. Aldose + OH = hemiacetal
glycoside: when a hemiacetal condenses with an alcohol. Hemiacetal + OH = glycoside
Lectins & viral infections
Some cells have glycoproteins on their surface
Some of those glycoproteins have a carbohydrate called neuraminic acid or sialic acid
Lectins are proteins that bind onto carbohydrates
*influenza virus has a lectin called hemagglutinin or HA which will bind onto neuraminic acid or sialic acid on the surface of the cells.
After binding, the virus now can go into the cell.
The enzyme neuraminidase clips off the neuraminic acid or sialic acid for the virus inside to be released
Neuraminidase Is a part of the virus!!!!
When exiting the cell, NA must clip the HA-sialic acid bound to become untethered from the cell membrane = infect other cells.
So what if we blocked neuraminidase? Virus can’t be set free!
DNA Methylation:
stops transcription so DNA does not go to RNA and therefore will not make proteins. This is a good way to regulate gene expression and this changes throughout development.
: also changes DNA structure and leads some DNA to assume the Z form
can also provide natural protection because some the cell can distinguish its own DNA (methylated) from foreign DNA (unmethylated).
can sometimes happens naturally
what is ™
the temp. Where 50% of the molecules are denatured
absorption relationship to denaturation
Absorbance increases as DNA denatures. Because light can get through the base pairing
cDNA
the DNA is from mRNA
That DNA is complementary to mRNA
These DNA fragments go into vectors
A, B, Z form of DNA
handedness
standard form
relative diameters
Rise per turn
relative “helix rise per base pair”
relative distance per turn for A, B and Z DNA.
handedness: right for A & B, left for Z
standard form: 3.4 A rise per base, 10.5 bases/turn, 36 A rise per turn
relative diameters: A is 26 A, (wider than B) B is 20 A, Z is 18 A (narrower than B).
Rise per turn: A Is more compact than B, Z is more elongated than B
relative “helix rise per base pair” (3.4 A rise per base)
relative distance per turn (28, 36 and 44 Å, respectively) for A, B and Z DNA - Z has the longest distance per turn, followed by B and then A has the shortest
