Bio Chemisity Flashcards
Chromatin structure
Chromatin structure
DNA exists in the condensed, chromatin form in order to fit into the nucleus. Negatively charged DNA loops twice around positively charged histone octamer to form nucleosome “bead.” Histones are rich in the amino acids lysine and arginine. H1 ties nucleosome beads together in a string.
In mitosis, DNA condenses to form chromosomes.
Think of “ beads on a string.”
H1 is the only histone that is not in the nucleosome core.
Heterochromatin
Heterochromatin
Condensed, transcriptionally inactive, sterically inaccessible.
HeteroChromatin = Highly Condensed.
Euchromatin
Euchromatin
Less condensed, transcriptionally active, sterically accessible.
Eu = true, so “truly transcribed.”
DNA methylation
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.
Histone methylation
Histone methylation
Inactivates transcription of DNA.
Methylation makes DNA Mute.
Histone acetylation
Histone acetylation
Relaxes DNA coiling, allowing for transcription
Acetylation makes DNA Active.
Nucleotides
Nucleotides
PURines (A, G)-2 rings. G has a double bond Oxygen. Think go go go G (Guanine) has an O
PYrimidines (C, U, T)-1 ring. Think CUT the pie (has one ring/circle
Guanine has a ketone. Thymine has a methyl.
Deamination of cytosine makes uracil.
Uracil found in RNA; thymine in DNA.
G-C bond (3 H bonds) stronger than A-T bond (2 H bonds).
higher amount of G-C content equals higher melting point.
- *PUR**e As Gold.
- *CUT** the PYrimidines (pie). Thymine has a methyl.
GAG-Amino acids necessary for purine synthesis :
Glycine
Aspartate
Glutamine
NucleoSide= base + ribose (Sugar).
NucleoTide= base + ribose + phosphaTe;
linked by 3’-5’ phosphodiester bond.
De novo purine synthesis
Purines
Start with sugar + phosphate (PRPP)
Add base
De novo pyrimidine synthesis
Pyrimidines
Make temporary base (orotic acid)
Add sugar + phosphate (PRPP)
Modify base
Ribonucleotides are synthesized first and are converted to deoxyribonucleotides by ribonucleotide reductase.
Carbamoyl phosphate is involved in 2 metabolic pathways: de novo pyrimidine synthesis and the urea cycle. Ornithine transcarbamoylase deficiency (OTC, key enzyme in the urea cycle) leads to an accumulation of carbamoyl phosphate, which is then converted to orotic acid.
Various antineoplastic and antibiotic drugs function by interfering with purine synthesis:
- Hydroxyurea inhibits ribonucleotide reductase
- 6-mercaptopurine (6-MP) blocks de novo purine synthesis
- 5-Auorouracil ( 5-FU) inhibits thymidylate synthase (decreased deoxythymidine monophosphate [dTMP])
- Methotrexate (MTX) inhibits dihydrofolate reductase decreased dTMP)
- Trimethoprim (TMP) inhibits bacterial dihydrofolate reductase (decreased dTMP)
Orotic aciduria
Orotic aciduria
Inability to convert orotic acid to UMP (de novo pyrimidine synthesis pathway) because ofdefect in UMP synthase (a bifunctional enzyme). Autosomal recessive.
FINDINGS: increased orotic acid in urine, megaloblastic anemia (does not improve with administration ofvitamin B12 or folic acid), failure to thrive. No hyperammonemia (vs. OTC deficiency- increased orotic acid with hyperammonemia).
TREATMENT: Oral uridine administration.
Purine salvage deficiencies
Purine salvage deficiencies
1) Adenosine deaminase deficiency: Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide reductase -> prevents DNA synthesis and thus decrease lymphocyte count. One of the major causes of SCID. Autosomal recessive.
Severe Combined Immunodeficiency Disease (SCID) happens to kids.
1st disease to be treated by experimental human gene therapy.
2) Lesch-Nyhan syndrome: Defective purine salvage owing to absence of HGPRT, which converts hypoxanthine to IMP and guanine to GMP. Results in excess uric acid production and de novo purine synthesis. X-linked recessive.
Findings: retardation, self-mutilation, aggression, hyperuricemia, gout, choreoathetosis.
learning aid: He’s Got Purine Recovery Trouble.
Genetic code features
Genetic code features
Unambiguous: Each codon specifies only l amino acid.
Degenerate/ redundant: Most amino acids are coded by multiple codons.
- Exceptions: methionine and tryptophan encoded by only l codon (AUG and UGG, respectively).
Commaless, nonoverlapping: Read from a fixed starting point as a continuous sequence of bases.
- Exceptions: some viruses.
Universal: Genetic code is conserved throughout evolution.
- Exception in humans: mitochondria.
Point mutations in DNA
Point mutations in DNA
Severity of damage: silent< missense< nonsense< frameshift.
Silent: Same amino acid, often base change in 3rd position of codon (tRNA wobble).
Missense: Changed amino acid (conservative-new amino acid is similar in chemical structure).
Nonsense: Change resulting in early stop codon.
- learning aid: Stop the nonsense!
Frameshift: Change resulting in misreading of all nucleotides downstream, usually resulting in a truncated, nonfunctional protein.
DNA replication
DNA replication
In both prokaryotes and eukaryotes, DNA replication is semiconservative and involves both continuous and discontinuous (Okazaki fragment) synthesis.
Origin of replication
Origin of replication
Particular consensus sequence ofbase pairs in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes).
DNA Replication fork
DNA Replication fork
Y-shapecl region along DNA template where leading and lagging strands are synthesized.
Helicase
Helicase unwinds DNA template at replication fork.
Single-stranded binding proteins
Single-stranded binding proteins prevent strands from reannealing.
DNA topoisomerases
DNA topoisomerases
Create a nick in the helix to relieve supercoils created during replication.
- Fluoroquinolones-inhibit DNA gyrase (prokaryotic topoisomerase II).
Primase
Primase makes an RNA primer on which DNA polymerase III can initiate replication.
DNA polymerase Ill
DNA polymerase Ill
Prokaryotic only. Elongates leading strand
by adding cleoxynucleoticles to the 3’ encl. Elongates lagging strand until it reaches primer of preceding fragment. 3’ -> 5’ exonuclease activity “proofreads” each aclclecl nucleotide.
- DNA polymerase III has 5’ -+ 3’ synthesis and proofreads with 3’ -+ 5’ exonuclease.
DNA polymerase I
DNA polymerase I
Prokaryotic only. Degrades RNA primer; replaces it with DNA.
- Has same functions as DNA polymerase III but also excises RNA primer with 5’ -+ 3’ exonuclease.
DNA ligase
DNA ligase
Catalyzes the formation ofphosphodiesterase bond within a strand of double-stranded DNA (i.e., joins Okazaki fragments).
- Seals.
Telomerase
Telomerase enzyme adds DNA to 3’ ends of chromosomes to avoid loss of genetic material with every duplication.
Types of RNA
Types of RNA
rRNA is the most abundant type.
mRNA is the longest type.
tRNA is the smallest type.
memory aid: Rampant, Massive, Tiny.
Start and stop codons
Start and stop codons
mRNA start codons: AUG (or rarely GUG).
- memory aid: AUG inAUGurates protein synthesis.
Eukaryotes: Codes for methionine, which may be removed before translation is completed.
Prokaryotes: Codes for formylmethionine (f-met).
mRNA stop codons: UAA, UAG, UGA.
- memory aid:
- U Are Awesome
- U Are Great
- U Gonna get an A!
or
- memory aid:
- UGA = U Go Away.
- UAA = U Are Away.
- UAG= U Are Gone.
Fundional organization of the gene
Fundional organization of the gene
Promoter
Promoter
Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (Kf-rich upstream sequence with TKfA and CAAT boxes).
- Promoter mutation commonly results in dramatic decrease in amount of gene transcribed.
Enhancer
Enhancer
Stretch of DNA that alters gene expression by binding transcription factors.
- Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene
Silencer
Silencer
Site where negative regulators (repressors) bind.
- whose expression it regulates.
Eukaryotes RNA polymerases
Eukaryotes RNA polymerases
RNA polymerase I makes rRNA (most numerous RNA, rampant).
RNA polymerase II makes mRNA (largest RNA, massive).
RNA polymerase III makes tR  A (smallest RNA, tiny).
No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promotor site
- I, II, and III are numbered as their products are used in protein synthesis.
- alpha-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes severe hepatotoxicity ifingested.
Prokaryotes Polymerases
Prokaryotes Polymerases
l RNA polymerase (multisubunit complex) makes all 3 kinds of RNA.
RNA processing (eukaryotes)
RNA processing (eukaryotes)
Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA destined for translation is called pre-mRA.
- Only processed RNA is transported out of the nucleus.
Processing occurs in nucleus. After transcription:
- Capping on 5’ end (addition of 7-methylguanosine cap)
- Polyadenylation on 3’ end (“” 200 1\s)
- Poly-A polymerase does not require a template.
- AAUAAA =polyadenylation signal.
- Splicing out of introns
- Capped, tailed, and spliced transcript is called mRNA.
Splicing of pre-mRNA
Splicing of pre-mRNA
1) Primary transcript combines with snRNPs and other proteins to form spliceosome.
2) Lariat-shaped (looped) intermediate is generated.
3) Lariat is released to remove intron precisely and join 2 exons.
Patients with lupus make antibodies to spliceosomal snRNPs.
lntrons vs. exons
lntrons vs. exons
Exons contain the actual genetic information coding for protein.
- Different exons can be combined by alternative splicing to make unique proteins in different tissues (e.g., beta-thalassemia mutations).
lntrons are intervening noncoding segments of DNA.
learning aid: lntrons are intervening sequences and stay in the nucleus, whereas exons exit and are expressed.
tRNA Structure
tRNA Structure
75-90 nucleotides, secondary structure, cloverleafform, anticodon end is opposite 3’ aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3’ end along with a high percentage of chemically modified bases. The amino acid is covalently bound to the 3’ end of the tRNA.
Memory aid: CCA: Can Carry Amino acids.
tRNA Charging
tRNA Charging
Aminoacyl-tRNA synthetase (1 per amino
acid, “matchmaker,” uses ATP) scrutinizes amino acid before and after it binds to tRNA. If incorrect, bond is hydrolyzed. The amino acid-tRNA bond has energy for formation of peptide bond. A mischarged tRNA reads usual codon but inserts wrong amino acid.
Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of amino acid selection.
Tetracyclines bind 30S subunit, preventing attachment of aminoacyl-tRNA.
tRNA wobble
tRNA wobble
Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codons differing in the 3rd “wobble” position may code for the same tRNA/amino acid (as a result of degeneracy of genetic code).
Initiation of protein synthesis
Initiation of protein synthesis
Activated by GTP hydrolysis, initiation factors (eukaryotic IFs) help assemble the 40S ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal subunit assemble with the complex.
Eukaryotes: 40S + 60S -+ 80S (Even).
PrOkaryotes: 30S + 50S – 70S (Odd).
ATP-tRNA Activation (charging).
- *G**TP-tRNA Gripping and Going places
(translocation) .
Elongation
Elongation
1) Aminoacyl-tRNA binds to A site (except for initiator methionine)
2) Ribosomal rRNA (“ribozyme”) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site.
3) Ribosome advances 3 nucleotides toward 3’ end of mRNA, moving peptidyl tRNA to P site (translocation)
Learning aid: Think of “going APE”:
A site = incoming Aminoacyl-tRNA.
P site = accommodates growing Peptide.
E site = holds Empty tRNA as it Exits.
Termination
Termination
Stop codon is recognized by release factor, and completed protein is released from ribosome.
Antibiotics that act as protein synthesis inhibitors:
Antibiotics that act as protein synthesis inhibitors:
• Aminoglycosides bind 30S and inhibit
formation of initiation complex and cause
misreading of mRNA
- Tetracyclines bind 30S and block aminoacyl tRNA from entering the acceptor site
- Chloramphenicol binds 50S and inhibits peptidyl transferase
- Macroslides bind 50S and prevent release of uncharged tRNA after it has donated its amino acid
Learning aid: The exam you took to apply to med school Macroslides Choramphenicol Aminoglycosides Tetracycline
Trimming (Posttranslational modifications)
Trimming (Posttranslational modifications)
Removal of N- or C-terminal propeptides from zymogens to generate mature proteins.
Covalent alterations (Posttranslational modifications)
Covalent alterations (Posttranslational modifications)
Phosphorylation, glycosylation, hydroxylation, methylation, and acetylation.
Learning aid: please give him my address!
Proteasomal degradation
(Posttranslational modifications)
Proteasomal degradation (Posttranslational modifications)
Attachment of ubiquitin to defective proteins to tag them for breakdown.
Cell cycle phases
Cell cycle phases
Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins, CDKs, and tumor suppressors. Mitosis (shortest phase): prophase-metaphase-anaphase-telophase. G1 and G0 are of variable duration.
CDKs
CDKs
Cyclin-dependent kinases; constitutive and inactive.
Cyclins
Cyclins
Regulatory proteins that control cell cycle
events; phase specific; activate CDKs.
Tumor suppressors (cell cycle)
Tumor suppressors (cell cycle)
p53 and hypophosphorylated Rb normally inhibit G1-to-S progression; mutations in these genes result in unrestrained cell division.
Permanent (cell type)
Permanent (cell type)
Remain in G0, regenerate from stem cells.
- Neurons, skeletal and cardiac muscle, RBCs.
Stable (quiescent) cell type
Stable (quiescent) cell type
Enter G1 from G0 when stimulated.
- Hepatocytes, lymphocytes.
Labile (cell type)
Labile (cell type)
Never go to G0, divide rapidly with a short G1.
- Bone marrow, gut epithelium, skin, hair follicles, germ cells.
Rough endoplasmic reticulum
Rough endoplasmic reticulum
- Site ofsynthesis ofsecretory (exported) proteins and of N-linked oligosaccharide addition to many proteins.
- Mucus-secreting goblet cells ofthe small intestine and antibody-secreting plasma cells are rich in RER.
- Nissl bodies (RER in neurons)- synthesize enzymes (e.g., ChAT [choline acetyltransferase] makes ACh) and peptide neurotransmitters.
- Free ribosomes-unattached to any membrane; site of synthesis of cytosolic and organellar proteins.
Smooth endoplasmic reticulum
Smooth endoplasmic reticulum
Site of steroid synthesis and detoxification of drugs and poisons.
- Liver hepatocytes and steroid hormone producing cells of the adrenal cortex are rich in SER.
Cell trafficking
Cell trafficking
Golgi is the distribution center for proteins and lipids from the ER to the vesicles and plasma membrane.
- It modifies N-oligosaccharides on asparagine.
- It adds O-oligosaccharides on serine and threonine.
-
It adds mannose-6-phosphate to proteins for trafficking to lysosomes.
- 1-cell disease (inclusion cell disease)-inherited lysosomal storage disorder; failure of addition of mannose-6-phosphate to lysosome proteins (enzymes are secreted outside the cell instead of being targeted to the lysosome). Results in coarse facial features, clouded corneas, restricted joint movement, and high plasma levels of lysosomal enzymes. Often fatal in childhood.
Endosomes are sorting centers for material from outside the cell or from the Golgi, sending it to lysosomes for destruction or back to the membrane/Golgi for further use.
Vesicular trafficking proteins
- COPI: Golgi -> Golgi (retrograde); Golgi -> ER.
- COPII: Golgi -> Golgi (anterograde); ER -> Golgi.
- Clathrin: trans-Golgi -> lysosomes; plasma membrane-> endosomes (receptor-mediated endocytosis.
Peroxisome
Peroxisome
Membrane-enclosed organelle involved in catabolism of very long fatty acids and amino acids.
- X-linked Adrenoleukodystrophy occurs when defective integral membrane protein responsible for transporting very long-chain fatty acids into peroxisomes for beta-oxidation. Accumulation of these long chain fatty acids in body fluids destroys the myelin sheath in nervous systems. Gene mutated: ABCD1
Proteasome
Proteasome
Barrel-shaped protein complex that degrades damaged or unnecessary proteins tagged for destruction with ubiquitin.
Microtubule
Microtubule
- Cylindrical structure composed of a helical array of polymerized dimers of alpha- and beta-tubulin.
- Each dimer has 2 GTP bound.
- Incorporated into flagella, cilia, mitotic spindles.
- Grows slowly, collapses quickly.
- Also involved in slow axoplasmic transport in neurons.
Microtubule molecular motor proteins
Microtubule molecular motor proteins
Molecular motor proteins is the transport cellular cargo toward opposite ends of microtubule tracks.
- Dynein= retrograde to microtubule (+ to -)
- Kinesin =anterograde to microtubule (- to +)
Drugs that act on microtubules:
- Mebenclazole/thiabenclazole (antihelminthic)
- Griseofulvin (antifungal)
- Vincristine/vinblastine (anti-cancer)
- Paclitaxel (anti-breast cancer)
- Colchicine (anti-gout)
Memory aid: Microtubule Growth Voiding Poisonous Chemicals!
Chediak-Higashi syndrome
Chediak-Higashi syndrome
Chediak-Higashi syndrome is mutation in the lysosomal trafficking regulator gene (LYST), whose product is required for the microtubule dependent sorting of endosomal proteins into late multivesicular endosomes. Results in recurrent pyogenic (pus) infections, partial albinism, and peripheral neuropathy (causing numbness or weakness).
Cilia structure
Cilia structure
9 + 2 arrangement of microtubules.
Axonemal dynein-ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets.
- Kartagener’s syndrome (primary ciliary dyskinesia) - immotile cilia clue to a dynein arm defect. Results in male infertility (immotile sperm) and decreased female fertility, bronchiectasis (abnormal widening of the bronchi or its branches leading to increased risk of respiratory infection), and recurrent sinusitis (bacteria and particles not pushed out); associated with situs inversus.
Cytoskeletal elements
Cytoskeletal elements
Actin and myosin: Microvilli, muscle contraction, cytokinesis, aclherens junctions.
Microtubule
Microtubule
For movement. Cilia, flagella, mitotic spindle, axonal trafficking, centrioles.
Intermediate filaments
Intermediate filaments
Structure. Vimentin, clesmin, cytokeratin, lamins, glial fibrillary acid proteins (GFAP), neurofilaments.
Plasma membrane composition
Plasma membrane composition
Asymmetric lipid bilayer.
Contains cholesterol, phospholipids, sphingolipicls, glycolipicls, and proteins.
Vimentin Stain
Vimentin Stain
Vimentin stain is an Immunohistochemical which stains for intermediate filaments
Vimentin stains for Connective tissue specifically
Desmin Stain
Desmin stain
Desmin stain is an Immunohistochemical which stains for intermediate filaments
Desmin stains for Muscle specifically
Cytokeratin Stain
Cytokeratin Stain
Cytokeratin stain is an Immunohistochemical which stains for intermediate filaments
Cytokeratin stains for Epithelial cells specifically
GFAP Stain
GFAP Stain
Neuroglia is an Immunohistochemical which stains for intermediate filaments
GFAP stains for NeuroGlia specifically
Neurofilaments Stain
Neurofilaments Stain
Neurofilaments is an immunohistochemical stain for intermediate filaments
Neurofilaments stains for neurons specially
sodium-potassium pump
sodium-potassium pump
Na+-K+ ATPase is located in the plasma membrane with ATP site on cytosolic side. For each ATP consumed, 3 Na+ go out and 2 K+ come in. During cycle, pump is phosphorylated.
Ouabain inhibits by binding to K+ site. Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+ ATPase, this leads to indirect inhibition of Na+/ Ca2+ exchange, which leads to increased [Ca2+]i -which leads to increased cardiac contractility.
Collagen
Collagen
Most abundant protein in the human body. Extensively modified by posttranslational modification.
Organizes and strengthens extracellular matrix.
learning aid: Be (So Totally) Cool, Read Books.
type l: Most common (90%)-Bone, Skin, Tendon, dentin, fascia, cornea, late wound repair.
- Type I: bone. Defective in osteogenesis imperfecta.
type ll: Cartilage (including hyaline), vitreous body, nucleus pulposus.
- memory aid: cartwolage
type lll: Reticulin-skin, blood vessels, uterus, fetal tissue, granulation tissue.
- Type III: defective in Ehlers-Danlos (ThreE D).
**Type IV: **Basement membrane or basal lamina.
- Type IV: under the floor (basement membrane). Defective in Alport syndrome.
collagen synthesis inside fibroblasts/ outside fibroblasts
collagen synthesis inside fibroblasts
1) Synthesis (RER): Translation of collagen a chains (preprocollagen) -usually Gly-X-Y (X and Y are proline or lysine).
2) Hydroxylation (ER): Hydroxylation ofspecific proline and lysine residues (requires vitamin C; deficiency leads to scurvy).
3) Glycosylation (ER): Glycosylation of pro-alpha-chain hyclroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen a chains). Problems forming triple helix leads to osteogenesis imperfecta.
4) Exocytosis: Exocytosis of procollagen into extracellular space.
collagen synthesis outside fibroblasts
5) Proteolytic processing: Cleavage of disulfide-rich terminal regions of procollagen, transforming it into insoluble tropocollagen.
6) Cross-linking: Reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysine cross-linkage (by Cu2+-containing lysyl oxidase) to make collagen fibrils. Problems with cross-linking leads to Ehlers-Danlos.
Osteogenesis imperfecta
Osteogenesis imperfecta- (collagen disease)
Genetic bone disorder (brittle bone disease) caused by a variety of gene defects.
Most common form is autosomal dominant with abnormal type I collagen (Most common (90%)-Bone, Skin, Tendon, dentin, fascia, cornea, late wound repair.), causing:
- Multiple fractures with minimal trauma;
- may occur during the birth process
- Blue sclera due to the translucency of the connective tissue over the choroidal veins
- Hearing loss (abnormal middle ear bones)
- Dental imperfections clue to lack of dentin
May be confused with child abuse. Incidence is 1 : 10,000.
Ehlers-Danlos syndrome
Ehlers-Danlos syndrome- (collagen disease)
Faulty collagen synthesis causing hyperextensible skin, tendency to bleed (easy bruising), and hypermobile joints.
There are 6 types.
- Type I (Be So Totally- Bone, Skin, Tendon, dentin, fascia, cornea, late wound repair.) or Type V collagen most frequently affected in severe classic Ehlers-Danlos syndrome.
Inheritance and severity vary. Can be autosomal dominant or recessive.
May be associated with joint dislocation, berry aneurysms, organ rupture.
Alport syndrome
Alport syndrome- (collagen disease)
Due to a variety of gene defects resulting in abnormal type IV collagen. Most common form is X-Iinked recessive.
- Type IV collagen is an important structural component of the basement membrane of the kidney, ears, and eyes.
- memory aid: Be (so totally) cool, read Books (basement membrane))
Characterized by progressive hereditary nephritis and deafness. May be associated with ocular disturbances.
Elastin
Elastin (collagen disease)
Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, ligamenta flava (connect vertebrae-+ relaxed and stretched conformations).
Rich in proline and glycine, nonhydroxylatecl forms.
Tropoelastin with fibrillin scaffolding.
- Madan’s syndrome-caused by a defect in fibrillin.
Cross-linking takes place extracellularly and gives elastin its elastic properties.
- Wrinkles of aging are clue to reduced collagen and elastin production.
Broken down by elastase, which is normally inhibited by alpha1-antitrypsin.
- Emphysema-can be caused by a1-antitrypsin deficiency, resulting in excess elastase activity.
Madan’s syndrome
Madan’s syndrome
Madan’s syndrome- caused by a defect in fibrillin.
Emphysema
Emphysema
Emphysema-can be caused by alpha 1-antitrypsin deficiency, resulting in excess elastase activity.
Polymerase chain reaction
Polymerase chain reaction
Molecular biology laboratory procedure used to amplify a desired fragment of DNA. Steps:
- Denaturation-DNA is denatured by heating to generate 2 separate strands
- Annealing-during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified.
- Elongation-heat-stable DNA polymerase replicates the DNA sequence following each primer.
These steps are repeated multiple times for DNA sequence amplification.
Agarose gel electrophoresis- used for size separation of PCR products (smaller molecules travel further); compared against DNA ladder.
Southern blot
Southern blot
A DNA sample is electrophoresed on a gel and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary strand. The resulting double-stranded, labeled piece of DNA is visualized when the filter is exposed to film.
learning aid: SNoW DRoP
Southern = DNA
Northern = RNA
Western = Protein
Northern blot
Northern blot
Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying mRNA levels.
note: I was taught that this method is used to identify tissue types.
learning aid: SNoW DRoP
Southern = DNA
Northern = RNA
Western = Protein
Western blot
Western blot
Sample protein is separated via gel electrophoresis and transferred to a filter. Labeled antibody is used to bind to relevant protein.
learning aid: SNoW DRoP
Southern = DNA
Northern = RNA
Western = Protein
Southwestern blot
Southwestern blot
Identifies DNA-binding proteins (e.g., transcription factors) using labeled oligonucleotide probes.
Microarrays
Microarrays
Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding.
Microarrays are used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments. Microarrays are able to detect single nucleotide polymorphisms (SNPs) for a variety of applications including genotyping, forensic analysis, predisposition to disease, cancer mutations, and genetic linkage analysis.
Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA)
A rapid immunologic technique testing for antigen-antibody reactivity.
Patient’s blood sample is probed with either:
l. Indirect ELISA: uses a test antigen to see if a specific antibody is present in the patient’s blood; a secondary antibody coupled to a color-generating enzyme is added to detect the first antibody; or
2. Direct ELISA: uses a test antibody coupled to a color-generating enzyme to see if a specific antigen is present in the patient’s blood.
If the target substance is present in the sample, the test solution will have an intense color reaction, indicating a positive test result.
Enzyme-linked immunosorbent assay (ELISA) is used in many laboratories to determine whether a particular antibody (e.g., anti-HIV) is present in a patient’s blood sample. Both the sensitivity and the specificity of ELISA approach 100%, but both false-positive and false-negative results do occur.
Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH)
Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes.
Used for specific localization of genes and direct visualization of anomalies (e.g., microdeletions) at molecular level (when deletion is too small to be visualized by karyotype).
Fluorescence = gene is present; no fluorescence =gene has been deleted.
Cloning methods
Cloning methods
Cloning is the production of a recombinant DNA molecule that is self-perpetuating.
Steps:
l. Isolate eukaryotic mRNA (post-RNA processing steps) of interest.
2. Expose mRNA to reverse transcriptase to produce cDNA.
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
4. Surviving bacteria on antibiotic medium produce cDNA library.
Gene expression modifications
Gene expression modifications
Transgenic strategies in mice involve:
- Random insertion ofgene into mouse genome
- Targeted insertion or deletion of gene through homologous recombination with mouse gene
Cre-lox system- Can inducibly manipulate genes at specific developmental points using an antibiotic-controlled promoter (e.g., to study a gene whose deletion causes embryonic death).
RNA interference (RNAi)- dsRNA is synthesized that is complementary to the mRNA sequence of interest. When transfected into human cells, dsRNA separates and promotes degradation of target mRNA, knocking down gene expression.
Karyotyping
Karyotyping
A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern. Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue. Used to diagnose chromosomal imbalances (e.g., autosomal trisomies, sex chromosome disorders).
Lesch-Nyhan syndrome
Lesch-Nyhan syndrome
Defective purine salvage due to absent HGPRT, which converts hypoxanthine to IMP and guanine to GMP.
Results in excess uric acid production and de novo purine synthesis. X-linked recessive.
Findings: intellectual disability, self-mutilation, aggression, hyperuricemia, gout, dystonia.
Treatment: allopurinol or febuxostat (2nd line).
learning aid: He’s Got Purine Recovery Trouble.
HGPRT:
- Hyperuricemia
- Gout
- Pissed off (aggression, self-mutilation)
- Retardation (intellectual disability)
- DysTonia (neuroloical movement disorder which cause sustained muscle contraction causing twisting and abnormal poster).
Adenosine deaminase deficiency
Adenosine deaminase deficiency
Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide reductase prevents DNA synthesis and thus decreases lymphocyte count.
One of the major causes of autosomal recessive
SCID.
Severe Combined Immunodeficiency Disease (SCID) happens to kids.
1st disease to be treated by experimental human gene therapy.
Codominance
Codominance
Both alleles contribute to the phenotype of the heterozygote.
example: Blood groups A, B, AB; α1-antitrypsin deficiency.
Variable expressivity
Variable expressivity
Phenotype varies among individuals with same genotype.
Example: 2 patients with neurofibromatosis type 1 (NF1) may have varying disease severity.
Incomplete penetrance
Incomplete penetrance
Not all individuals with a mutant genotype show the mutant phenotype.
Example: BRCA1 gene mutations do not always result in breast or ovarian cancer.
Pleiotropy
Pleiotropy
One gene contributes to multiple phenotypic effects.
Example: Untreated phenylketonuria (PKU) manifests with light skin, intellectual disability, and musty body odor.
Anticipation
Anticipation
Increased severity or earlier onset of disease in succeeding generations.
Example: Trinucleotide repeat diseases (e.g., Huntington disease).
Loss of heterozygosity
Loss of heterozygosity
If a patient inherits or develops a mutation in a tumor suppressor gene, the complementary allele must be deleted/mutated before cancer develops. This is not true of oncogenes.
Retinoblastoma and the “two-hit hypothesis.”
Dominant negative mutation
Dominant negative mutation
Exerts a dominant effect. A heterozygote produces a nonfunctional altered protein that also prevents the normal gene product from functioning.
Example: Mutation of a transcription factor in its allosteric site. Nonfunctioning mutant can still bind DNA, preventing wild-type transcription factor from binding.
Linkage disequilibrium
Linkage disequilibrium
Tendency for certain alleles at 2 linked loci to occur together more often than expected by chance. Measured in a population, not in a family, and often varies in different populations.
Mosaicism
Mosaicism
Presence of genetically distinct cell lines in the same individual. Arises from mitotic errors after fertilization.
Somatic mosaicism—mutation propagates through multiple tissues or organs.
Gonadal mosaicism—mutation only in egg or sperm cells.
Example: McCune-Albright syndrome is lethal if the mutation is somatic, but survivable if mosaic.
Locus heterogeneity
Locus heterogeneity
Mutations at different loci can produce a similar phenotype.
Example: Albinism.
Allelic heterogeneity
Allelic heterogeneity
Different mutations in the same locus produce the same phenotype.
Example: ß-thalassemia.
Heteroplasmy
Heteroplasmy
Presence of both normal and mutated mtDNA, resulting in variable expression in mitochondrial inherited disease.
Uniparental disomy
Uniparental disomy
Offspring receives 2 copies of a chromosome from 1 parent and no copies from the other parent.
Heterodisomy (heterozygous) indicates a meiosis I error.
Isodisomy (homozygous) indicates a meiosis II error or postzygotic chromosomal duplication of one of a pair of chromosomes, and loss of the other of the original pair.
Example: Uniparental is eUploid (correct number of chromosomes), not aneuploid. Most occurrences of UPD lead to normal phenotype. Consider UPD in an individual manifesting a recessive disorder when only one parent is a carrier.
Hardy-Weinberg population genetics
Hardy-Weinberg population genetics
If a population is in Hardy-Weinberg equilibrium and if p and q are the frequencies of separate alleles, then: p2 + 2pq + q2 = 1 and p + q = 1, which implies that:
p2 = frequency of homozygosity for allele p q2 = frequency of homozygosity for allele q 2pq = frequency of heterozygosity (carrier frequency, if an autosomal recessive disease). The frequency of an X-linked recessive disease in males = q and in females = q2.
Example: Hardy-Weinberg law assumptions include:
- No mutation occurring at the locus
- Natural selection is not occurring
- Completely random mating
- No net migration
Imprinting
Imprinting
At some loci, only one allele is active; the other is inactive (imprinted/inactivated by methylation). With one allele inactivated, deletion of the active allele leads to disease.
Example: Both Prader-Willi and Angelman syndromes are due to mutation or deletion of genes on chromosome 15.
Can also occur as a result of uniparental disomy.
Prader-Willi syndrome
Prader-Willi syndrome
Maternal imprinting: gene from mom is normally silent and Paternal gene is deleted/ mutated. Results in hyperphagia (excessive hunger), obesity, intellectual disability, hypogonadism, and hypotonia.
example: 25% of cases due to maternal uniparental disomy (two maternally imprinted genes are received; no paternal gene received).
Angelman syndrome
AngelMan syndrome
Paternal imprinting: gene from dad is normally silent and Maternal gene is deleted/mutated. Results in inappropriate laughter (“happy puppet”), seizures, ataxia, and severe intellectual disability.
Autosomal dominant
Autosomal dominant
Often due to defects in structural genes. Many generations, both male and female, affected.
Often pleiotropic (one gene effecting multiple seemingly unrelated phenotypes). Family history crucial to diagnosis.
Legend:
purple= uninfected
pink= infected
Autosomal recessive
Autosomal recessive
25% of offspring from 2 carrier parents are affected. Often due to enzyme deficiencies. Usually seen in only 1 generation.
Commonly more severe than dominant disorders; patients often present in childhood. Increased risk in consanguineous families.
X-linked recessive
X-linked recessive
Sons of heterozygous mothers have a 50% chance of being affected. No male-to-male transmission.
Commonly more severe in males. Females usually must be homozygous to be affected.
X-linked dominant
X-linked dominant
Transmitted through both parents. Mothers transmit to 50% of daughters and sons; fathers transmit to all daughters but no sons.
Hypophosphatemic rickets—formerly known as vitamin D–resistant rickets. Inherited disorder resulting inphosphate wasting at proximal tubule. Results in rickets-like presentation.
Mitochondrial inheritance
Mitochondrial inheritance
Transmitted only through the mother. All offspring of affected females may show signs of disease.
Variable expression in a population or even within a family due to heteroplasmy.
Mitochondrial myopathies—rare disorders; often present with myopathy, lactic acidosis and CNS disease. 2° to failure in oxidative phosphorylation. Muscle biopsy often shows “ragged red fibers.”
Autosomal dominant polycystic kidney disease (ADPKD)
Autosomal dominant polycystic kidney disease (ADPKD)
Formerly known as adult polycystic kidney disease. Always bilateral, massive enlargement of kidneys due to multiple large cysts. 85% of cases are due to mutation in PKD1 (chromosome 16; 16 letters in “polycystic kidney”); remainder due to mutation in PKD2 (chromosome 4).
Autosomal dominant diseases
Familial adenomatous polyposis
Familial adenomatous polyposis
Colon becomes covered with adenomatous polyps after puberty. Progresses to colon cancer unless colon is resected. Mutations on chromosome 5 (APC gene); 5 letters in “polyp.”
Autosomal dominant diseases
Familial hypercholesterolemia
Familial hypercholesterolemia
Elevated LDL due to defective or absent LDL receptor. Leads to severe atherosclerotic disease early in life, and tendon xanthomas (classically in the Achilles tendon).
Autosomal dominant diseases
Hereditary hemorrhagic telangiectasia
Hereditary hemorrhagic telangiectasia
Inherited disorder of blood vessels. Findings: telangiectasia, recurrent epistaxis, skin discolorations, arteriovenous malformations (AVMs), GI bleeding, hematuria. Also known as Osler-Weber-Rendu syndrome.
Autosomal dominant diseases
Hereditary spherocytosis
Hereditary spherocytosis
Spheroid erythrocytes due to spectrin or ankyrin defect; hemolytic anemia; increased MCHC.
Treatment: splenectomy.
Autosomal dominant diseases
Huntington disease
Huntington disease
Findings: depression, progressive dementia, choreiform movements, caudate atrophy, andlevels of GABA and ACh in the brain. Gene on chromosome 4; trinucleotide repeat disorder: (CAG)n increased repeats lead to lower age of onset.
Autosomal dominant diseases
Memory aid: HUNTING 4 food
Marfan syndrome
Marfan syndrome
Fibrillin-1 gene mutation lead to connective tissue disorder affecting skeleton, heart, and eyes. Findings: tall with long extremities, pectus excavatum, hypermobile joints, and long, tapering fingers and toes (arachnodactyly); cystic medial necrosis of aortaaortic incompetence and dissecting lead to aortic aneurysms; floppy mitral valve. Subluxation of lenses, typically upward and temporally.
Autosomal dominant diseases
Multiple endocrine neoplasias (MEN)
Multiple endocrine neoplasias (MEN)
Several distinct syndromes (1, 2A, 2B) characterized by familial tumors of endocrine glands, including those of the pancreas, parathyroid, pituitary, thyroid, and adrenal medulla. MEN 2A and 2B are associated with ret gene.
Autosomal dominant diseases
Neurofibromatosis type 1 (von Recklinghausen disease)
Neurofibromatosis type 1 (von Recklinghausen disease)
Neurocutaneous disorder characterized by café-au-lait spots and cutaneous neurofibromas. Autosomal dominant, 100% penetrance, variable expression. Caused by mutations in the NF1 gene on chromosome 17; 17 letters in “von Recklinghausen.”
Autosomal dominant diseases
Neurofibromatosis
type 2
Neurofibromatosis
type 2
Findings: bilateral acoustic schwannomas, juvenile cataracts, meningiomas, and ependymomas. NF2 gene on chromosome 22; type 2 = 22.
Autosomal dominant diseases
Tuberous sclerosis
Tuberous sclerosis
Tuberous sclerosis: Neurocutaneous disorder with multi-organ system involvement, characterized by numerous benign hamartomas. Incomplete penetrance, variable expression.
Autosomal dominant diseases
von Hippel-Lindau disease
von Hippel-Lindau disease
Disorder characterized by development of numerous tumors, both benign and malignant. Associated with deletion of VHL gene (tumor suppressor) on chromosome 3 (3p). Von Hippel-Lindau = 3 words for chromosome 3.

Autosomal dominant diseases
