Exam 3: Biology

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 graphic depicts the most likely effect of the HIF-1α inhibitor on emodin?  (Note: control = cells cultured under normoxic conditions.)

The bones of humans and other vertebrates are alike in that they:

Passage Overexpression of the miR-17~92 gene cluster occurs in many human cancers involving c-Myc, a gene coding for a transcription factor that regulates expression of proteins such as p21, an inhibitor of cell cycle progression.  miR-17~92 encodes six distinct microRNAs (miRNAs) that can be divided into four families based on sequence identity at positions 2-7 (Table 1).Table 1  Sequence Identities of miRNA Families Encoded by the mir-17∼92 Gene Cluster Experiment 1Two independent, diffuse B-cell lymphoma cell lines were used to study the functional relationship between miR-17~92 transcripts and c-Myc in cancer pathogenesis.  The first cell line was derived from Eμ-Myc mice (Eμ), which overexpress a c-Myc transgene under the control of a B-cell-specific enhancer that induces early development of B-cell lymphoma.  The second cell line was derived from Eμ-Myc mice carrying a miR-17∼92 knockout allele (Δ/Δ).  As shown in Figure 1, researchers measured the growth of both cell lines, as well as that of Δ/Δ cells infected with a retrovirus expressing the entire miR-17∼92 cluster (Δ/Δ + miR-17∼92). Figure 1  Growth curves of Eμ cells, Δ/Δ cells, and Δ/Δ + miR-17∼92 cells (error bars = standard deviation)Experiment 2To assess the relative contribution of each miRNA to the oncogenic ability of the miR-17∼92 cluster, Δ/Δ cell lines were transduced with an empty vector or with miR-17∼92 mutant allele constructs that were engineered to lack expression of miRNA from at least one of the four families.  In each of the constructs, the family that was knocked out of the vector is indicated by the Δ symbol.  After verifying that the transduced cells correctly expressed the desired miRNA, the fraction of apoptotic cells in each line was measured (Figure 2). Figure 2  Percentage of apoptotic cells in Eμ cells and Δ/Δ cells transduced with the indicated miR-17~92 construct (error bars = standard deviation) Adapted from Mu P, Han YC, Betel D, et al. Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 2009;23(24):2806-11. -Based on the passage, what conclusions can be made about c-Myc and the miR-17∼92 gene cluster?c-Myc induces miR-17∼92 expression.miR-17~92 induces c-Myc expression.miR-17~92 suppresses apoptosis.

Passage Trypanosoma brucei is a eukaryotic parasite that causes sleeping sickness in Sub-Saharan Africa.  The cell cycle of T. brucei differs from that of higher eukaryotes.  For example, the chromatin does not condense during mitosis, and the nuclear envelope remains intact.  In addition, the nucleus divides before cell division, and the cell divides along the longitudinal axis (Figure 1). Figure 1  Mitosis in T. bruceiA culture of T. brucei was arrested in S phase of the cell cycle by overnight incubation with the reversible inhibitor hydroxyurea.  The cells were then washed, and their progression through the cell cycle was monitored by flow cytometry and microscopy.  A subset of the culture was first exposed to the drug VX-680, which arrests cells upon completion of metaphase.  The results are shown in Figure 2. Figure 2  Percentages of a population of T. brucei cells in each phase of the cell cycle, measured at different time points in the (A) absence and (B) presence of VX-680 Adapted from Li Z, Umeyama T, Wang CC. The Aurora Kinase in Trypanosoma brucei plays distinctive roles in metaphase-anaphase transition and cytokinetic initiation. PLoS Pathog. 2009;5(9):e1000575. -Based on Figure 2, in the absence of VX-680, how long does it take T. brucei cells released from S phase at 0 hours to complete one full cell cycle?

The genetic code is said to be degenerate because there are 64 different codons, but translation produces only 20 unique amino acids.  The degeneracy of the genetic code is due to which mechanism?

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. -Based on the information given in Table 1, the results of this examination may be explained by all of the following EXCEPT:

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. -The image below shows a population of unicellular organisms.  In each organism, the shaded mitochondria express a deleterious mutant allele of a mitochondrial gene, and the nonshaded mitochondria express the wild-type allele of that same gene. Which of the following events most likely occurred between Generation 1 and Generation 2?

Passage Adeno-associated virus 2 (AAV2) is a nonpathogenic, nonenveloped DNA virus that requires helper viruses such as adenovirus (AV) or herpes simplex virus (HSV) to replicate efficiently.  Because it can transduce human cells, including quiescent (nondividing) cells, without causing disease, AAV2 is attractive as a gene therapy vector.  In clinical trials, a modified AAV2 virus has successfully treated the retinal disease Leber's congenital amaurosis as well as the blood clotting disorder hemophilia B.  AAV2 transduces cells via a multistep process, shown in Figure 1. Figure 1  Schematic showing the mechanism of transduction by AAV2Studies indicate that unlike AV and HSV, AAV2 inhibits pathways other than the endosomal pathway, such as trafficking to the Golgi, increasing the percentage of invading virus particles that arrive at the nucleus. Adapted from Xiao PJ, Samulski RJ. Cytoplasmic trafficking, endosomal escape, and perinuclear accumulation of adeno-associated virus type 2 particles are facilitated by microtubule network. J Virol. 2012;86(19):10462-73. -Which of the following is true concerning the proteins to which AAV2 binds in Figure 1, Step 1?

Passage Saliva, a secreted fluid with multiple physiologic functions, is composed of water (~99%), proteins, and electrolytes.  Together, these components facilitate nutrient consumption, respiration, and communication.  For example, saliva aids in digestion by serving as a lubricant during mastication and by initiating carbohydrate and fat metabolism.  In addition, the calcium and phosphate present in saliva promote tooth remineralization, and antimicrobial and chemical buffering agents give saliva protective properties.The gustatory-salivary reflex, the secretion of saliva from the salivary glands in response to gustation (taste), is primarily mediated by the parasympathetic division of the nervous system.  In general, parasympathetic pathways originate from the brain stem or the sacral spinal cord.  Fibers originating from nuclei in these regions synapse onto ganglia that lie in close proximity to the target organ.  Both preganglionic neurons (those originating from the nuclei) and postganglionic neurons (those originating from the ganglia) release acetylcholine (ACh).In the gustatory-salivary reflex, activation of taste receptors in the tongue sends impulses to the superior and inferior salivatory nuclei in the brainstem.  Local processing within these nuclei activates the parasympathetic pathways that innervate the salivary glands.  Ultimately, ACh released from postganglionic neurons acts on the muscarinic acetylcholine receptors (mAChRs) on the secretory cells of the salivary gland to stimulate salivation (Figure 1). Figure 1  Parasympathetic regulation of salivary secretionSympathetic stimulation of the salivary glands also promotes salivation but to a lesser degree than parasympathetic stimulation.  This may contribute to what is often perceived to be decreased salivation when the sympathetic division is more active than its counterpart.  Unlike parasympathetic pathways, sympathetic pathways originate in the thoracic and lumbar spinal segments, and the ganglia lie in close proximity to the spinal cord.  Although preganglionic neurons of the sympathetic nervous system release ACh, the postganglionic neurons often release norepinephrine. -Which of the following neurons correspond to the gustatory-salivary reflex's afferent and efferent components, respectively?

Passage In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers.  To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells.  The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray.  The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes.  Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure.  The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI.  Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI. Figure 1  Overview of the pSKII vector (plasmid) Figure 2  The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes. To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections.  One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons.  The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons. -What observation could have led researchers to conclude that cldnk is expressed in oligodendrocytes?

Passage Osteoarthritis (OA) is a disorder marked by the deterioration of articular (hyaline) cartilage, the connective tissue lining the ends of bones at most movable joints.  The progressive erosion of articular cartilage in OA is thought to be caused by an inflammatory response induced by years of biomechanical stress due to excessive compression and friction.  This response involves the release of proteolytic enzymes that break down cartilaginous proteins.  The progressive breakdown of articular cartilage ultimately results in complete exposure of the cortical portion of the underlying subchondral bone with narrowing of the joint space.  Repeated and abnormal joint stress can also cause the development of osteophytes, or bony growths, that form on the subchondral bone.  These degenerative processes cause pain and restrict joint movement.Stem cell therapy, in which injected or grafted stem cells are induced to differentiate into chondrocytes (cartilage cells), is a potential new treatment for OA.  To further understand chondrogenesis, researchers have been studying the molecules that regulate the expression and activity of the transcription factor Sox9, which has been shown to promote chondrocyte differentiation.  For example, researchers have found that binding of the growth factor TGF-β to its receptor results in Sox9 activation.MicroRNAs (miRNAs), which are known to alter gene expression by acting at the translational level, are also potential regulators of Sox9.  In particular, miRNA-145 is thought to regulate Sox9 expression by binding to the 3′-UTR of the Sox9 mRNA transcript.  After chondrocyte differentiation was induced using TGF-β, researchers used Western blotting to generate an expression profile of Sox9 and β-actin (a protein not regulated by miRNA-145) in mouse stem cells transfected with miRNA-145, anti-miRNA-145, or neither (Figure 1). Figure 1  Expression of Sox9 in the presence and absence of miRNA-145 or anti-miRNA-145 following induction of chondrocyte differentiation Adapted from Yang B, Guo H, Zhang Y, Chen L, Ying D, Dong S. MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS ONE. 2011;6(7):e21679. -Osteoporosis, a disease marked by decreased bone density and mass, is similar to OA in that it is a degenerative bone disorder correlated with old age.  Which two skeletal system functions would OA and osteoporosis impair, respectively?

As the two anatomical divisions of the adrenal gland, the adrenal cortex and adrenal medulla share a common feature in that both:

Passage In 1906, the Nobel Prize in Physiology or Medicine was awarded to two biologists for their groundbreaking work in the field of neuroanatomy.  Despite using the same technique to visualize neurons, the two biologists formed opposing theories regarding nervous system organization.Hypothesis 1:  Reticular TheoryBiologist 1 developed a neural staining technique that was carried out by fixing brain tissue in potassium dichromate (K₂Cr₂O₇) before treating it with silver nitrate (AgNO₃).  A silver chromate (Ag₂CrO₄) precipitate deposited within the neurons, making them appear black.  The silver impregnation method was revolutionary because of two features.  First, only a random subset of neurons within a given tissue sample (~1% of the total cell population) was subject to silver impregnation.  Second, the impregnated cells were stained in their entirety, allowing for visualization of the complete neuron.  Biologist 1 concluded that his findings supported the idea that the nervous system is a single reticulum of fused neural processes that transmit information via cytoplasmic continuity.Hypothesis 2:  Neuron DoctrineBiologist 2 modified the silver impregnation method by first subjecting the brain tissue to two rounds of staining ("double impregnation") rather than one.  Second, where Biologist 1 studied the brain tissue of older animals with myelinated nerve fibers, Biologist 2 employed the silver stain on brain tissue from younger animals, which made it easier for the silver to impregnate the neural processes due to relative lack of myelination.  Biologist 2 reasoned that the nervous system is not a single web of interconnected fibers but rather is made up of discrete neural units that communicate via close, but not continuous, contact.The neuron doctrine was later proven correct with the advent of electron microscopy, which allowed scientists to visualize the synapse (the point of communication between two distinct neurons).  Further investigation allowed researchers to ultimately discover that there are two types of synapses:  electrical and chemical. -A neuron is exposed to a compound that prevents the passage of Na⁺ ions through voltage-gated Na⁺ channels.  Which of the following graphs best represents the local membrane potential change of this neuron upon injection of a positive current that allows the membrane to reach the threshold value?  (Note:  The solid line represents the action potential after normal stimulation; the dashed line represents the action potential after exposure to the compound.)

Passage Saliva, a secreted fluid with multiple physiologic functions, is composed of water (~99%), proteins, and electrolytes.  Together, these components facilitate nutrient consumption, respiration, and communication.  For example, saliva aids in digestion by serving as a lubricant during mastication and by initiating carbohydrate and fat metabolism.  In addition, the calcium and phosphate present in saliva promote tooth remineralization, and antimicrobial and chemical buffering agents give saliva protective properties.The gustatory-salivary reflex, the secretion of saliva from the salivary glands in response to gustation (taste), is primarily mediated by the parasympathetic division of the nervous system.  In general, parasympathetic pathways originate from the brain stem or the sacral spinal cord.  Fibers originating from nuclei in these regions synapse onto ganglia that lie in close proximity to the target organ.  Both preganglionic neurons (those originating from the nuclei) and postganglionic neurons (those originating from the ganglia) release acetylcholine (ACh).In the gustatory-salivary reflex, activation of taste receptors in the tongue sends impulses to the superior and inferior salivatory nuclei in the brainstem.  Local processing within these nuclei activates the parasympathetic pathways that innervate the salivary glands.  Ultimately, ACh released from postganglionic neurons acts on the muscarinic acetylcholine receptors (mAChRs) on the secretory cells of the salivary gland to stimulate salivation (Figure 1). Figure 1  Parasympathetic regulation of salivary secretionSympathetic stimulation of the salivary glands also promotes salivation but to a lesser degree than parasympathetic stimulation.  This may contribute to what is often perceived to be decreased salivation when the sympathetic division is more active than its counterpart.  Unlike parasympathetic pathways, sympathetic pathways originate in the thoracic and lumbar spinal segments, and the ganglia lie in close proximity to the spinal cord.  Although preganglionic neurons of the sympathetic nervous system release ACh, the postganglionic neurons often release norepinephrine. -The gustatory-salivary reflex can be modulated (dampened or enhanced) by input from higher centers in the central nervous system.  To modulate the salivary response, descending pathways from the brain would most likely synapse onto which part of the reflex arc?

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. -A gene expression assay shows that the SRY gene is expressed in male embryos during the early stages of development.  Which of the following best describes the DNA methylation pattern exhibited by the SRY gene in embryonic gonadal cells compared to embryonic cardiac cells?

Passage In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers.  To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells.  The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray.  The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes.  Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure.  The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI.  Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI. Figure 1  Overview of the pSKII vector (plasmid) Figure 2  The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes. To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections.  One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons.  The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons. -Which of the following diagrams shows the structure of the cloned cldnk gene after digestion with EcoRI and XhoI?

Passage Eukaryotic organelles such as mitochondria and chloroplasts are believed to be relics of formerly free-living prokaryotes.  The transition from a hypoxic (low O₂) to an oxic atmosphere (21% O₂) is said to have enabled primitive eukaryotic anaerobes to engulf ancient aerobic prokaryotes and consequently acquire the ability to produce energy through oxidative phosphorylation.  This endosymbiotic theory of eukaryotic evolution also postulates that endosymbiosis resulted in larger eukaryotic genomes, which originated from the partial transfer of mitochondrial genes to the nuclear genome.  On integration into the host genome, mitochondria-derived genes became indistinguishable from the original nuclear genes.Researchers have alternatively proposed that after a prolonged period of symbiosis, there is a possibility of gene transfer from eukaryotes to prokaryotes.  This hypothesis was initially supported when copper/zinc (Cu/Zn) superoxide dismutase (SOD), a metalloprotein confined to the cytosol of eukaryotic cells, was found in Photobacterium leiognathi.  The free-living bacterium P. leiognathi is also a known symbiont of ponyfish, a small fish species native to the Indian and Pacific Oceans.SODs are antioxidant enzymes that serve as the cell's first line of defense against reactive oxygen species (ROS).  ROS produced by the electron transport chain damage proteins by oxidizing amino acid residues and metal ions on prosthetic groups, but can accumulate during times of biochemical and environmental stress.  Superoxide (O₂⁻) radicals, a form of ROS, are sequestered by SODs and converted into less toxic hydrogen peroxide (H₂O₂) and O₂ gas. Adapted from Bannister JV, Parker MW. The presence of a copper/zinc superoxide dismutase in the bacterium Photobacterium leiognathi: a likely case of gene transfer from eukaryotes to prokaryotes. Proc Natl Acad Sci USA. 1985;82(1):149-52. -SOD mRNA in P. leiognathi encodes a signal sequence that directs the transport of the SOD protein for secretion.  This signal sequence will direct SOD proteins to which structure in P. leiognathi?

Passage The lac operon encodes the proteins that metabolize lactose and is a key biochemical feature that differentiates between members of the Enterobacteriaceae family.  For example, the severely virulent Salmonella typhimurium can be distinguished from its less virulent relative Escherichia coli by the absence of a lac operon.  Researchers propose that the evolutionary loss of the lac operon may contribute to S. typhimurium's ability to invade epithelial cells.To test the hypothesis, wild-type (WT) S. typhimurium cells were transformed with plasmids encoding components of the lac operon (Figure 1): β-galactosidase (lacZ⁺), lactose permease (lacY⁺), transacetylase (lacA⁺), or all three (lac⁺).  Each plasmid also contained genes for the lac repressor (lacI), which can inhibit lac operon transcription in the absence of lactose; the cAMP receptor protein (crp), which induces lac expression in low glucose; and a chloramphenicol resistance gene (cat). Figure 1  Plasmid PYM-LACZYA (lac⁺), used to transform WT S. typhimurium.  Similar plasmids containing only one of the three lacZ, lacY, or lacA genes were also used.Virulence assays were performed in mice by inoculating with WT or lac⁺ S. typhimurium.  The assays confirmed that lac⁺ bacteria were less virulent than their WT counterparts, and that both WT and lac⁺ bacteria were more virulent in the presence of glucose.  Subsequently, several genes related to invasion were analyzed for their expression levels in the presence or absence of lac plasmids (Table 1; Figure 2).Table 1  Transcription Levels of Invasion-Associated Genes in lac⁺ S. typhimurium Relative to WT Figure 2  Transcript concentrations of two S. typhimurium flagellar genes, fljB and fliC, in bacteria transformed with lacZ⁺, lacY⁺, lacA⁺, or lac⁺ plasmids.  Concentrations are expressed as a percentage of WT levels. Adapted from Jiang L, Ni Z, Wang L, Feng L, Liu B. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis. Curr Microbiol. 2015;70(3):315-23. -The reduced virulence of lac⁺ S. typhimurium has been correlated with an inability to generate the torque that turns the flagellum.  Which structure in the flagellum is most likely being affected by lac genes?

Passage Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a bacterium responsible for causing foodborne illnesses.  EHEC virulence, or the ability to cause disease, is acquired through quorum sensing, a form of bacterial cell-cell communication dependent on population density.  EHEC O157:H7 bacteria monitor population density by detecting changes in the concentration of autoinducer-3 (AI-3), an extracellular signaling molecule secreted by many bacterial species.  AI-3 generally acts on neighboring bacteria to activate the genes on the enterocyte effacement (LEE) locus.  These genes code for type III secretion proteins (EspA, EspB, EspD, Tir), which promote entry of Shiga-like toxins secreted by EHEC into host intestinal epithelial cells.  This ultimately leads to cell death, lesions, and symptoms such as bloody diarrhea and abdominal cramps.It has been reported that a mutation in S-ribosylhomocysteine lyase (LuxS), an enzyme involved in quorum sensing, may result in diminished production of AI-3.  However, researchers predict that certain peptide hormones can activate LEE gene transcription in the LuxS.  To test this hypothesis, mutant EHEC bacteria deficient in LuxS (LuxS⁻) were cultured and separately exposed to the hormones epinephrine (E), norepinephrine (NE), gastrin (G), and secretin (S) at 37°C.  Total RNA was extracted from the treated bacterial cultures, and LEE mRNA was quantified (Figure 1). Figure 1  Concentration of LEE mRNA in mutant LuxS⁻ bacteria supplemented with purified peptide hormones (Note:  C = control.)Western blot was performed to compare the presence and concentration of type III secretion proteins from wild-type (WT), LuxS⁻, and LuxS⁻ cultured in preconditioned media (PCM).  PCM was generated by culturing uninfected intestinal epithelial cells in growth media for 24 hours.  After 24 hours, the cells were removed and the PCM was used to supplement the growth of LuxS⁻ bacteria. Figure 2  Western blot results of type III secretion proteins isolated from WT, mutant LuxS⁻, and LuxS⁻ in PCM -Based on the passage, what best explains the western blot results of type III secretion proteins in LuxS⁻ E. coli cells cultured in PCM?