Biochemistry Past Paper FA 2023 PDF

32 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular ` BIOCHEMISTRY—MOLECULAR Chromatin structure DNA exists in the condensed, chromatin form to DNA has ⊝ charge from phosphate groups. fit into the nucleus. DNA loops twice around a Histones are large and have ⊕ charge from histone octamer to form a nucleosome (“beads lysine and arginine. on a string”). H1 binds to the nucleosome In mitosis, DNA condenses to form and to “linker DNA,” thereby stabilizing the chromosomes. DNA and histone synthesis chromatin fiber. occurs during S phase. Mitochondria have their own DNA, which is circular and does not utilize histones. DNA doub le-hel ix H1 histone (linker) DNA Nucleosome (H2A, H2B, H3, H4) 2 Euchromatin Supercoiled structure Heterochromatin Metaphase chromosome Heterochromatin Condensed, appears darker on EM (labeled H in A ; Nu, nucleolus). Sterically inaccessible, thus transcriptionally inactive. methylation, acetylation. Heterochromatin = highly condensed. Barr bodies (inactive X chromosomes) may be visible on the periphery of nucleus. Euchromatin Less condensed, appears lighter on EM (labeled E in A ). Transcriptionally active, sterically accessible. Eu = true, “truly transcribed.” Euchromatin is expressed. DNA methylation Changes the expression of a DNA segment without changing the sequence. Involved with aging, carcinogenesis, genomic imprinting, transposable element repression, and X chromosome inactivation (lyonization). DNA is methylated in imprinting. Methylation within gene promoter (CpG islands) typically represses (silences) gene transcription. CpG methylation makes DNA mute. Dysregulated DNA methylation is implicated in Fragile X syndrome. Histone methylation Usually causes reversible transcriptional suppression, but can also cause activation depending on location of methyl groups. Histone methylation mostly makes DNA mute. Lysine and arginine residues of histones can be methylated. Histone acetylation Removal of histone’s ⊕ charge relaxed DNA coiling transcription. Thyroid hormone synthesis is altered by acetylation of thyroid hormone receptor. Histone acetylation makes DNA active. Histone deacetylation Removal of acetyl groups tightened DNA coiling transcription. Histone deacetylation may be responsible for altered gene expression in Huntington disease. Histone deacetylation deactivates DNA. A E H Nu FAS1_2023_01-Biochem.indd 32 11/17/22 7:12 PM Biochemistry  BIOCHEMISTRY—Molecular Nucleotides Nucleoside = base + (deoxy)ribose (sugar). Nucleotide = base + (deoxy)ribose + phosphate; linked by 3′-5′ phosphodiester bond. Purines (A,G)—2 rings. Pyrimidines (C,U,T)—1 ring. Uracil found in RNA; thymine in DNA. Methylation of uracil makes thymine. Purine (A, G) Glycine N C C C N Carbamoyl phosphate Aspartate C N C C THF Amino acids necessary for purine synthesis (cats purr until they GAG): Glycine Aspartate Glutamine Pyrimidine (C, U, T) CO2 N 5′ end of incoming nucleotide bears the triphosphate (energy source for the bond). α-Phosphate is target of 3′ hydroxyl attack. Pure As Gold. CUT the pyramid. Thymine has a methyl. C-G bond (3 H bonds) stronger than A-T bond (2 H bonds). C-G content melting temperature of DNA. “C-G bonds are like Crazy Glue.” Deamination reactions: Cytosine uracil Adenine hypoxanthine Guanine xanthine 5-methylcytosine thymine Aspartate C THF C C N 33 SEC TION II N Glutamine FAS1_2023_01-Biochem.indd 33 11/17/22 7:12 PM 34 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular De novo pyrimidine and purine synthesis Various immunosuppressive, antineoplastic, and antibiotic drugs function by interfering with nucleotide synthesis: Pyrimidine base production (requires aspartate) Purine base production or reuse from salvage pathway (de novo requires aspartate, glycine, glutamine, and THF) Ribose 5-P Glutamine + CO₂ 2 ATP 2 ADP + Pi + Glutamate CPS2 (carbamoyl phosphate synthetase II) Carbamoyl phosphate PRPP (phosphoribosyl pyrophosphate) synthetase Aspartate Leflunomide PRPP Orotic acid Impaired in orotic aciduria UMP Ribo n reduucleot ctas ide e Hydroxyurea Dihydrofolate reductase MTX, TMP, pyrimethamine DHF AMP CTP dUMP Thymidylate synthase THF IMP UDP dUDP N5N10methylene THF 6-MP, MTX, azathioprine dTMP 5-FU Mycophenolate, ribavirin GMP Pyrimidine synthesis: Leflunomide: inhibits dihydroorotate dehydrogenase 5-fl orouracil (5-FU) and its prodrug capecitabine: form 5-F-dUMP, which inhibits thymidylate synthase ( dTMP) Purine synthesis: 6-mercaptopurine (6-MP) and its prodrug azathioprine: inhibit de novo purine synthesis (guanine phosphoribosyltransferase); azathioprine is metabolized via purine degradation pathway and can lead to immunosuppression when administered with xanthine oxidase inhibitor Mycophenolate and ribavirin: inhibit inosine monophosphate dehydrogenase Purine and pyrimidine synthesis: Hydroxyurea: inhibits ribonucleotide reductase Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine: inhibit dihydrofolate reductase ( deoxythymidine monophosphate [dTMP]) in humans (methotrexate), bacteria (trimethoprim), and protozoa (pyrimethamine) CPS1 = m1tochondria, urea cycle, found in liver CPS2 = cytwosol, pyrimidine synthesis, found in most cells FAS1_2023_01-Biochem.indd 34 11/17/22 7:12 PM Biochemistry  BIOCHEMISTRY—Molecular 35 SEC TION II Purine salvage deficiencie Catabolism Nucleic acids (DNA and RNA) Nucleic acids Ribose 5-phosphate De novo synthesis PRPP synthetase Salvage Nucleotides Nucleosides GMP Guanosine IMP Lesch-Nyhan syndrome HGPRT Nucleic acids AMP Cladribine, pentostatin ADA Inosine SCID Free bases Guanine PRPP APRT Adenosine PRPP Hypoxanthine XO Xanthine XO Adenine Allopurinol Febuxostat Degradation and salvage Uric acid Urate oxidase (rasburicase)a Allantoin Excretion Absent in humans. ADA, adenosine deaminase; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine guanine phosphoribosyltransferase, XO, xanthine oxidase; SCID, severe combined immune deficiency (autosomal recessive inheritance) a Adenosine deaminase deficiency ADA is required for degradation of adenosine and deoxyadenosine. ADA dATP ribonucleotide reductase activity DNA precursors in cells lymphocytes. One of the major causes of autosomal recessive SCID. Lesch-Nyhan syndrome Defective purine salvage. Absent HGPRT GMP (from guanine) and IMP (from hypoxanthine) formation. Compensatory in purine synthesis ( PRPP amidotransferase activity) excess uric acid production. X-linked recessive. Findings: intellectual disability, self-mutilation, aggression, hyperuricemia (red/orange “sand” [sodium urate crystals] in diaper), gout, dystonia, macrocytosis. HGPRT: Hyperuricemia Gout Pissed off (aggression, self-mutilation) Red/orange crystals in urine Tense muscles (dystonia) Treatment: allopurinol, febuxostat. Genetic code features Unambiguous Each codon specifies only 1 amino acid. Degenerate/ redundant Most amino acids are coded by multiple codons. Wobble hypothesis—first 2 nucleotides of codon are essential for anticodon recognition while the 3rd nucleotide can differ (“wobble”). 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. Exceptions: some viruses. Universal Genetic code is conserved throughout evolution. Exception in humans: mitochondria. FAS1_2023_01-Biochem.indd 35 11/17/22 7:12 PM 36 SEC TION II DNA replication Biochemistry  BIOCHEMISTRY—Molecular Occurs in 5′ 3′ direction (“5ynth3sis”) in continuous and discontinuous (Okazaki fragment) fashion. Semiconservative. More complex in eukaryotes than in prokaryotes, but shares analogous enzymes. Origin of replication A Particular consensus sequence in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes). Replication fork B Y-shaped region along DNA template where leading and lagging strands are synthesized. Helicase C Unwinds DNA template at replication fork. Single-stranded binding proteins D Prevent strands from reannealing or degradation by nucleases. DNA topoisomerases E Creates a single- (topoisomerase I) or double(topoisomerase II) stranded break in the helix to add or remove supercoils (as needed due to underwinding or overwinding of DNA). Primase F Makes RNA primer for DNA polymerase III to initiate replication. DNA polymerase III G Prokaryotes only. Elongates leading strand by adding deoxynucleotides to the 3′ end. Elongates lagging strand until it reaches primer of preceding fragment. DNA polymerase III has 5′ 3′ synthesis and proofreads with 3′ 5′ exonuclease. Drugs blocking DNA replication often have a modified 3′ OH, thereby preventing addition of the next nucleotide (“chain termination”). DNA polymerase I H Prokaryotes only. Degrades RNA primer; replaces it with DNA. Same functions as DNA polymerase III, also excises RNA primer with 5′ 3′ exonuclease. DNA ligase I Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA. Joins Okazaki fragments. Ligase links DNA. Telomerase Eukaryotes only. A reverse transcriptase (RNAdependent DNA polymerase) that adds DNA (TTAGGG) to 3′ ends of chromosomes to avoid loss of genetic material with every duplication. Upregulated in progenitor cells and also often in cancer; downregulated in aging and progeria. Telomerase TAGs for Greatness and Glory. AT-rich sequences (eg, TATA box regions) are found in promoters (often upstream) and origins of replication (ori). Helicase halves DNA. Deficient in Bloom syndrome (BLM gene mutation). In eukaryotes: irinotecan/topotecan inhibit topoisomerase (TOP) I, etoposide/teniposide inhibit TOP II. In prokaryotes: fluoroquinolones inhibit TOP II (DNA gyrase) and TOP IV. 3' E Topoisomerase 5' A Origin of replication C Helicase Leading strand B Replication fork A Origin of replication Lagging strand Area of interest Leading strand Fork movement Lagging strand FAS1_2023_01-Biochem.indd 36 Fork movement Leading strand D Single-stranded binding protein G DNA polymerase Lagging strand Okazaki fragment 3' 5' RNA primer I DNA ligase F Primase H DNA polymerase I 11/17/22 7:12 PM Biochemistry  BIOCHEMISTRY—Molecular 37 SEC TION II DNA repair Double strand Nonhomologous end joining Homologous recombination Brings together 2 ends of DNA fragments to repair double-stranded breaks. Homology not required. Part of the DNA may be lost or translocated. May be dysfunctional in ataxia telangiectasia. Double strand break 5´ 3´ 3´ 5´ Double strand break 5´ 3´ 5´ 3´ Nonhomologous end joining DoubleAstrand break Requires 2 homologous DNA duplexes. 5´ 3´ strand from damaged dsDNA is repaired using a complementary strand from intact homologous dsDNA as a template. Nonhomologous end joining Defective in breast/ovarian cancers with BRCA1 or BRCA2 mutations and in Fanconi anemia. Restores duplexes accurately without loss of nucleotides. 3´ 5´ Double strand break 5´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ Homologous recombination Homologous recombination Single strand Nucleotide excision repair Specific endonucleases remove the oligonucleotides containing damaged bases; DNA polymerase and ligase fill and reseal the gap, respectively. Repairs bulky helix-distorting lesions (eg, pyrimidine dimers). Occurs in G1 phase of cell cycle. Defective in xeroderma pigmentosum (inability to repair DNA pyrimidine dimers caused by UV exposure). Presents with dry skin, photosensitivity, skin cancer. Base excision repair Base-specific Glycosylase removes altered base and creates AP site (apurinic/apyrimidinic). One or more nucleotides are removed by AP-Endonuclease, which cleaves 5′ end. APLyase cleaves 3′ end. DNA Polymerase-β fills the gap and DNA ligase seals it. Occurs throughout cell cycle. Important in repair of spontaneous/toxic deamination. “GEL Please.” Mismatch repair Mismatched nucleotides in newly synthesized strand are removed and gap is filled and resealed. Occurs predominantly in S phase of cell cycle. Defective in Lynch syndrome (hereditary nonpolyposis colorectal cancer [HNPCC]). UV exposure Pyrimidine dimer Deaminated C T T A A Endonucleases remove damaged segment T T U G G A AP site G A A G U Glycosylase removes base Endonuclease and lyase remove backbone segment G Mismatched segment removed A Newly replaced segment FAS1_2023_01-Biochem.indd 37 T T U T A A G A Nucelotide excision repair Base excision repair Mismatch repair A B C 11/17/22 7:12 PM 38 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular Mutations in DNA Degree of change: silent << missense < nonsense < frameshift. Single nucleotide substitutions are repaired by DNA polymerase and DNA ligase. Types of single nucleotide (point) mutations: Transition—purine to purine (eg, A to G) or pyrimidine to pyrimidine (eg, C to T). Transversion—purine to pyrimidine (eg, A to T) or pyrimidine to purine (eg, C to G). Single nucleotide substitutions Silent mutation Codes for same (synonymous) amino acid; often involves 3rd position of codon (tRNA wobble). Missense mutation Results in changed amino acid (called conservative if new amino acid has similar chemical structure). Examples: sickle cell disease (substitution of glutamic acid with valine). Nonsense mutation Results in early stop codon (UGA, UAA, UAG). Usually generates nonfunctional protein. Stop the nonsense! Other mutations Frameshift mutation Deletion or insertion of any number of nucleotides not divisible by 3 misreading of all nucleotides downstream. Protein may be shorter or longer, and its function may be disrupted or altered. Examples: Duchenne muscular dystrophy, Tay-Sachs disease, cystic fibrosis. Splice site mutation Retained intron in mRNA protein with impaired or altered function. Examples: rare causes of cancers, dementia, epilepsy, some types of β-thalassemia, Gaucher disease, Marfan syndrome. Original sequence Coding DNA 5´ mRNA codon 5´ Silent mutation Missense mutation Nonsense mutation Frameshift insertion T Frameshift deletion G GAG GAA GTG TAG GA GAG GAA GUG UAG GAU GAC Glu Glu Val Stop Asp Asp Amino acid G GA C 3´ 3´ Altered amino acids Lac operon 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 from ATP activation of catabolite activator protein (CAP) transcription. High lactose unbinds repressor protein from repressor/operator site transcription. CAP cAMP Adenylate cyclase Glucose ATP Binds CAP site, induces transcription Lac operon DNA genes Lacl CAP site Promoter Operator o r, e ra t o n Binds opnscripti blocks tra Repressor protein STATE Low glucose Lactose available LacZ LacY LacA FAS1_2023_01-Biochem.indd 38 Inactivated repressor Lac genes strongly expressed Repressor protein High glucose Lactose unavailable Lac genes not expressed Low glucose Lactose unavailable High glucose Lactose available Allolactose (inducer) RNA CAP polymerase CAP site P Lac genes not expressed O Very low (basal) expression 11/17/22 7:12 PM Biochemistry  BIOCHEMISTRY—Molecular Functional organization of a eukaryotic gene Enhancer/ silencer Promoter 5´ UTR DNA (coding strand) 5´ CAAT CAAT Box 3´ UTR Open reading frame Exon Intron Exon Intron TATAAA GT AG GT Silencer Exon AG TATA Box Transcription start 39 SEC TION II AATAAA 3´ Polyadenylation signal Transcription Exon Intron Exon Intron Pre-mRNA Splicing GU 5´ cap Mature mRNA AG GU AG Exon AAUAAA Protein coding region AAUAAA AAAAAA AUG start codon Stop Poly-A tail Translation Protein Regulation of gene expression 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, which differ between eukaryotes and prokaryotes). Promoters increase ori activity. Promoter 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. Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene whose expression they regulate. Silencer DNA locus where regulatory proteins (“repressors”) bind, decreasing expression of a gene on the same chromosome. Epigenetics Changes made to gene expression (heritable mitotically/meiotically) without a change in underlying DNA sequence. FAS1_2023_01-Biochem.indd 39 Primary mechanisms of epigenetic change include DNA methylation, histone modification, and noncoding RNA. 11/17/22 7:12 PM 40 SEC TION II RNA processing (eukaryotes) Biochemistry  BIOCHEMISTRY—Molecular Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA is then modified and becomes mRNA. The following processes occur in the nucleus: Capping of 5′ end (addition of 7-methylguanosine cap; cotranscriptional) Polyadenylation of 3′ end (∼200 A’s poly-A tail; posttranscriptional) Splicing out of introns (posttranscriptional) Capped, tailed, and spliced transcript is called mRNA. mRNA is transported out of nucleus to be translated in cytosol. mRNA quality control occurs at cytoplasmic processing bodies (P-bodies), which contain exonucleases, decapping enzymes, and microRNAs; mRNAs may be degraded or stored in P-bodies for future translation. Poly-A polymerase does not require a template. AAUAAA = polyadenylation signal. Mutation in polyadenylation signal early degradation prior to translation. Kozak sequence—initiation site in most eukaryotic mRNA. Facilitates binding of small subunit of ribosome to mRNA. Mutations in sequence impairment of initiation of translation protein synthesis. Eukaryotes RNA polymerase I makes rRNA, the most common (rampant) type; present only in nucleolus. RNA polymerase II makes mRNA (massive), microRNA (miRNA), and small nuclear RNA (snRNA). RNA polymerase III makes 5S rRNA, tRNA (tiny). No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promoter site. I, II, and III are numbered in the same order that their products are used in protein synthesis: rRNA, mRNA, then tRNA. α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes dysentery and severe hepatotoxicity if ingested. Dactinomycin inhibits RNA polymerase in both prokaryotes and eukaryotes. Prokaryotes 1 RNA polymerase (multisubunit complex) makes all 3 kinds of RNA. Rifamycins (rifampin, rifabutin) inhibit DNAdependent RNA polymerase in prokaryotes. Cap 5' 3' Coding Gppp HO-AAAAA Tail RNA polymerases FAS1_2023_01-Biochem.indd 40 11/17/22 7:12 PM Introns vs exons Introns are intervening sequences and stay in the nucleus, whereas exons exit and are expressed. Alternative splicing—can produce a variety of protein products from a single hnRNA (heterogenous nuclear RNA) sequence (eg, transmembrane vs secreted Ig, tropomyosin variants in muscle, dopamine receptors in the brain, host defense evasion by tumor cells). Exons contain the actual genetic information coding for protein or functional RNA. Introns do not code for protein, but are important in regulation of gene expression. Different exons are frequently combined by alternative splicing to produce a larger number of unique proteins. 5′ 3′ DNA 41 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular Exon 1 Exon 2 Exon 3 1 2 3 Exon 4 Exon 5 Exon 6 5 6 3′ 5′ Transcription hnRNA 5′ 4 Alternative splicing Splicing mRNA 5′ 1 2 4 5 6 3′ 5′ 1 3 5 6 3′ 5′ 1 3 3′ 4 5 6 3′ Translation 4 Splicing of pre-mRNA 5 1 Proteins 5 1 6 4 3 2 5 1 6 6 3 Part of process by which precursor mRNA (pre-mRNA) is transformed into mature mRNA. Introns typically begin with GU and end with AG. Alterations in snRNP assembly can cause clinical disease; eg, in spinal muscular atrophy, snRNP assembly is affected due to SMN protein congenital degeneration of anterior horns of spinal cord symmetric weakness (hypotonia, or “floppy baby syndrome”). snRNPs are snRNA bound to proteins (eg, Smith [Sm]) to form a spliceosome that cleaves premRNA. Anti-U1 snRNP antibodies are associated with SLE, mixed connective tissue disease, other rheumatic diseases. Primary transcript combines with small nuclear ribonucleoproteins (snRNPs) and other proteins to form spliceosome. 5′ splice site O 5′ P GU Exon 1 3′ splice site Branch point U2 snRNP U1 snRNP OH A Intron AG P O 3′ Exon 2 Spliceosome Cleavage at 5′ splice site; lariatshaped (loop) intermediate is generated. UG OH 3′ P A AG P O Exon 1 Cleavage at 3′ splice site; lariat is released to precisely remove intron and join 2 exons. FAS1_2023_01-Biochem.indd 41 Exon 2 Mature mRNA P Exon 1 Exon 2 + UG P A AG OH 3′ 11/17/22 7:12 PM 42 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular tRNA Structure 75–90 nucleotides, 2º 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 tRNAribosome binding. T-arm Tethers tRNA molecule to ribosome. D-arm: contains Dihydrouridine residues necessary for tRNA recognition by the correct aminoacyltRNA synthetase. D-arm allows Detection of the tRNA by aminoacyl-tRNA synthetase. Attachment site: 3′-ACC-5′ is the amino acid ACCeptor site. Charging Aminoacyl-tRNA synthetase (uses ATP; 1 unique enzyme per respective amino acid) and binding of charged tRNA to the codon are responsible for the accuracy of amino acid selection. Aminoacyl-tRNA synthetase matches an amino acid to the tRNA by scrutinizing the amino acid before and after it binds to tRNA. If an incorrect amino acid is attached, the bond is hydrolyzed. A mischarged tRNA reads the usual codon but inserts the wrong amino acid. Structure Charging (aminoacylation) Amino acid Attachment site OH A C C 3´ O 5´ T-arm Pairing (codon-anticodon) Amino acid A C C O 3´ 3´ A C C 5´ D-arm D C Ψ T D ATP 5´ IF2 (initiation factor) AMP + PPi Aminoacyl-tRNA synthetase D C Ψ T D C Ψ T D D Variable arm Anticodon loop U A C Wobble position U A C Anticodon (5´-CAU-3´) mRNA C C U A C C A U G A U A C Codon (5´-AUG-3´) Start and stop codons mRNA start codon AUG. AUG inAUGurates protein synthesis. Eukaryotes Codes for methionine, which may be removed before translation is completed. Prokaryotes Codes for N-formylmethionine (fMet). fMet stimulates neutrophil chemotaxis. UGA, UAA, UAG. Recognized by release factors. UGA = U Go Away. UA A = U Are Away. UAG = U Are Gone. mRNA stop codons FAS1_2023_01-Biochem.indd 42 11/17/22 7:12 PM 43 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular Protein synthesis Initiation 1. Eukaryotic initiation factors (eIFs) identify the 5′ cap. 2. eIFs help assemble the 40S ribosomal subunit with the initiator tRNA. 3. eIFs released when the mRNA and the ribosomal 60S subunit assemble with the complex. Requires GTP. Elongation Eukaryotes: 40S + 60S 80S (even). Prokaryotes: 30S + 50S 70S (prime). Synthesis occurs from N-terminus to C-terminus. A minoacyl-tRNA binds to A site (except for initiator methionine, which binds the P site), requires an elongation factor and GTP. rRNA (“ribozyme”) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site. Ribosome advances 3 nucleotides toward 3′ end of mRNA, moving peptidyl tRNA to P site (translocation). Termination Eukaryotic release factors (eRFs) recognize the stop codon and halt translation completed polypeptide is released from ribosome. Requires GTP. ATP—tRNA Activation (charging). GTP—tRNA Gripping and Going places (translocation). Think of “going APE”: A site = incoming Aminoacyl-tRNA. P site = accommodates growing Peptide. E site = holds Empty tRNA as it Exits. Elongation factors are targets of bacterial toxins (eg, Diphtheria, Pseudomonas). Shine-Dalgarno sequence—ribosomal binding site in prokaryotic mRNA. Recognized by 16S RNA in ribosomal subunit. Enables protein synthesis initiation by aligning ribosome with start codon so that code is read correctly. 60/50S 80/70S 40/30S R M M Initiator tRNA H U A C 5´ A U G C A U G A U U A C mRNA M M E P U A C G U A 3´ A 5´ A U G C A U G A U Initiation E P 3´ A S Ribosome moves left to right along mRNA H Elongation M Q E A U G C A U G A U P A H U G A U A C G U A U A C 5´ M G U A 3´ 5´ Termination A U G C A U G A U E P 3´ A Posttranslational modifi ations 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. Chaperone protein FAS1_2023_01-Biochem.indd 43 Intracellular protein involved in facilitating and maintaining protein folding. In yeast, heat shock proteins (eg, HSP60) are constitutively expressed, but expression may increase with high temperatures, acidic pH, and hypoxia to prevent protein denaturing/misfolding. 11/17/22 7:12 PM 44 SEC TION II Biochemistry  BIOCHEMISTRY—Cellular ` BIOCHEMISTRY—CELLULAR Cell cycle phases 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 is of variable duration. REGULATION OF CELL CYCLE Cyclin-dependent kinases Constitutively expressed but inactive when not bound to cyclin. Cyclin-CDK complexes Cyclins are phase-specific regulatory proteins that activate CDKs when stimulated by growth factors. The cyclin-CDK complex can then phosphorylate other proteins (eg, Rb) to coordinate cell cycle progression. This complex must be activated/inactivated at appropriate times for cell cycle to progress. Tumor suppressors p53 p21 induction CDK inhibition Rb hypophosphorylation (activation) G1-S progression inhibition. 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. CELL TYPES Permanent Remain 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. Cell cycle arrest es th ow Gr is G1 Rb, p53 modulate G1 restriction point p21 Cy p53 BCL-2 BCL-XL Rb DNA P P M E t he S AS P Rb E2F Sy n BAX/BAK IN TERPH HPV E6 GO Cyclin CDK sis Mito Li-Fraumeni syndrome (loss of function) Cyclin to DNA damage CDK kin p21 s is Caspase activation Apoptosis (intrinsic pathway) FAS1_2023_01-Biochem.indd 44 E2F G2 Gene transcription 11/17/22 7:12 PM Biochemistry  BIOCHEMISTRY—Cellular 45 SEC TION II Rough endoplasmic reticulum Site of synthesis of secretory (exported) proteins and of N-linked oligosaccharide addition to lysosomal and other proteins. Nissl bodies (RER in neurons)—synthesize peptide neurotransmitters for secretion. Free ribosomes—unattached to any membrane; site of synthesis of cytosolic, peroxisomal, and mitochondrial proteins. N-linked glycosylation occurs in the eNdoplasmic reticulum. Mucus-secreting goblet cells of small intestine and antibody-secreting plasma cells are rich in RER. Proteins within organelles (eg, ER, Golgi bodies, lysosomes) are formed in RER. Smooth endoplasmic reticulum Site of steroid synthesis and detoxification of drugs and poisons. Lacks surface ribosomes. Location of glucose-6-phosphatase (last step in both glycogenolysis and gluconeogenesis). Hepatocytes and steroid hormone–producing cells of the adrenal cortex and gonads are rich in SER. Cell traffi ing Golgi is distribution center for proteins and lipids from ER to vesicles and plasma membrane. Posttranslational events in GOlgi include modifying N-oligosaccharides on asparagine, adding O-oligosaccharides on serine and threonine, and adding mannose-6-phosphate to proteins for lysosomal and other proteins. 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. I-cell disease (inclusion cell disease/mucolipidosis type II)—inherited lysosomal storage disorder (autosomal recessive); defect in N-acetylglucosaminyl-1-phosphotransferase failure of the Golgi to phosphorylate mannose residues ( mannose-6-phosphate) on glycoproteins enzymes secreted extracellularly rather than delivered to lysosomes lysosomes deficient in digestive enzymes buildup of cellular debris in lysosomes (inclusion bodies). Results in coarse facial features, gingival hyperplasia, corneal clouding, restricted joint movements, claw hand deformities, kyphoscoliosis, and plasma levels of lysosomal enzymes. Symptoms similar to but more severe than Hurler syndrome. Often fatal in childhood. Key: sma Pla Clathrin COPI COPII Retrograde Anterograde brane mem Late endosome Lysosome trans Golgi apparatus cis Secretory vesicle Early endosome Signal recognition particle (SRP)—abundant, cytosolic ribonucleoprotein that traffics polypeptide-ribosome complex from the cytosol to the RER. Absent or dysfunctional SRP accumulation of protein in cytosol. Vesicular trafficking proteins COPI: Golgi Golgi (retrograde); cis-Golgi ER. COPII: ER cis-Golgi (anterograde). “Two (COPII) steps forward (anterograde); one (COPI) step back (retrograde).” Clathrin: trans-Golgi lysosomes; plasma membrane endosomes (receptor-mediated endocytosis [eg, LDL receptor activity]). Rough endoplasmic reticulum Nuclear envelope FAS1_2023_01-Biochem.indd 45 11/17/22 7:12 PM 46 SEC TION II Peroxisome Biochemistry  BIOCHEMISTRY—Cellular Membrane-enclosed organelle involved in: β-oxidation of very-long-chain fatty acids (VLCFA) (strictly peroxisomal process) α-oxidation of branched-chain fatty acids (strictly peroxisomal process) Catabolism of amino acids and ethanol Synthesis of bile acids and plasmalogens (important membrane phospholipid, especially in white matter of brain) Zellweger syndrome—autosomal recessive disorder of peroxisome biogenesis due to mutated PEX genes. Hypotonia, seizures, jaundice, craniofacial dysmorphia, hepatomegaly, early death. Refsum disease—autosomal recessive disorder of α-oxidation buildup of phytanic acid due to inability to degrade it. Scaly skin, ataxia, cataracts/night blindness, shortening of 4th toe, epiphyseal dysplasia. Treatment: diet, plasmapheresis. Adrenoleukodystrophy—X-linked recessive disorder of β-oxidation due to mutation in ABCD1 gene VLCFA buildup in adrenal glands, white (leuko) matter of brain, testes. Progressive disease that can lead to adrenal gland crisis, progressive loss of neurologic function, death. Proteasome Cytoskeletal elements Barrel-shaped protein complex that degrades polyubiquitin-tagged proteins. Plays a role in many cellular processes, including immune response (MHC I–mediated). Defects in ubiquitin-proteasome system also implicated in diverse human diseases including neurodegenerative diseases. A network of protein fibers within the cytoplasm that supports cell structure, cell and organelle movement, and cell division. TYPE OF FILAMENT PREDOMINANT FUNCTION EXAMPLES Microfilaments Muscle contraction, cytokinesis Actin, microvilli. Intermediate filaments Maintain cell structure Vimentin, desmin, cytokeratin, lamins, glial fibrillary acidic protein (GFAP), neurofilaments. Microtubules Movement, cell division Cilia, flagella, mitotic spindle, axonal trafficking, centrioles. Cylindrical outer structure composed of a helical array of polymerized heterodimers of α- and β-tubulin. Each dimer has 2 GTP bound. Incorporated into flagella, cilia, mitotic spindles. Also involved in slow axoplasmic transport in neurons. Molecular motor proteins—transport cellular cargo toward opposite ends of microtubule. Retrograde to microtubule (+ −)—dynein. Anterograde to microtubule (− +)—kinesin. Clostridium tetani toxin, poliovirus, rabies virus, and herpes simplex virus (HSV) use dynein for retrograde transport to the neuronal cell body. HSV reactivation occurs via anterograde transport from cell body (kinesin mediated). Slow anterograde transport rate limiting step of peripheral nerve regeneration after injury. Drugs that act on microtubules (microtubules get constructed very terribly): Mebendazole (antihelminthic) Griseofulvin (antifungal) Colchicine (antigout) Vinca alkaloids (anticancer) Taxanes (anticancer) Negative end near nucleus. Positive end points to periphery. Microtubule Positive end (+) Heterodimer Protofilament Negative end (–) FAS1_2023_01-Biochem.indd 46 Ready? Attack! 11/17/22 7:12 PM Biochemistry  BIOCHEMISTRY—Cellular Cilia structure 47 SEC TION II Motile cilia consist of 9 doublet + 2 singlet arrangement of microtubules (axoneme) A . Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets B with no central microtubules. Nonmotile (primary) cilia work as chemical signal sensors and have a role in signal transduction and cell growth control. Dysgenesis may lead to polycystic kidney disease, mitral valve prolapse, or retinal degeneration. Axonemal dynein—ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets. Gap junctions enable coordinated ciliary movement. A B Dynein arm Microtubule A Microtubule B Nexin Doublets Primary ciliary dyskinesia A R L Sodium-potassium pump Triplets Autosomal recessive. Dynein arm defect immotile cilia dysfunctional ciliated epithelia. Most common type is Kartagener syndrome (PCD with situs inversus). Developmental abnormalities due to impaired migration and orientation (eg, situs inversus A , hearing loss due to dysfunctional eustachian tube cilia); recurrent infections (eg, sinusitis, ear infections, bronchiectasis due to impaired ciliary clearance of debris/pathogens); infertility ( risk of ectopic pregnancy due to dysfunctional fallopian tube cilia, immotile spermatozoa). Lab findings: nasal nitric oxide (used as screening test). Na+/K+-ATPase is located in the plasma membrane with ATP site on cytosolic side. For each ATP consumed, 2 K+ go in to the cell (pump dephosphorylated) and 3 Na+ go out of the cell (pump phosphorylated). Extracellular space 2 strikes? K, you’re still in. 3 strikes? Nah, you’re out! Digoxin directly inhibits Na+/K+-ATPase indirect inhibition of Na+/Ca2+ exchange [Ca2+]i cardiac contractility. 3Na+ 2K+ Plasma membrane Cytosol FAS1_2023_01-Biochem.indd 47 P 3Na+ ATP ADP P 2K+ 11/17/22 7:13 PM 48 SEC TION II Biochemistry  BIOCHEMISTRY—Cellular Collagen Most abundant protein in the human body. Extensively modified by posttranslational modification. Organizes and strengthens extracellular matrix. Types I to IV are the most common types in humans. Type I - Skeleton Type II - Cartilage Type III - Arteries Type IV - Basement membrane SCAB Type I Most common (90%)—Bone (made by osteoblasts), Skin, Tendon, dentin, fascia, cornea, late wound repair. Type I: bone, tendone. production in osteogenesis imperfecta type I. Type II Cartilage (including hyaline), vitreous body, nucleus pulposus. Type II: cartwolage. Type III Reticulin—skin, blood vessels, uterus, fetal tissue, early wound repair. Type III: deficient in vascular type of EhlersDanlos syndrome (threE D). Type IV Basement membrane/basal lamina (glomerulus, cochlea), lens. Type IV: under the floor (basement membrane). Defective in Alport syndrome; targeted by autoantibodies in Goodpasture syndrome. Myofibroblasts are responsible for secretion (proliferative stage) and wound contraction. Collagen synthesis and structure Preprocollagen Fibroblast Pro α-chain backbone (Gly-X-Y) Nucleus OH Collagen mRNA OH Hydroxylation of proline and lysine (requires vitamin C) Sugar Cytoplasm OH RER OH Glycosylation Triple helix formation Procollagen Golgi Exocytosis Extracellular space Cleavage of procollagen C- and N-terminals Tropocollagen Self assembly into collagen fibrils Formation of cross-links (stabilized by lysyl oxidase) Collagen fiber FAS1_2023_01-Biochem.indd 48 S ynthesis—translation of collagen α chains (preprocollagen)—usually Gly-X-Y (X is often proline or lysine and Y is often hydroxyproline or hydroxylysine). Collagen is 1/3 glycine; glycine content of collagen is less variable than that of lysine and proline. H ydroxylation—hydroxylation (“hydroxCylation”) of specific proline and lysine residues. Requires vitamin C; deficiency scurvy. Glycosylation—glycosylation of pro-α-chain hydroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen α chains). Problems forming triple helix osteogenesis imperfecta. Exocytosis—exocytosis of procollagen into extracellular space. Proteolytic processing—cleavage of disulfide-rich terminal regions of procollagen insoluble tropocollagen. Assembly and alignment—collagen assembles in fibrils and aligns for cross-linking. C ross-linking—reinforcement of staggered tropocollagen molecules by covalent lysinehydroxylysine cross-linkage (by coppercontaining lysyl oxidase) to make collagen fibers. Cross-linking of collagen with age. Problems with cross-linking Menkes disease. 11/17/22 7:13 PM Biochemistry  BIOCHEMISTRY—Cellular Osteogenesis imperfecta 49 SEC TION II Genetic bone disorder (brittle bone disease) caused by a variety of gene defects (most commonly COL1A1 and COL1A2). Most common form is autosomal dominant with production of otherwise normal type I collagen (altered triple helix formation). Manifestations include: Multiple fractures and bone deformities (arrows in A ) after minimal trauma (eg, during birth) Blue sclerae B due to the translucent connective tissue over choroidal veins Some forms have tooth abnormalities, including opalescent teeth that wear easily due to lack of dentin (dentinogenesis imperfecta) Conductive hearing loss (abnormal ossicles) May be confused with child abuse. Treat with bisphosphonates to fracture risk. Patients can’t BITE: Bones = multiple fractures I (eye) = blue sclerae Teeth = dental imperfections Ear = hearing loss Ehlers-Danlos syndrome Faulty collagen synthesis causing hyperextensible skin A , hypermobile joints B , and tendency to bleed (easy bruising). Multiple types. Inheritance and severity vary. Can be autosomal dominant or recessive. May be associated with joint dislocation, berry and aortic aneurysms, organ rupture. Hypermobility type (joint instability): most common type. Classical type (joint and skin symptoms): caused by a mutation in type V collagen (eg, COL5A1, COL5A2). Vascular type (fragile tissues including vessels [eg, aorta], muscles, and organs that are prone to rupture [eg, gravid uterus]): mutations in type III procollagen (eg, COL3A1). Can be caused by procollagen peptidase deficiency. A Menkes disease X-linked recessive connective tissue disease caused by impaired copper absorption and transport due to defective Menkes protein ATP7A (Absent copper), vs ATP7B in Wilson disease (copper Buildup). Leads to activity of lysyl oxidase (copper is a necessary cofactor) defective collagen cross-linking. Results in brittle, “kinky” hair, growth and developmental delay, hypotonia, risk of cerebral aneurysms. A Upper extremity FAS1_2023_01-Biochem.indd 49 B B 11/17/22 7:14 PM 50 SEC TION II Biochemistry  Biochemistry—Laboratory Techniques Elastin Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, epiglottis, ligamenta flava (connect vertebrae relaxed and stretched conformations). Rich in nonhydroxylated proline, glycine, and lysine residues, vs the hydroxylated residues of collagen. Tropoelastin with fibrillin scaffolding. Cross-linking occurs extracellularly via lysyl oxidase and gives elastin its elastic properties. Broken down by elastase, which is normally inhibited by α1-antitrypsin. α1-Antitrypsin deficiency results in unopposed elastase activity, which can cause COPD. Single elastin Stretch Relax Cross-link molecule Marfan syndrome—autosomal dominant (with variable expression) connective tissue disorder affecting skeleton, heart, and eyes. FBN1 gene mutation on chromosome 15 (fifteen) results in defective fibrillin-1, a glycoprotein that forms a sheath around elastin and sequesters TGF-β. Findings: tall with long extremities; chest wall deformity (pectus carinatum [pigeon chest] or pectus excavatum A ); hypermobile joints; long, tapering fingers and toes (arachnodactyly); cystic medial necrosis of aorta; aortic root aneurysm rupture or dissection (most common cause of death); mitral valve prolapse; risk of spontaneous pneumothorax. A Homocystinuria—most commonly due to cystathionine synthase deficiency leading to homocysteine buildup. Presentation similar to Marfan syndrome with pectus deformity, tall stature, arm:height ratio, upper:lower body segment ratio, arachnodactyly, joint hyperlaxity, skin hyperelasticity, scoliosis, fair complexion (vs Marfan syndrome). Marfan syndrome Homocystinuria INHERITANCE Autosomal dominant Autosomal recessive INTELLECT Normal Decreased VASCULAR COMPLICATIONS Aortic root dilatation Thrombosis LENS DISLOCATION Upward/temporal (Marfan fans out) Downward/nasal ` BIOCHEMISTRY—LABORATORY TECHNIQUES Polymerase chain reaction Molecular biology lab procedure used to amplify a desired fragment of DNA. Useful as a diagnostic tool (eg, neonatal HIV, herpes encephalitis). 5' 5' 3' 3' 5' 3' 5' 3' 5' 3' 3' DNA primer 5' dNTP Repeat 5' 3' Double-stranded DNA 3' 5' 3' 5' 3' 5' enaturation—DNA template, DNA primers, a heat-stable DNA polymerase, and D deoxynucleotide triphosphates (dNTPs) are heated to ~ 95ºC to separate the DNA strands. Annealing—sample is cooled to ~ 55ºC. DNA primers anneal to the specific sequence to be amplified on the DNA template. Elongation—temperature is increased to ~ 72ºC. DNA polymerase adds dNTPs to the strand to replicate the sequence after each primer. Heating and cooling cycles continue until the amount of DNA is sufficient. FAS1_2023_01-Biochem.indd 50 11/17/22 7:14 PM CRISPR/Cas9 51 SEC TION II Biochemistry  Biochemistry—Laboratory Techniques A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) , which is complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single- or double-strand break at the target site . Imperfectly cut segments are repaired by nonhomologous end joining (NHEJ) accidental frameshift mutations (“knock-out”) , or a donor DNA sequence can be added to fill in the gap using homology-directed repair (HDR) . Potential applications include removing virulence factors from pathogens, replacing disease-causing alleles of genes with healthy variants (in clinical trials for sickle cell disease), and specifically targeting tumor cells. Cas9 gRNA NHEJ 3A 3B HDR Donor DNA + Frameshift/inactivation (”knock-out”) Edited sequence (”knock-in”) Blotting procedures I: Parents PEDIGREE 1. DNA sample is enzymatically cleaved into smaller pieces, which are separated by gel electrophoresis, and then transferred to a membrane. 2. Membrane is exposed to labeled DNA probe that anneals to its complementary strand. 3. Resulting double-stranded, labeled piece of DNA is visualized when membrane is exposed to film or digital imager. Useful to identify size of specific sequences (eg, determination of heterozygosity [as seen in image], # of CGG repeats in FMR1 to diagnose Fragile X syndrome) II: Children Aa Aa SOUTHERN BLOT Southern blot aa Aa AA Genotype Mutant Normal SNoW DRoP: Southern = DNA Northern = RNA Western = Protein Northern blot Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying mRNA levels and size, which are reflective of gene expression. Detects splicing errors. Western blot Sample protein is separated via gel electrophoresis and transferred to a membrane. Labeled antibody is used to bind relevant protein. This helps identify specific protein and determines quantity. Southwestern blot Identifies DNA-binding proteins (eg, c-Jun, c-Fos [leucine zipper motif]) using labeled doublestranded DNA probes. Southern (DNA) + Western (protein) = Southwestern (DNA-binding protein). FAS1_2023_01-Biochem.indd 51 11/17/22 7:15 PM SEC TION II Flow cytometry Biochemistry  Biochemistry—Laboratory Techniques 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 a 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. Data are plotted either as histogram (one measure) or scatter plot (any two measures, as shown). In illustration: Cells in left lower quadrant ⊝ for both CD8 and CD3. Cells in right lower quadrant ⊕ for CD8 and ⊝ for CD3. In this example, right lower quadrant is empty because all CD8-expressing cells also express CD3. Cells in left upper quadrant ⊕ for CD3 and ⊝ for CD8. Cells in right upper quadrant ⊕ for both CD8 and CD3. Commonly used in workup of hematologic abnormalities (eg, leukemia, paroxysmal nocturnal hemoglobinuria, fetal RBCs in pregnant person’s blood) and immunodeficiencies (eg, CD4+ cell count in HIV). Fluorescent label Antibody Anti-CD3 Ab Cell Anti-CD8 Ab Fluorescence is detected; labeled cells are counted Laser Laser makes label fluoresce r cto te De 104 103 CD3 52 102 101 100 100 101 102 103 104 CD8 Microarrays Array consisting of thousands of DNA oligonucleotides arranged in a grid on a glass or silicon chip. The DNA or RNA samples being compared are attached to different fluorophores and hybridized to the array. The ratio of fluorescence signal at a particular oligonucleotide reflects the relative amount of the hybridizing nucleic acid in the two samples. Used to compare the relative transcription of genes in two RNA samples. Can detect single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for genotyping, clinical genetic testing, forensic analysis, and cancer mutation and genetic linkage analysis when DNA is used. Enzyme-linked immunosorbent assay Immunologic test used to detect the presence of either a specific antigen or antibody in a patient’s blood sample. Detection involves the use of an antibody linked to an enzyme. Added substrate reacts with the enzyme, producing a detectable signal. Can have high sensitivity and specificity, but is less specific than Western blot. Often used to screen for HIV infection. Direct ELISA Substrate Enzyme Detectable signal Labeled 1° antibody Antigen Substrate Indirect ELISA Labeled 2° antibody 1° antibody Antigen FAS1_2023_01-Biochem.indd 52 11/17/22 7:15 PM Biochemistry  Biochemistry—Laboratory Techniques 53 SEC TION II Karyotyping Colchicine is added to cultured cells to halt chromosomes in metaphase. Chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern (arrows in A point to extensive abnormalities in a cancer cell). 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). A Fluorescence in situ hybridization Fluorescent DNA or RNA probe binds to specific gene or other site of interest on chromosomes. Used for specific localization of genes and direct visualization of chromosomal anomalies. Microdeletion—no fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome. Translocation— A fluorescence signal (from ABL gene) that corresponds to one chromosome (chromosome 9) is found in a different chromosome (chromosome 22, next to BCR gene). Duplication—a second copy of a chromosome, resulting in a trisomy or tetrasomy. A Molecular cloning Production of a recombinant DNA molecule in a bacterial host. Useful for production of human proteins in bacteria (eg, human growth hormone, insulin). Steps: 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) recombinant plasmid into bacteria. 5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA). FAS1_2023_01-Biochem.indd 53 11/17/22 7:15 PM 54 SEC TION II Gene expression modifi ations RNA interference Biochemistry  BIOCHEMISTRY—Genetics Transgenic strategies in mice involve: Random insertion of gene into mouse genome Targeted insertion or deletion of gene through homologous recombination with mouse gene Knock-out = removing a gene, taking it out. Knock-in = inserting a gene. Random insertion—constitutive expression. Targeted insertion—conditional expression. Process whereby small non-coding RNA molecules target mRNAs to inhibit gene expression. MicroRNA Naturally produced by cell as hairpin structures. Loose nucleotide pairing allows broad targeting of related mRNAs. When miRNA binds to mRNA, it blocks translation of mRNA and sometimes facilitates its degradation. Abnormal expression of miRNAs contributes to certain malignancies (eg, by silencing an mRNA from a tumor suppressor gene). Small interfering RNA Usually derived from exogenous dsRNA source (eg, virus). Once inside a cell, siRNA requires complete nucleotide pairing, leading to highly specific mRNA targeting. Results in mRNA cleavage prior to translation. Can be produced by transcription or chemically synthesized for gene “knockdown” experiments. ` BIOCHEMISTRY—GENETICS Genetic terms TERM DEFINITION EXAMPLE Codominance Both alleles contribute to the phenotype of the heterozygote. Blood groups A, B, AB; α1-antitrypsin deficiency; HLA groups. Variable expressivity Patients with the same genotype have varying phenotypes. Two patients with neurofibromatosis type 1 (NF1) may have varying disease severity. Incomplete penetrance Not all individuals with a disease show the disease. % penetrance × probability of inheriting genotype = risk of expressing phenotype. BRCA1 gene mutations do not always result in breast or ovarian cancer. Pleiotropy One gene contributes to multiple phenotypic effects. Untreated phenylketonuria (PKU) manifests with light skin, intellectual disability, musty body odor. Anticipation Increased severity or earlier onset of disease in succeeding generations. Trinucleotide repeat diseases (eg, Huntington disease). Loss of heterozygosity If a patient inherits or develops a mutation in a tumor suppressor gene, the wild type allele must be deleted/mutated/eliminated before cancer develops. This is not true of oncogenes. Retinoblastoma and the “two-hit hypothesis,” Lynch syndrome (HNPCC), Li-Fraumeni syndrome. Epistasis The allele of one gene affects the phenotypic expression of alleles in another gene. Albinism, alopecia. Aneuploidy An abnormal number of chromosomes; due to chromosomal nondisjunction during mitosis or meiosis. Down syndrome, Turner syndrome, oncogenesis. FAS1_2023_01-Biochem.indd 54 11/17/22 7:15 PM Biochemistry  BIOCHEMISTRY—Genetics 55 SEC TION II Genetic terms (continued) TERM DEFINITION EXAMPLE Dominant negative mutation Exerts a dominant effect. A heterozygote produces a nonfunctional altered protein that also prevents the normal gene product from functioning. A single mutated p53 tumor suppressor gene results in a protein that is able to bind DNA and block the wild type p53 from binding to the promoter. Linkage disequilibrium Tendency for certain alleles to occur in close proximity on the same chromosome more or less often than expected by chance. Measured in a population, not in a family, and often varies in different populations. Mosaicism Presence of genetically distinct cell lines in the same individual. Somatic mosaicism—mutation arises from mitotic errors after fertilization and propagates through multiple tissues or organs. Germline (gonadal) mosaicism—mutation only in egg or sperm cells. If parents and relatives do not have the disease, suspect gonadal (or germline) mosaicism. McCune-Albright syndrome—due to Gs-protein activating mutation. Presents with unilateral café-au-lait spots A with ragged edges, polyostotic fibrous dysplasia (bone is replaced by collagen and fibroblasts), and at least one endocrinopathy (eg, precocious puberty). Lethal if mutation occurs before fertilization (affecting all cells), but survivable in patients with mosaicism. Locus heterogeneity Mutations at different loci result in the same disease. Albinism, retinitis pigmentosa, familial hypercholesteremia. Allelic heterogeneity Different mutations in the same locus result in the same disease. β-thalassemia. Heteroplasmy Presence of both normal and mutated mtDNA, resulting in variable expression in mitochondrially inherited disease. mtDNA passed from mother to all children. 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. Uniparental is euploid (correct number of chromosomes). Most occurrences of uniparental disomy (UPD) normal phenotype. Consider isodisomy in an individual manifesting a recessive disorder when only one parent is a carrier. Examples: Prader-Willi and Angelman syndromes. CONCEPT DESCRIPTION EXAMPLE Bottleneck effect Fitness equal across alleles natural disaster that removes certain alleles by chance new allelic frequency (by chance, not naturally selected). The founder effect is a type of bottleneck when cause is due to calamitous population separation. Natural selection Alleles that increase species fitness are more likely to be passed down to offspring and vice versa. Human evolution. Genetic drift Also called allelic drift or Wright effect. A dramatic shift in allelic frequency that occurs by change (not by natural selection). Founder effect and bottleneck effect are both examples of genetic drift. A Population genetics FAS1_2023_01-Biochem.indd 55 11/17/22 7:16 PM 56 SEC TION II Hardy-Weinberg principle A (p) a (q) A (p) AA (p2) Aa (pq) a (q) Aa (pq) aa (q2) Biochemistry  BIOCHEMISTRY—Genetics In a given population where mating is at random, allele and genotype frequencies will be constant. If p and q represent the frequencies of alleles A and a in a population, respectively, then p + q = 1, where: p2 = frequency of homozygosity for allele A q2 = frequency of homozygosity for allele a 2pq = frequency of heterozygosity (carrier frequency, if an autosomal recessive disease) Therefore the sum of the frequencies of these genotypes is p2 + 2pq + q2 = 1. The frequency of an X-linked recessive disease in males = q and in females = q2. Hardy-Weinberg law assumptions include: No mutation occurring at the locus Natural selection is not occurring Completely random mating No net migration Large population If a population is in Hardy-Weinberg equilibrium, then the values of p and q remain constant from generation to generation. In rare autosomal recessive diseases, p ≈ 1. Example: The prevalence of cystic fibrosis (an autosomal recessive disease) in the US is approximately 1/3200, which tells us that q2 = 1/3200, with q ≈ 0.017 or 1.7% of the population. Since p + q = 1, we know that p = 1 – √1/3200 ≈ 0.982, which gives us a heterozygous carrier frequency of 2pq = 0.035 or 3.5% of the population. Notice that since the disease is relatively rare, we could have approximated p ≈ 1 and obtained a similar result. Disorders of imprinting One gene copy is silenced by methylation, and only the other copy is expressed parent-of-origin effects. The expressed copy may be mutated, may not be expressed, or may be deleted altogether. Prader-Willi syndrome Angelman syndrome WHICH GENE IS SILENT? Maternally derived genes are silenced Disease occurs when the paternal allele is deleted or mutated Paternally derived UBE3A is silenced Disease occurs when the maternal allele is deleted or mutated SIGNS AND SYMPTOMS Hyperphagia, obesity, intellectual disability, hypogonadism, hypotonia Hand-flapping, Ataxia, severe Intellectual disability, inappropriate Laughter, Seizures. HAILS the Angels. CHROMOSOMES INVOLVED Chromosome 15 of paternal origin UBE3A on maternal copy of chromosome 15 NOTES 25% of cases are due to maternal uniparental disomy 5% of cases are due to paternal uniparental disomy POP: Prader-Willi, Obesity/overeating, Paternal allele deleted MAMAS: Maternal allele deleted, Angelman syndrome, Mood, Ataxia, Seizures Normal P M Mutation P M P = Paternal M = Maternal Active gene Silenced gene (imprinting) Gene deletion/mutation Prader-Willi syndrome Angelman syndrome FAS1_2023_01-Biochem.indd 56 11/17/22 7:16 PM Biochemistry  BIOCHEMISTRY—Genetics 57 SEC TION II Modes of inheritance Autosomal dominant Autosomal recessive X-linked recessive carrier X-linked dominant Mitochondrial inheritance = unaffected male; FAS1_2023_01-Biochem.indd 57 Often due to defects in structural genes. Many generations, both males and females are affected. A a a Aa aa a Aa aa With 2 carrier (heterozygous) parents, on average: each child has a 25% chance of being affected, 50% chance of being a carrier, and 25% chance of not being affected nor a carrier. A a A

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