Bio Exam 2 study guide answers

Chapter 6: DNA Replication and Repair

Q: How did the work of Meselson and Stahl support the semiconservative model of DNA replication? A: They used isotope labeling of nitrogen in DNA and density gradient centrifugation to show that each new DNA molecule consists of one old and one new strand.

Q: What is a replication bubble? A: A region where the DNA double helix is unwound to allow replication.

Q: What do initiator proteins do? A: They bind to the replication origin and help separate the DNA strands to initiate replication.

Q: What does helicase do during DNA replication? A: Unwinds the DNA double helix at the replication fork.

Q: What does topoisomerase (gyrase) do? A: Relieves supercoiling tension ahead of the replication fork by cutting and rejoining DNA strands.

Q: What is a replication fork? A: The Y-shaped region where the DNA strands are separated and replication occurs.

Q: Where does DNA primase build an RNA primer? A: On the leading strand once and on the lagging strand multiple times to provide a starting point for DNA polymerase.

Q: Where does DNA polymerase attach, and why? A: It attaches to the RNA primer to begin synthesizing the new DNA strand.

Q: What is the direction of DNA synthesis? A: 5’ to 3’ direction, since nucleotides can only be added to the 3’ end of the growing strand.

Q: What is the leading vs. lagging strand during replication? A: The leading strand is synthesized continuously, while the lagging strand is synthesized in short Okazaki fragments.

Q: What problem occurs with the lagging strand, and how does telomerase help? A: The lagging strand shortens over time; telomerase extends the ends of chromosomes to prevent genetic loss.

Q: What are the key enzymes in DNA replication and their functions? A: Helicase (unwinding), Topoisomerase (relieves tension), Primase (makes primers), DNA Polymerase III (elongates strands), DNA Polymerase I (replaces RNA primers), Ligase (joins fragments), Telomerase (extends chromosome ends).

Q: What provides energy for building DNA? A: The cleavage of phosphate bonds in deoxynucleoside triphosphates (dNTPs).

Q: Define Okazaki fragments. A: Short DNA fragments synthesized on the lagging strand during replication.

Q: What effect does UV light have on DNA? A: It causes thymine dimers, which can lead to mutations.

Q: Define depurination. A: The loss of a purine base (adenine or guanine) from DNA, leading to mutations.

Q: Define deamination. A: The removal of an amino group from cytosine, converting it to uracil.

Q: How does mismatch repair work? A: It corrects base-pair mismatches by excising incorrect nucleotides and replacing them with the correct ones.

Q: How are double-stranded breaks repaired? A: By non-homologous end joining (NHEJ) or homologous recombination.

Chapter 7: Transcription and Translation

Q: What is the central dogma of molecular biology? A: DNA → RNA → Protein.

Q: How does RNA differ from DNA? A: RNA has ribose sugar, uracil instead of thymine, and is single-stranded.

Q: Define ssRNA. A: Single-stranded RNA, as opposed to double-stranded DNA.

Q: What is RNA polymerase and how does it function? A: It synthesizes RNA from a DNA template; unlike DNA polymerase, it does not require a primer.

Q: How is transcription initiated and terminated? A: Initiated at a promoter sequence and terminated by a stop signal or hairpin loop structure.

Q: What are enhancer and silencer sequences? A: Enhancers increase transcription, silencers decrease transcription.

Q: What is the role of the promoter in transcription? A: The promoter is a DNA sequence that signals RNA polymerase where to begin transcription.

Q: What is a codon? A: A three-nucleotide sequence that codes for an amino acid.

Q: How do you read a codon table correctly? A: Identify the first nucleotide on the left, the second across the top, and the third on the right.

Q: What is the start codon? A: AUG (Methionine).

Q: What are the stop codons? A: UAA, UAG, UGA.

Q: What is a reading frame? A: A way of dividing nucleotide sequences into sets of three codons for translation.

Q: What role does tRNA play in translation? A: tRNA carries amino acids to the ribosome and matches its anticodon to the mRNA codon.

Q: How are amino acids added to tRNAs? By what enzyme? A: Aminoacyl-tRNA synthetase attaches amino acids to their respective tRNAs.

Q: What are the terms for the large and small ribosomal subunits in prokaryotes and eukaryotes? A: Prokaryotes: 30S (small) and 50S (large); Eukaryotes: 40S (small) and 60S (large).

Q: What are the functions of the A, P, and E sites in ribosomes? A: A (accepts tRNA), P (holds growing peptide), E (exit for empty tRNA).

Q: How does a ribosome form a polypeptide chain, and how does the process terminate? A: The ribosome reads mRNA, tRNA delivers amino acids, peptide bonds form, and termination occurs at a stop codon.

Q: Define ribozyme. A: An RNA molecule with enzymatic activity, such as rRNA in ribosomes.

Q: What role does rRNA play in evolutionary research? A: rRNA sequences are highly conserved and used to study evolutionary relationships.

Q: How does transcription differ between eukaryotes and prokaryotes? A: Eukaryotes have transcription in the nucleus with modifications like splicing; prokaryotes transcribe and translate simultaneously in the cytoplasm.

Q: What is the 5’ cap? A: A modified guanine nucleotide added to the 5’ end of eukaryotic mRNA for stability and translation initiation.

Q: What is a poly-A tail? A: A stretch of adenine nucleotides added to the 3’ end of eukaryotic mRNA for stability and nuclear export.

Q: How are introns removed and exons spliced? A: The spliceosome, made of snRNPs, removes introns and joins exons.

Q: What are snRNPs and what is a spliceosome? A: snRNPs are small nuclear ribonucleoproteins that form the spliceosome, which splices pre-mRNA.

Q: What are alternative splice isoforms? A: Different mRNA versions from the same gene due to alternative splicing.

Q: Define polycistronic and how it applies to prokaryotic mRNAs. A: A single mRNA encoding multiple proteins, common in prokaryotes.

Q: Name the mutation types and their consequences. A:

• Silent: No effect on protein.

• Missense: Alters one amino acid.

• Nonsense: Introduces a stop codon.

• Frameshift: Shifts reading frame, altering protein sequence.

Chapter 8: Gene Regulation and Expression

Q: What is the preferred food source for E. coli? A: Glucose.

Q: Which two proteins must E. coli express to utilize lactose? A: Lactose permease and beta-galactosidase.

Q: Define operon. A: A cluster of genes under the control of a single promoter, common in prokaryotes.

Q: How does the Lac operon work? A: It is activated when lactose is present and glucose is low, allowing the breakdown of lactose.

Q: What does a repressor protein do? A: It binds to the operator sequence to block RNA polymerase and prevent transcription.

Q: What is an operator sequence? A: A DNA segment where a repressor binds to regulate gene transcription.

Q: What is an activator protein? A: A protein that enhances gene transcription by assisting RNA polymerase binding.

Q: How is the activator protein used in the Lac operon regulated? A: It binds in the absence of glucose and presence of cAMP to activate transcription.

Q: How does gene expression affect the development of specialized cells? A: Gene expression patterns determine cell differentiation by activating specific genes.

Q: What are the regulatory elements in eukaryotes and what do they do? A: Promoters, enhancers, silencers, and insulators control transcription by interacting with transcription factors.

Q: What are transcription factors? A: Proteins that bind to DNA to regulate gene transcription.

Q: How do transcription factors bind to DNA? A: Through specific DNA-binding domains that recognize promoter or enhancer sequences.

Q: How do regulatory elements control gene expression? A: By promoting or inhibiting transcription factor binding and RNA polymerase activity.

Q: Why are some regulatory elements far from the genes they control? A: DNA looping allows distant enhancers to interact with promoters.

Q: What are CTCF proteins and what do they do? A: They are insulator-binding proteins that help organize chromatin structure and regulate gene expression.

Q: Define “topologically associated domains” and describe how they function. A: 3D chromosome regions that facilitate coordinated gene regulation by keeping certain genes together.

Q: How do chromatin remodeling complexes function? A: They reposition or modify nucleosomes to make DNA more or less accessible for transcription.

Q: How do histone-modifying enzymes function? A: They add or remove chemical groups on histones to alter chromatin structure and gene expression.

Q: Why is acetylation of lysine important for transcription? How does it work? A: It neutralizes histone charge, loosening DNA to allow transcription.

Q: How are histone proteins modified? A: Through acetylation, methylation, phosphorylation, and ubiquitination.

Q: What effect does methylation of cytosines in DNA have on gene expression? How does it work? A: It silences genes by recruiting proteins that compact chromatin.

Q: Define epigenetic inheritance and describe the three ways patterns of gene expression pass from a cell to its progeny. A: Heritable gene expression changes without altering DNA sequence, via DNA methylation, histone modification, and regulatory RNAs.

Q: What are some ways by which gene expression and epigenetic changes occur? A: Environmental factors, developmental signals, and cellular stress can trigger these changes.

Q: What are pluripotent stem cells and why are they important? How are they useful? A: Cells that can differentiate into any cell type; useful for regenerative medicine.

Q: What are master transcriptional regulators and why are they important? A: Proteins that control cell fate by activating key developmental genes.

Q: What are microRNAs? How do they interact with RISC? For what purpose? A: Small RNAs that bind to RISC to degrade or block translation of target mRNAs.

Chapter 10: DNA Technology and Genetic Engineering

Q: What role do restriction enzymes play in the process of amplifying DNA fragments? A: They cut DNA at specific sequences to create fragments for cloning and analysis.

Q: In what organism did restriction enzymes evolve? For what purpose? A: They evolved in bacteria to protect against viral infections by cutting foreign DNA.

Q: What role does DNA ligase play in the process of amplifying DNA fragments? A: It joins DNA fragments by forming phosphodiester bonds.

Q: Define recombinant DNA. A: DNA that has been artificially combined from different sources.

Q: How is the term “vector” used when discussing amplification of DNA fragments? A: A vector is a DNA molecule used to transport foreign genetic material into a host cell.

Q: What three essential elements should a vector contain? Why? A: An origin of replication, a selectable marker, and a multiple cloning site; these elements allow replication, selection of transformed cells, and insertion of DNA.

Q: By what process is a recombinant plasmid inserted into a bacterial cell? A: Transformation.

Q: How are bacterial cells with the correct recombinant plasmid selected using antibiotic media? A: Only bacteria containing the plasmid with an antibiotic resistance gene survive on antibiotic-containing media.

Q: What are genomic DNA libraries? How are they created? A: Collections of DNA fragments representing an entire genome; created by cutting DNA with restriction enzymes and cloning into vectors.

Q: What is complementary DNA (cDNA)? Why is a cDNA library useful? A: cDNA is synthesized from mRNA and represents expressed genes; useful for studying gene expression.

Q: What are two ways to find a particular gene in a DNA library? A: Hybridization with a labeled probe or PCR amplification.

Q: What is a complementation screen? What types of genes does it help find? A: A method to identify genes that restore function in a mutant background; used for functional gene identification.

Q: What is a protein expression screen? How does it use antibodies? A: A technique to detect proteins expressed from a DNA library using labeled antibodies.

Q: Why does DNA migrate toward the positive electrode in gel electrophoresis? A: DNA is negatively charged due to its phosphate backbone.

Q: Why do DNA fragments stop at different places in a gel during electrophoresis? A: Smaller fragments move faster and farther than larger fragments.

Q: What is agarose and why is it useful for gel electrophoresis? A: A gel matrix that separates DNA fragments by size.

Q: Why is the gel electrophoresis technique useful? A: It allows visualization and size determination of DNA fragments.

Q: What is a dideoxynucleotide and how is it used in the lab? A: A modified nucleotide that terminates DNA synthesis in Sanger sequencing.

Q: Describe the steps involved in Sanger sequencing. How do the steps differ when using gel electrophoresis vs. sequencing machines? A: DNA polymerase incorporates dideoxynucleotides to generate fragments; gel electrophoresis separates fragments by size, while sequencing machines use fluorescence and capillary electrophoresis.

Q: Why is PCR useful? A: It amplifies specific DNA sequences for analysis.

Q: What polymerase is used during PCR and why? A: Taq polymerase, because it is heat-stable and withstands the high temperatures required for DNA denaturation.

Q: What are the steps necessary for PCR to be successful? A: Denaturation, annealing, and extension.

Q: How are viruses, such as coronavirus, identified by PCR? A: By amplifying viral RNA using reverse transcription PCR (RT-PCR).

Q: What is next-generation sequencing? Why is it important? A: A high-throughput method for sequencing DNA rapidly and cost-effectively.

Q: What is nanopore sequencing? Why is it important? A: A sequencing technique that passes DNA through a nanopore to detect base changes; important for long-read sequencing.

Q: Give examples of the best research uses for each type of sequencing. A: Sanger sequencing for small DNA segments, next-generation sequencing for whole genomes, and nanopore sequencing for long-read applications.

Q: Where was Cas9 originally isolated? A: From the bacterial CRISPR system.

Q: How does CRISPR-Cas9 target and replace genes? A: It uses a guide RNA to direct the Cas9 enzyme to a specific DNA sequence for cutting and repair.

Q: How is Cas9 used to activate a dormant gene? A: A modified Cas9 fused with activator domains binds to a gene promoter to enhance transcription.

Q: How is Cas9 used to turn off an actively expressed gene? A: A catalytically inactive Cas9 (dCas9) blocks transcription by binding to the gene's promoter.