Molecular Biology Flashcards
Nucleotides are composed of three subunits:
- Pentose sugar
- Ribose
- Deoxyribose - Nitrogenous base
- Purine
- Pyrimidine - Phosphate group: Forms the linkages between nucleotides.
How are nucleotides linked?
By a 3′–5′ phosphodiester bond
What are effective carriers?
Effective carriers are molecules that are relatively stable as leaving groups. These include phosphoryl (ATP), electrons (NADH, NADPH, FADH2), sugars (UDP glucose), methyl (SAM), 1 carbon (THF), CO2 (biotin), and acyl (coenzyme A).
Which bond increases the melting temperature?
G-C bonds (3 H-bonds) are stronger than A-T bonds (2 H-bonds). Increased G-C content increases the melting temperature (Tm), which is the
temperature at which half of the DNA base-pair hydrogen bonds are broken.
Purine nucleotide synthesis pathways
De novo or salvage
- De novo synthesis utilizes elemental precursors and is used primarily for rapidly dividing cells.
- The salvage pathway recycles the nucleosides and nitrogenous bases that are released from degraded nucleic acids; it is considered the major route for synthesis in adults.
Purine analogs that inhibit PRPP amidotransferase
Allopurinol and 6-mercaptopurine
PRPP
5-phosphoribosil-1-pyrophosphate
Purine de novo synthesis
- Rate-limiting step by glutamine PRPP amidotransferase
- PRPP amidotransferase is inhibited by downstream products (IMP, GMP, AMP) and purine analogs
- Required cofactors: tetrahydrofolate, glutamine, glycine, aspartate
Reciprocal substrate effects in purine de novo synthesis
GTP and ATP are substrates in AMP and GMP synthesis, respectively. For example, ↓ GTP → ↓ AMP → ↓ ATP.
This allows for balanced synthesis of adenine and guanine nucleotides.
Purine Salvage pathway
-Recycles ~90% of the preformed purines that are released when cells’ nucleases degrade endogenous DNA and RNA and make new purine nucleotides.
-Catalyzed by hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which is inhibited by IMP and GMP.
-Nitrogenous base (guanine, hypoxanthine) + PRPP → GMP/IMP + PPi
.
What is Lesch-Nyhan Syndrome?
X-linked recessive disorder of failed purine salvage due to the absence of HGPRT. HGPRT converts hypoxanthine → IMP and guanine → GMP. The inability to salvage purines leads to excess purine synthesis and consequent excess uric acid production.
Lesch-Nyhan Syndrome Presentation
Retardation, cerebral palsy, self-mutilation, aggression, gout, choreoathetosis, arthritis, nephropathy
Lesch-Nyhan Syndrome Diagnosis
Orange crystals in diaper, difficulty with movement, self-injury, hyperuricemia
Lesch-Nyhan Syndrome Treatment
Allopurinol, which inhibits xantine oxidase. Treatment does not ameliorate neurologic symptoms
Lesch-Nyhan Syndrome Prognosis
Urate nephropathy, death in the first decade, usually as a result of renal failure
What is Gout?
Disorder associated with hyperuricemia, due to either overproduction or underexcretion of uric acid. Uric acid is less soluble than hypoxanthine and xanthine, and, therefore, sodium urate crystals deposit in joints and soft tissues, leading to arthritis.
Primary gout?
Due to hyperuricemia without evident cause. Affected individuals may have a familial disposition. May occur in association with PRPP synthetase hyperactivity or HGPRT deficiency of Lesch-Nyhan syndrome; most common form.
Secondary (acquired) gout?
Uric acid overproduction can be caused by leukemia, myeloproliferative syndrome, multiple myeloma, hemolysis, neoplasia, psoriasis, and alcoholism and is more common in men. Secondary gout due to urate underexcretion can be caused by kidney disease and drugs such as aspirin, diuretics, and alcohol.
Gout Presentation
Monoarticular arthritis of distal joints (eg, podagra—gout of the great toe), often with history of hyperuricemia for > 20–30 years, precipitated by a sudden change in urate levels (eg, due to large meals, alcohol), eventually leads to nodular tophi (urate crystals surrounded by fibrous connective tissue) located around the joints and Achilles tendon.
Gout Diagnosis
Arthritis, hyperuricemia, detection of negatively bifringent crystals from articular tap. Negatively birefringent crystals will be yeLLow when paraLLel to polarized light, blue when perpendicular. Note: positively birefringent crystals are characteristically found in pseudogout.
Gout Treatment
Normalize uric acid levels (allopurinol, probenecid for chronic gout), decrease pain and inflammation (colchicines, nonsteroidal anti-inflammatory drug for acute gout), avoid large meals and alcohol.
Differential diagnosis for increased uric acid and gout:
+ Lesch-Nyhan
+ Alcoholism
+ G6P deficiency, hereditary fructose intolerance, galactose 1P uridyl transferase deficiency—all disorders with increased
accumulation of phosphorylated sugars, increased degradation products (eg, AMP)
What is Severe Combined (T and B) immunodeficiency (SCID)?
Autosomal recessive disorder caused by a deficiency in adenosine deaminase (ADA). Excess ATP and dATP causes an imbalance in the nucleotide pool via inhibition of ribonucleotide reductase (catalyzes ribose → deoxyribose). This prevents DNA synthesis and decreases the lymphocyte count. It is not understood why the enzyme deficiency devastates lymphocytes in particular.
SCID Presentation
Children recurrently infected with bacterial, protozoan, and viral pathogens, especially Candida and Pneumocystis jiroveci.
SCID Diagnosis
No plasma cells or B or T lymphocytes on complete blood count (CBC), no thymus.
SCID Treatment
Gene therapy, bone marrow transplantation
Poor Prognosis
Pyrimidine nucleotide synthesis pathways
De novo and salvage
+The salvage pathway relies on pyrimidine phosphoribosyl transferase enzyme, which is responsible for recycling orotic acid, uracil, and thymine, but not cytosine.
+De novo synthesis relies on a different set of enzymes.
What is Hereditary Orotic Aciduria?
Deficiency in orotate phosphoribosyl transferase and/or OMP decarboxylase (pyrimidine metabolism).
Hereditary Orotic Aciduria Presentation
Retarded growth, severe anemia
Hereditary Orotic Aciduria Diagnosis
Low serum, leukopenia, megaloblastosis, white precipitate in urine
Hereditary Orotic Aciduria Treatment
Synthetic cytidine or uridine given to maintain pyrimidine nucleotide levels for DNA and RNA synthesis
Products of purine and pyrimidine degradation
+Purine - uric acid
+Pyrimidine - beta-amino acids, CO2 and NH4
Hydroxyurea inhibits…
ribonucleotide reductase
5-fluorocil (5-FU) inhibits…
thymidylate synthase
Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine inhibit…
dihydrofolate reductase
Bacterial dihydrofolate reductase is inhibited by…
The antimetabolite trimethoprim.
+It is often used in combination with sulfonamides (eg, sulfamethoxazole) to sequentially block folate synthesis.
Conditions that lead to denaturation of the DNA helix:
Heat, alkaline pH, formamide, urea
Inhibit bacterial topoisomerase IV:
Quinolone antibiotics
Levels of DNA protein organization:
DNA -> Histones -> Nucleosome 10nm fiber -> 30nm fiber
Heterochromatin
Condensed, appears darker on EM. Transcriptionally inactive, sterically inaccessible.
- HeteroChromatin = Highly Condensed.
- Barr bodies (inactive X chromosomes) are heterochromatin
Euchromatin
Less condensed, appears lighter on EM.
Transcriptionally active, sterically accessible.
DNA methylation
+Template strand cytosine and adenine are methylated in DNA replication, which allows mismatch repair enzymes to distinguish between old and new strands in prokaryotes.
+DNA methylation at CpG islands represses transcription.
+CpG Methylation Makes DNA Mute
Histone methylation
+Usually reversibly represses DNA transcription, but can activate it in some cases depending on methylation location.
+Histone Methylation Mostly Makes DNA Mute.
Histone acetylation
+Relaxes DNA coiling, allowing for transcription. Histone +Acetylation makes DNA Active.
Leflunomide inhibits…
dihydroorate dehydrogenase
Mycophenolate and ribavirin inhibit…
IMP dehydrogenase
6-mercaptopurine (6-MP) and its prodrug azathioprine inhibit…
de novo purine synthesis
