MCBM Final Exam - Steven Lin

List advantages and disadvantages of adult stem cells/iPSCs

*Adult stem cells* *Advantages*: (1) No destruction of embryo or therapeutic cloning (2) No immune rejection (3) Growing evidence of plasticity (4) Already somewhat specialized so easier to induce (transdifferentiate) into desired targeted tissue (5) easier to procure (6) non-tumorigenic (7) no harm to donor *Disadvantages*: 1) Limited capacity for developing pluripotent lines 2) Present in such small quantities in body 3) Hard to multiply 4) More DNA abnormalities 5) Finite- shorter life than embryonic stem cells in culture *Successfully used in therapeutic applications*

What does alcoholism do to levels of NADH and fatty acids in the liver?

*Alcohol increases NADH and accumulates fatty acids in the liver* - Alcohol -> significant reduction in NAD available -> excess NADH results (oxidation of ethanol to form acetaldehyde and subsequent oxidation to make acetic acid consumes NAD+ and make NADH) - *Excess NADH inhibits TCA* cycle and *FA oxidation* and leads to *accumulation of FA in liver* (fatty liver) and rise in ketone bodies 1) TCA cycle inhibited due to depletion of oxaloacetate 2) Gluconeogenesis inhibited due to depletion of pyruvate 3) Glycolysis inhibited due to depletion of NAD+ - Cirrhosis = acetaldehyde damaging liver - Fatty liver due to accumulation of FA

*Allele specific PCR*

*Allele specific PCR* - helps *distinguish between homozygotes and heterozygotes* Example process: 1) Denature alleles and PCR with ASP 1 determine homozygosity or heterozygosity in cells 2) If heterozygote w/ two alleles: perfect match- strand extension and amplification occur 3) If imperfect - no strand extension or amplification can occur Homozygte: amplification with ONLY ONE OF ASP 1 OR ASP 2 Heterozygote: amplification with both ASP 1 and ASP 2 (20)

Define allele, allele frequency, genotype, genotype frequency homozygote, heterozygote, dominant and recessive.

*Allele*: different forms (DNA sequences) of a gene in population *Allele frequency*: frequency (0-1) of allele in population *Genotype*- individual's allele composition at a locus *Genotype frequency* - proportion of individuals w/ specific genotype in pop *Homozygote* - individuals w/ 2 identical alleles at locus *Heterozygote* - individuals w/ 2 different alleles at locus *Dominant* - allele expressed in homozygotes and heterozygotes *Recessive* - allele expressed only in homozygotes

Array CGH

*Array CGH (array comparative genomic hybridization)*: label DNA with different fluorophores and hybridize array to look at entire genome...very high resolution...can even look at 1 kb rearrangement! - Control DNA = red, pt. DNA = green - Yellow = equal amounts of patient and control DNA - Red signal = DNA loss in pt (deletion) - Green signal = gain in pt (duplication) *Detects*: Duplications/Deletions (Gain/Loss of DNA) *Limitations*: -*CANNOT* detect abnormalities *NOT involving changes in amount of DNA* (like inversions and balanced translocations). - *CANNOT* detect triploidy (presence of extra chromosome) . Anything not involving gain or loss, you cannot see.

3) List the assumptions required for Hardy-Weinberg Equilibrium to hold.

*Assumptions for HWE to hold*: 1) *Random mating* (cannot be from consanguineous relationships) 2) *No selection for any genotype* 3) *No population migration* (i.e. no gene flow), 4) *Large population size* 5) *No new mutations* If any of them are true, we cannot use HWE to calculate frequencies Helpful in calculating carrier frequencies

*Autosomal dominant (AA, Aa)* Pedigree & Punnett Square

*Autosomal dominant (AA, Aa)* - There are no carriers - Parent to child transmission (vertical transmission -does not skip generations) - Unaffected parents do not transmit - Equal XX XY - 50% of children affected

Draw a pedigree for traits displaying the following modes of inheritance: autosomal dominant, autosomal recessive, X-linked dominant and X-linked recessive. $$$ (Will be on exam!) Determine the mode of inheritance of a trait when given a pedigree that describes the transmission of the trait. Draw Punnett squares and be able to derive the genotypes and ratios (or frequencies) of genotypes of offspring (when given the genotype of the parents) for: autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive traits.

*Autosomal dominant (AA, Aa)* - There are no carriers - Parent to child transmission (vertical transmission -does not skip generations) - Unaffected parents do not transmit - Equal XX XY - 50% of children affected *Autosomal recessive (aa)* - Carriers present - Skip generation - Unaffected parents can have affected children - Equal XX XY - 25% of children affected - Cansanguineous matings occurring among relatives increase chance for homozygous children *X-linked dominant (X^X, X^Y)* - No male or female carriers - XX, XY equal - Mother transmits to daughters & sons - Father transmits only to daughters - Every generation affected *X-linked recessive* - Males always affected if they have allele - Unaffected males do not transmit - Carrier women transmit to affected sons - Unaffected males do not transmit - Carrier women transmit to affected sons - All daughters of affected males are carriers

*Autosomal recessive (aa)* Pedigree & Punnett Square

*Autosomal recessive (aa)* - Carriers present - Skip generation - *Unaffected parents can have affected children* - Equal XX XY - 25% of children affected - Cansanguineous matings occurring among relatives increase chance for homozygous children

Discuss bioethical issues surrounding stem cell research

*Bioethical issues*: 1) Destruction of embryos (what defines life?) 2) Fate of unused in vitro fertilized embryos (rights?, who owns these?) 3) Financial gain, donor safety, cloning, confidentiality - Reproductive cloning is illegal - Property/legal: usually embryos from IVF clinics are considered property of the couple *Majority of ethical issues concerns when is the start of life and end of life* Diff definitions: - Metabolism: when is it functional? - Genetics - conception - Embryological: Gastrulation - Ecological - when fetus is viable Very important -> patient centered care - must consider patient's views

Describe the role of gluconeogenesis in the homeostasis of blood glucose levels.

*Blood glucose levels maintained through*: 1) Glycogenolysis in liver (18-24 hr supply) during fasting 2) *Gluconeogenesis in liver and kidney: synthesis of glucose from non-carbon precursors* during starvation Fasting (24 hrs): erythrocytes can only use glucose. Liver glycogen stores used to release glucose. TAGs accessed. Starvation: liver glycogen stores depleted, gluconeogenesis is the only source of glucose heavily relying on TAGs *The glucose generated by liver CANNOT be oxidized by the liver bc glycolysis is off in the liver during fasting/starvation via glucagon!*

Discuss CIRM

*CIRM = California institute for regenerative medicine* - state agency to make grants and provide loans for stem cell research, research facilities, and other vital research opportunities Prop 71: California stem cell research and cures act - cannot use bonds, pluripotent stem cells emphasized and priority for therapies and cures, safety and ethical, reduce future health care costs

Describe how changes in chromosome ploidy are generated.

*Changes in ploidy occur due to non-disjunction in meiosis during gamete formation* Non-disjunction at Meiosis I - all daughter cells affected (2 cells disomic, 2 nullisomic) Non-disjunction at Meiosis II - half of daughter cells affected (1 disomic, 1 nullisomic, 2 monosomic) Disomic = n+1 (basically two haploids in one) Nullisomic = n-1 (empty egg) Monosomic = n (normal) Changes in chromosome number (ploidy): - *Aneuploidy* - *change in # of one or more chromosomes* . Only Trisomies 13, 18, 21 are viable. *Trisomy 16 is most common cause of 1st trimester spontaneous abortion*. - *Aneuploidies in gametes*: nullisomic (n-1), disomic (n+1) - *Polyploidy* - *gain of entire haploid chromosome set* - *Euploidy* - normal number 2n=46 for somatic cells (n=23 for gametes) - *Different from changes in chromosome structure: translocations, deletions etc. which doesn't change the number of chromosomes*

Describe how banding, FISH, chromosome painting, M-FISH, SKY and array CGH are used to detect chromosomal abnormalities and what types of chromosomal abnormalities each method can detect.

*Chromosome banding*: staining & display of chromosomes from metaphase spread - Giemsa (G-banding) or Quinacrine (Q-banding) stains yields diff. bands and resolutions (*Karyotype*) - *Detects*: changes in chromosome # and rearrangements (deletions, duplications, inversions, translocations) *FISH (fluorescent in situ hybridization)*: *hybridization of complementary nucleic acid* sequences via fluorescently labeled probes to *ONE* specific *region or centromere or telomere* after denaturing. - *Detects*: complex and small cytogenetic aberrations (translocations, deletions, inversions and duplications) BUT higher resolution -> can detect smaller rearrangements than karyotype banding Types of FISH: A) *Chromosome painting* - "BIG FISH" - *visualize entire single chromosome* (in *one color*) when mixture of many probes are added. B) *M-FISH (multiplex FISH)/SKY (spectral karyotyping)*: Use different fluorescent probes for every single chromosome for *many colors*. Allows for simultaneous visualization of ALL chromosomes!

Be Able to Describe the Roles of the Stomach, Gallbladder, Small Intestine, and Liver in the Solubilization and Absorption of Dietary Fats

*Dietary fatty acids are primarily taken up by adipose or muscle (first dibs!) or transported in plasma by albumin* Mouth: lipase secreted from gland at back of tongue *Stomach* Solubilization: acidic digestion of short chain fatty acids. *Small Intestine* *Solubilization*: (1) Bile acids and lipases (pancreas) work here to hydrolyze fatty acids (2) *Enterocytes* resynthesize TAG and cholesterol esters (fatty acids). Poorly soluble lipid products repackaged into chylomicrons for exocytosis into lymph and deliver to adipose and muscle tissues. *Absorption*: lipids (fatty acids, monoacylglycerol, and cholesterol) absorbed by intestinal mucosal cells (*enterocytes*). *Gall bladder* Solubilization: secretes bile salts made by liver to emulsify fats *Liver* *Solubilization*: (1) bile salts made in liver (2) makes albumin that carries free fatty acids not taken up by adipose and muscle tissues. *Absorption*: Chylomicron remnants rich in cholesterol and phospholipids are taken up by liver through receptor mediated endocytosis - *exclusively uses released glycerol$$* Pancreas - produce lipases that hydrolyze ester bonds of TAGs to form fatty acids and monoacylglycerol

Describe how embryonic stem (ES) cells are isolated & cultured from in vitro fertilization (IVF) blastocysts and by somatic cell nuclear transfer (SCNT).

*ES cells* : from inner cell mass in *blastocyst stage* (not embryo) - Mouse cells are gold standard - Must be able to form *embryoid bodies* (similar to embryo) or can form chimera when put in early embryo *From IVF*: eggs from donor fertilized and allowed to develop in vitro. Fertilized eggs implanted in mother but extra embryos used to harvest ES cells. Inner cell mass can be harvested from blastocyst (30 cell stage) and then cultured. *Somatic Cell Nuclear Transfer* (SCNT) - genetic cloning (dolly); nucleus from somatic cell transferred into enucleated eggs. Early embryo can be used to create ES.

List advantages and disadvantages of embryonic (ES) stem cells

*Embryonic stem cells* *Advantages*: 1) Pluripotent 2) Easily grow large numbers in culture 4) Immortal cell lines 5) Large available resource (IVF clinics) *Disadvantages*: 1) Potenttially cause transplant rejection 2) Not yet determined in human experimentation 3) Limited number of cell lines 4) Must destroy embryos or therapeutic cloning to obtain 5) Opens the door to major ethical debate and consideration 6) Difficult to differentiate uniformly and homogeneously into specific target tissue 7) Some capacity to be tumorgenic

2) Using the Hardy-Weinberg formula, *determine the risk* for *two unrelated parents to have a child that is a carrier or affected with an autosomal recessive disorder when the frequency of individuals affected with the disorder in the population is known.

*Estimate risk for unrelated parents to have affected offspring for autosomal recessive disorder* Example: 1/10,000 in population is affected (aa) *Assume unrelated parents are carriers* Probability parent is carrier= 2pq =0.0198 (1.98%) Probability *child affected*= *(Probability mom carrier) x (Probability dad carrier) x 1/4*= *2pq x 2pq x 1/4* Probability child affected 1/10,200 Probability *child carrier*= (Probability mom carrier) x (Probability dad carrier) x 2/4= *2pq x 2pq x 2/4 Probability child carrier 1/5,100*

Briefly review previous barriers with previous stem cell lines and discuss the new administration position

*Executive order 2007: "Expanding Approved Stem Cell Lines in Ethically Responsible Ways."* - Previous barriers - only 71 lines New order: research on alternative sources of pluripotent stem cells - New administration position - *2009 - EO 13505* - removed barriers to previous executive orders, results in recent guidelines for embryoinic research - New NIH stem cell guidelines July 2001- House of Reps- Therapeutic cloning and human cloning a criminal offense Current problems: possible contamination of cell lines with non-human molecules

Exome Sequencing

*Exome sequencing*/Next generation parallel sequencing - Identify *novel/unknown mutations* - Sequence *all human exons* within single assay in a few hours - Method: fragment DNA, ligate sequences to linkers (linkers = known sequences) on array that is complementary to all exons in human (isolates all exons) → exons eluted and sequenced in machine - *We can get the sequence of all the exons in a patient*

Familial Hypercholesterolemia (Not in LO but important for understanding everything else)

*Familial hypercholesterolemia (FH)* (1:500 people) - two-fold increase in LDL particles from birth, see heart attacks in early 30s - Patients homozygous for familial hypercholesterolemia insensitive to cholesterol as an allosteric inhibitor of HMG CoA reductase - LDL particles bind to surface but not internalized in some patients homozygous for FHH - When LDL is not internalized, cells keep making more LDL while there is a lot of LDL in the blood.

Features of human genome sequence & Gene knock out strategy for identifying gene function

*Features of human genome sequence* - 3.1647 billion base pairs - 25,000 genes - Only 1.5% of genome is coding (G+C rich regions) *Gene knock out strategy for identifying gene function* -disturb normal gene and see what happens phenotypically: 1) Make vector by interrupting gene of interest with antibiotic resistance + negative selection marker. 2) Place in ES cells 3) Cells with homologous recombination will grow in the presence of antibiotic and negative selection marker. 4) Once that cell is found, can be injected into early embryo (mice) 5) *Need to breed for second generation to get rid of chimeric genes* Ex. FgF5 gene in Mice for long hair

Genomic Cloning

*Genomic cloning*: cloning using chromosomal DNA (includes exons and introns so it can be converted to proteins as well) Formation of recombinant DNA molecules: 1) *Restriction endonucleases *- recognize 4,6,8 bp sequences and make restriction fragments with cohesive ends 2) *Ligase* - joins two cohesive restriction fragments into a recombinant molecule Ex: EcoRI GAATTC - EcoRI cuts, ligase repastes them.

What is the basic building block for the formation of TAGs?

*Glycerol = basic building block for formation of TAGs.* Glycerol-phosphate origins: (1) from DHAP during glycolysis (2) Dietary (but can only be used *ONLY IN THE LIVER* since glycerol kinase is only in liver which phosphorylates glycerol)

Hematopoietic stem cells

*Hematopoietic stem cell treatment*: includes bone marrow and stem cells from non-marrow sources (umbilical cord blood) - *Hematopoietic stem cell grafts can repopulate marrow* Process: incomplete destruction of host cells and using graft-versus-leukemia effect to destroy tumor cells Research: - Small molecule (prostaglandin DMPGE) used to to amplify stem cell populations to enhance rates of graft formation - Focus on umbilical cord blood but unfit due to low stem cell count - Transcriptional profiling used to optimize treatment (mRNA gene expression increases show upregulation for pathways involved in proliferation, migration, receptor signaling)

How does insulin regulate fatty acid biosynthesis?

*Insulin activates Acetyl Co-A carboxylase* via protein phosphatase, *upregulating fatty acid biosynthesis* Other roles of insulin: - Insulin enhances availability of extracellular fatty acids to permit adipose tissue uptake and formation of TAGs - Insulin blocks lipolysis through activation of phosphodiesterase to enhance synthesis of fats - Glucagon/adrenaline inhibits fatty acid biosynthesis via activation of cAMP dependent protein kinase

Know Relationships Between Classes of Apolipoproteins and Respective Densities, as Well as Reasons For Differences in Density (i.e., Lipid-to-Protine Ratio) $$$

*Low density lipoproteins* are *LARGER* and have *high lipid-to-protein ratio* (less proteins). *High density lipoproteins* are *SMALLER* and *low lipid-to-protein ratio* (more proteins) *Chylomicrons* - carry *dietary lipids to body cells*, largest *VLDL* - *carry lipids synthesized by liver to body cells*, very large *LDL* - *carry cholesterols around the body*, large *HDL* - absorb *cholesterol from body and return to liver* for breakdown and excretion. Small and highest density of proteins. Albumin - lipoprotein complex, carry fatty acids (cannot pass thru blood-brain barrier so brain cannot use them) *MEMORIZE SLIDE this $$$* - lipids are less dense than proteins

6) List disorders that are transmitted by mitochondrial inheritance and describe how they are transmitted.

*Mitochondrial disorders*: - Mutations in *mitochondrially encoded genes* - Mother to child transmission (*ALL children of affected mother are affected$$$*) - Affected father does not transmit - Mitochondrial genes involved in energy metabolism (OxPhos). Clinical symptoms in muscle, heart, CNS - Number of mutant mitochondria within a cell has to exceed a threshold for disease manifestation *Mitochondrial disorders* 1) *NARP/Leigh disease* 2) *Melas* 3) *MeRRF* - *Homoplasmy*: all mitochondria within a cell have identical sequence (no disease) - *Heteroplasmy*: leads to the disease -> mixture of wild-type & mutant mitochondria within a cell (more mutant mitochondria = phenotypic severity)

Describe how tissue specific stem cells can be used therapeutically (especially hematopoietic stem cells for grafting into bone marrow).$$$

*Only tissue specific stem cells* Tissue Subtypes • *Post-mitotic* (*muscle and neuronal*): non-differentiating nondividing cells soon after birth and contain *few or NO* stem cells. Skeletal muscle contains satellite cells critical for regeneration (not true stem cells). • *Expanding* (*liver, kidney, and thyroid*):undergo an expansion only during juvenile growth and possess *some* stem cells that can generate new cells to repopulate tissues (stem cells typically are not clearly identified). • *Renewal* (*epithelial lining of intestine, stomach, epidermis of skin, testes*, and *hematopoietic system*): *undergo continual replacement* (involving transit amplifying cells that cease dividing in basal layers and then progressively differentiate).

4) Define penetrance, expressivity, locus heterogeneity and mutational heterogeneity in Mendelian disorders.

*Penetrance*: fraction of individuals with *same genotype* that show expected phenotype - Measured across individuals of same genotype not within an individual - Fully penetrant (*always expressed* CFTR) vs. incompletely penetrant (*sometimes expressed*) *Expressivity*: *range of phenotypes* produced by a specific genotype - Phenotype *variable* among individuals of same genotype *Locus heterogeneity*: mutations in *different loci (genes)* produce *same phenotype*/disorder (Retinitis pigmentosa, BRCA1,2,, sensorineural hearing loss) *Mutational heterogeneity*: *different mutations (alleles) in same locus* (gene) produce the *same disorder* (ex. beta thalassemia)

Describe the differences in plasticity of each type of stem cell (e.g., pluripotent vs. multipotent). Be able to distinguish between *tissue specific stem cells* (multipotent), *embryonic stem cells* (pluripotent), and *induced pluripotent stem cells* (iPS cells) which are engineered cells derived from somatic cells that resemble embryonic stem (ES) cells (pluripotent). *$$$ Will be on exam for sure!*

*Pluripotent* - give rise to *all cell types* *Multipotent* - small number of cell types *induced Pluripotent (iPS)*: enginerred cells derived from somatic cells that resemble ES cells. *Advantage*: *perfect immunological match* to donor and treatment for genetic diseases/cancers. *Disadvantage*: tumor formation concern bc of viral gene, silenced genes reactivated later Totipotentcy: potential to develop into whole organism, refers to *fertilized egg at 4 cell stage*

Analyze potential uses for stem cells

*Potential uses of stem cells*: 1) Research on human development 2) Testing new drugs 3) Generation of cells for cell-based therapies (differentiated tissues used to repair damage) Therapeutic cloning: cloning for the isolation of human embryonic stem cells. Injecting nucleus into a somatic cell? - Ethical consideration: It is morally correct to create the precursor of human life simply for destruction?

Define reciprocal and Robertsonian translocations

*Reciprocal translocation*: - breaking & exchange between 2 chromosomes - formation of 2 new derivative chrom (balanced or unbalanced) *Robertsonian translocation*: translocation involving 2 acrocentric chromosomes (chrom with centromere at end) - Familial forms of down syndrome - Acrocentric chrom fuse at centromere and formation of new derivative chromosome (2 chromosomes become 1) - Loss of satellite material from arms of acrocentric chroms *Translocations*: *transfer of genetic material* from one chrom to another, *non-homologous chromosomes involved* - *Translocations involve abnormal pairing and segregation of chromosomes during meiosis & affect daughter cells $$$* Unbalanced: involve loss or gain of chromosome material Balanced: no gain or loss of genetic material

Describe how Robertsonian translocations are associated with heritable forms of Down syndrome

*Robertsonian translocation and association with heritable Down syndrome* - Robertsonian, caused by *translocation* during meiosis and is *inheritable*. - Trisomy 21 = non-disjunction during meiosis, not heritable - Involves *chromosomes 14 and 21* . Ends up with 46 chro with extra copy from 21. - Parent asymptomatic, but due to abnormal segregation during meiosis, the offspring can have trisomy 21 - Normal: separate 14 and 21 - Carrier = 14&21 on one chromosome - Down: 14&21 + extra 21

Sanger Sequencing

*Sanger sequencing* - Used when *mutation is unknown* and identify *novel* mutations - Identify/detect SNPs/point mutations, deletions, insertions - Method: chain terminating nucleotide analogs, size fractionation

Know How Statin Work and Be Able to Describe How Overall Cholesterol Biosynthesis is Controlled Given that Statin-Dependent Decreases in Cholesterol Abundance Upregulate the Transcription of Genes Encoding HMG CoA Reductase and LDL Receptor (Hint: Statins Are Selectively Taken Up By the Liver).

*Statins* = *competitive inhibitors of HMG CoA Reductase* Direct effect of statins: 1) *Inhibits HMG CoA Reductase in liver* thus preventing cholesterol synthesis Important side effect: 2) Decrease in liver cholesterol *upregulates the transcription* of *LDL receptors* to take more cholesterol in *AND HMG CoA Reductase* (gets rid of cholesterol from blood-double whammy!)

Therapeutic cloning, ex vivo and in vivo gene therapy, various methods and vehicles for gene therapy.

*Therapeutic cloning*: use ES cells from blastocyst and differentiated for transplantation *Ex-vivo*: correct defect in patient's cells outside of body and then inject corrected cells (SCID) In-vivo therapy: Directly inject cloned gene Methods for gene therapy - somatic cell correction : gene augmentation, replacement, targeted inhibition or killing Gene therapy by repairing mutant genes/inactivation of pathogenic gene: *Homologous recombination* - mutant gene replaced by normal one *Antisense Oligonucleotides* - inactivate disease causing gene *Ribozyme* - mRNA transcript of disease degraded RNA interference- mRNA of target degraded Vehicles for gene therapy: viruses (adenovirus, lentivirus), nonviral (liposomes, injection, receptor mediated)

Discuss different types of stem cells

*Totipotent*- produced at the fusion of an egg and sperm. Can differentiate into embryonic and extraembryonic cell types *Pluripotent*- can differentiate into cells from any of the three germ layers *Multipotent*- can only produce cells of a closely related family (hematopoietic stem cells can produce different types of blood cells) *Unipotent*-can only produce one cell line, but have the property for self-renewal Desired qualities: 1) Must be pluripotent (able to differentiated into all 3 primary germ layers) 2) Must be genetically stable 3) Long-lived and self-renewing

5) Describe how triplet repeat disorders are transmitted and define anticipation.

*Triplet repeat disorders* = disorders that result because of an increase (expansion) in the number of trinucleotide repeats in a gene (in successive generations) - Occurs during gametogenesis & expanded # of repeats transmitted to offspring *Anticipation*= *the higher number the # of repeats, the earlier the onset and severity.* *ANTICIPATION = TRINUCLEOTIDE REPEAT DISORDERS!!!$$$* - Possible mech: slippage by DNA polymerase during replication in gametes. Ex. Huntington disease (CAG) Spinocerebellar ataxia type I (CAG) Machado-Joseph disease (CAG) Friedrich ataxia (CAA) Fragile X syndrome (CGG) Myotonic dystrophy (CAG)

7) Define multifactorial inheritance, liability and risk.

*Vast majority of disorders are multifactorial* Multifactorial inheritance = *a lot of genes (alleles at multiple loci) cause a single disorder*. *Multifactorial = polygenic disorders* *Interplay of genes x enviro* 1) *Monogenic/single gene*: disorders & traits involve alleles at single locus (NOT MULTIFACTORIAL) - 3 possible genotypes that produce 3 distinct phenotypes in population - Finite number of phenotypes always 2) *Polygenic*= *disorders & traits involve alleles at multiple loci*; Genes have additive effect on phenotype - *Many possible genotypes result in many phenotypes* following continuous distribution - Genes + environment interaction results in even greater phenotypic diversity

Molecular defects/deficiencies in autosomal and X-linked SCID, and gene therapy of these disorders.

*X-linked SCID* (Bubble boy disease) - Most common - X-SCID due to *IL2RG* gene mutation and *IL2R deficiency* - Defect in T and B lymphocytes - Bone marrow CD34+ cells treated ex vivo and reinserted *Autosomal recessive SCID* - Due to *adenosine deaminase* deficiency Somatic cell correction techniques: *Gene augmentation*- introduce functional copy: for loss-of function diseases *Gene replacement (in situ correction)*- for gain-of function diseases *Targeted inhibition* of activated oncogenes or pathogens *Targeted killing*- cancer treatments.

*X-linked dominant (X^X, X^Y)* Pedigree & Punnett Square

*X-linked dominant (X^X, X^Y)* - No male or female carriers - XX, XY equal - Mother transmits to daughters & sons - *Father transmits only to daughters* - Every generation affected

*X-linked recessive* Pedigree & Punnett Square

*X-linked recessive* - Males always affected if they have allele - Unaffected males do not transmit - Carrier women transmit to affected sons - Unaffected males do not transmit - Carrier women transmit to affected sons - *All daughters of affected males are carriers*

Determine the chromosomal aberration when given the karyotype of an individual.

*Xq12* - chromosome #, Arm, Region, Band - *46, XY, del(4)(p14) = Total number of diploid chromosomes, sex, specific chromosome, area deleted* (should understand this...highly likely to be on test) $$$ - 2 *arms* (p,q) - 2 *regions* for each arm - Many *bands* in one region - Many *sub-bands* in one band Cytogenic abnormalities - *del*: deletion - *dup*: duplication - *inv*: inversion - *t*: translocation - *ter*: terminal (pter/qter) Examples: Turner syndrome 45,X Klinefelter syndrome 47, XXY Trisomy 21 47, XX, +21 Trisomy 18 47, XY, +18

cDNA library & cDNA cloning

*cDNA library*: collection of many clones of a specific fragment/fragments of cDNAs (converted fromo RNA via reverse transcriptase) which have been ligated into a vector. Can make multiple copies (clone) and use them. *mRNA + reverse transcriptase + oligo dT + nucleotides + appropriate buffer* Process: (1) PolyT primer (Oligo dt) to polyA tail of mRNA (2) Once hybridized, put in reverse transcriptase (primer, enzyme, RNA independent) (3) Degrade RNA with RNase H (4) Put in polymerase to complete the DNA strand using leftovers of the RNA primers from the previous degradation

What does fatty acid synthase make and how?

- *Fatty acid synthase* (2 active sites) *makes palmitate 16:0* *Fatty acid (TAG) synthesis is the creation of fatty acids from acetyl-CoA and malonyl-CoA via fatty acid synthase* - 2 active sites which involve *priming with acetyl-CoA* and *condensation with malonyl-CoA* to make *palmitate* - *Fatty acid synthesis occurs in cytosol and uses NADPH (not NADH)* while beta oxidation of fatty acids occur in mitochondrial matrix. A) *Priming rxn*: Acetyl CoA primes enzyme (fatty acid synthase with thiol groups) B) *Decarboxylation of Malonyl* CoA lets it condense with initial fatty acid synthase/acetyl CoA, growing the fatty acyl chain by two carbons C) *Condensation*: ketone reduction, dehydration, reduction D) Cleavage by Palmitoyl thiesterase +further elongation and desaturation in Smooth ER

Understand Central Role of the Liver in Regulating Cholesterol Availability

- *Liver takes up cholesterol in apolipoprotein particles (HDL)* - HMG CoA reductase is localized to liver and is the rate limiting step of cholesterol biosynthesis - *However it is the last to receive chylomicrons. First two are adipose tissue and muscles.* - Therapeutic targeting of statins is directed against HMG CoA reductase in liver.

Describe how ethanol inhibits gluconeogenesis.

- Alcohol induced hypoglycemia - Ethanol inhibits gluconeogenesis! - Why: Alcohol metabolism required alcohol dehydrogenase which *USES NAD+* to metabolize ethanol to acetaldehyde, thus *there won't be enough NAD+ to convert malate back to oxaloacetate* Vitamin B3 precursor for NAD+, will it work? probably not fast enough lol

How many fragments are produced from each?

- Dideoxy A will truncate at Ts, Cs on Gs, Gs on Cs, and Ts on As - Ribose has 2' as well as 3' position occupied with OH. - Deoxyribose (dATP) has only 3' positions occupied with OH. - Dideoxyribose (ddATP) has no OH at 2' and 3' positions. Clockwise: 1) 2 fragments 2) 1 fragment 3) 1 fragment 4) 2 fragments

Importance of Hardy-Weinberg equilibrium for risk determination in Mendelian diseases.

- Frequency of first generation = *p2 (AA); (aa) q2; 2pq (Aa)* *Affected child's parent producing another affected* = *Parent 1 carrier risk x other person's carrier risk x 1/4* = 1 x 1/50 x ¼ = 1/200 (from previous case) - ¼ bc this is a recessive disease Dominant = ½ chance

Understand Mechanism of LDL-Receptor in Cholesterol Uptake

- Process: LDL receptor binds to LDL, coated by clathrin, endocytosed and becomes lysosome. Becomes cholesterol and amino acids after degradation (LDL receptor also degraded in the process) - A lot of cholesterol -> LDL receptors downregulated to prevent any more cholesterols taken in -- In presence of ACAT, more cholesterols made (cholesterol Acyl Transferase - stabilizes intracell cholesterol)

Recombination as a tool for determining location of a gene, different types of genetic disorders, and patterns of inheritance in pedigrees.

- Recombination is an important tool for locating the position of genes since the frequency of recombination is related to where the gene is Example in picture: - *No recombination* = *AB, ab* inherited together - *Recombination* = *Ab and aB* inherited together Gene Mapping: - Is disease traveling with marker gene? - How close or far is the disease gene from marker? - estimated by frequency of recombinations - *Infrequent (less chance) recombinations* = *shorter distance* - *Frequent (more chance) recombinations* = *longer distance* Ex. 100 individuals, 20 meioses w/ recombinants. 20/100 = *20% recombinant fraction = 20cM (centi morgans)* 1 cM = 1 million base pairs

Understand the Physical Reasons for Using Chylomicrons as Transport Vehicles for Dietary Fats.

- Repackaging dietary fats into chylomicrons *enhance solubility* and thus is easier to transport 1) Hydrolysis (removal) of TAG fatty aids increase their solubility 2) Bile salts made in liver, secreted by gall bladder emulsify TAGs 3) Fats absorbed 4) Fatty acids re-esterified and *repackaged into chylomicrons* before being to be transported back to *lymph → blood → adipose tissues and muscle → liver*

Be Able to Describe Role of CoA-SH Thioester Bond in Activation of FA for Biosynthesis.

- The CoA-SH *thioester bond* allows the *transfer of acetyl-CoA upon fatty acid synthase energetically favorable* which is the *priming step*? of fatty acid synthesis. Steps 1) Activation of FA by CoA-SH 2) Sequential condensation at positions 1, 2, 3, 4 - Each time what is driving the rxn is the cleaving of the thiol-ester bond (12) Released fatty acids carried by albumin in blood to tissues

The engineering of a therapeutic construct in relation to various viral genes and packaging cells.

- Viral genes deleted to prevent infectivity - *Viral proteins provided by helper cell* Gene transfer by retroviral vectors: - By *substituting* *gag, pol, env* sequences with the *therapeutic gene*, virus is rendered noninfectious - *Psi Ψ sequences* are *retained* - important for packaging of viral particles

Be Able to Describe the Degradative Route and Apolipoprotein Carriers Used to Move Dietary TAG to Adipose and Muscle Tissue.

1) *TAGs must be hydrolyzed* to fatty acids or mono/diacylglcerols *to become soluble in water for absorption* 2) After absorption, *condensation rxns reform TAGs and transport fats to adipose tissues and muscle for storage and use* Bile acids solubilize dietary fats → chylomicrons → lymph → blood → tissues. Apolipoprotein carriers: *Chylomicrons, VLDL, LDL, HDL, Albumin* Dietary lipids = TAGs, phospholipids, cholesterol (90% is TAG)

9) Describe the two uses of SNPs in molecular medicine.

1) SNPs can *serve as markers for association studies* to identify disease genes. This can happen esp if they are linked close to certain genes. ex. Allele 1 of SNPX in linkage with disease gene Y. Allele 2 linkage with WT gene Y. Tells us which one is the culprit. 2) *Can be causative agents for disease or causes of non-disease phenotypic variation* (diff in drug metabolism, drug responses) - SNPs are bi-allelic (having 2 alleles) with a major one (found at higher frequencies) and a minor - Can find linkage SNPs via genome-wide association studies (scans SNPs in entire genome)

Analyze Federal directives that regulate stem cell research (previous and current administration)

2001- Federal funds given if stem cells are from before aug 2001 - Must have been derived from from reproductive purposes and embryo no longer needed - Informed consent - Only 71 cell lines

Describe the chromosomal abnormalities and main symptoms associated Down, Patau, Edwards, Turner and Klinefelter syndromes.

3 Vital autosomal trisomies: - correlations with advanced maternal age *Down (47, XX/XY, +21)* - craniofacial, single palmar crease, septal defects, patent ductus arteriosus, mental retardation (Iq 45), short stature. Increase in non-disjunction during meiosis in older women. 50-60 years. 1/800 *Patau (47, XX/XY, +13)* - seizures, microcephaly, polydactyly, cryptorchidism, serious congenital heart defects, cleft lip and palate, holoprocencephaly (brain does not divide into two hemispheres). Death by 1 month. 1/5000 - Patau = unlucky 13 *Edwards (47, XX/XY, +18)* - Micrognathia (small jaw), micropthalmia (small eyes), intrauterine growth retardation, clenced feet. Death in 1 year. 1/5000 - Edward dated Bella who was 18 years old Sex chromosomes: *Turner (45, X/X0)* - only one X chromosome. Short stature metatarsals, widely spaced niipples and shield-like chest, webbing skin at neck, sterile, no secondary sexual characteristics. Increased incidence of DM and inflammatory bowel disease. Detection by ultrasound in 2nd trimester as nuchal cyst or edema. 1/2500 *Klinefelter syndromes (47, XXY)* - female body shape, breasts, infertile, increased risk of osteoporosis and breast cancer. IQ and life expectancy same as general pop. 1/1000 Screening: MaterniT21 or Panorama testing, triple serum screen, amniocentesis, karyotyping Changes in chromosome # = ploidy Changes in chromosome structure = translocations/deletions/duplications/inversions (ex. criduchat)

List the precursors for gluconeogenesis & describe how they are generated.

3 different precursors*: 1) *Lactate* - from *anaerobic glycolysis in exercising muscle* and *RBCs* "Cori Cycle". 28% 2) *Glycerol* - from *lipolysis in adipose tissues of triacylglycerides* - *TAGS* → glycerol + fatty acids. 12% 3) *Aminoacids* - *major source* of glucose from *degradation of muscle protein* due to drop in insulin levels. 60%

Describe how the different precursors enter the gluconeogenic pathway.

3 ways to enter gluconeogenic pathway: DHAP, Pyruvate, Oxaloacetate Precursors: 1) *Lactate → pyruvate* (lactate dehydrogenase) 2) *Glycerol → DHAP* - Glycerol → Glycerol-3P (by *glycerol kinase* - only in liver) → Dihodroxyacetone phosphate "*DHAP*" (by *glycerol phosphate dehydrogenase) 3) Glucogenic aminoacids → TCA cycle intermediates → oxaloacetate (OR) Glucogenic amino acids → pyruvate

α-thallasemia (hemoglobinopathy)

3) *Hemoglobinopathy (a-thalassemia)* - Normal *Chromosome 16* has 2 copies of normal a-globin gene, pseudo a-globin, and repetitive DNA. - *Unequal crossovers* between *misaligned X or Z-repeat* produce chromosomes with 1-3 active α or no active α-globin genes. *More 4 α-globin = good!* Memorize: - *4 α-globin present = normal phenotype* - 3 active - silent - 2 active - α-thallasemia - 1 alpha gene = HbH disease serious - NO alpha genes= hydrops fetalis , non viable!!!!

Understand Role of Acetyl CoA Carboxylase in Regulating Fatty Acid Biosynthesis, and the Function Role of Malonyl CoA.

A lot of ATP (and insulin) will activate *Acetyl Co-A carboxylase* so that: *Acetyl-CoA → Malonyl-CoA* via *Biotin* carboxylation. *Functional role of Malonyl-CoA*: Needed for elongation of fatty acids How: *Decarboxylation of Malonyl CoA extends growing fatty acyl chain by two carbons* - Thioester bond in malonyl CoA maintained following transfer to fatty acid synthase

Know the Names of the Ketone Bodies and Understand Why Fatty Acids Cannot be Used by the Brain in the Absence of Glucose.

Acetyl-CoA = two carbon thiol ester leads to *3 ketone bodies*: 1) *Acetone* 2) *Acetoacetic acid* 3) *β-OH-butyrate* Fatty acids = TAGs, cholesteryl esters, and phospholipids Free fatty acid cannot be used by the brain because it is not actually free but bound to albumin and cannot cross the blood-brain barrier. - Also cannot be taken up by RBCs bc they don't have mitos.

Understand the Advantages of Storing Energy in the Form of Fats, As Opposed to the More Readily Available Carbohydrate Form of Energy Storage.

Advantages of storing energy in fat: - *More energy per unit* because it is so reduced, *Very light*, less hydrated Disadvantages: - Problems w/ transport bc not easily soluble like glycogen - Takes longer to mobilize - Fats store 6 times more energy per unit, carbs weigh a lot more

1) Using the Hardy-Weinberg formula, *determine the carrier frequency* for an *autosomal recessive disorder* when the frequency of individuals affected with the disorder in the population is known.

Alleles A & a *p*= frequency of A (0-1) *q*= frequency of a (0-1) *p+q=1* Genotype frequencies AA = *p²* Aa = *2pq* aa= *q²* Example: If incidence of disease in population is 1/10,000 then estimate carrier frequency 1/10,000 in population is affected (aa) *aa*= *q²*= 1/10,000 *q*= 1/100 *p*= *1-q*= 99/100 Aa= *2pq*= 2 x (99/100) x (1/100) = 0.0198 *Carrier frequency is 1.98% (appr. 1:50)*

Describe how the energy required for gluconeogenesis is supplied.

Anabolic process that requires ATP, GTP, NADH Source of ATP for gluconeogenesis: *FATTY ACID OXIDATION*!!! - Lipolysis: fatty acids generate lots and lots of ATP and used as fuel - Fatty acid generation also produces NADH *Remember, ATP cannot be provided from glycolysis during fasting/starvation!!* Place where energy is used (each occurs twice x2): 1) Pyruvate carboxylase - 2 ATP 2) PEPCK - 2 GTP 3) Phosphoglycerate kinase - 2 ATP 4) G3p-dehydrogenase - 2 NADH Total: *4 ADP, 2 GDP, 2 NAD+* generated NB: Ketone bodies, fatty acids, and AcCoA *cannot* be used to generate glucose

Be Able to Articulate How Bile Salts Emulsify Fats, and Aid is Subsequent Absorption of Fatty Acids by Intestinal Mucosal Cells.

Bile salts emulsifying fats to smaller droplets to increase surface area for absorption Mechanism: Bile salts (*cholic acid, deoxycholic acid*) have hydrophobic sides and hydrophilic sides. The hydrophobic sites interact with the fat and the hydrophilic sites interact with water - The is *not digestion/chemical process, just physical process$$$*

Be Able to Indicate the Sites of Action and Role of the Hormones Cholecystokinin and Secretin in Aiding Digestion of Dietary Fats.

Both secreted in the presence of lipids in upper SI by intestinal mucosa: *Cholecystokinin* Site of action: stomach, pancreas, gall bladder Functions: (1) decrease gastric mobility (2) stimulates bile release by gall bladder (3) activates release of pancreatic enzymes $$$ *Secretin* Site of action: Circulation & intestinal lumen Functions: (1) Causes pancreas and liver to release bicarbonate solution to neutralize pH of partially digested food (chyme) in intestine $$$

Frequency calculations for genotypes.

Calculating gene frequency: *p2 (AA); (aa) q2; 2pq (Aa)* Example: Autosomal recessive Incidence = 1/10,000 p = normal, q = mutant q^2 = 1/10,000 Therefore: q gene = 1/100 individuals , p=99/100 Carrier frequency = 2pq = 1/50

Be Able to Describe What Role Citrate Plays in Fatty Acid Biosynthesis, and the Role of Cytosolic Enzymes.

Citrate is the only means by which Acetyl-CoA can cross the mitochondrial membrane into cytosol *Acetyl-CoA + OAA → Citrate* (Citrate synthase) - After cross - *Fatty acid biosynth occurs primarily in liver cytosol* Key point: - *Excess [Citrate] only when cell energy needs are met* (fed state, lots of ATP, inhibition of isocitrate dehydrogenase) - *The requirement of Acetyl-CoA to become Citrate for transport for FA synthesis is in itself the regulatory mechanism $$$*

Understand importance of ES cells in research (role in creation of genetically modified mice).

ES cells can receive modified DNA and be inserted into early embryo to show that phenotype. First generation will be a chimeric though so need to breed to get rid of those chimeric genes. - *Knockouts*: use homologous recombination to inactivate genes - *Knock-ins*: insertion of genes (ex. - Modifies mouse genome in vitro and introduce changes into animals - Use of transgenic animals to isolate therapeutic agents

Describe how AcCoA coordinately regulates PDH & Pyruvate Carboxylase in the liver & what the outcome of this regulation is.

Excess *AcCoA* during fasting/starvation from lipolysis regulates both PDH and Pyruvate carboxylase to ENABLE gluconeogenesis: - *Inhibits PDH* → diverts pyruvate away from TCA cycle - *Activates Pyruvate Carboxylase* → lets pyruvate be used for glucose synthesis - PDH during fed state -> ATP - Pyruvate carboxylase during fasting/starvation -> glucose

Understand differences between existing (proven) stem cell treatments and aspirational stem cell therapies.

Existing therapies (tissue specific stem cells): bone marrow transfer, IVF Aspirational treatments: - *Diabetes*: form differentiated beta cells and introduced into hepatic portal vein to lodge in liver (not applicable to type I DM). Slow progress bc existing therapies are largely effective. - *Parkinson's Disease* - neurons must be replaced in proper context at position (must form neuronal connections). May form teratomas. Alternative therapies like L-DOPA and deep brain stimulation may prove more effective. - *Spinal repair* - remyelinating cells to treat spinal trauma. tumor risks. - *Vision (Macular degeneration*) $ - recent clinical trial , *immunoprivilaged nature of eye* avoids issues of tolerance to foreign antigens. Low doses possible due to local focus of disease. (Currently only in stage of study to determine safety - first evidence of *medium* safety) - 150 uL injected into subretinal space and delivered to area of atrophic photoreceptor pigment epithelium -> small improvement in visual acuity (many people not convinced)

Understand the Steps Associated with the Activation of Fatty Acids for TAG Biosynthesis

Fatty acids must be activated for TAG biosynthesis via: 1) Formation of thioester bond with CoA (CoA-SH) Acetyl-CoA = two carbon thiol ester Next: 2) Condensation of activated fatty acids 3) Hydrolysis of the phosphoester bond via phosphatase

Sequencing principle and use of dideoxy nucleotides, features of human genome sequence and gene knock out strategy for identifying gene function.

Gene sequencing: *In vitro synthesis* in the presence of *chain terminators* (PCR with truncating ddATPs) *Need both normal dATPs for elongation and labeled ddATPs for truncation* Process: 1) Denaturation of DNA 2) Spike solution with small amount of ddATP and mostly normal dATP 3) Regular incorporation of dATP or *random truncation at every position upon incorporation of ddATP* 4) DNA fragments separated by gel electrophoresis and can use fluorescently labeled ddATP to determine sequence

List the reactions that encompass the three bypasses of gluconeogenesis and the enzymes that catalyze them.

Gluconeogenesis = pyruvate → oxaloacetate → PEP →→glucose *Bypass 1*: Glycolysis: Phosphoenolpyruvate (PEP)→ Pyruvate (Pyruvate kinase) (How?) *Gluconeogenesis: Pyruvate → Oxaloacetate → PEP* In *Mitochondria*: *Pyruvate → Oxaloacetate* (Pyruvate carboxylase), requires *biotin* as cofactor - also an anapleurotic rxn for TCA cycle In Cytoplasm: *Oxaloacetate → Phosphoenolpyruvate* (PEP carboxykinase "*PEPCK*". Requires GTP. *Bypass 2*: Glycoslysis: Fructose-6-P → Fructose-1,6-BP (PFK-1) (How?) Gluconeogenesis: *Fructose-1,6-BP → Fructose-6P* (*F-1,6-Bpase*) *Bypass 3*: Glycolysis: Glucose → Glucose-6P (Glucokinase) - slowest step (How?) Gluconeogenesis: *Glucose 6P → Glucose (Glucose-6 phosphatase)* NP: - Glucose-6P only present in the liver and extremely important!!! That's why we can't use it in muscles - *Between Bypass 1 & 2*: A lot of reversible rxns glycolysis. Importantly, *ATP hydrolysis + NADH used up* - DHAP enters at around bypass 2 as well

Gluconeogenesis summary

Gluconeogenesis is NOT glycolysis in reverse Glut 1-4: glucose transportation Glut 2: transport glucose from liver into blood Pyruvate gets converted to lactate when it idsn't onverted fast enough Cori Cycle - glucose to lactate (muscle/rbcs) - lactate to glucose (liver) Gluconeogenesis = pyruvate -> oxaloacetate -> glucose Ketone bodies, fatty acids, and AcCoA are not glucogenic (They can't enger gluconeogenesis) That's why these guys are used instead to make ATP - No direct route from acetyl CoA to pyruvate

Describe how gluconeogenesis is stimulated in diabetes mellitus.

Gluconeogenesis is stimulated in DM because insulin fails to repress PEPCK In Diabetes: *insulin resistance or lack of insulin* → *lose repression of PEPCK* →*Gluconeogenesis activated* Thus Gluconeogensis is stimulated and glucose is produced contributing to hyperglycemia - Diabetes is characterized by hyperglycemia Worse: Liver producing glucose when there is already glucose in blood - New meds: inhibitors of F-1,6-BPase

Know that HMG CoA Reductase Catalyzes the Rate Limiting Step in Cholesterol Biosynthesis from Acetyl CoA.

HMG CoA reductase catalyzes: *HMG-CoA → Mevaolonic acid* Low cholesterol levels upregulate HMG-CoA reductase High cholesterol levels reduce HMG-CoA reductase transcription - First committed step of the pathway and highly regulated (rate limiting step) - Controls cholesterol synthesis - Target of statins

Be Able to Describe the Regulation of HMG CoA Activity in Cells (i.e., Feedback Inhibition by Cholesterol and Transcriptional Regulation by Sterol Regulatory Element Binding Protein (SREBP).

HMG CoA regulated by: 1) Cholesterol (feedback inhibition) 2) Transcriptional regulation In the presence of cholesterol: - *Cholesterol is a feedback inhibitor of HMG CoA reductase* but that first requires LDL cholesterol to be internalized via receptors - *Delivery of cholesterol to golgi transcriptionally downregulates HMG CoA reductase and LDL receptor*. - Patients with *familial hypercholesterolemia* *do not inhibit HMG CoA Reductase with extracellular LDL* In the absence of cholesterol: - *Coordinated transcriptional regulation of LDL receptors*: 1) SREPB protein (sterol regulatory element binding protein) transported to golgi and cleaved into SCAB 2) Activates SREBP (transcription factor) in nucleus which upregulates LDL receptor protein, HMG CoA reductase, and genes encoding steps in cholesterol biosynth.

8) Define Single Nucleotide Polymorphism (SNP), and describe the different possible effects of SNPs on protein function.

Humans are 99.9% similar 0.1% different (sequence variation) and the most frequent variation are SNPs *SNPs* = most prevalent sequence variation - Single bp changes & bi-allelic (major & minor alleles) - The SNPs that occur in genic regions are the ones that have potential to cause disease *Different possible effects of SNPs on protein function*: - *Synonymous cSNPs* = SNPs that do not change the amino acid (occur in 3rd codon position, redundancy). - *Non-synonymous cSNPs* = coding SNPs that change amino acid

Understand the Requirements for Bacterial Metabolism in the Intestine to Maintain the Solubility of Bile Salts.

Important role of intestinal bacteria: *recycling of primary bile salts into secondary bile salts to promote their reuptake* (99% recycled) - Bile salts are conjugated by liver (by glycine or taurine) to enhance solubility in acidic duodenum - *Bacteria remove the conjugates of bile salts after their work is done into secondary bile salts in colon to promote reuptake by liver* - *Gut microbiome metabolizes carnitine and choline to form timethylamine* (and methane) Carnitine important for fatty acid transport into tissues.

Be Able to Calculate How Many ATP are Formed for Each Round of Beta Oxidation and for the Complete Oxidation of Palmitate. $$$

Investment phase: 2 ATP used (Fatty acid activation by Fatty Acyl CoA synthetase consumes 2 ATP equivalents) (Missing slide after 15) Palmitate = 16 carbons and each round takes off 2 carbons One round of TCA cycle = 12 ATP FADH2 = 2 ATP NADH = 3 ATP = *17 ATP each round*- but last round only splits it (no FADH2 and NADH created) so total 8 rounds of creating Acetyl Co-A and 7 rounds of extra FADH2/NADH 16 carbons = 7 rounds of of beta oxidation (bc the final bond cleavage yields 2 Acetyl-Coa?) Net ATP per palmitate = 129 ATP (131 total) (16) How many ATPs created from 3 rounds? (possible question on test)$$$ 17 x 3 = 51 ATP GO THROUGH SLIDE 17, REWATCH IF NEEDED , need to understand this

Linkage, LOD score, and the utility of recombinant fraction for disease gene mapping.

Linkage: gene linked to a specific chromosome How to find chromosome location = *determine mode of inheritance (pedigree analysis) -> determine linkage of gene to specific chromosome via linkage mapping using polymorphic markers* Process: - *Use restriction fragment length polymorphism (RFLP) markers that are digested with specific restriction enzymes*. The person with a mutation would have a different fragment length from normal people. In the case of Neurofibromatosis, it was the 4.7kb fragment. Ex. Neurofibromatosis (von Reclinghausen) = chromosome 17 linked - Autosomal dominant - Everyone affected had the 4.7 kb fragment so it tells that the disease is traveling with the 4.7 kb fragment LOD score: ratio of likelihoods If ratio is 3, odds are 1000 times in favor of linkage Log 1000 =3 *LOD more than 3* = loci linked *LOD less than 3* = loci not linked

10) Describe how Southern blots, microarray hybridization, Sanger sequencing, exome sequencing, PCR-RFLP and ARMS/ASP PCR can detect or identify changes in DNA sequence.

Overall: Patient sample → Purification of *genomic DNA* → Assay for mutation → Collect and interpret *Sanger sequencing* - Used when *mutation is unknown* and identify *novel* mutations - Identify/detect SNPs/point mutations, deletions, insertions *Exome sequencing*/Next generation parallel sequencing - Identify *novel mutations* - Sequence *all human exons* within single assay in a few hours - *We can get the sequence of all the exons in a patient* *Microarray hybridization* - *Novel* and *known* mutations - *Screen entire genome in single assay* *Southern Blot* - Mutation *must be known* - Detecting *larger rearrangements* *PCR Amplification* - Generally used in conjunc with other assays - Rapidly amplify desired segment of patient's DNA → high yield amplicon A) *PCR-RFLP* - Detect *known* mutations only, takes advantage of SNPs affected by restriction enzymes B) *ARMS PCR* - used exclusively for *known* mutations. - It uses primers instead of restriction enzymes - will only get PCR amplification if patient has allele specific to primer (no mismatch at 3' end)

Describe how oxaloacetate produced in the mitochondria by pyruvate carboxylase is exported into the cytoplasm to complete the reactions of bypass 1.

Oxaloacetate can't be transported out of mitochondria directly during bypass 1 *2 step transport*: 1) *Oxaloacetate -> malate* (mitochondrial Malate dehydrogenase) Malate transported into cytoplasm 2) *Malate -> Oxaloacetate* (cytoplasmic Malate dehydrogenase) *Requires NAD+*!!! - alcohol affects this

Be Able to Describe the Structures of Palmitic Acid, Stearic Acid, Oleic Acid, and Arachidonic Acid.

PSOA 16-18-18-20 *Palmitic acid = 16:0* (16 C, 0 double bonds) *Stearic acid = 18:0* *Oleic acid = 18: 1(9)* (18 C, 1 double bond at position 9) *Arachidonic acid = 20:4 (5, 8, 11, 14)* - important signaling molecule and products involved in inflammatory responses Number before colon = length Number after colon = # double bonds (Parenthesis) = positions of unsaturation Saturated = no double bonds (long shelf lives bc they can't be oxidized/hydrolyzed - margarine) Unsaturated = double bonds present with kinks

Briefly review potential alternative sources for embryonic stem cells

Potential alternative sources of human pluripotent stem cells 1) Dead embryos 2) Living embryos via non-destructive biopsy 3) Bioengineered embryo-like artifacts 4) Reprogrammed adult somatic cells

Describe how insulin & glucagon reciprocally regulate glycolysis & gluconeogenesis via the allosteric effector fructose-2,6-biphosphate and by enzyme induction.

Reciprocal regulation of glycolysis & gluconeogenesis is via: 1) F-2,6-Biphosphate via the bi-functional enzyme PFK2+F-2,6-BPase 2) PEPCK *F-2,6-BP* turns *PFK-1 ON* (allosteric activator) *allowing glycolysis* AND *Fructose-1,6-BPase OFF* (allosteric inhibitor) *inhibiting glucogenesis* *To allow gluconeogenesis is to remove F-2,6-BP via Glucagon and inducing PEPCK* *Enzyme Induction* *Fasting/Starvation state*$$: *Glucagon* → stimulates Protein Kinase A → *Turns ON Fructose-2,6-Bpase* → Removes F-2,6-BP 1) Inactivates PFK-1 → *Inhibits glycolysis* 2) Activates F-1,6-BPase → *Allows Gluconeogenesis* - PKA also turns OFF PFK-2 which prevents the formation of F-2,6-BP (double whammy) - *Glucagon induces PEPCK turning gluconeogenesis ON* *Fed state*: *Insulin* turns PFK-2 ON (glycolysis on), F-2,6-Bpase OFF via Protein phosphatase (gluconeogenesis & Glycogenolysis off) - *Insulin represses PEPCK, turning gluconeogenesis OFF*

Recombination as a cause of diseases such as leukemia, lymphoma and a-thalassemia, and for generating antibody diversity. $$$

Recombination = reciprocal exchange of DNA sequences during gametogenesis - recombination can also lead to deleterious effects CML → Translocation ABL(9) + BCR (22) = Phil chrom→ ABL oncogene active [Gleevec] Burkitt → Translocation MYC(8) to IGH (14-very active location) → MYC overexpression a-thalassemia → Chrom 16 unequal crossovers X-Z repeats → 5 phenotypes (4-0: normal→carrier→a-thalassemia→HbH→Hydrops fetalis) Recombination of genes/enzymes (light/heavy chains) → Antibody diversity in B-cells (10^15) 1) *Chronic myeloid leukemia* - Patients have Philadelphia chromosome Cause: *Translocation* joins *ABL* (Chrom *9*) and 5' part of *BCR* gene (Chrom *22*) - *ABL kinase* becomes *constitutively active* after translocation (leads to *overexpression of oncogene products*) - *Gleevec = drug used to treat CML is an inhibitor of ABL!!!* - 2) *Burkitt's lymphoma* Cause: *MYC oncogene* (Chrom *8*) is *translocated* to *IGH* locus (Chrom *14*) - Myc gene placed in chromatin domain that is *highly active in B-cells*, the antibody producing cells (IGH locus) -> causes *MYC oncogene to be perpetually active* 4) *Antibody diversity* - chromosomal recombination leads to antibody diversity in B-cells - 10^15 different antibodies - Light chain - VJC - Heavy chain - VDJC (can generate large numbers of permutations and combinations)

Describe how gluconeogenesis & glycolysis are reciprocally regulated by energy charge.

Remember this is in the LIVER! 1) *Low energy charge* A) Gluconeogenesis *OFF* (requires ATP) glycolysis *ON* (to generate ATP) B) (ADP & AMP) -> PEPCK & PC & Fructose-1,6-Bpase *OFF* PFK-1 *ON* 2) *High energy charge* A) Glycolysis *OFF* (sufficient ATP) gluconeogenesis *ON* (requires ATP) B) ATP -> Pyruvate Kinase & PFK-1 *OFF* NB: Fructose-2,6-BP is allosteric effector for: PFK-1 *ON* Fructose-1,6-Biphosphatase *OFF*

Unique symptoms of Trisomies 18, 13, 21 $$$

Some symptoms are common among the 3 trisomies (mental retardation) and some are shared among 2 of the trisomies (ex. micrognathia in trisomies 13 & 18). However certain sets of symptoms are unique to each trisomy such as the *Trisomy 18* = *rocker bottom feet* *Trisomy 13* = *polydactyly* *Trisomy 21* = characteristic facial structure (brachycephaly, epicanthic folds, upward sloping palpebral fissure​) The presence of these unique symptoms should allow you to differentiate between the 3 trisomies in a patient.

Hybridization applications for Southern, northern and western blotting

Steps for all kinds of hybridization = *denaturation, probe hybridization, renaturation, complementary strand annealing* *Southern hybridization*: gene structure, making genomic maps of DNA - digest DNA, electrophoresis, and hybridizing with fluorescent gene specific probes (probing target DNA with fragment which can be rna or dna) *Northern hybridization* - *determines abundance of specific mRNA* - target mRNA is hybridized to gene-specific RNA or DNA *Western blot* - *determine protein expression*. - Proteins separated by electrophoresis, transferred onto membrane, *antibody* binding, *color development*.

Summary

Sums up ethics: "For those who believe that the embryo has the moral status of a person from the moment of conception, research or any other activity that would destroy it is wrong. For those who believe the human embryo deserves some measure of respect, but disagree that the respect due should equal that given to a fully formed human, it could be considered immoral not to use embryos that would otherwise be destroyed to develop potential cures for disease affecting millions of people. An additional concern related to public policy is whether federal funds should be used for research that some Americans find unethical."

Understand the Correlation Between Markers of Cardiovascular Disease Trimethylamine (TMA) and the Presence of Carnitine and Choline in Diet.

Timethylamine (TMA) & TMAO (trimethylamine-N-oxide) = markers linked as risk factors for heart disease Carnitine = from red meats Choline = dietary fat Gut microbiome metabolizes: (1) *Choline* → *TMA*/*TMAO* (2) *Carnitine* → *TMA*/*TMAO* - *Choline in diet correlates with cardiovascular disease and trimethylamine oxide* - *Dietary carnitine accelerates atherosclerosis* - Mechanism= *TMAO results (1) less cholesterol absorption, (2) large reduction in bile acid pool (3) increasd macrophage foam cell formation (contributes to plaque formation) (4) small allosteric modulator of protein function* - Trimethylamine (TMAO) dependent on consumption of red meat, *red meat rich in carnitine* for transport of fatty acids into mitochondria is *suppressed after administration of antibiotic to kill gut bacteria*. - Individuals that are vegan have diff. gut microbiome that did not convert carnitine into TMA or TMAO

Know Functions of Different Lipoprotein Complexes and Relative Sizes

What they do what organs do they target • Chylomicrons carry diet-derived lipids to body cells (largest-least protein-dense) • VLDLs carry lipids synthesized by the liver to body cells • LDLs carry cholesterol around the body • HDLs carry cholesterol from the body back to the liver for breakdown and excretion (smallest-most dense)

Describe how induced pluripotent stem cells (iPS) cells are generated.

adult stem cells → pluripotent genes inserted via vector (transcription factors and oncogenes) into cell→ plated onto feeder cells → subculture individual colonies → freeze or induce phenotype

List the research and therapeutic applications of different types of stem cells.

• *Resident (Multipotent)* Stem Cells Are Therapeutically Robust, and *Represent Major Forms of Treatment*. *ES cells* (*Pluripotent*): Embryonic stem cells *not used to cure diseases*. • Use of *ES and iPS* (Pluripotent) Stems Cells Represent *Important Research Tools* (Cell Culture and Genetically Modified Organisms). • Major Value of Human Pluripotent Stem Cells (iPS) is to Study Human Embryonic Development and Assess Drug Screening without Needing Embryos.

Liability and Risk

• *Risk* = disease susceptibility conferred by *ONLY GENES* • *Liability* = encompasses *ALL* factors affecting disease development→ *genes AND environment (G X E)!!!* - Liability graph is continuous (would look like normal distribution with a *distinct threshold* which when crossed will lead to disease) - Liability/Threshold model = liability of *majority population is below threshold*. *Relatives of affected individual have more risk genes and higher incidence* of disorder than general pop. *Diff bet. Mendelian vs. Multifactorial Inheritance* 1) *Mendelian* = all or nothing, if you have gene you have disease, risk does not increase during lifetime 2) *Multifactorial *= additive varying phenotype, lot of contributing genes but disease occurs when person passes threshold, can acquire more liability through life