Exam 3: Biology

Human cardiac muscle differs from skeletal muscle because it:

Passage The lumen of the human gut is lined by a monolayer of epithelial cells that acts as a selectively permeable barrier, preventing the passage of harmful intraluminal foreign antigens, flora, and toxins into the circulation while allowing digestion and absorption of essential dietary nutrients along with the transfer of electrolytes and water.Proteins in the tight junctions of intestinal epithelial cells maintain barrier integrity, but barrier dysfunction occurs when these cells are damaged in the setting of infection, burns, shock, or hypoxia (low oxygen levels).  The transcription factor HIF-1, a heterodimer composed of the macromolecules HIF-1α and HIF-1β, regulates the adaptive cellular response to hypoxia and the consequent expression of tight junction proteins.Researchers assessed the concentration of HIF-1 heterodimer components in human intestinal Caco-2 cells subjected to hypoxia/reoxygenation (H/R) in vitro.  Caco-2 cells, a colon-derived cell line, were cultured under specific conditions to mimic the functional and morphological phenotype of wild-type enterocytes lining the small intestine.  These cells were prepared and grown as a monolayer on a collagen-coated membrane.Next, the monolayer was cultured in hypoxic conditions and then exposed to atmospheric oxygen levels (normoxia) for 30, 60, and 120 minutes.  Protein levels were quantified using direct enzyme-linked immunosorbent assay (ELISA), in which an antibody linked to a reporter enzyme was utilized to bind and detect expression of the target molecule (analyte) in a sample.  When the colorless substrate of the reporter enzyme was added, the enzyme generated a visible colored product that could be quantified based on color intensity. Figure 1  Concentration of (A) HIF-1α and (B) HIF-1β in Caco-2 cells subjected to H/R (Note:  0 minutes = cells cultured in hypoxic conditions)Emodin, an anthraquinone compound that prevents hypoxia-induced epithelial cell disruption, was used in conjunction with a chemical HIF-1α inhibitor (HIF-1α-I) to treat Caco-2 cells in a separate experiment.  Transepithelial electrical resistance (TEER) was assessed as a measure of barrier function, with higher TEER values indicating a more intact epithelial cell barrier.  HIF-1α-I was found to block emodin's protective effect on epithelial barrier integrity. Adapted from Lei Q, Qiang F, Chao D, et al. Int J Mol Med. 2014;34(6):1629-39. -Based on the passage, Caco-2 cells originate from a segment of the human gut that mainly functions to:

Assume L410 is an acquired mutation that occurs only in hepatocytes of adults who were exposed to a certain mutagen.  A woman with the L410 mutation has a child with an unaffected man.  What is the probability that this child has the L410 mutation?

Passage The Bloom syndrome helicase (BLM) transcript is catalytically inactive in Bloom syndrome (BS), a genetic disorder that leads to genomic instability characterized by excessive somatic recombination events.  Research studies have shown that BLM may be involved in DNA synthesis repair of stalled replication forks during replication stress (arrest).  To investigate this further, an experiment was conducted using cell lines derived from a patient with BS.  One set of cells differed only by BLM status:  PSNF5 (BLM⁺, vector with BLM cDNA transfected) and PSNG13 (BLM⁻, empty vector transfected).  Additional mutations, BLM-K695T and BLM-T99A, were studied in stable transfected cells.The nucleoside analogs iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) were used to label and track genomic regions of active replication.  Cells were initially treated with IdU for 15 minutes.  Subsequently, either 4 mM of hydroxyurea (HU) or 30 µM of aphidicolin (Aph) were used to induce replication stress for 4 hours, after which cells were treated with CldU for 85 minutes to assess replication recovery.  Cells were mounted on microscope slides, lysed to isolate chromosome fibers, and then fixed on the slides.  Sites of IdU and CldU incorporation into the growing DNA strand were then detected by immunostaining the isolated chromosome fibers with analog-specific, fluorescent-coupled antibodies.  Replication activity was visualized via microscopy and measured. Figure 1  (A) Experimental protocol and (B) fluorescent imaging of a microscope slide showing incorporation of IdU (black lines) and CldU (gray lines) into DNA fibers isolated from a group of BLM⁺ and BLM⁻ cells, with single fibers depicted in each row Figure 2  Replication fork recovery in cells expressing BLM⁺, BLM⁻, and the mutation BLM-K695T (antagonistic to wild-type) Figure 3  New sites of replication in cells expressing BLM⁺, BLM⁻, and the mutation BLM-T99A (T99 phosphorylated after replication arrest) Adapted from Davies SL, North PS, Dart A, Lakin ND, Hickson ID. Mol Cell Biol. 2004;24(3):1279-91. -During HU treatment of PSNF5 cells, replication stress response factors would act at DNA fibers that resemble which rows in Figure 1B?

Passage Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells.  During FRAP, the fluorescence of a green fluorescent protein (GFP)-tagged molecule is first measured and then photodestroyed in targeted cell regions.  Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region.  The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins.  HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure. Figure 1  Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP.  FRAP was performed on euchromatin and heterochromatin.  Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively.  The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1  Effect of HMGB1 and HMGA1 on H1-GFP Mobility Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10):4321-8. -If HMGA1 were injected into cells expressing HMGB1-GFP instead of H1-GFP, which of the following FRAP analysis graphs would show conclusively that HMGA1 binds the same sites as HMGB1 on chromatin?  [The t80 value for HMGB1-GFP is indicated by the arrows].

Passage The lumen of the human gut is lined by a monolayer of epithelial cells that acts as a selectively permeable barrier, preventing the passage of harmful intraluminal foreign antigens, flora, and toxins into the circulation while allowing digestion and absorption of essential dietary nutrients along with the transfer of electrolytes and water.Proteins in the tight junctions of intestinal epithelial cells maintain barrier integrity, but barrier dysfunction occurs when these cells are damaged in the setting of infection, burns, shock, or hypoxia (low oxygen levels).  The transcription factor HIF-1, a heterodimer composed of the macromolecules HIF-1α and HIF-1β, regulates the adaptive cellular response to hypoxia and the consequent expression of tight junction proteins.Researchers assessed the concentration of HIF-1 heterodimer components in human intestinal Caco-2 cells subjected to hypoxia/reoxygenation (H/R) in vitro.  Caco-2 cells, a colon-derived cell line, were cultured under specific conditions to mimic the functional and morphological phenotype of wild-type enterocytes lining the small intestine.  These cells were prepared and grown as a monolayer on a collagen-coated membrane.Next, the monolayer was cultured in hypoxic conditions and then exposed to atmospheric oxygen levels (normoxia) for 30, 60, and 120 minutes.  Protein levels were quantified using direct enzyme-linked immunosorbent assay (ELISA), in which an antibody linked to a reporter enzyme was utilized to bind and detect expression of the target molecule (analyte) in a sample.  When the colorless substrate of the reporter enzyme was added, the enzyme generated a visible colored product that could be quantified based on color intensity. Figure 1  Concentration of (A) HIF-1α and (B) HIF-1β in Caco-2 cells subjected to H/R (Note:  0 minutes = cells cultured in hypoxic conditions)Emodin, an anthraquinone compound that prevents hypoxia-induced epithelial cell disruption, was used in conjunction with a chemical HIF-1α inhibitor (HIF-1α-I) to treat Caco-2 cells in a separate experiment.  Transepithelial electrical resistance (TEER) was assessed as a measure of barrier function, with higher TEER values indicating a more intact epithelial cell barrier.  HIF-1α-I was found to block emodin's protective effect on epithelial barrier integrity. Adapted from Lei Q, Qiang F, Chao D, et al. Int J Mol Med. 2014;34(6):1629-39. -Which of the following statements about HIF-1 is most likely to be true?At low oxygen levels, HIF-1 is nonfunctional.At low oxygen levels, HIF-1 is functional.At normal oxygen levels, HIF-1 concentration is decreased.At normal oxygen levels, HIF-1 concentration is increased.

In a drug trial, one patient receives an injection containing inactivated antigen A and another patient receives a saline injection.  The immune system can mount a response against inactive antigen A.  If each patient was exposed to antigen A twice after receiving the initial injection, which of the following graphs shows the expected changes in the levels of serum antibodies against antigen A across time?

Passage The sex chromosomes X and Y are involved in mammalian sex determination.  Organisms with two wild-type (WT) X chromosomes develop as females (XX), but those with a combination of WT X and Y chromosomes develop as males (XY) (Table 1).  Both sexes also inherit a set of autosomes (nonsex chromosomes).  Humans inherit 22 pairs of autosomes (44 total).  In contrast, rabbits inherit 21 pairs of autosomes (42 total).Table 1  Chromosome Types Inherited by a Male and Female Rabbit (Note: The Y chromosome has fewer alleles than the X chromosome.) Specifically, the SRY gene, located on the mammalian Y chromosome, is crucial for male sex determination and induction of testis development.  Studies in mammals have shown that regardless of an organism's combination of sex chromosomes, fetuses will always develop as female in the absence of the SRY protein.  In addition, one of the X chromosomes becomes inactivated in WT,XX females to prevent them from having double the amount of X-chromosome gene products as WT,XY males.Sp1 is a transcription factor with demonstrated regulatory control of SRY gene expression, and XY individuals with mutations affecting the Sp1 binding site on the SRY gene have been shown to develop female sex characteristics.  Unlike SRY, the gene for Sp1 is located on an autosome.Researchers knocked out (KO) Sp1 binding sites on the SRY gene in rabbit embryos (Sp1 KO,XY rabbits) and observed their sexual development following implantation in surrogate mothers.  The relationship between the SRY gene and sexual development can be explained by the following experimental observations: Most Sp1 KO,XY rabbits had female genitalia and displayed normal mating behaviors. Sp1 KO,XY rabbits had significantly smaller ovaries and fewer follicles than WT,XX rabbits. No significant differences were observed in the cervix and uterus between Sp1 KO,XY and WT,XX rabbits. There was significantly reduced SRY expression in Sp1 KO,XY rabbits when compared to WT,XY males. WT,XY male rabbits had significantly higher testosterone levels when compared to Sp1 KO,XY rabbits. Sp1 KO,XY rabbits were infertile when mated with WT,XY males. Sp1 KO,XY rabbits could become pregnant via embryo transfer. Song Y et al. Mutation of the Sp1 binding site in the 5' flanking region of SRY causes sex reversal in rabbits. Oncotarget. 2017; 8(24):38176-83. -If researchers were to artificially express the SRY gene in WT,XX female rabbit embryos, then these rabbits would most likely develop:

Given that a species diverged from its ancestor 80 million years ago, which of the following graphs most likely illustrates the pattern of mutation accumulation in the genome of this species over time?

In an isolated population of mice, 10,000 mice are randomly sampled every 10 generations and their coat colors are recorded for a scientific study.  The data for 100 generations of mice are shown in the graph below. Which of the following conclusions about the black and white mice in this population is best supported by the graph?

Passage Proteins known as cancer/testis antigens (CTAs) are normally expressed exclusively in germline cells but may become reactivated in cancer cells due to chromatin remodeling (eg, gene activation due to DNA demethylation).  Because of their normally restricted expression, CTAs can elicit an immune response when expressed by cancer cells derived from other tissue types.  Several CTAs are known to inhibit apoptosis or promote cell division in cancer cells, making them more resistant to traditional chemotherapy.  Accordingly, cancer research instead has focused on immunotherapy treatments targeting CTAs.In a study, researchers isolated peripheral blood lymphocytes (B lymphocytes, T lymphocytes, natural killer [NK] cells) from two healthy individuals.  These lymphocytes were cultured with signaling factors known to promote helper T cell (T_h cell) proliferation.  After 7 days, cultures from both individuals were enriched for T_h cells, which expressed proteins generally involved in activating other lymphocytes.  A subset of the cultures from each individual was treated with 5-Aza-CdR, a DNA-demethylating agent.  Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze expression of several CTAs (ie, MAGEs) in both untreated and 5-Aza-CdR-treated lymphocyte cultures, as well as in untreated melanoma (skin cancer) and breast cancer cell lines (Figure 1).  GAPDH, a protein expressed in most cell types, served as a control. Figure 1  Gels showing RT-PCR results for MAGE expressionAdditional peripheral blood lymphocytes were isolated from the same healthy individuals and incubated with the T_h cells that had been treated with 5-Aza-CdR.  After 11 days, these cultures consisted primarily of an increased number of NK cells and cytotoxic T lymphocytes (CTLs).  Researchers found that these CTLs were able to induce apoptosis in breast cancer cell lines but did not affect healthy cells.Researchers used a similar procedure to induce CTL proliferation in lymphocytes isolated from a breast cancer patient.  The CTLs were then incubated with cells obtained from a biopsy of the patient's cancerous tissue.  Following this experiment, researchers found that apoptosis was induced in the patient's cancer cells. Kirkin et al. Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells. Nature Communications. 2018; 9:785 -Scientists find that the tumor cells of some breast cancer patients are resistant to CTLs generated using the methods described in the passage.  The best explanation for this finding would be:

Passage Alzheimer disease (AD) is a progressive condition affecting memory and cognition that can be pathologically characterized by the presence of neurofibrillary tangles (NFTs).  NFTs, aggregates of hyperphosphorylated tau proteins that have dissociated from microtubules, are believed to contribute to neuronal degradation and subsequent AD symptoms.  In general, ribosomes initiate translation of tau mRNA by binding the 7-methylguanosine (m7Gpp) cap present at the end of the 5′ untranslated region (UTR) of the mRNA molecule.  However, researchers believe that tau protein translation initiation can occur without recognition of the m7Gpp cap.  This alternate mechanism, referred to as cap-independent translation, indicates that ribosomes are recruited by internal ribosome entry sites (IRES) generally located in the 5′ UTR of the mRNA sequence.To determine the initiation mechanism of tau mRNA translation, neuroblastoma cells of AD mice were transfected with two different DNA constructs (Figure 1).  Construct 1 contained the 5′ UTR of tau mRNA isoforms (v1 and v2) extracted from cells displaying NFTs.  Construct 2 included the 5′ UTR of the β-globulin gene, which normally codes for a plasma protein.  The luciferase reporter genes, Renillla and Photinus, were also included in the two constructs. /_ /_ Figure 1  DNA constructs containing β-globulin or tau gene used to transfect neuroblastoma cellsLuciferase gene expression from the constructs was monitored in the absence and presence of a synthetic m7Gpp cap analog (Figure 2).  Translation of Renillla luciferase mRNA depended on ribosomal binding to the m7Gpp cap of the mRNA, but translation of Photinus luciferase required ribosome recruitment by an IRES in the 5′ UTR of the adjacent gene. Figure 2  Luciferase activity in neuroblastoma cells exposed to m7Gpp cap analogIn a second study, researchers used siRNA to target initiation factor eIF4E, a peptide translated in a cap-dependent manner that facilitates the binding of ribosomes to the m7Gpp cap.  Expression of factor eIF4E, elongation factor eEF2K, and tau v2 protein was assessed in siRNA-treated cells (Figure 3). Figure 3  Western blot of eIF4E, eEF2K, and tau v2 extracted from non-AD cells treated with eIF4E siRNA Adapted from Veo BL, Krushel LA. Translation initiation of the human tau mRNA through an internal ribosomal entry site. J Alzheimers Dis. 2009; 16(2):271-5. -The cytotoxic protein X.23A induces conformational changes in the large ribosomal subunit, reducing its catalytic activity.  Treating neuronal AD mice cells that overexpress tau protein with X.23A would most likely interfere with the binding of the:

Passage Mitochondria are thought to have been independent bacterial organisms that were engulfed and integrated into eukaryotic cells approximately two billion years ago.  Most of the mitochondrial genome was lost or transferred into the large central genome of the eukaryotic nucleus, leaving only a residual genome within each mitochondrion.Because multiple mitochondria are found in the cell, mitochondrial DNA (mtDNA) mutations result in a condition known as heteroplasmy, or the intracellular mixture of wild-type mtDNA and mutant mtDNA.  Just as the cellular ratio of wild-type to mutant mtDNA varies, the overall mtDNA content within a tissue or organ is also highly variable.  For this reason, disease-causing mutant mtDNA manifests phenotypically only when the majority of mitochondria within a given tissue express the deleterious allele.  In addition, the variability in mtDNA content makes it possible for individuals with the same mitochondrial mutation to display drastically different clinical symptoms.To investigate how heteroplasmy influences disease manifestation, researchers analyzed an A to G substitution at nucleotide 3243 in the tRNA^leu gene of mtDNA.  This mutation has been associated with hereditary hearing loss, diabetes mellitus, and a syndrome characterized by mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes.Family members who were confirmed to have the A3243G mutation were tested using an audiometry examination to assess hearing impairment, as shown in Table 1.  The subjects carried no other genetic mutations known to cause hearing loss.Table 1  Audiometry Results From Family Members with the A3243G Mutation The blood samples of these subjects were collected, and the 3243 region of the tRNA^leu gene was amplified via standard PCR.  The PCR products were subsequently treated with the restriction enzyme ApaI and visualized on a PAGE gel.  The normal undigested PCR product is 161 bp in length, but the PCR product amplified from the mutant A3243G is digested into two fragments by ApaI. Figure 1  PAGE visualization of PCR products following ApaI digestion Adapted from Hadjivasiliou Z, Pomiankowski A, Seymour RM, Lane N. Selection for mitonuclear co-adaptation could favour the evolution of two sexes. Proc Biol Sci. 2012;279(1734):1865-72. -Which of the following would be true regarding the inheritance of the A3243G mutation?  (Note: Assume affected individuals inherited the mutation.)

In an assay, researchers assessed activity levels of an oxidative enzyme present on the inner mitochondrial membrane of skeletal muscle fibers.  Enzyme activity was visualized by reacting the product of the enzymatic reaction with a dark-colored salt, which resulted in dark-colored staining of the muscle fibers.  Assay results in two different microscopic cross sections of muscle fibers are shown below. Given these results, muscle fibers in cross section 1 most likely:

Passage Parathyroid hormone (PTH) is an important regulator of calcium and phosphate homeostasis.  Specifically, PTH functions to increase plasma calcium concentration and reduce plasma phosphate levels.  PTH secretion is closely regulated by changes in plasma calcium concentration, which are detected by the human calcium-sensing receptor (hCaSR), a G protein-coupled receptor located on chief cells of the parathyroid gland.  hCaSR activation ultimately inhibits PTH release by inducing a second messenger cascade in which intracellular calcium levels rise due to the hydrolysis of the membrane phospholipid phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG) by the membrane-bound enzyme phospholipase C (PLC).Secondary hyperparathyroidism is a condition characterized by excessive PTH release in response to depressed serum calcium or elevated serum phosphate levels.  The novel peptide AMG 416, an hCaSR agonist, is being investigated as a possible treatment for secondary hyperparathyroidism caused by end-stage renal disease (ESRD).  IP-1, a downstream metabolite of IP₃, is a marker of hCaSR activity.  Researchers used embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells stably transfected with hCaSR (hCaSR) to measure IP-1 levels following AMG 416 treatment (Figure 1). Figure 1  IP-1 concentration in embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells transfected with hCaSR (hCaSR) following AMG 416 treatmentThe dependence of AMG 416 on extracellular calcium was investigated by incubating hCaSR cells with increasing concentrations of AMG 416 in the presence or absence of calcium treatment (Figure 2). Figure 2  IP-1 levels in hCaSR cells incubated with and without calcium and AMG 416 peptide  (Note:  IP-1 levels are normalized to the number of cells present in each culture.)In addition, PTH concentration was measured in 5/6 Nx mice, an animal model of secondary hyperparathyroidism in human ESRD.  Blood samples were taken from male and female 5/6 Nx mice for 8 hours following administration of varying concentrations of AMG 416 (Figure 3), and PTH levels were measured. Figure 3  Serum PTH concentration following administration of AMG 416 to 5/6 Nx mice -Which of the following conclusions is NOT consistent with the experimental findings presented in the passage?

Passage Lipopolysaccharides (LPS) found in gram-negative bacterial cells can elicit a lethal degree of adjuvanticity, or immune activation, in LPS-responsive host organisms.  In mice, responses may be modulated by abnormalities in the toll-like receptor (TLR) encoded by the Tlr4 gene closely linked to coat color on Chromosome 4.  To investigate the effect of a particular Tlr4 mutation on LPS response, three experiments were performed following a cross between fully responsive He-N mice [tlr4(+)/tlr4(+)] and He-J mice homozygous for the mutation [tlr4(d)/tlr4(d)].Experiment 1Researchers isolated spleen cells for analysis of protein expression and LPS response.  In cells isolated from F1 mice, both wild-type and mutant TLRs were distinguishable by protein electrophoresis.  Following this first analysis, spleen cells from each cohort were exposed to 2 × 10⁵ SRC, an antigen formed by sheep erythrocytes.  Cultures were then incubated in the presence or absence of Ph-LPS, with LPS extracted from Escherichia coli K345 cells using a phenol-water mixture.  Titers of anti-SRC antibody were recorded on incubation day 5, with the results shown in Table 1.Table 1  Mean anti-SRC titers with standard error Experiment 2Serum samples were assayed for interferon production 2 hours post LPS injection.  Interferon was quantified using a plaque reduction assay on mouse cells challenged with vesicular stomatitis virus. Figure 1  Average serum interferon levels in mice cohortsExperiment 3Mice were injected with varying doses of Ph-LPS and monitored for 4 days.  The lethal dose (LD₅₀) was defined as the minimum dose causing death in 50% of injected mice.  Compared to He-N mice, results reflected a 10-fold increase in LD₅₀ for F1 mice and a 100-fold increase in LD₅₀ for He-J mice. -In mice, a brown coat color is a dominant trait.  Assuming that the He-N mice in the experiments were homozygous for the white allele and the He-J mice were homozygous for the brown allele, which of the following is true with regard to coat color and LPS response in F1 and F2 mice?

Passage Mitochondria are thought to have been independent bacterial organisms that were engulfed and integrated into eukaryotic cells approximately two billion years ago.  Most of the mitochondrial genome was lost or transferred into the large central genome of the eukaryotic nucleus, leaving only a residual genome within each mitochondrion.Because multiple mitochondria are found in the cell, mitochondrial DNA (mtDNA) mutations result in a condition known as heteroplasmy, or the intracellular mixture of wild-type mtDNA and mutant mtDNA.  Just as the cellular ratio of wild-type to mutant mtDNA varies, the overall mtDNA content within a tissue or organ is also highly variable.  For this reason, disease-causing mutant mtDNA manifests phenotypically only when the majority of mitochondria within a given tissue express the deleterious allele.  In addition, the variability in mtDNA content makes it possible for individuals with the same mitochondrial mutation to display drastically different clinical symptoms.To investigate how heteroplasmy influences disease manifestation, researchers analyzed an A to G substitution at nucleotide 3243 in the tRNA^leu gene of mtDNA.  This mutation has been associated with hereditary hearing loss, diabetes mellitus, and a syndrome characterized by mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes.Family members who were confirmed to have the A3243G mutation were tested using an audiometry examination to assess hearing impairment, as shown in Table 1.  The subjects carried no other genetic mutations known to cause hearing loss.Table 1  Audiometry Results From Family Members with the A3243G Mutation The blood samples of these subjects were collected, and the 3243 region of the tRNA^leu gene was amplified via standard PCR.  The PCR products were subsequently treated with the restriction enzyme ApaI and visualized on a PAGE gel.  The normal undigested PCR product is 161 bp in length, but the PCR product amplified from the mutant A3243G is digested into two fragments by ApaI. Figure 1  PAGE visualization of PCR products following ApaI digestion Adapted from Hadjivasiliou Z, Pomiankowski A, Seymour RM, Lane N. Selection for mitonuclear co-adaptation could favour the evolution of two sexes. Proc Biol Sci. 2012;279(1734):1865-72. -What conclusion can be drawn from the results shown in Figure 1?

Passage Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease affecting 1 in 10,000 live births.  The pathological hallmark of SMA is the loss of α-motor neurons in the spinal cord, which causes progressive paralysis and, ultimately, premature death in infants.  SMA is caused by insufficient levels of survival motor neuron (SMN), a regulatory protein required for the maintenance of the spinal cord nerve cells that control voluntary muscle movement.The SMN protein is generated from the SMN1 and SMN2 genes, which differ in sequence by only a single nucleotide in exon 7.  In healthy individuals, the gene product of SMN1 is the full-length (FL) SMN protein, and the gene products of SMN2 are both the FL-SMN protein as well as a truncated unstable protein (SMNΔ7) that is rapidly degraded.  Approximately 90% of the SMN2 gene product is SMNΔ7, and only 10% is FL-SMN (Figure 1).  In most patients with SMA, SMN1 is mutated such that its protein product is nonfunctional or deleted.  The disease phenotype arises because the SMN2 gene cannot fully compensate for the deficiency in FL-SMN protein synthesis. Figure 1  Structure of SMN1 and SMN2 transcriptsAntisense oligonucleotides (ASOs) that bind pre-mRNA were injected into SMA mice (SMN1^−/−, SMN2^+/+) exhibiting progressive tail necrosis, a characteristic measure of SMA severity.  ASO therapy effectiveness was assessed by measuring the tail length of wild-type (WT) mice and SMA mice exposed to increasing concentrations of ASOs (Figure 2). Figure 2  Tail length progression of WT mice compared to SMA mice treated with either saline or ASOs Adapted from Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA. 1999;96(11):6307-11. -Based on the data shown in Figure 2, which statement best explains the function of antisense oligonucleotides (ASOs) in SMA mice?  ASOs affect:

Passage Alzheimer disease (AD) is a progressive condition affecting memory and cognition that can be pathologically characterized by the presence of neurofibrillary tangles (NFTs).  NFTs, aggregates of hyperphosphorylated tau proteins that have dissociated from microtubules, are believed to contribute to neuronal degradation and subsequent AD symptoms.  In general, ribosomes initiate translation of tau mRNA by binding the 7-methylguanosine (m7Gpp) cap present at the end of the 5′ untranslated region (UTR) of the mRNA molecule.  However, researchers believe that tau protein translation initiation can occur without recognition of the m7Gpp cap.  This alternate mechanism, referred to as cap-independent translation, indicates that ribosomes are recruited by internal ribosome entry sites (IRES) generally located in the 5′ UTR of the mRNA sequence.To determine the initiation mechanism of tau mRNA translation, neuroblastoma cells of AD mice were transfected with two different DNA constructs (Figure 1).  Construct 1 contained the 5′ UTR of tau mRNA isoforms (v1 and v2) extracted from cells displaying NFTs.  Construct 2 included the 5′ UTR of the β-globulin gene, which normally codes for a plasma protein.  The luciferase reporter genes, Renillla and Photinus, were also included in the two constructs. /_ /_ Figure 1  DNA constructs containing β-globulin or tau gene used to transfect neuroblastoma cellsLuciferase gene expression from the constructs was monitored in the absence and presence of a synthetic m7Gpp cap analog (Figure 2).  Translation of Renillla luciferase mRNA depended on ribosomal binding to the m7Gpp cap of the mRNA, but translation of Photinus luciferase required ribosome recruitment by an IRES in the 5′ UTR of the adjacent gene. Figure 2  Luciferase activity in neuroblastoma cells exposed to m7Gpp cap analogIn a second study, researchers used siRNA to target initiation factor eIF4E, a peptide translated in a cap-dependent manner that facilitates the binding of ribosomes to the m7Gpp cap.  Expression of factor eIF4E, elongation factor eEF2K, and tau v2 protein was assessed in siRNA-treated cells (Figure 3). Figure 3  Western blot of eIF4E, eEF2K, and tau v2 extracted from non-AD cells treated with eIF4E siRNA Adapted from Veo BL, Krushel LA. Translation initiation of the human tau mRNA through an internal ribosomal entry site. J Alzheimers Dis. 2009; 16(2):271-5. -During the interval between 30 and 60 minutes in Figure 1A, which of the following will be observed in WT mice?

Excess post-exercise oxygen consumption is the extra amount of oxygen consumed after the completion of exercise and before respiration returns to resting levels.  This extra oxygen is used to restore muscle fibers to their pre-exercise state by doing all of the following EXCEPT: