Molecular Diagnostics Flashcards
DNA and RNA are polymers of
mononucleotides
A mononucleotide consists of
sugar, PO4-, and a nitrogenous base
A nucleoside consists of
a sugar and a nitrogenous base
The two purines are
adenine and guanine
The three pyrimidines are
thymine, uracil, and cytosine
What bonds a base to a sugar?
Glycosidic bond
Purine synthesis of AMP and GMP requires
Many amino acids (glycine, glutamine, arginine), Nitrogens, Oxygens, and folate
Purine synthesis of AMP and GMP is an energy consuming process, that requires how many ATPs?
~7-8 ATPs for 1 purine
Describe the degradation of purines.
- Adenosine has its nitrogens removed by adenosine deaminase to become inosine
- Inosine gets de-phosphorylated, becomes Hypoxanthine
- Hypoxanthine has H2O2 removed by Xanthine oxidase, becomes Xanthine
- Xanthine has H2O2 removed by Xanthine oxidase (again), becomes uric acid
In degradation of purines, Hypoxanthine-guanine phospho-ribosyltransferase (HGPRT) enzyme INHIBITS de-phosphorylation causing
hypoxanthine to go back to inosine –> adenosine –> AMP
Xanthanine oxidase deficiency is a purine metabolism disease that causes
hypouricemia
HGPRTase, PRPP deficiency (salvage pathway enzymes) is a purine metabolism disease that causes
Hyperuricemia (Lesch-Nyhan syndrome/Juvenile gout)
Adenosine deaminase deficiency is a purine metabolism disease that causes:
Severe combined immunodeficiency disease (SCID) (Missing body defense of B and T-cells)
DNA is linear and the 3’ to 5’ BOND is formed due to ribose becoming deoxyribose via
Ribonucleotide reductase
Pyrimidine synthesis is different than purine synthesis in the sense that it does not
- require large energy
- No significant salvage in eukaryotes
Pyrimidine synthesis requires
Amino acids (glutamine and aspartate), Carbons, and Nitrogens
Cytosine pyrimidine is deaminated to ________ which is degraded to ________,
Uracil; B-alanine
Thymidine (pyrimidine) is degraded to __________, which is then degraded to _____.
beta-aminoisobutyrate (aa derivative); urea
dTMP (thymidine monophosphate) is important for DNA synthesis.
Describe the process of thymidylate synthesis.
dUMP is converted to dTMP by thymidine synthase + Folate + B12
Thymidine synthase just adds a Methyl group
Thymidylate synthesis is inhibited by cancer therapy drugs. What are the drugs and their exact methods of inhibition?
Methotrexate and Aminopterin inhibit DHFR so DHF cannot become MTHF (Inihibits one carbon metabolism)
5-fluorouracil (F-FU) pyrimidine analog inhibits thymidine synthase by inhibiting dUMP
Folic acid (B9/folate) works with what vitamin to do what?
Works with B12 to make RBCs and help iron function
In order for inactive folic acid to be activated, it must be converted by di-hydrofolate reductase (DHFR) to:
Tetrahydrofolic acid (THF) –> Methyltetrahydrofolate (MTHF) (Which can be used for DNA synthesis)
THF and MTHF are folic acid congeners that are transported where they are needed to: (5)
- maintain normal erythropoiesis
- Interconvert amino acids
- Methylate tRNA
- generate and use formate
- Synthesize purine and thymidylate nucleic acid
Vitamin B12 (Cyanocobalamin) is not made by the body, so it must be obtained via diet. What are the two forms used by the body?
- Methylcobalamin: Cofactor for homocysteine –> Methionine (Required for DNA methylation)
- 5-deoxyadenosyl cobalamin: cofactor for I-methylmalonyl CoA –> succinyl CoA
In DNA synthesis (primary structure), chain growth is always ____, while nucleobases are linked by ________.
Chain growth: 5’-3’
Nucleobases are linked by 3’-5’ phosphodiester ends
In DNA synthesis, the secondary structure is double stranded and bases are paired through hydrogen bonds. What are the rungs and the legs of the ladder?
Rungs: Nitrogenous bases
Legs: Phosphate and sugar backbone
What is the direction of the leading and lagging strand?
Leading: 5’-3’ (Continuous replication)
Lagging: 3’-5’ (Discontinuous replication)
DNA synthesis is semiconservative meaning both strands are synthesized
simultaneously
What are the 3 prokaryotic polymerase in prokaryotic DNA synthesis?
DNA polymerase I: replication and repair
DNA polymerase 2: implicated and repair
DNA polymerase 3: MAIN processive replicative enzyme (de novo synthesis of DNA) (aka makes it longer) 3’-5’
Prokaryotic DNA synthesis sequence of events: (7)
- Topoisomerase
- Helicase
- SSB
- Primase
- DNA polymerase III
- Ligase
- Gyrase (type 2 isomerase)
Topoisomerase function:
uncoils/separates strands and cuts 1 or more phopshate backbones
Helicase functions:
Breaks hydrogen bonds and forms replication fork
Single-strand binding protein (SSB) function:
prevents rejoining of DNA single strand
RNA primase function
synthesized RNA primers used in DNA daughter strands formation
DNA primase function
RNA polymerase that generates RNA primers (templates for DNA replication)
DNA polymerase function
synthesizes DNA daughter strands by adding nucleotides to leading and lagging strands
DNA gyrase
rewraps and recoils DNA strands
DNA ligase
links newly synthesized DNA fragments (okazaki fragments) by forming phosphodiester bonds
Exonucleases:
group of enzymes that remove nucleotide bases from the end of DNA chain
Eukaryotic DNA synthesis consists of
-Replication in S phase of cell cycle
-Unwraping DNA from histones
-Multiple origins of replication on each chromosome (~100,000)
-DNA polymerases (alpha, beta, gamma, and epsilon)
Describe the eukaryotic DNA polymerases necessary for polymerization
DNA poly alpha: acts as a primase, synthesizing an RNA primer, and repair
DNA poly beta: repair
DNA poly gamma: mitochondrial polymerase
DNA poly delta AND epsiol: Elongation of leading strand (synthesis) and lagging strand (gap filling)
Inhibitors of DNA synthesis include
- quinolones (norafloxin, ciprofloxacin): inhibit DNA gyrase
- (Chemo) Methotrexate, aminopterin, trimethoprim: inhibit DHFR which inhibits DNA synthesis via thymidylate synth
- (Chemo) 5’ fluoracil: inhibits thymidylate synthesis
- AZT: DNA chain terminator (no 3’ OH)
AZT and 5FU are uracil
analogs
Describe replication process
DNA –> DNA replication –> Transcription –> mRNA nucleus –> mRNA cytoplasm –> Translation –> protein
Prokaryotic DNA is
circular
How are eukaryotic chromosomes organization?
- DNA helix wraps around 4 pairs of histones (forms nucleosomes)
- Linear DNA connects nucleosomes (forms thick fiber)
- Fibers form loops to condense chromatin (forms chromosome)
- Each chromosome becomes two sister chromatids that attach at centromere
Each homologous set of chromosomes is made up of
2 homologues
What phase of mitosis is best for viewing chromosomes?
Metaphase; they;re highly coiled and very thick
Metaphase chromosomes make up the Karyotype picture (Set of chromosmes per individual)
Euchromatin in the _____ type of chromatin. Describe this type.
Active type that helps transcribe RNA to proteins
-Less condensed –> stains lighter
-Gene expressing/gene rich (high G/C)
Heterochromatin is the _____ type of chromatin, Describe this type.
-inactive
-More condensed (especially at centromeres and telomeres)
-Gene poor (high A/T content)
-Stains
Telomere are made up of
repetitive nucleotide sequences in buffer zone that serve as a termination signal (TTAGGG; ‘cap sequence’)
Telomerase enzyne adds more nucleotides to telomeres to
regenerate the protective “CAP” DNA sequence (avoid DNA damage)
Lost/reduced telomerase activity causes _________, while over expression _______
premature cell aging and death; helps cancer cells grow fast/live longer
Chromosome nomeclature.
- Chromosome #
- Location on short or long arm
- Region of arm )specific band)
Check points of the cell cycle
G1, G2, and M (metaphase)
What is the Go phase of the cell cycle?
Resting phase; Cell has left cycle and stopped dividing
What is G1 in the cell cycle?
CHECKPOINT! makes sure everything is ready for DNA synth
-Cells increase in size
What is the S phase in the cell cycle?
DNA replication occurs!!
What is the G2 phase of the cell cycle?
CHECKPOINT Make sure everything is ready to go into mitosis
-Cells still growing
What is the M (mitosis) phase of the cell cycle?
Cell growth stops and division into two daughter cells occurs
METAPHASE CHECKPOINT makes sure cell is ready to complete cell division
Major proteins that control the cell cycle? These proteins are depended on activation via:
- Cyklin-dependent protein kinases (Cdks)
- Cyclins
- Cdk-cyklin complex: ability of cdl to phosphorylate target depends on cyclin that it forms with
Phosphorylation/dephospho-rylation
SnoRNA and miRNA are dual regulatory non-coding RNAs. What are their characteristics?
-Regulatory roles in cells
-Non-coding
What are the three major types of RNA and their dominance (%)?
rRNA (80%)
tRNA (15%)
mRNA (5%)
RNA synthesis requires
- DNA template
- RNA polymerase* (1 polymerase/6 subunits; alpha-2, beta, beta-prime, sigma, and omega)
The sigma (σ) subunit is important for RNA synthesis because
it recognizes the promoter region!
DNA polymerase
DNA –> DNA
RNA polymerase
DNA –> RNA
RNA replicase
RNA –> RNA
Reverse transcriptase
RNA –> DNA
Rifampin inhibits RNA synthesis by
inhibiting B unit of polymerase
Steps in prokaryotic transcription
- RNA polymerase binds to promoter region
- Promotor has specific sequences (CAAT, GC, TATA)
- DNA coding strand=sense
(same sequence as message except U instead of T) - Non-coding strand (template)= anti-sense (Used in the 3’-5’ direction so RNA chain is synthesized 5’-3’ and makes a coding strand)
Enzymes in eukaryotic transcription
Poly 1: nucleolus: synth 45s rRNA
Poly 2: nucleoplasm, synth mRNA (recognized promoter sequence)
Poly 3: nucleus, synthesizes tRNA, 5sRNA, miRNA
Mitochondrial polymerase: synthesized mitochondrial RNA
TATA mutation results in
B-thalassemia
Upstream of gene consists of
Hormone binding receptors, transcription binding receptos, enhancers, and promoter region
Eukaryotic primary transcript (pre) RNA contains Introns and exons. To form a function mRNA what must occur?
Splicing must occur to remove the introns and combine the exons
What are the 3 types of processing for mRNA?
- 5’ cap: add 7-Methyl-guanosine to 5’ end
- 3’ poly-A tail: add many adenosines for stability
- Splicing: removal of introns
-Alternative splicing
Inhibitors of RNA synthesis (5)
- Actinomycin D: inhibit template binding (at transcription initiation)
- Rifampin: inhibits B subunit of polymerase binding
- A amanitin (mushroom toxin): Inhibit euk pol II and III
- AZT: inhibits reverse transcription
- DDIL inhibits RT
3 human diseases related to mRNA synthesis
- Thalassemia: abnormal formation of hemoglobin (inherited)
- a-thalassemia: prod. of alpha globin chain is affected (deletions)
- b-thalassemia: prod. of beta chain is affected (mutations)
Genes can be regulated at any point of gene expression, but most commonly at
transcription by transcription factors, enhancers, suppressors, or during certain developmental stages
Peptide bonds are formed via peptide linkage. When two amino acids are formed,
water is removed
Carboxyl terminal is always linked to:
incoming or next amino acid
In the amino acid, the central carbon is covalently bonded to
- amine group
- hydrogen
- a carboxyl group
- variable R- group
A genetic code is made up of
3 nucleotides
What is a degenerate (wobble base codon)?
First 2 positions of mRNA have Watson base pairing rules, but 3rd position exhibits wobble
What is the initiation codon/amino acid?
AUG (Met)
What are the stop codons?
UAA, UAG, UGA
Translation is a high energy consuming process that consumes ____% of cells energy and ___ ATP
90%; 4 ATP
Protein synthesis players consist of
Ribosome –> rough ER –> rER (rRNA + protein)
tRNA=anticodon w/ aa
mRNA= codon
2 ribosome sites:
P-side: initiation
A-site: elongation
Protein synthesis steps in detail (3)
- Initiation
**First aa is always methionine at P side
-Codon is read by anticodon or tRNA - Elongation
New aa brought to match new codons and peptide bonds are formed - Termination
Stop codon
mRNA function
carries instructions from DNA to rest of ribosome (tells ribosome what protein to make(
tRNA function
A go getter!! gets the right parts to make right protein for mRNA
**ANTICODON NEEDS TO BE COMP. W/ mRNA codon sequencE
rRNA function and its two types.
-part of ribosome structure
-helps in protein production
-Contains two rRNAs and 50 or more proteins
-Prokaryotic rRNA (size 70s): 50s, 30s
Eukaryotic rRNA (size: 80s): 60s, 40s
Proteins (post-translation) can be modified and regulated by
Glycosylation, proteolysis cleavage, folding, R-group modifications, etc.
R-group modifications:
1. phosphorylation
2. Methylation
3. acetylation
4. isopentation
5. hydroxylation
Describe insulin maturation
- Pre-pro insulin:
-single-peptide sequence (~30 AA) REQUIRED for transfer into ER
-B chain, alpha chain, and C. peptide
-Cleaved - Pro-insulin
-Folds
-No signal sequence - Insulin
-A and B chain (No-C peptide)
C-peptide levels are measured instead of insulin levels because:
C-peptide can assess a persons own insulin secretions EVEN if they’ve had insulin injections
(Liver does not metabolize C-peptide, so blood C-peptide is best)
Genetic codon changes that cause mutations in proteins
- point mutations
1a. no change: silent due to alteration of wobble base
1b. missense: change in base leads to change in aa
1c. nonsense: formation or modification or termination codon - frameshift: insertion or deletion of a nucleotide
Alpha thalassemia is caused by a mutation and has many variants. What are they?
Alpha thalassemia (nonsense mutation):
Normally 142 aa long, but becomes 172 aa
Variants:
Constant spring –> glutamine @ 142
Thalassemia is caused by what mutation? What is abnormal hemoglobin wayne?
Thalassemia is caused by frameshift mutation
Abnormal hemoglobin Wayne: everything after 138 is incorrect (Goes to 147 before it stops)
Inhibitors of protein synthesis (7)
- streptomycin/gentamicin: 30S prokaryotic initiation
- Erythromycin: 50S pro elongation in gram +/-
- Chloramphenicol: 70S, elongation, broad spectrum, none marrow suppression
- tetracycline: inhibits incoming tRNA to site A at 30S
- Cyclohexidide: 80 S and fungus
- puromycin: mimic tRNA binds at A site in pros and euks
- Diptheria toxin: euk elongation factor II inhibitor
The following antibiotics bind to the 30S subunit of the ribosome
Aminoglycoside
Tetracycline
The following antibiotics bind to the 50S ribosomal subunit
Chloramphenicol
Erythromycin
Streptogramins – a group of cyclic peptide antibiotics that inhibit, like macrolides and lincosamides, the synthesis of bacterial proteins
4 types of exogenous DNA damage
- Thermal disruption
- UV light exposure
- Ionizing radiation
- Exposure to mutagens, carcinogens, viruses
3 types of endogenous DNA damages
- cellular metabolism
- hyrdolysis
- nuclease digestion
Agents that damage DNA (3)
Certain wavelengths of radiation (UV-B, UV-C) (~260)
Chemicals in the environment: hydrocarbons, aflatoxins
Intrinsic spontaneous mutation:
Reactive oxidative species
Error during DNA replication
MMR enzyme mutation caused by mismatch repair failure
How can damaged or inappropriate bases be repaired?
- Direct chemical reversal of the damage
- Excision repair: damaged base(s) are moved and then replaced with the correct one
-Three modes: Base excision repair (BER), Nucleotide excision repair (NER), Mismatch repair (MMR)
Direct repair genes
- DNA photolyase:
-natural repair for pyrimidine dimers caused by UV damage
-Directly reverse CPD via photochemical directions - ## O6- methylguanine- DNA- methyl-transferase (MGMT):
