Question 1

Yeast expression systems — commonly used S. cerevisiae promoters Which promoters are widely used to drive gene expression in Saccharomyces cerevisiae laboratory constructs?

Accepted Answer

Introduction / Context: Promoter choice is central to designing yeast expression systems for basic research and biotechnology. S. cerevisiae provides strong constitutive and inducible promoters that control timing and strength of transcription. This question checks recognition of three classic workhorse promoters. Given Data / Assumptions: Organism: Saccharomyces cerevisiae. Goal: high, regulated, or condition-specific expression. Promoters listed are standard in yeast cloning vectors. Concept / Approach: GAL1 is an inducible promoter activated by galactose and repressed by glucose, ideal for controlled induction. ADH2 is repressed by glucose (derepressed during growth on non-fermentable carbon sources), enabling metabolic state-dependent expression. PGK is a strong constitutive promoter driving robust expression during glycolysis. Together, they represent the spectrum of inducible and constitutive control used in yeast work. Step-by-Step Solution: Identify each listed promoter as a commonly used yeast promoter.Recognize that the set covers inducible (GAL1), derepressible (ADH2), and constitutive (PGK) strategies.Therefore, select the inclusive option. Verification / Alternative check: Yeast toolkits and commercial vectors frequently include GAL1, PGK, and ADH family promoters for modular cloning and expression studies. Why Other Options Are Wrong: A single promoter choice ignores the others, all of which are widely used. None of the above is incorrect given extensive literature use of all three. Common Pitfalls: Confusing ADH1 (often stronger constitutive) with ADH2 (glucose-repressible); both ADH promoters are common in yeast vectors but have different regulation. Final Answer: All of the above

Question 2

Baculovirus biology — role of polyhedrin protein In baculoviruses, what is the functional significance of the polyhedrin protein?

Accepted Answer

Introduction / Context: Baculoviruses are widely used as expression vectors in insect cells. Understanding the roles of structural proteins is crucial for vector design. Polyhedrin is a hallmark protein forming occlusion bodies in infected insect larvae and plays a special role distinct from genome replication. Given Data / Assumptions: Two phenotypic forms: budded virus for cell-to-cell spread, and occlusion-derived virus protected by polyhedrin matrix for environmental persistence. Cell culture expression systems often delete polyhedrin to insert foreign genes. Concept / Approach: Polyhedrin forms crystalline occlusion bodies (polyhedra) that encase virions, protecting them in the environment and facilitating oral infection in insect hosts. However, polyhedrin is not required for virus replication in cultured cells, enabling replacement of the polyhedrin locus with heterologous genes under strong promoters for high-level expression. Step-by-Step Solution: Acknowledge polyhedrin as structural matrix protein of occlusion bodies.Note that deletion of polyhedrin does not abolish replication in vitro, allowing expression vector design.Therefore, both nonessential status for replication in culture and matrix role are correct. Verification / Alternative check: Commercial baculovirus systems (e.g., using AcMNPV) routinely leverage the polyhedrin locus; viable replication in Sf9/Sf21 cells without polyhedrin is well documented. Why Other Options Are Wrong: Absolutely essential for replication: contradicted by polyhedrin-minus vectors. DNA polymerase subunit: polyhedrin is a structural protein, not a replication enzyme. Common Pitfalls: Assuming all abundant viral proteins are replication enzymes; here abundance serves environmental stability and transmission in larvae. Final Answer: Both (a) and (b)

Question 3

SV40 vector systems — requirements for late-region replacement constructs SV40 cloning vectors in which late-region coding sequences are replaced generally require what for productive packaging?

Accepted Answer

Introduction / Context: SV40 (Simian Virus 40) has been used historically as a small DNA virus vector. Different designs either delete early or late regions to make room for inserts. Understanding which host or helper functions are required informs how to produce vector stocks and which cells to use. Given Data / Assumptions: Late-region replacement removes capsid protein coding sequences (VP1–VP3). Early region (e.g., large T antigen) may be intact or complemented by host cells like COS. Packaging of vector genomes into virions requires late gene products. Concept / Approach: Late-region replacement vectors lack structural proteins needed for virion assembly. Therefore, a helper SV40 providing the late gene products in trans is required for packaging. By contrast, vectors with early-region deletions can replicate in COS cells expressing large T antigen. Thus, the specific requirement depends on which region is deleted. Here, with late-region replacement, helper virus supply is the key. Step-by-Step Solution: Identify that late genes encode capsid proteins.Recognize that without capsid proteins, packaging cannot occur autonomously.Conclude that a helper SV40 must provide late functions in trans for productive packaging. Verification / Alternative check: Classical SV40 vector literature distinguishes early-deletion vectors (require T antigen in COS cells) from late-deletion vectors (require helper for capsid proteins). Why Other Options Are Wrong: COS cells supply T antigen (early function), not late capsid proteins. pBR322 is a bacterial plasmid backbone; it does not supply viral proteins. Adenovirus E1A is irrelevant to SV40 late gene provision. None of these is incorrect because helper is needed. Common Pitfalls: Memorizing COS cell requirement and applying it generally; always match the deleted region to the required complementing system. Final Answer: SV40 helper virus to supply late gene functions in trans

Question 4

SV40 minichromosome composition — histone association Which histone is notably absent from the SV40 virion-associated minichromosome?

Accepted Answer

Introduction / Context: SV40 packages its circular DNA with host histones, forming a minichromosome that resembles nucleosome organization in eukaryotic chromatin. However, the histone composition is distinctive compared with bulk chromatin. Given Data / Assumptions: Standard core histones: H2A, H2B, H3, H4. Linker histone: H1 stabilizes higher-order chromatin in many eukaryotes. SV40 virions contain nucleosome-like particles. Concept / Approach: Analyses of SV40 chromatin show association with core histones H2A, H2B, H3, and H4 that form nucleosomes. The linker histone H1 is not a stable component of the packaged viral minichromosome inside virions. This difference is often tested to distinguish core nucleosome assembly from higher-order chromatin organization. Step-by-Step Solution: Recall that nucleosomes comprise core histones H2A, H2B, H3, H4.Recognize that H1 is the linker histone and is notably absent in SV40 virions.Choose H1 as the missing histone. Verification / Alternative check: Biochemical isolation of SV40 chromatin demonstrates core histones present, with H1 largely absent from mature virions. Why Other Options Are Wrong: H2A, H2B, H3, H4 are core histones detected in SV40 chromatin. Common Pitfalls: Assuming viral chromatin mirrors all aspects of host chromatin; virions streamline components for packaging. Final Answer: H1

Question 5

Genome type of retroviruses — central dogma reversal Retroviruses are characterized by which type of genome inside the virion?

Accepted Answer

Introduction / Context: Retroviruses are iconic for using reverse transcriptase to convert their RNA genome into DNA that integrates into the host genome. Recognizing the virion genome type is foundational for understanding their replication strategy and the basis for retroviral vectors. Given Data / Assumptions: Virions contain the genetic material that first enters the host cell. Reverse transcription occurs after entry. Integration as proviral DNA happens post reverse transcription. Concept / Approach: Retroviruses package two copies of a positive-sense single-stranded RNA genome along with reverse transcriptase and other enzymes. After infection, reverse transcription generates double-stranded DNA, which is then integrated as a provirus. Therefore, the genome inside the virion is RNA, not DNA. Step-by-Step Solution: Recall hallmark: RNA genome + reverse transcriptase.Note that cDNA arises after entry, not in the virion.Select RNA genome as correct. Verification / Alternative check: Classic retroviral biology (e.g., HIV, MLV) demonstrates two RNA strands per virion and packaged RT; only after infection is a DNA intermediate synthesized. Why Other Options Are Wrong: dsDNA/ssDNA: not packaged by retroviruses (parvoviruses package ssDNA; hepadnaviruses have relaxed circular DNA). cDNA pre-integrated: integration occurs in host nucleus after reverse transcription. Protein-only genome: describes prions, not viruses. Common Pitfalls: Confusing the proviral DNA state (host-integrated) with the virion contents. Final Answer: RNA genome

Question 6

Vector classification — AcMNPV as an expression platform Autographa californica nuclear polyhedrosis virus (AcMNPV), commonly used for recombinant protein expression in insect cells, is an example of which vector type?

Accepted Answer

Introduction / Context: Baculovirus expression vectors are a mainstay for producing complex eukaryotic proteins with proper folding and post-translational modifications in insect cell lines (e.g., Sf9). Recognizing the viral origin of popular expression systems is essential for vector selection in biotechnology. Given Data / Assumptions: AcMNPV is the standard laboratory baculovirus backbone. Expression often uses the strong polyhedrin or p10 promoters. Infection is performed in lepidopteran cell lines. Concept / Approach: AcMNPV belongs to the Baculoviridae family. It is not a poxvirus (vaccinia), papillomavirus, or retrovirus. Its large dsDNA genome, insect host range, and capacity for high-level expression under late promoters identify it as a baculovirus. Step-by-Step Solution: Recall that nuclear polyhedrosis viruses are baculoviruses infecting insects.Associate AcMNPV with standard baculovirus-based expression systems.Select baculovirus. Verification / Alternative check: Vendor manuals and protocols (BEVS systems) explicitly cite AcMNPV as the baculovirus used for recombinant protein expression. Why Other Options Are Wrong: Vaccinia: poxvirus used for recombinant vaccines; mammalian host range. Papillomavirus: small circular DNA viruses (e.g., HPV), not used for insect expression vectors. Retrovirus/AAV: mammalian gene delivery vectors, different biology. Common Pitfalls: Confusing baculovirus with vaccinia because both can produce high protein levels; host range and genome organization differ. Final Answer: Baculovirus

Question 7

Permissivity and lytic infection in SV40 In which cell type does a lytic infection by SV40 fail to occur?

Accepted Answer

Introduction / Context: Small DNA tumor viruses such as SV40 show host-range and cell-type specificity. Whether infection proceeds to lytic replication or is abortive depends on availability of host factors and compatibility for viral gene expression. This concept underlies the use of permissive cell lines and transformation studies. Given Data / Assumptions: SV40 requires specific host machinery and temperature/condition suitability for productive replication. Permissive cells support full lytic cycle and virion production. Non-permissive cells restrict productive replication. Concept / Approach: By definition, permissive cells allow productive viral replication and lytic infection. Non-permissive cells do not permit the complete lytic cycle; infection may result in limited gene expression, integration, or transformation-like outcomes without virion production. Therefore, lytic infection does not occur in non-permissive cells. Step-by-Step Solution: Define permissivity in virology.Apply to SV40: permissive cells (e.g., certain monkey kidney lines) enable lytic virus production.Conclude that non-permissive cells fail to sustain lytic infection. Verification / Alternative check: Classic SV40 studies distinguish permissive monkey cells (productive lytic replication) from non-permissive rodent cells (abortive replication and transformation). Why Other Options Are Wrong: Permissive cells: by definition support lytic growth. Both/none: contradict core definitions. Large T overexpression: tends to enhance early functions; not a category where lytic infection is absent by definition. Common Pitfalls: Equating viral entry with successful lytic replication; entry can occur without permissive replication. Final Answer: Non-permissive cells

Question 8

Yeast Artificial Chromosome (YAC) vectors: Why can they form artificial chromosomes in eukaryotic hosts? Identify the essential DNA elements they carry that allow stable replication and segregation.

Accepted Answer

Introduction / Context: Yeast Artificial Chromosomes (YACs) are engineered cloning vectors that allow very large DNA inserts to be propagated like mini-chromosomes in yeast. This question checks whether you know the three essential chromosomal elements that make a YAC behave like a natural chromosome inside a eukaryotic cell. Given Data / Assumptions: The vector is designed for replication and stable inheritance in yeast. Chromosome-like behavior requires specific sequence elements. We must decide which features are present in YAC vectors. Concept / Approach: For any linear eukaryotic chromosome to replicate and segregate correctly, it needs: (1) an origin of replication to initiate DNA synthesis, (2) a centromere to attach to the spindle apparatus for faithful segregation, and (3) telomeres to protect the ends from degradation and end-to-end fusions. In yeast, the origin is an ARS (autonomously replicating sequence). Therefore, a complete YAC contains telomere sequences at both ends, a functional centromere (CEN) element, and an ARS for replication. Step-by-Step Solution: List the three required components for a eukaryotic chromosome: ARS, CEN, and TEL.Match these with the options: ARS = autonomously replicating sequence; centromere; telomere.Recognize that YAC vectors are built with all three to allow stable maintenance of very large inserts.Therefore, the correct consolidated choice is “all of these”. Verification / Alternative check: When YAC elements are incomplete (e.g., missing telomeres), constructs circularize or become unstable. Inclusion of CEN and TEL elements yields mitotic stability and proper partitioning, confirming the necessity of all three components. Why Other Options Are Wrong: Centromere only: allows segregation but without ARS and telomeres, replication/ends fail. Telomere only: protects ends but no replication origin or segregation element. ARS only: replication occurs, but DNA is unstable and mis-segregates without CEN and TEL. None of these: contradicts the observed design and function of YACs. Common Pitfalls: Confusing ARS (yeast origin) with bacterial ori; forgetting that telomeres are essential because YACs are linear, not circular. Final Answer: all of these

Question 9

Yeast cloning systems: Which yeast plasmid types are typically maintained at high copy number inside Saccharomyces cerevisiae?

Accepted Answer

Introduction / Context: Different yeast vector backbones differ in stability and copy number. Understanding YEp, YRp, and YCp behavior is essential for designing experiments that need either strong expression (high copy) or tight control (low copy) of cloned genes in Saccharomyces cerevisiae. Given Data / Assumptions: YEp vectors derive from the 2μ plasmid system. YRp vectors rely on ARS elements for replication. YCp vectors carry a centromere (CEN) and behave like mini-chromosomes (low copy). Concept / Approach: YEp vectors typically contain the 2μ origin and associated functions, supporting high copy numbers (often 20–50+ per cell). YRp vectors include ARS sequences that also allow autonomous replication, commonly resulting in relatively higher copy numbers than centromere-based plasmids, though sometimes less stable. In contrast, YCp vectors incorporate CEN and ARS, are segregated like chromosomes, and are maintained at a low copy (usually ~1–2 per cell). Step-by-Step Solution: Identify high-copy systems: 2μ-based YEp; ARS-only YRp often high but less stable.Recognize low-copy system: YCp (CEN-based).Select the combined answer acknowledging both YEp and YRp. Verification / Alternative check: Expression studies routinely use YEp for overexpression and YCp for near-genomic expression levels, confirming the copy-number distinction. Why Other Options Are Wrong: YEp only or YRp only: incomplete; both can be high copy. YCp: intentionally low copy due to centromere segregation. YAC: not a plasmid; behaves as a chromosome for very large inserts. Common Pitfalls: Equating stability with copy number; YRp may be high copy yet less stable than YEp. Final Answer: Both (a) and (b)

Question 10

Adenoviral vectors: What is the typical maximum foreign DNA insert size that first-generation adenovirus vectors can accommodate?

Accepted Answer

Introduction / Context: Adenovirus-based vectors are widely used in gene delivery because they efficiently transduce many cell types. Their packaging capacity is limited by the viral capsid and genome requirements. This question targets recall of the commonly accepted insert capacity for first-generation designs. Given Data / Assumptions: We are considering first-generation adenoviral vectors with standard deletions (e.g., E1/E3). Insert size refers to foreign DNA beyond the essential viral sequences that must remain. Concept / Approach: First-generation adenoviral vectors typically allow insertion of approximately 6–8 kilobases of foreign DNA while keeping the total genome length within the capsid packaging constraints. Although “gutless” or helper-dependent adenoviral systems can package larger payloads (approaching 30–36 kb), those are specialized constructs and not implied in standard, first-generation vectors. Step-by-Step Solution: Recall packaging constraint of typical first-generation systems.Recognize that 6–8 kb is the standard foreign insert range after E1/E3 deletions.Exclude larger numbers that pertain to helper-dependent designs.Select “6–8 kb”. Verification / Alternative check: Gene therapy manuals and vector datasheets frequently cite a working capacity near 7–8 kb for first-generation adenovirus; more than this risks inefficient packaging or instability. Why Other Options Are Wrong: 4–6 kb: too conservative for standard E1/E3-deleted vectors. 10–12 kb or 20 kb: exceed first-generation norms. ≥30 kb: applies to helper-dependent systems, not the basic format implied by the question. Common Pitfalls: Confusing capacities of different adenoviral generations; mixing adenovirus with AAV capacities (AAV ~4.7 kb). Final Answer: 6–8 kb

Question 11

Bovine papillomavirus (BPV)–derived vectors in mammalian cells: How are recombinant DNAs maintained in permanent cell lines established with BPV vectors?

Accepted Answer

Introduction / Context: BPV-derived vectors exploit viral elements that support replication as episomes in mammalian cells, enabling long-term maintenance without immediate integration. Understanding their behavior helps in choosing systems for stable expression versus genomic integration strategies. Given Data / Assumptions: BPV sequences can drive extrachromosomal replication in some mammalian cells. Permanent lines may contain episomal forms and, in some cases, integrated forms. Copy number can be high when episomal replication is supported. Concept / Approach: BPV vectors can persist as multicopy episomes due to viral replication functions, supporting high-level expression. During cell line establishment, recombination or selection can also yield integration into the host genome. Reports indicate high copy maintenance for either form, especially under selection, enabling robust expression of the recombinant cassette. Step-by-Step Solution: Recognize BPV's capacity for episomal replication in mammalian cells.Acknowledge that integration can also occur during selection.Note that both modes can support high copy numbers.Choose the option that captures both possibilities at high copy number. Verification / Alternative check: Empirical studies with BPV-based shuttle vectors show maintained episomes and, less commonly, integrated forms; both can reach high copy numbers under drug selection. Why Other Options Are Wrong: Episomal only / integrated only: too restrictive; both states are documented. Low copy number: contradicts typical multicopy maintenance with BPV elements. Not maintained: contradicted by the use of BPV in stable line generation. Common Pitfalls: Assuming episomal maintenance precludes integration; cell line derivation can generate a mixture of states. Final Answer: Either episomally or integrated at high copy number

Question 12

SV40-based delivery: Recombinant SV40 viruses introduce foreign DNA into mammalian cells in what way?

Accepted Answer

Introduction / Context: Simian Virus 40 (SV40) is a DNA tumor virus historically used as a cloning and expression vector in mammalian systems. Recombinant SV40 particles enter cells using viral entry mechanisms, bypassing classical chemical or physical transfection procedures. Given Data / Assumptions: Recombinant SV40 vectors are packaged into infectious virions. Viruses naturally deliver their genomes to host cells. “DNA-mediated transfection” refers to methods like calcium phosphate, lipofection, or electroporation. Concept / Approach: Infection by an SV40 virion introduces the recombinant DNA directly through receptor-mediated entry, uncoating, and nuclear import of the viral genome. Therefore, one does not require separate DNA transfection protocols to achieve delivery—viral infection accomplishes it. Step-by-Step Solution: Identify that the vector is delivered as an infectious particle.Recall that infection replaces the need for chemical/physical transfection methods.Choose the option that states delivery occurs without DNA-mediated transfection. Verification / Alternative check: Standard SV40 vector protocols rely on generating high-titer stocks and infecting cells, confirming that infection is the delivery route. Why Other Options Are Wrong: With DNA-mediated transfection: unnecessary when using infectious virus. RNA-mediated transfection: irrelevant for a DNA virus system. Lipid fusion only or microinjection: not the typical SV40 delivery route. Common Pitfalls: Confusing transfection (non-viral) with transduction (viral delivery). Final Answer: Without DNA-mediated transfection (via viral infection)

Question 13

Selectable markers in Saccharomyces cerevisiae: Which of the following are commonly used auxotrophic markers for yeast molecular genetics?

Accepted Answer

Introduction / Context: Yeast genetics routinely uses auxotrophic markers to select transformants on defined media. Recognizing standard markers speeds vector choice and experimental design in cloning and genome engineering. Given Data / Assumptions: Common yeast lab strains carry auxotrophic mutations (e.g., his3Δ, ura3Δ, trp1Δ). Complementation with a wild-type marker gene restores growth on selective media. Concept / Approach: HIS3 complements histidine auxotrophy, URA3 complements uracil auxotrophy, and TRP1 complements tryptophan auxotrophy. Vectors often include one of these markers to select for successful transformation, and LEU2 is another common marker (not exclusive). Therefore, HIS3, URA3, and TRP1 are all correct examples of commonly used markers. Step-by-Step Solution: Recall standard selectable markers in yeast cloning toolkits.Match HIS3, URA3, and TRP1 to their respective amino acid/nucleotide biosynthesis pathways.Select the inclusive option “All of these”. Verification / Alternative check: Yeast vector catalogs and classical protocols list HIS3, URA3, TRP1, LEU2, and others as routine markers. Why Other Options Are Wrong: Any single marker alone is incomplete; multiple are in common use. “LEU2 only” ignores the prevalence of HIS3/URA3/TRP1 markers. Common Pitfalls: Assuming one marker dominates; in practice, marker choice depends on host genotype and experimental design. Final Answer: All of these

Question 14

SV40 genome basics: What is the approximate size of the Simian Virus 40 (SV40) genome?

Accepted Answer

Introduction / Context: SV40 is a classic small DNA virus used in molecular biology as a model for eukaryotic replication and as the source of powerful regulatory elements (e.g., SV40 promoter). Knowing its genome size is foundational knowledge for vector design and literature comprehension. Given Data / Assumptions: Genome size is requested in kilobases (kb). SV40 is a small, circular, double-stranded DNA virus. Concept / Approach: The SV40 genome is approximately 5.2 kilobases in length (about 5,243 base pairs). This compact genome encodes early and late genes and regulatory elements widely adapted for expression constructs. Larger sizes listed in the distractors correspond to entirely different viral systems or are unrealistic for SV40. Step-by-Step Solution: Recall SV40 as a ~5.2 kb circular dsDNA virus.Compare options and select the closest correct value.Exclude implausible larger sizes (e.g., 69 kb, 110 kb).Choose “5.2 kb”. Verification / Alternative check: Genome maps and reference sequences consistently report ~5.2 kb for SV40, validating the answer. Why Other Options Are Wrong: 7.2 kb: too large for SV40; closer to some other small viral genomes. 69 kb, 110 kb: sizes more typical of large DNA viruses, not SV40. 2.6 kb: too small to encode SV40 functions. Common Pitfalls: Confusing SV40 with adenovirus or herpesvirus sizes; those are much larger. Final Answer: 5.2 kb

Question 15

Why are retroviruses useful as vectors for animal cells? Identify the principal advantages that make them attractive for gene delivery.

Accepted Answer

Introduction / Context: Retroviral and lentiviral vectors are staples in animal-cell gene delivery. Their biology offers several advantages that facilitate stable and efficient gene transfer, particularly for long-term expression in dividing cells (retrovirus) or even non-dividing cells (lentivirus derivatives). Given Data / Assumptions: The question addresses general advantages of retroviral systems as vectors. We consider common properties exploited in vector engineering. Concept / Approach: Engineered retroviruses often have broad tropism (host range) after pseudotyping (e.g., VSV-G), can establish persistent infection without immediate cytolysis, and use potent Long Terminal Repeat (LTR) promoters or internal promoters for robust expression. These features collectively make retroviral systems versatile for stable integration and gene expression in many animal cell types. Step-by-Step Solution: Recall broad host range achievable via envelope pseudotypes.Note non-lytic infection profile enabling sustained vector presence.Recognize strong promoter/enhancer activity enabling good transgene expression.Select the comprehensive option “All of the above”. Verification / Alternative check: Standard vector manuals list these benefits; clinical and research applications exploit integration for stable lines and in vivo gene transfer. Why Other Options Are Wrong: Single advantages alone are incomplete; retroviruses combine several useful traits. “None of the above” contradicts extensive empirical use in gene delivery. Common Pitfalls: Ignoring biosafety modifications; modern vectors are replication-defective and split into packaging/helper components. Final Answer: All of the above

Question 16

Properties of Yeast Episomal plasmids (YEp): Which statement correctly describes a typical YEp vector?

Accepted Answer

Introduction / Context: Multiple classes of yeast vectors exist, each optimized for different performance characteristics. YEp vectors are designed for high-copy episomal maintenance by leveraging the endogenous 2μ plasmid system of Saccharomyces cerevisiae. Given Data / Assumptions: YEp stands for Yeast Episomal plasmid. We compare YEp with YRp (ARS-based), YCp (CEN-based), and chromosome-like constructs (YAC). Concept / Approach: YEp plasmids contain the 2μ origin of replication and rep genes, enabling stable, often high-copy episomal replication in yeast. YRp vectors rely primarily on ARS elements and can be higher copy but less stable. YCp vectors carry a centromere (CEN) and are maintained at low copy with chromosome-like segregation. Telomeres are not typical for plasmids; those appear in YACs. Homologous recombination (crossing over) is a mechanism for integration, not for episomal maintenance, and is not required for YEp replication. Step-by-Step Solution: Identify YEp as 2μ-based.Exclude ARS-only description (fits YRp more closely).Exclude CEN/TEL description (fits YCp/YAC).Select the option describing the 2μ ori and rep functions. Verification / Alternative check: Standard yeast molecular cloning guides describe YEps as 2μ-derived, high copy, and selectable via auxotrophic markers or drug resistance. Why Other Options Are Wrong: Crossing over: pertains to integrative vectors, not episomal YEps. ARS-only: aligns with YRp vectors. CEN/TEL: belongs to YCp (CEN) or YAC (TEL). “Cannot replicate without integration”: false for episomal YEps. Common Pitfalls: Confusing ARS-only vectors with 2μ-based episomal systems; both can be high copy but have distinct backbones. Final Answer: Has a 2μ origin of replication and associated rep functions

Question 17

Yeast Replicating plasmids (YRp): Which key sequence do they carry to enable autonomous replication in yeast?

Accepted Answer

Introduction / Context: YRp plasmids are one of the core yeast vector classes. Knowing which functional element confers replication competency is essential for selecting the right backbone in cloning strategies that balance copy number, stability, and segregation. Given Data / Assumptions: YRp stands for Yeast Replicating plasmid. We distinguish YRp from YEp (2μ-based), YCp (centromere-based), and YAC (chromosome-like). Concept / Approach: YRp vectors carry ARS (autonomously replicating sequence) elements. ARS acts as an origin of replication in yeast, allowing plasmids to replicate without integration. They generally lack a centromere, so segregation can be less faithful and stability lower than YCp vectors. Telomeres are not a feature of circular plasmids like YRp. Step-by-Step Solution: Recall that ARS is the yeast origin of replication.Recognize that YRp uses ARS to replicate autonomously.Exclude CEN/TEL options, which correspond to different vector types.Choose “autonomously replicating sequence (ARS)”. Verification / Alternative check: Yeast cloning manuals consistently define YRp as ARS-bearing plasmids, typically higher copy but less stable than CEN-based YCp vectors. Why Other Options Are Wrong: CEN: defines YCp, not YRp. TEL: used in YACs to cap linear constructs, not in circular YRp. Both (a) and (c): incorrect because YRp lack CEN. Integration requirement: not necessary for ARS-bearing plasmids. Common Pitfalls: Assuming all autonomously replicating yeast vectors have CEN; that would classify them as YCp. Final Answer: autonomously replicating sequence (ARS)

Question 18

Recombinant adenovirus design — functional role of E1A/E1B deletions  In constructing replication-defective recombinant adenovirus vectors for gene delivery, the E1A/E1B region is commonly deleted. What is the principal reason this deletion prevents…

Accepted Answer

Introduction / Context: Adenoviral vectors are widely used in gene therapy and vaccine development because they can infect dividing and non-dividing cells and yield high transgene expression. To ensure safety and control, these vectors are often rendered replication-defective by deleting specific early genes, notably E1A and E1B. Given Data / Assumptions: E1A and E1B are early genes expressed soon after adenovirus entry. Replication-defective vectors lack E1 functions and therefore require complementation in producer cell lines (for example, HEK 293 cells). The question asks why deleting E1A/E1B blocks autonomous replication. Concept / Approach: E1A acts as a master transcriptional regulator that drives the expression of other early viral genes and pushes host cells into a replication-permissive state by modulating cell-cycle regulators. E1B stabilizes viral mRNAs and helps counteract host defenses, including apoptosis. Together, E1A/E1B establish the molecular environment for efficient viral DNA replication. Removing this region prevents the virus from activating the necessary early transcriptional program. Step-by-Step Solution: Identify the role of E1A: transcriptional activation of early genes and cell-cycle modulation.Identify the role of E1B: protecting viral replication by antagonizing host responses and supporting late gene expression.Infer consequence of deletion: no early gene cascade, no viral DNA replication, and no productive infection.Relate to practice: producer cell lines provide E1 in trans to allow vector production but target tissues do not. Verification / Alternative check: In laboratory production, HEK 293 cells supply E1 functions, enabling growth of E1-deleted adenoviral vectors. Outside such cells, the vectors enter but cannot replicate, confirming the essential role of E1A/E1B. Why Other Options Are Wrong: Nonessential genes (option b) can be deleted without blocking replication—this is untrue for E1. Capsid-only roles (option c) and packaging-only roles (option d) mischaracterize E1. Latency-only function (option e) is incorrect; adenoviruses do not have a true latent program like herpesviruses. Common Pitfalls: Confusing structural genes with regulatory early genes; assuming any early region is dispensable. E1 is uniquely critical to launch the replication program. Final Answer: They encode transcriptional regulators essential for initiating and sustaining viral replication

Question 19

COS cell line — origin and purpose in SV40-based expression  What accurately describes the COS (COS-1/COS-7) mammalian cell line commonly used for transient expression from SV40-origin plasmids?

Accepted Answer

Introduction / Context: COS cells (COS-1 or COS-7) are workhorse mammalian cell lines used for high-level, transient protein expression. They are particularly useful for plasmids carrying the SV40 origin of replication because they provide the SV40 large T antigen in trans. Given Data / Assumptions: COS cells derive from CV-1 African green monkey kidney cells. They express SV40 large T antigen, which recognizes and drives replication from SV40 ori. The question contrasts COS cells with the phage λ cos sequence and unrelated cell backgrounds. Concept / Approach: SV40 large T antigen is necessary to initiate bidirectional DNA replication at the SV40 origin. By stably expressing large T, COS cells amplify SV40-ori plasmids episomally, boosting transgene expression without needing viral infection. This distinguishes the cell line from the similarly named λ cos DNA packaging site. Step-by-Step Solution: Identify COS lineage: CV-1 monkey kidney origin.Recognize engineered feature: stable expression of SV40 large T antigen.Connect function: supports replication of SV40-ori plasmids, enabling high transient expression.Select the description aligning with these properties. Verification / Alternative check: Standard expression protocols specify COS-7 for SV40-ori plasmids; omission of large T antigen support in other cell lines yields lower copy number, confirming the unique benefit of COS cells. Why Other Options Are Wrong: Phage λ cos DNA sites (options b and c) are sequence elements, not cell lines. Insect cells (option d) pertain to baculovirus systems. Human fibroblasts with telomerase (option e) are unrelated to SV40-ori dependence. Common Pitfalls: Confusing COS (cell line) with cos (λ DNA packaging site). The similar names refer to very different concepts. Final Answer: Derivative of permissive CV-1 African green monkey cells engineered to express SV40 large T antigen

Question 20

Construction of yeast episomal plasmids (YEp)  YEp cloning vectors were historically engineered from which key components?

Accepted Answer

Introduction / Context: Yeast episomal plasmids (YEp) are shuttle vectors that replicate in both Escherichia coli and Saccharomyces cerevisiae. Their design allows convenient DNA manipulation in bacteria and functional expression or selection in yeast. Given Data / Assumptions: 2μm plasmid is a natural, high-copy yeast plasmid providing replication functions in S. cerevisiae. pBR322 provides bacterial origin of replication and antibiotic resistance for propagation in E. coli. Inclusion of yeast nuclear DNA fragments can supply selectable markers (for example, LEU2, URA3). Concept / Approach: By combining the 2μm sequences (for yeast replication), yeast marker genes (for selection), and a bacterial backbone (for cloning and amplification), YEp vectors function as classic shuttle plasmids. This architecture enables rapid cloning in E. coli and subsequent transformation and high-copy maintenance in yeast. Step-by-Step Solution: Identify yeast replication element: 2μm origin.Identify bacterial backbone: pBR322 origin and antibiotic resistance.Add selectable yeast markers from nuclear DNA.Match to option listing these three components. Verification / Alternative check: Textbook diagrams of YEp vectors consistently show 2μm-derived sequences, a yeast marker, and pBR322-derived sequences, validating the composition. Why Other Options Are Wrong: Options omitting pBR322 (or substituting pMB9) or the 2μm element fail to provide dual-host replication/selection; 12μm plasmid is not a standard yeast replicon; mitochondrial or T7 components are not typical for YEp. Common Pitfalls: Confusing YEp with YRp (ARS-based) or YIp (integrative) vectors; YEp specifically leverages 2μm for stable episomal maintenance. Final Answer: 2μm yeast plasmid, fragments of yeast nuclear DNA, and E. coli vector pBR322

Vectors Uses for Animal Cell Culture Questions