Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
277 Cards in this Set
- Front
- Back
|
How many a.a. are found in proteins?
|
20
|
|
|
What are proteins coded by?
|
DNA
|
|
|
How do a.a.differ from one another?
|
Their R groups.
|
|
|
What are the four types of R groups and their characteristics?
|
Nonpolar (hydrophobic)
Polar (uncharged, unequal e- distribution) Acidic (negative charge) Basic (positive charge) |
|
|
Which a.a. does not exhibit chirality?
|
Glycine
|
|
|
What is a zwitterion?
|
A zwitterion is a chemical compound that is electrically neutral but carries formal positive and negative charges on different atoms. They are polar and usually have a high solubility in water and a poor solubility in most organic solvents.
|
|
|
Amino Acids can act as acids AND bases are called? These are the most common type of zwitterion.
|
Ampholytes (they are amphoteric)
|
|
|
4 a.a not found in proteins
|
citruillene
L-Ornithine SAM Homocystiene |
|
|
Basic a.a.
|
HLA
Histidine Lysine Arginine |
|
|
Acidic a.a.
|
Aspartic acid
Glutamic acid |
|
|
a.a with polar side groups (6)
|
CT TAGS
Cystiene Threonine Tyrosine Asparagine Glutamine Serine |
|
|
NOTE
|
a.a. with nonpolar side groups are all of the remaining!
|
|
|
#1) Small Ka = ______ acid
#2) Small pka = ________ acid |
1 = Weaker
2 = Stronger |
|
|
what determines the structure (shape) of a protein?
|
The specific a.a. sequence
|
|
|
Exhibits partial double bond character. What type of bond am I?
|
Peptide bond (forms amide linkages; prevents free rotation; links a alpha-carboxyl group to the alpha-amino group w/in the primary protein structure
|
|
|
What type of bonds link the alpha-amino group with the alpha-carboxy group in primary structure?
|
peptide bonds (for amide linkages)
|
|
|
Exhibits partial double bond character. What type of bond?
|
Peptide bond, prevents free rotation
|
|
|
Peptide bonds have what type of configuration?
|
Trans (prevents steric hinderance)
|
|
|
What occurs in a cis bond to prevent steric hinderance?
|
kink in the polypeptide
|
|
|
Protein purification - at what point on a gel electrophoresis will a protein stop?
|
At the point in which pH = pI
|
|
|
Determining protein sequence my edman degredation results in two different sequences. What are they are what is their fn?
|
Tryptic peptide - These are separate peptide sequences that determine order.
Chymotryptic peptide - Identifies the overlapping in the above two tryptic sequences. |
|
|
Two types of secondary sequences are called what?
|
Alpha helix
Beta-pleated sheet |
|
|
How many amino acid residues per turn of alpha helix?
|
3.6
|
|
|
L or R-handed helices are more common? why?
|
R b/c more energetically favorable
|
|
|
Myosin and tropomyosin, fibrin and keratin are examples of what type of secondary structure?
|
They represent two or more combined alpha-helices
|
|
|
What are motifs?
|
Super-secondary structures. They are an intermediate between secondary and tertiary structures.
|
|
|
Composition of tertiary structure proteins?
|
Composed of domains each ave the same or different functions.
|
|
|
Proteins vs membrane proteins
|
Proteins behave in a typical manner meaning that hydrophobic a.a. are located on the inside, however, membrane proteins are atypical and as such have hydrophobic a.a. on the outside.
|
|
|
How are extracellular proteins stabilized?
|
By disulfide bonds (intra- or inter-chain)
|
|
|
Quarternary Proteins
|
Two or more different polypeptide chains held by non-covalent forces.
|
|
|
Protein folding is random. T or F?
|
False, it is done sequentially, in an ordered fashion.
|
|
|
Mad cow is aka ____ in humans?
|
CJD (Creutzfeldt–Jakob disease)
|
|
|
What is the cause of CJD?
|
Fatal neurodegenerative disorders due to deposition of insoluble protein aggregates in neural cells (protein misfolding)
|
|
|
Amyloidoses are caused by what mechanism?
|
Abnormal cleavage leads to a change in conformation causing the formation of long fibrillar protein assemblies (of β sheets)
|
|
|
Classic example of amyloidose disease?
|
Alzheimers
|
|
|
Defective protein in alzheimers?
|
Also defective Tau protein which causes neurofibrillary tangles.
|
|
|
Composition of Nucleic acids
|
Linear unbranched polymers
|
|
|
Nucleic acid monomers are referred to as?
|
nucleotides
|
|
|
Purines
|
AG
Adenine Guanine |
|
|
Pyrimidines
|
TCU
Thymine Cytosiene Uracil |
|
|
Fundamental units of genetic information
|
genes (made up of DNA)
|
|
|
Central Dogma
|
DNA--(transcription)-->RNA--(translation)-->proteins
|
|
|
Three components of necleotides (monomers)?
|
A sugar, a phosphate, and a base
|
|
|
How are the sugars in nucleic acids linked?
|
The sugars in nucleic acids are linked to one another by 3 C’,5 C’ –phosphodiester bonds.
|
|
|
Define: Nucleoside
|
A unit consisting of a base + sugar
|
|
|
Define: Nucleotide
|
nucleoside + phosphate groups linked by an ester linkage
|
|
|
DNA has polarity...T or F?
|
True
|
|
|
DNA Structure: Where are the sugar phosphates located on the helix? Where are the purines/pyrimidines located on the helix?
|
Sugar phosphates are on the outside.
Purines/pyrimidines are on the inside. |
|
|
How many bases per turn?
Right or left-handed twist? a)B-DNA b)A-DNA c)Z-DNA |
B-DNA normal form right-hand twist 10 bases per turn
A-DNA dehydrated form right-hand twist 11 bp per turn (also found in DNA-RNA hybrids and RNA-RNA pairing) Z-DNA found in poly GC regions left-hand twist 12 bp per turn |
|
|
How many H-bonds are between A-T & C-G?
|
A-T => 2 bonds
C-G => 3 bonds |
|
|
Each human cell contains approximately ___ meters of DNA
|
2
|
|
|
The complex of DNA and protein is called _________.
|
chromatin
|
|
|
The highly condensed chromosomes in a dividing cell are known as _________ chromosomes.
|
mitotic
|
|
|
What are the components of "beads on a string"?
|
The string = DNA that wraps around the beads
The beads = nucleasome core particle with DNA wrapped around it. Linker DNA is responsible for attachment of beads! |
|
|
To which end of DNA are nucleotides added?
|
3' end
|
|
|
DNA Replication: T or F
DNA polymerases require a primer. |
True, they require a primer with a free 3'-OH already base paired to the growing strand.
|
|
|
OriC
|
Origin of replication in prokaryotes (E. Coli)
|
|
|
Prokaryotes:
OriC is rich in what type of base pairs? |
A-T
|
|
|
Prokaryotes:
OriC is the binding site for which initiation protein |
dnaA
|
|
|
Prokaryotes:
What is the site of DNA synthesis called? |
Replication fork
|
|
|
Prokaryotes:
What direction does DNA synthesis take place? |
Bidirectional
|
|
|
Prokaryotes:
Fn. of DNA helicases? |
To unwind via ATP
|
|
|
Prokaryotes:
What is the prepriming complex? How is it made? |
prepriming complex makes single-stranded DNA accessible for other enzymes to begin synthesis of the complementary strands. The preprimin complex is resultant from binding of dna A & unwinding of DNA by DNA helicases.
|
|
|
Prokaryotes:
What primes the synthesis of DNA? |
RNA (RNA polymerase, aka a primase syn. ~5-10 nucleotides)
|
|
|
Prokaryotes:
Direction of DNA synthesis? |
Synthesis of DNA proceeds in the 5'=>3' direction.
|
|
|
How is DNA able to be synthesized simultaneously & bidirectionally?
|
Because it forms a leading strand and lagging strand (discontinuous - called Okazaki Fragments which are 100-200bp long)
|
|
|
Prokaryotes:
DNA elongation enzyme? |
DNA pol III holoenzyme - Has high catalytic potency, fidelity, and processivity (ability to syn. many strands w/out releasing its substrate).
|
|
|
Prokaryotes:
Fn. of 5'=>3' exonuclease? |
Allows removal of RNA primer, and is also used during DNA repair
|
|
|
Prokaryotes:
Fn. of 3'=>5' exonuclease? |
During replication allows DNA polymerase to remove mismatched bases Polymerase,proof reading, and ensures high fidelity.
|
|
|
Prokaryotes:
Fn. of topoisomerases? |
To relieve torsional stress into the duplex ahead of the replication fork.
|
|
|
Prokaryotes:
What type of topoisomerase is DNA gyrase? |
Topo. II
|
|
|
Prokaryotes:
What type of antibiotics target DNA gyrases? |
Fluoroquinolone antibiotics target DNA gyrases in many gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin).
|
|
|
Prokaryotes:
Fn. of DNA pol I? |
The RNA primers of the leading strands and Okazaki fragments are removed by DNA polymerase I replacing with deoxyribonucleotides
|
|
|
Prokaryotes:
After DNA pol I what seals the gaps? |
DNA ligase
|
|
|
Eukaryotes:
What two polymerases fn. in repair? |
Pol. β & Pol. ε
|
|
|
Eukaryotes:
What polymerases have proofreading capabilities? |
Pol. γ
Pol. δ Pol. ε |
|
|
Eukaryotes:
Fn. of Pol. δ |
Elongation
|
|
|
Eukaryotes:
Fn. of Pol. γ |
Replicates mtDNA.
|
|
|
Eukaryotes:
What are the 2 Fn's of Pol. α |
Contains primase & initiates replication (primase).
|
|
|
Eukaryotes:
What is special about the ends of DNA? |
Telomeres
|
|
|
Eukaryotes:
Fn. of Telomerase? |
Elongates the lagging-strand template from its 3’ -hydroxyl end.
Note: Telomerase is a modified reverse transcriptase. |
|
|
What are the four types of eukaryotic DNA Repair mechanisms?
|
Mismatch Repair
Base Excision Repair Nucleotide Excision Repair ds Break Repair |
|
|
Eukaryotes:
What is a exinuclease? |
A UV-specific endonuclease
|
|
|
What condition is resultant from DNA mismatch repair?
|
HNPCC (AD) - Affected cells with a mutated mismatch repair enzyme are unable to remove small loops of unpaired DNA (Termed microsatellite instability).
|
|
|
Which Polymerase is utilized during mismatch repair?
|
Pol I
|
|
|
How does NER work?
|
Cleaves the mutation on the 5' and 3' end removing up to 30 nucleotides.
|
|
|
In eukaryotic NER what pol. are used to replace the removed bases?
|
Pol. δ/ε
|
|
|
Defect in NER causes what disease?
|
XP (AR)
|
|
|
Symptoms of XP?
|
Sensitivity to UV light, increase pyrimidine dimers after exposure, increased risk of skin cancer.
|
|
|
Base excision Repair =>
Mechanism, enzymes, pol used, etc? |
flipping the mutated base out of the DNA helix and repairing the base alone. There are two main enzymes used, DNA glycosylases and AP endonucleases. The DNA glycosylase is used to break the β-N glycosidic bond to create an AP site. AP endonuclease recognizes this site and nicks the damaged DNA on the 5' side (upstream) of the AP site creating a free 3'-OH. DNA polymerase, Pol I, extends the DNA from the free 3'-OH using its exonuclease activity to replace the nucleotide of the damaged base, as well as a few downstream, followed by sealing of the new DNA strand by DNA ligase.
|
|
|
2 mech. of ds break repair?
|
Homologous recombination repair
Nonhomologous end-joining repair |
|
|
T or F:
Transcription is the similar in eukaryotes & prokaryotes. |
False
|
|
|
Transcription is the process by which DNA mediates the synthesis of what?
|
RNA
|
|
|
What are the three types of RNA & their corresponding fn's?
|
mRNA - plays a role in translation
tRNA - ensures that the proper insertion of a.a. into the elongating strand. rRNA - assemble together with ribosomal proteins to form ribosomes. |
|
|
hnRNA is a precursor to _____?
|
mRNA
|
|
|
snRNA plays a role in what process?
|
mRNA processing
|
|
|
Describe the secondary structure of tRNA.
|
Cloverleaf structure.
5' end (terminal phosphate) => D loop => anticodon => TΨC loop (where Ψ is pseudouridine) => 3' end (has a signature CCA sequence at this end that is important for the recognition of tRNA by enzymes critical in translation; it is the site of a.a. attachment. In prokaryotes, the CCA sequence is transcribed. In eukaryotes, the CCA sequence is added during processing and therefore does not appear in the tRNA gene). |
|
|
Direction of RNA Polymerase in RNA synthesis?
|
RNA Pol moves along the template strand in a 3'=>5' direction so that RNA is syn. in the 5'=>3' direction.
|
|
|
What are the four ways in which RNA transcription differs from DNA transcription?
|
1. Initiation, at many more sites than replication.
2. Many more molecules of RNA polymerase per cell than DNA polymerase. 3. RNA pol. rxn proceeds at a rate much slower than DNA polymerase. 4. Fidelity of RNA polymerization is much lower than DNA. |
|
|
Regulatory step in RNA transcription?
|
Initiation
|
|
|
What subunit is req'd for RNA pol to start transcription?
|
Sigma subunit
|
|
|
Three steps in transcription.
|
Initiation
Elongation Termination |
|
|
Two common promoters (recognized by RNA pol holoenzyme) on the 5’ side of the start site in prokaryotes?
|
-10 sequence (pribnow box) and
the -35 sequence. NOTE: Transcription start site is 7 bp upstream of the -10 site. |
|
|
Sense vs antisense
|
Antisense (-) = the template strand.
Sense (+) = the coding strand |
|
|
T or F:
RNA synthesis does not require a primer. |
True
|
|
|
What are the two methods used by prokaryotes to terminated transcription?
|
Rho-independent & Rho-dependent
|
|
|
Mechanism of Rho-independent transcription termination
|
Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop, followed by a run of U's, which makes it detach from the DNA template.
|
|
|
Mechanism of Rho-dependent transcription termination
|
In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex; thus acting as a RNA-DNA helicase
|
|
|
These antibiotics inhibit prokaryotic transcription but do not affect eukaryotic transcription.
|
Rifampicin
Actinomycin D |
|
|
Three types of RNA polymerase
a.) template specificity, b.) location in the nucleus, c.) susceptibility to inhibitors |
i.) RNA polymerase I transcribes the tandem array of genes for 18S, 5.8S, and 28S ribosomal RNA
ii.) RNA polymerase II transcribes precursors of messenger RNA as well as snRNA molecules, such as those of the splicing apparatus. This is inhibited by α-amanitin from Amanita phalloides. iii.) RNA polymerase III makes 5S rRNA and all the transfer RNA molecules |
|
|
Eukaryotic vs Prokaryotic transcription promotors
|
Eukaryotic - TATA box (Hogness box), located -30bp downstream of the start site; also includes the CAAT promotor region (between -70 & -150).
Prokaryotic - promotor is -10 sequence |
|
|
How are primary transcripts of mRNA in eukaryotes modified in order to become functional?
|
Removal of Introns by splicing
|
|
|
Eukaryotic rRNA includes _____, ______ & ______.
|
18S
28S 5.8S |
|
|
Prokaryotics rRNA includes ____, _____ & ____.
|
16S
23S 5S |
|
|
Characteristic feature of 3' end of tRNA?
|
CCA sequence
|
|
|
Approximately __% of tRNA bases have been modified.
|
10%
|
|
|
Three major modification made to pre-tRNA, yielding Mature tRNA.
|
1. Removal of sequence from 5'-P end by RNAseP.
2. Removal of uracil from 3'-OH end and replacement with characteristic CCA sequence. 3. Removal of intron from anticodon loop. |
|
|
DNA --> mRNA requires what cofactor?
|
RNA Pol.
|
|
|
5 steps req'd to take transcribed pre-mRNA out of the nucleus for translation?
|
1. 5' cap
2. Splicing 3. Editing 4. Polyadenylation 5. Transport out of nucleus for translation. |
|
|
Mech of add'n of 5' cap in eukaryotic mRNA processing.
|
7-methylguanosine is added to 5' end after transcritpion by guanylyl transferase; methyl groups (from SAM) are added by Guanine-7-methyl transferase.
|
|
|
What is the purpose of the 5' cap in eukaryotic mRNA?
|
The 5’ cap aligns eukaryotic mRNAs on the ribosome during translation
|
|
|
NOTE
|
Prokaryotic mRNA is mature almost immediately after transcription & does NOT require the extensive modification as is req'd by eukaryotic mRNA.
|
|
|
RNA molecules that are capable of catalyzing their own splicing are called?
|
Ribozymes
|
|
|
Purpose of euk. mRNA editing?
|
Change nucleotide composition, sometimes resulting in a shorter mRNA molecule.
|
|
|
Mech. of euk mRNA polyadenylation?
|
Polyadenylation involves the add'n of a poly-A tail to euk. mRNA by Poly-A-polymerase.
|
|
|
What type of elements can be found up- & Down-stream to eukaryotic poly-A sequence?
|
G-U rich elements.
|
|
|
How many A's in poly-A-tail?
|
200
|
|
|
Fn. of Poly-A tail in eukaryotic mRNA
|
In translation & mRNA stability.
|
|
|
Fn. & composition of splicesosomes.
|
Fn: Splicing to remove Introns
Composition: Proteins and snRNAs (U1, U2, U4-6). |
|
|
Abnormal processing of eukaryotic mRNA results in that classic disease?
|
Beta-thalassaemia
NOTE: 15% of all genetic diseases are the result of eukaryotic mRNA processing. |
|
|
Termination (stop or nonsense) codons?
|
UAA, UAG, UGA
|
|
|
A total of __ combinations of bases are possible in genetic code
|
64
|
|
|
The genetic code is universal, however there are three exceptions in mitochondria. What are they?
|
AUA is Met not Ile
UGA is Trp not stop AGA and AGG are stop not Arg |
|
|
Point Mutations: Transition vs Tranversion
|
Transition - purine -> purine or pyrimidine -> pyrimidine
Transversion - purine -> pyrimidine or pyrimidine -> purine |
|
|
Point Mutations (base substitutions) can result in three types of mutations. What are they?
|
Silent (synonmous) - not detected
Missense - code for another a.a. Nonsense - intro a stop codon prematurely |
|
|
Three components req'd for translation.
|
mRNA
tRNA - intermediate adapter molecules that recognize a specific nucleotide sequence as well as a specific a.a. sequence. A.A. - if an essetial a.a. is absent translation stops |
|
|
when is tRNA said to be charged?
|
When there is an activated a.a. attached to the 3' CCA of the acceptor stem.
|
|
|
Where is the anticodon located in tRNA?
|
in the middle at the bottom.
|
|
|
How is the activated a.a. attached to a tRNA molecule?
|
via aminoacyl-transfer RNA synthetases
|
|
|
Mech by which a.a. are attached to tRNA.
|
1. formation of an aminoacyl adenylate (aminoacyl-AMP) from an amino acid and ATP.
2. transfer of the aminoacyl group to a particular tRNA molecule to form aminoacyl-tRNA. |
|
|
T or F: Aminoacyl-Transfer RNA Synthetases have proof reading abilities?
|
True
|
|
|
Primary site of gene expression in prokaryotes and eukaryotes?
|
Transcription
|
|
|
Regulation of transcription (2)
|
1. Regulatory sequences in DNA
Commonly in non-coding regions -> cis-acting elements. 2. Trans-acting factors -> Regulatory proteins |
|
|
Unit of transcription
|
Operon
|
|
|
polycistronic mRNA
|
a single mRNA molecule that codes for more than one protein
|
|
|
What are the units of an operon (3)?
|
1. Structural genes -> code for the enzymes
2. Regulatory genes -> code for regulatory proteins 3. Control elements (operators) -> sites on DNA near structural genes at which the regulatory proteins act |
|
|
Three structural genes that make up the lac operon?
|
Lac Z
Lac Y Lac A |
|
|
What does Lac Z code for? and what is the fn.?
|
lac Z -> β-galctosidase -> hydrolysis of lactose to glucose and galactose
|
|
|
What does Lac Y code for? and what is the fn.?
|
lac Y -> permease -> transport of lactose into cell
|
|
|
What does Lac A code for? and what is the fn.?
|
lac A -> thiogalactosidase transacetylase -> function unknown
|
|
|
When is the lac operon suppressed?
|
In the presence of glucose or the absence of lactose
|
|
|
Fn. of promotor in lac operon?
|
Binds RNA Pol
|
|
|
When are lac ZYA expressed?
|
when Operator is empty & CAP is bound by a complex of cAMP with catabolite gene activator protein (CAP) –also called cAMP regulatory protein (CRP)
|
|
|
Regulatory gene of lac operon and fn.?
|
lacI –codes for repressor protein that binds to operator site
|
|
|
When lactose is present and glucose nearly absent cAMP binds to CAP to perform what fn.?
|
Increase the efficieny of transcription initiation by RNA pol.
|
|
|
What enzyme converts ATP -> cAMP?
|
Adenylate cyclase
|
|
|
Tryptophan operon is made up of?
|
5 structural genes, a promotor and an operator
|
|
|
Tryptophan operon is generally always on unless suppressed by a corepressor. What is the corepressor?
|
The product of the tryptophan operon pathway which binds to a repressor and facilitates its binding to the operator.
|
|
|
Effect os attenuation on the tryptophan operon?
|
Attenuation is another regulator of gene transcription. It works by forming a stem-loop structure in the attenuator region when enough tryptophan is present which leads to the release of RNA pol from the DNA.
|
|
|
Why is attenuation only possible in prokaryotes and not eukaryotes?
|
transcription and translation take place in separate compartments in eukaryotes.
|
|
|
What is the stringent response?
|
The stringent response occurs in all prokaryotes and some plants in reaction to amino-acid starvation or carbon starvation. The stringent response is capable of modulating up to 1/3 of all genes in a cell. It causes the cell to divert resources away from growth and division and toward amino acid synthesis.
|
|
|
How are ribosomal proteins regulated?
|
self-regulated
|
|
|
T or F:
Eukaryotes are polycistronic? |
False they are monocistronic, thus they do not have operons.
|
|
|
4 DNA binding domains involved with eukaryotic transcription in trans-acting regulatory elements?
|
Zinc finger motif, leucine zipper motif, Helix turn Helix and Helix loop helix motif
|
|
|
In cis-acting molecules of eukaryotic transcription what are the promotors?
|
TATA box(hogness), CAAT box, GC box
|
|
|
Cis-acting regulatory elements: What are some examples of signals mediated by intracellular receptors?
|
steroid hormones, vitaminD , retinoic acid thyroid hormone
|
|
|
Cis-acting regulatory elements: Examples of signals mediated by cell-surface receptors?
|
peptide hormones like glucagon, epinephrine insulin
|
|
|
How can different mRNAs be produced from the same gene?
|
most pre-mRNAs contain multiple introns, different mRNAs can be produced from the same gene by different combinations of 5 and 3 splice sites.
|
|
|
Regulation of gene expression by posttranslational processing includes what mech?
|
RNA editing
|
|
|
Phosphorylation of eIF2 controls which rxn?
|
Transcription. Inhibits if insufficient amt of Heme is present by phosphorylation of eIF2 which blocks the exchange of GTP for GDP or promotes in the presence of sufficient heme.
|
|
|
chromatin remodeling
|
Regulation of gene expression by methylation of cytosines.
|
|
|
What are Transposons?
|
Mobile segments of DNA that move from one site to another in genome; mediated by transpoases.
|
|
|
Role of restriction enzymes?
|
cut dsDNA into more manageable pieces.
|
|
|
Restriction enzymes cut at specific site recognizable by what?
|
A 4 to 6bp sequence called a palindrome (read fwd & backward).
|
|
|
Result of cleaving by Restriction Enzymes?
|
Sticky (overlapping/staggered)ends by cleavage with TaqI & blunt ends via cleavage with HaeIII.
|
|
|
What is a clone?
|
A large population of identical molecules, bacteria or cells that arise from a common ancestor.
|
|
|
What is a vector? Give some examples.
|
Vector is a molecule of DNA to which the fragment of DNA to be cloned is joined.
Ex. plasmids, phages, cosmids. |
|
|
Plasmid vector.
|
Small circular dsDNA that confers antibiotic resistance to host cells.
|
|
|
Phage vector.
|
Linear DNA; viruses that infect bacteria
|
|
|
Cosmid vector
|
Capable if independant replication and are able to carry the largest DNA inserts in comparison to the other vectors.
|
|
|
YAC (Yeast artificial carriers)
|
Are capable of carrying the largest DNA inserts.
|
|
|
DNA library
|
A collection of recombinant clones.
|
|
|
Genomic library
|
A set of clones, packaged in the same vector, that together represents all regions of the genome of the organism in question.
|
|
|
cDNA library
|
represents all of the mRNA present in a particular tissue, which has been converted back to a DNA template by the use of the enzyme reverse transcriptase. NOTE: It does not represent the entire genome (like a genomic library) rather only the DNA being transcribed!
|
|
|
What is a probe?
|
It is a ss piece of DNA that is complimentary to the DNA of interst.
|
|
|
4 blotting techniques include?
|
Southern Blot => for DNA
Northern Blot => for RNA Western Blot => for protein Southwestern Blot => for protein/DNA interactions |
|
|
Fn. of allele specific oligonucleotide probes
|
Detect a single bp mismatch between probe & target. E.g. sickle cell disease.
|
|
|
In what position is the mutation for sickle cell?
|
Glu -> Val in 6th position of B-globin chain.
|
|
|
Steps in PCR (3)
|
1. Denaturing
2. Annealing 3. Chain extension or polymerization -> by adding excess dNTP’s and heat stable DNA polymerase (Taq polymerase) |
|
|
What will show on a PCR analysis of a.) a carrier of CFTR and b.) on a homo for CFTR mutation?
|
a.) Two bands
b.) one band (at a lower kb b/c missing 3 bases with CFTR mutation) |
|
|
What is a RFLP?
|
Restriction Fragment Length Polymorphism -> an inherited difference in the pattern of restriction.
|
|
|
RFLP can be used for prenatal testing of sickle cell. hat characteristic features will be seen on this analysis if the fetus is positive for sickle cell?
|
single base mutation
MstII restriction site is lost due to T>A transversion normal produces a 1150bp, SS 1350bp (b/c normal restriction site is cut out). |
|
|
RFLP is used indriectly to test for PKU...why not directly?
|
B/C mutations in the gene do not affect the restriction site. Thus, analyze (RFLP) DNA of the family instead & then compare it to the fetus.
|
|
|
Purpose of a Microarray?
|
To detect patterns of mRNA production, gene expression, etc. and is performed by converting mRNA -> cDNA -> DNA chip -> measure amt of fluorescence.
|
|
|
How do you test for HIV?
|
ELISA (protein), then confirm with Western Blot
|
|
|
What are knockout mice?
|
Mice that have their normal gene replaced by a mutant gene to express human disease(s).
|
|
|
First Law of Thermodynamics
|
Energy cannot be created or destroyed
|
|
|
2nd Law of Thermodynamics
|
randomness or entropy of the universe increases in all natural processes.
|
|
|
Gibbs free energy eq'n.
|
ΔG = ΔH - T ΔS
ΔG = gibbs free energy ΔH = heat T = Temp ΔS = Entropy(disorder) |
|
|
If Keq =1, then ΔG0 =0
|
A <=> B
|
|
|
If Keq >1, ΔG0 <1
|
A -> B more readily than B -> A
|
|
|
If Keq <1, ΔG0 >0
|
B -> A more readily than A-> B
|
|
|
Structure of ATP includes what components?
|
adenosine
ribose 3 phosphate groups |
|
|
Globular vs Fibrous proteins
|
Globular: carry out all of the chemical mech's =>syn, transport, metablosim
Fibrous: Protective, connective or supportive functions |
|
|
Hemeproteins are what type of protein?
|
Globular
|
|
|
Hemeproteins include (4)...
|
Myoglobin & Hemoglobin -> reversible binding to oxygen
Cytochromes -> electron carrier Catlase -> breakdown H2O2 |
|
|
Center of Heme has what form of iron?
|
Ferrous (Fe2+)
|
|
|
Structure & Fn. of myoglobin
|
Single polypeptide chain with eight α-helices (A through H)
Acts as a reservoir & carrier of O2 |
|
|
Myoglobin: nonpolar amino acids inside and polar amino acids outside...except?
|
His E7 and His F8 which lie in the interior
|
|
|
What prevents the irreversible oxidation of Fe2+ to Fe3+ in myoglobin?
|
apoprotein structure
|
|
|
Chief fn of Hb?
|
Carry oxygen from lungs to tissues.
|
|
|
Hb: Major adult type
|
HbA
|
|
|
Structure of HbA?
|
Has 4 polypeptide chains: 2A & 2B held together by hydrophobic bonds (a1b1 & a2b2). The two dimers are then held together with polar bonds (free to rotate).
|
|
|
How many O2 binding sites does myoglobin have?
|
one
|
|
|
O2 dissociation curve for Mb vs Hb
|
Mb is rectangular
Hb is sigmoidal |
|
|
O2 transportation: Mb vs Hb?
|
Mb cannot release to tissues where Hb can.
|
|
|
Allosteric effectors of Hb are?
|
pH, pCO2 and 2,3 BPG
|
|
|
Decrease in pH has what effect on the oxygen dissociation curve?
|
Shifts it to the right.
|
|
|
T state is stabilized -> oxygen released to tissues. Under what scenario would this likely occur?
|
Increase in CO2
|
|
|
Effect of 2,3 BPG on the oxygen dissociation curve? Under what conditions would this occur?
|
Shift to the right, more O2 released, T state is stabilized.
In an individual at high altitudes. |
|
|
What common diseases cause an increase in 2,3-BPG?
|
High Altitudes
Anemia Chronic hypoxia |
|
|
Effect of carbon monoxide (CO)?
|
Binding shifts the equilibrium to R state -> O2 binds to other 3 sites with high affinity -> cannot be released to tissues
|
|
|
What diseases are associated with increased amounts of hemoglobin F?
|
Sickle cell anemia, aplastic anemia, and leukemia.
|
|
|
Mechanism of sickle RBC shape.
|
HbS polymerises reversibly when deoxygenated -> gelatinous network of fibrous polymers -> potassium leakage and calcium influx stiffen RBC membrane, -> viscosity and dehydration -> sickle shape
|
|
|
What is the effect of a sticky membrane of the sickle cells?
|
adhere to endothelium -> microvascular occlusion and premature RBC destruction (mainly in spleen)
|
|
|
Clinical features of sickle cell anemia
|
Vaso-occlusive crises with infarction of spleen, brain, marrow, kidney, lung
Aseptic necrosis of bones Splenomegaly -> Repeated infection |
|
|
Individuals hetero for sickle cell trait can experience sickling under what conditions?
|
low O2 (hypoxic)
|
|
|
Variables that increase sickling.
|
Increased pCO2, decreased pH, and Increased 2,3 BPG.
|
|
|
Defect in Hb C is caused by what mutation?
|
Glu -> Lys
|
|
|
Methemoglobinemias: what happens to iron?
|
Remains in the ferric (fe3+) form -> unable to bind O2.
|
|
|
This enzyme is responsible for the maintenance of Heme iron in reduced state.
|
methemoglobin reductase
|
|
|
Two minor hemoglobins.
|
HbF & HbA2
|
|
|
Methemoglobinemias
|
Present with chocolate cyanosis, anxiety, headache and dyspnoea
|
|
|
Thalassaemias
|
Disorder of syn of Hb
|
|
|
α Thalassemia vs β Thalassemia
|
In α Thalassemia – the synthesis of α chain is decreased or absent.
In β Thalassemia – the β chain synthesis is decreased or absent. |
|
|
Mechanism which leads to anemia in thalassemia patients
|
The chain that is produced at normal rate is in relative excess -> precipitates in cell (Heinz Bodies) -> damage to cell membrane -> premature red cell destruction -> hemolytic anemia
|
|
|
Most common cause of B-thalassemia?
|
single bp substitution
|
|
|
Why is B-thalassemia seen a few months after birth and not right away?
|
B/C HbF still remains functional for a few months, thus not showing the deficit until that time!
|
|
|
Most common cause of A-thalassemia?
|
gene deletions
|
|
|
NOTE
|
A-Thalassemia affect both fetus and adult Hb, whereas B-Thalassemia only affects adult.
|
|
|
What occurs when infants have encountered severed intrauterine hypoxia?
|
Infants have severe intrauterine hypoxia -> massive generalized fluid accumulation -> Hydrops Fetalis
|
|
|
Decreased formation of Hemoglobin
|
Microcytic hypochromic anemia
|
|
|
Elevated HbA2
|
Beta-Thalassemias
|
|
|
Key features of thalassemias (3).
|
Bone marrow expansion
Extramedullary erythropoeisis Calorie diversion to erythropoeisis |
|
|
Classic e.g. of fibrous proteins
|
Collagen & Elastin
|
|
|
Shape of fibrous proteins?
|
Secondary structure.
|
|
|
Structure of collagen.
|
Triple stranded
Composed of A-helices (repeating sequence of 3 amino acids, viz., Gly-X-Y (X = proline, Y = hydroxyproline, or hydroxylysine) R-handed superhelix |
|
|
Match the following with their respective number, may be used once, more than once or not at all…
Fibril-forming (fibrillar)/Fibril Associates/Network forming I/II/III/IX/XII/IV/VII |
Fibril-forming (fibrillar) - I, II, III
Fibril Associates IX, XII Network forming IV VII |
|
|
A-chain of collagen: R or L-handed helix?
|
L
|
|
|
How many a.a. per turn of collagen a-helix?
|
3
|
|
|
What a.a. are important in the formation of the triple-stranded collagen helix?
|
proline &glycine
|
|
|
FN. Of Proline in Collagen.
|
ring structure stabilizes the helical conformation in each α chain –confer rigidity
|
|
|
Where would you find Glycine in the structure of collagen?
|
in the center
|
|
|
Why does collagen appear to be banded?
|
b/c Collagen fibers are assembled by lateral association of the triple helices in a quarter staggered fashion.
|
|
|
1. Collagen is synthesized by ______ in CT;
2. Collagen is synthesized by ______ in Bone 3. Collagen is synthesized by ______ in Embryonic Cartilage 4. Collagen is synthesized by ______ in Teeth |
1. fibroblasts
2. osteoblasts 3. chondrocytes 4. odontoblasts |
|
|
Where are Individual collagen polypeptide chains syn.?
|
on membrane-bound ribosomes as prepro-A-chains
|
|
|
Post-translational modification of collagen requires O2 and ascorbic acid. If deficient in ascorbic acid...what disease?
|
Scurvy
|
|
|
Where does procollagen syn. take place?
|
In the Golgi
|
|
|
Mech. of pro-collagen syn.
|
Process begins with formation of disulfide bonds at C-terminal extensions of pro-α chains -> Each pro-α chain then combines with two others to form a hydrogen-bonded, triple-stranded, helical molecule known as procollagen
|
|
|
What collagen biosyntheis steps take place in the extracellular space (4)?
|
1. Cleavage by N and C procollagen peptides
2. Formation of collagen fibrils by regular, staggered, spontaneous association 3. Each overlapping its neighbour by ¾ of a molecule 4. Crosslinking |
|
|
How is N and C procollagen peptides cleaved?
|
By N and C collagen peptidases.
|
|
|
What enzyme is responsible for collagen crosslinking?
|
lysyl oxidase
|
|
|
Lysine hydroxylase vs lysyl oxidase?
|
Lysine hydroxylase (Vit C) vs lysyl oxidase (collagen)
|
|
|
Fn of matrix metalloproteiases?
|
Degradation of collagen
|
|
|
Clinical features: brittle bones, blue sclera, hearing loss, dental deformity
|
OI
|
|
|
What type of mutations cx. OI?
|
OI is due to point mutations (null or missense).
|
|
|
NOTE
|
Difference in severity Mutation at N terminal is mild disorder however, if mutation at C-terminal end this leads to a severe disorder.
|
|
|
NOTE
|
Whether the C term end or N term end is involved, when gly is sibstituted by a bulky grp will lead to a sever for of OI.
|
|
|
hyper extensible skin, hyper mobile joints, ocular fragility
|
Ehler-Danlos Syndrome
|
|
|
Enzyme cx. of Ehlers-Danlos?
|
lysyl oxidase
|
|
|
Mutations in gene for Type III Collagen
|
Ehlers-Danlos Syndrome
|
|
|
Found in lungs, walls of large arteries, elastic ligaments
|
Elastin
|
|
|
What feat. Differentiate elastin from collagen (3)?
|
Gly not present at every 3rd a.a.
Not glycosylated Contains hydroxy-proline NOT hydroxylysine. |
|
|
Crosslinking in elastin is known as ______ link. Give elastic properties to elastic molecule
|
Desmosine
|
|
|
Most important physiologic function is to inhibit the neutrophil elastase
|
A-1 antitrypsin
|
|
|
Where does mutated A-1 antitrypsin polymerize?
|
Polymerizes in ER (not in extracellular space) leading to liver cirrhosis.
|
|
|
Most common mutation cx A-1 Antitrypin deficiency?
|
Glu -> lys
|
