Biochemistry - Molecular, Cellular, Techniques

Double strand DNA repair - Brings together 2 ends of DNA fragments to replair double-stranded breaks. - No requirement for homology. - Some DNA may be lost. Defective in ataxia telangiectasia and Fanconi anemia.

Double strand DNA repair - Requires two homologous DNA duplexes.-  A strand from the damaged dsDNA is repaired using a complementary strand from the intact homologous dsDNA as a template. - Restores duplexes accurately without loss of nucleotides. Defective in breast/ovarian cancers with BRCA1 mutation.

Promoter: Site where RNA polymerase II and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence with TATA and CAAT boxes).- Promotor mutation commonly results in dramatic ↓ in level of gene transcription. Enhancer: DNA locus where regulatory proteins ("activators") bind → increasing expression of a gene on the same chromosome. Silencer: DNA locus where regulatory proteins ("repressors") bind → decreasing expression of a gene on the same chromosome.- Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene whose expression it regulates.

Exons contain the actual genetic information coding for protein.Introns are intervening noncoding segments of DNA. Different exons are frequently combined by alternative splicing to produce a larger number of unique proteins.Alternative splicing can produce a variety of protein products from a single hnRNA sequence (eg, transmembrane vs secreted Ig, tropomyosin variants in muscle, dopamine receptors in the brain). Variants in which splicing occurs abnormally are implicated in oncogenesis and many genetic disorders (eg, β-thalassemia, Gaucher disease, Tay-Sachs disease, Marfan syndrome). Introns stay in the nucleus. Exons exit and are expressed.

MicroRNAs (miRNAs) are small, conserved, noncoding RNA molecules that posttranscriptionally regulate gene expression by targeting the 3' untranslated region of specific mRNAs for degradation or translational repression. Abnormal expression of miRNAs contributes to certain malignancies (eg, by silencing an mRNA from a tumor suppressor gene).

75-90 nucleotides, 2° structure, cloverleaf form, 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.CCA Can Carry Amino acids. T-arm: contains the TψC (ribothymidine, pseudouridine, cytidine) sequence necessary for tRNA-ribosome binding. T-arm tethers tRNA molecule to ribosome.D-arm: contains dihydrouridine residues necessary for tRNA recognition by the correct aminoacyl-tRNA synthetase. D-arm detects the tRNA by aminoacyl-tRNA synthetase.Acceptor stem: the 5-CCA-3' is the amino acid receptor site. Aminoacyl-tRNA synthetase (1 per amino acid; "matchmaker", uses ATP) scrutinizes amino acid before and after it binds to tRNA. If incorrect, bond is hydrolzed. 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.

Trimming: Removal of N- or C-terminal propeptides from zymogen to generate mature protein (eg, trypsinogen to trypsin). Covalent alterations: Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.

Intracellular protein involved in facilitating and/or maintaining protein folding.  Eg, in yeast, heat shock proteins (eg, HSP60) are expressed at high temperatures to prevent protein denaturing/misfolding.

Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins, cyclin-dependent kinases (CDKs), and tumor suppressors. M phase (shortest phase of cell cycle) includes mitosis (prophase, prometaphase, metaphase, anaphase, telophase) and cytokinesis (cytoplasm splits in two). G1 and G0 are of variable duration. Cyclin-dependent kinases: Constitutive and inactive.Cyclins: Regulatory proteins that control cell cycle events; phase specific; activate CDKs.Cyclin-CDK complexes: Phosphorylate other proteins to coordinate cell cycle progression; must be activated and inactivated at appropriate times for cell cycle to progress.Tumor suppressors: p53 induces p21, which inhibits CKDs → hypophosphorylation (activation) of Rb → inhibition of G1-S progression. Mutations in tumor suppressor genes can result in unrestrained cell division (eg, Li-Fraumeni syndrome). Growth factors (eg, insulin, PDGF, EPO, EGF) bind tyrosine kinase receptors to transition the cell from G1 to S phase.

Permanent: Remian in G0, regenerate from stem cells.- Neurons, skeletal and cardiac muscle, RBCs. Stable (quiescent): Enter G1 from G0 when stimulated.- Hepatocytes, lymphocytes, PCT, periosteal cells. Labile: Never go to G0, divide rapidly with a short G1. Most affected by chemotherapy.- Bone marrow, gut epithelium, skin, hair follicles, germ cells.

Site of synthesis of secretory (exported) proteins and of N-linked oligosaccharide addition to many proteins. Nissl bodies (RER in neurons) – synthesize peptide neurotransmitters for secretion.Free ribosomes – unattached to any membrane; site of synthesis of cytosolic and organellar proteins. Mucus-secreting goblet cells of the small intestine and antibody-secreting plasma cells are rich in RER.

Site of steroid synthesis and detoxification of drugs and poisons. Lacks surface ribosomes. Liver hepatocytes and steroid hormone-producing cells of the adrenal cortex and gonads are rich in SER.

Golgi is the distribution center for proteins and lipids from the ER to the vesicles and plasma membrane. - Modifies N-oligosaccharides on aspargine. - Adds O-oligosaccharides on serine and threonine. - Adds mannose-6-phosphate to proteins for trafficking to lysosomes. 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.

Abundant, cytosolic ribonucleoprotein that traffics proteins from the ribosome to the RER. Absent or dysfunctional SRP → proteins accumulate in the cytosol.

COPII: ER → cis-Golgi (anterograde). COPI: Golgi → Golgi (retrograde); cis-Golgi → ER Clathrin: trans-Golgi → lysosomes; plasma membrane → endosomes (receptor-mediated endocytosis [eg, LDL receptor activity]).

Barrel-shaped protein complex that degrades damaged or ubiquitin-tagged proteins. Defects in ubiquitin-proteasome system have been implicated in some cases of Parkinson disease.

Cylindrical outer structure composed of a helical array of polymerized heterodimers of α- and β-tubulin. - Each dimer hase 2 GTP bound. - Incorporated into flagella, cilia, mitotic spindles.- Also involved in slow axoplasmic transport in neurons. - Grows slowly, collapses quickly. Molecular motor proteins – transport cellular cargo towards opposite ends of microtubule tracks.- Dynein – retrograde to microtubule (+ → -)- Kinesin – anterograde to microtubule (- → +) Drugs that act on microtubles: Metronidazole, griseofulvin, vincristine/vinblastine, paclitaxel, colchicine.

9 doublet + 2 singlet arrangement of microtubules. Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets with no central microtubules. Axonemal dynein – ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets. Gap junctions enable coordinated ciliary movement.

Scurvy: Deficient hydroxylation secondary to ascorbate deficiency.- Petechiae, ecchymoses, loose teeth, bleeding gums, poor wound healing, poor bone development Osteogenesis imperfecta: Mutations in collagen genes, problems in forming triple-helix- Skeletal deformities, fractures, blue sclera Ehlers-Danlos syndromes: Mutations in collagen genes and proline and lysyl hydroxylases (cross-linking)- Hyperextensible, fragile skin, hypermobile joints, dislocations, varicose veins, ecchymoses, arterial, intestinal ruptures Menkes disease: Deficient cross-linking secondary to functional copper deficiency- Depigmented (steely) hair, arterial tortuosity, rupture, cerebral degeneration, osteoporosis, anemia

Autosomal-dominant connective tissue disorder affecting skeleton, heart, and eyes. FBN1 gene mutation on chromosome 15 results in defective fibrillin, a glycoprotein that forms a sheath around elastin.- Fibrillin-1 is abundant in blood vessels, periosteum, and the suspensory ligaments of the lens. Findings: - Tall with long extremities- Pectus carinatum (more specific) or excavatum- Hypermobile joints- Long, tapering fingers and toes (arachnodactyly)- Cystic medial necrosis of aorta- Aortic incompetence and dissecting aortic aneurysms; floppy mitral valve- Subluxation of lenses (typically upward and temporally)

Molecular biology lab procedure used to amplify a desired fragment of DNA.  1. Denaturation – DNA is heated to ~95°C to separate the strands.2. Annealing – Sample is cooled to ~55°C. DNA primers, a heat-stable DNA polymerase (Taq), and deoxynucleotide triphosphates (dNTPs) are added. DNA primers anneal to the specific sequene to be amplified on each strand.3. Elongation – Temperature is increased to ~72°C. DNA polymerase attaches dNTPs to the strand to replicate the sequence after each primer.

A genome editing tool, derived from bacteria. Composed of an endonuclease (Cas9, which cleaves dsDNA) and a guide RNA (gRNA) sequence that binds to a complementary target DNA sequence. Cell DNA repair machinery (nonhomologous end joining) fills in the gap introduced by the system (knock-out) or a donor DNA can be added to the system to fill the gap (knock-in). The gRNA can be designed to target any DNA sequence.

Southern blot:1. DNA sample is enzymatically cleaved into smaller pieces, which are separated on a gel by electrophoresis, and then transferred to a filter.2. Filter is exposed to radiolabeled DNA probe that recognizes and anneals to its complementary strand.3. Resulting double-stranded, labeled piece of DNA is visualized when filter is exposed to film. Northern blot: Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying mRNA levels, which are reflective of gene expression. Western blot: Sample protein is separated via gel electrophoresis and transferred to a membrane. Labeled antibody is used to bind to relevant protein. Southwestern blot: Identifies DNA-binding proteins (eg, transcription factors) using labeled oligonucleotide probes.

Laboratory technique to assess size, granularity, and protein expression (immunophenotype) of individual cells in a sample. Cells are tagged with antibodies specific to surface or intracellular proteins. Antibodies are then tagged with unique fluorescent dye. Sample is analyzed one cell at a time by focusing a laser on the cell and measuring light scatter and intensity of fluorescence. Commonly used in workup of hematologic abnormalities (eg, paroxysmal noctural hemoglobinuria, fetal RBCs in mother's blood) and immunodeficiencies (eg, CD4 cell count in HIV). Data are plotted either as histogram (one measure) or scatter plot (any two measures).

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. Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments. Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) for a variety of applications including genotyping, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis.

Immunologic test used to detect the presence of either a specific antigen (eg, HBsAg) or antibody (eg, anti-HBs) in a patient's blood sample. Detection involves the use of an antibody linked to an enzyme. Added substrate reacts with enzyme, producing a detectable signal. Can have high sensitivity and specificity, but is less specific than Western blot. Direct ELISA tests for the antigen directly, while indirect ELISA tests for the antibody (thus indirectly testing for the antigen).

A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length raio, and banding pattern. Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue. Used to diagnose chromosomal imbalances (eg, autosomal trisomies, sex chromosome disorders).

Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes. Used for specific localization of genes and direct visualization of chromosomal anomalies at the molecular level. - Microdeletion – no fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome.- Translocation – fluorescence signal that corresponds to one chromosome is found in a different chromosome- Duplication – a second copy of a chromosome, resulting in a trisomy or tetrasomy

Production of a recombinant DNA molecule in a bacterial host. 1. Isolate eukaryotic mRNA (post-RNA processing) of interest.2. Add reverse transcriptase (an RNA-dependent DNA polymerase) to produce complementary DNA (cDNA, lacks introns).3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.4. Transform (insert) plasmids into bacteria.5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA).

Transgenic strategies in mice involve:- Random insertion of gene into mouse genome. Random insertion = constitutive.- Targeted insertion or deletion of gene through homologous recombination with mouse gene. Targeted insertion = conditional. Cre-lox system – Can inducibly manipulate genes at specific developmental points (eg, to study a gene whose deletion causes embryonic death). RNA interference – 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.

Unambiguous: Each codon specifies only 1 amino acid. Degenerate/redundant: Most amino acids are coded by multiple codons.- Wobble – codons that differ in 3rd, "wobble" position may code for the same tRNA/amino acid. Specific base pairing is usually required only in the first 2 nucleotide positions of mRNA.- Exceptions: methionine (AUG) and tryptophan (UGG) encoded by only 1 codon. Commaless, nonoverlapping: Read from a fixed starting point as a continuous sequence of bases.- Exception: some viruses Universal: Genetic code is conserved throughout evolution- Exception in humans: mitochondria

Classic example of a genetic response to an environmental change. Glucose is the preferred metabolic substrate in E coli, but when glucose is absent and lactose is available, the lac operon is activated to switch to lactose metabolism. Mechanism of shift:- Low glucose → ↑ adenylate cyclase activity → ↑ generation of cAMP → activation of catabolite activator protein (CAP) → ↑ transcription of LacZ, LacY, LacA- High lactose → unbinds repressor protein from repressor/operator site → ↑ transcription. Lac I repressor protein is the product of lacI gene that lays upstream of the lac operon and is constitutively expressed. If lactose is present, it will bind to the repressor protein. CAP binds to the promotor, which induces transcription by RNA polymerase. LacZ gene codes for β-galactosidase, which is responsible for the hydrolysis of lactose to glucose and galactose.LacY gene codes for permease, which allows lactose to enter the bacterium.

Disrupt pyrimidine synthesis Inhibit dihydrofolate reductase (↓ deoxythymidine monophosphate [dTMP]) in humans, bacteria, and protozoa, respectively