From Gene to Protein I
1. How did evidence from the study of metabolic defects contribute to the fundamental relationship between genes and proteins?
Studies of metabolic defects, especially in organisms with inherited diseases (like alkaptonuria), suggested that specific genes are responsible for specific enzymes involved in metabolic pathways. Mutations that led to the absence or malfunction of these enzymes resulted in observable phenotypic consequences, supporting the idea that genes encode proteins that perform cellular functions.
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2. Describe the experiment by Beadle and Tatum which helped to develop the one gene – one enzyme hypothesis.
Beadle and Tatum (1941) exposed Neurospora crassa (a bread mold) to X-rays to induce mutations. They then grew the mutants on minimal medium and observed which ones could not grow unless supplemented with specific nutrients. They discovered that each mutant had a defect in a single gene that corresponded to a missing enzyme in a metabolic pathway. This led to the "one gene–one enzyme" hypothesis, later refined to "one gene–one polypeptide."
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3. Why has the one gene – one enzyme hypothesis been restated to be more accurate?
The original hypothesis was revised because:
* Not all proteins are enzymes.
* Some proteins are made from multiple polypeptide chains, each coded by a different gene.
* A single gene can produce multiple proteins via alternative splicing.
Thus, the modern understanding is "one gene–one polypeptide," though even this is an oversimplification in some contexts.
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4. Describe the basic principles of transcription and translation.
* Transcription: DNA is transcribed into mRNA by RNA polymerase in the nucleus (eukaryotes). The enzyme reads the DNA template strand and builds a complementary RNA strand.
* Translation: The mRNA is translated into a polypeptide at the ribosome in the cytoplasm. tRNAs bring amino acids matching the mRNA codons, forming the protein chain.
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5. Compare and contrast prokaryotic and eukaryotic transcription and translation.
Feature
Prokaryotes
Eukaryotes
Location
Cytoplasm
Transcription in nucleus, translation in cytoplasm
RNA Processing
Minimal or none
Extensive: capping, splicing, polyadenylation
Timing
Concurrent transcription and translation
Separated processes
Gene organization
Often operons (polycistronic mRNA)
Typically monocistronic mRNA
RNA Polymerases
One type
Three types (I, II, III)
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6. How are the instructions for assembling amino acids into proteins encoded into DNA?
DNA uses sequences of three-nucleotide codons to encode amino acids. Each codon in the mRNA corresponds to a specific amino acid or a stop signal. This genetic code is used during translation to assemble the amino acid sequence of a protein.
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7. How is the genetic code redundant but not ambiguous?
* Redundant: Most amino acids are encoded by more than one codon (e.g., Leucine has 6 codons).
* Not ambiguous: Each codon specifies only one amino acid, ensuring the correct protein is produced.
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8. Describe the molecular components of transcription.
* DNA template strand
* RNA polymerase: Synthesizes RNA
* Transcription factors: Help RNA polymerase bind in eukaryotes
* Promoter region: Signals where transcription starts
* mRNA: The transcript produced
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9. How do enhancers and promoters contribute to transcription?
* Promoters: DNA sequences near the transcription start site where RNA polymerase binds.
* Enhancers: Distal DNA elements that bind activator proteins to increase transcription levels. They can act at a distance through DNA looping.
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10. How do eukaryotic cells modify RNA?
After transcription, eukaryotic pre-mRNA undergoes:
* 5' capping: A modified guanine added to the 5' end
* 3' polyadenylation: A tail of adenines (poly-A tail) added to the 3' end
* Splicing: Removal of introns and joining of exons
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11. How is RNA splicing carried out?
Splicing is performed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and proteins. It:
* Recognizes splice sites at intron/exon boundaries
* Cuts out introns
* Joins exons together to form mature mRNA
From Gene to Protein II
1. What is the functional and evolutionary importance of introns?
Functional Importance:
* Alternative splicing: Introns allow for different combinations of exons to be joined together, enabling a single gene to produce multiple proteins.
* Gene regulation: Some introns contain regulatory elements like enhancers or silencers that influence gene expression.
* RNA transport and stability: Introns influence the export and stability of mRNA.
Evolutionary Importance:
* Introns facilitate exon shuffling, a mechanism that can create new proteins by recombining functional domains.
* They may increase genomic flexibility, allowing more rapid evolution of new gene functions.
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2. How do transfer RNAs (tRNAs) contribute to polypeptide elongation?
tRNAs bring the correct amino acids to the ribosome during translation. Each tRNA has:
* An anticodon that base-pairs with a specific mRNA codon.
* An amino acid attached to its 3' end, corresponding to its anticodon.
During elongation:
1. A tRNA enters the A site of the ribosome.
2. The ribosome catalyzes peptide bond formation between the new amino acid and the growing chain at the P site.
3. The tRNA exits from the E site.
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3. How does each specific tRNA pair with codons of mRNA?
Each tRNA has an anticodon loop with three nucleotides that are complementary and antiparallel to an mRNA codon. For example, if the mRNA codon is 5’-AUG-3’, the tRNA anticodon will be 3’-UAC-5’. This ensures codon-specific translation, aided by wobble pairing at the third codon position, allowing some flexibility.
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4. How do ribosomes facilitate protein synthesis?
Ribosomes are the molecular machines that:
* Bind mRNA and read codons in sequence.
* Recruit tRNAs and ensure proper codon-anticodon matching.
* Catalyze peptide bond formation between amino acids.
* Move along the mRNA (translocation), adding amino acids until a stop codon is reached.
They consist of a large and small subunit, each made of rRNA and proteins.
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5. How is protein synthesis terminated?
Termination occurs when a stop codon (UAA, UAG, UGA) is reached on the mRNA. These codons do not code for amino acids.
* A release factor binds to the ribosome at the A site.
* It triggers the hydrolysis of the polypeptide from the tRNA.
* The ribosome dissociates, releasing the mRNA and completed protein.
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6. How are polypeptides shuttled to the endoplasmic reticulum (ER) for secretion?
Proteins destined for secretion have an N-terminal signal peptide.
* As the protein is synthesized, the signal recognition particle (SRP) binds the signal peptide and halts translation.
* The complex docks at the SRP receptor on the rough ER.
* Translation resumes, and the growing polypeptide is threaded into the ER through a translocon.
* The signal peptide is often cleaved, and the protein is folded inside the ER.
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7. Describe the different types of DNA mutations that can occur.
* Point mutations – change in a single nucleotide:
* Substitution (e.g., A → G)
* Insertions or deletions (indels):
* May cause frameshifts, altering the reading frame
* Duplication – repeated DNA segment
* Inversion – a segment is flipped in orientation
* Translocation – segments from different chromosomes swap places
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8. What are the differences between silent, missense, and nonsense mutations?
Mutation Type
Description
Effect
Silent
Changes a codon to another that codes for the same amino acid
No change in protein sequence
Missense
Changes a codon to one that codes for a different amino acid
May affect protein function
Nonsense
Changes a codon to a stop codon
Leads to premature termination and truncated protein
Prokaryotic Gene Expression
1. How do bacteria regulate the production of enzymes?
Bacteria regulate enzyme production primarily at the transcriptional level using operons—clusters of genes under the control of a single promoter. Regulation occurs in response to environmental changes to conserve energy. The mechanisms include:
* Negative regulation: A repressor protein binds to DNA to block transcription.
* Positive regulation: An activator protein enhances transcription.
* Feedback inhibition: The end product of a pathway inhibits enzyme activity or gene expression.
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2. Define the components of an operon.
An operon typically includes:
* Promoter: DNA sequence where RNA polymerase binds to initiate transcription.
* Operator: A segment of DNA where a repressor can bind to block transcription.
* Structural genes: Genes that code for proteins (e.g., enzymes in a pathway).
* Regulatory gene (not part of operon proper): Encodes the repressor or activator protein.
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3. How does a repressor prevent gene transcription?
A repressor protein binds to the operator region of the operon. This physically blocks RNA polymerase from transcribing the structural genes. Whether the repressor is active depends on interaction with specific molecules:
* In an inducible system, the repressor is active until inactivated by an inducer.
* In a repressible system, the repressor is inactive until activated by a corepressor.
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4. Compare and contrast repressible and inducible operons.
Feature
Inducible Operon (e.g., lac operon)
Repressible Operon (e.g., trp operon)
Default state
OFF (genes are normally not expressed)
ON (genes are normally expressed)
Triggered by
Presence of a substrate (e.g., lactose)
Presence of an end product (e.g., tryptophan)
Repressor activity
Active until inactivated by an inducer
Inactive until activated by a corepressor
Biological role
Catabolic pathways (breaking down substances)
Anabolic pathways (building substances)
Goal
Make enzymes only when substrate is available
Stop making enzymes when product is sufficient
Eukaryotic Gene Expression
1. Describe the different stages where gene expression is regulated in eukaryotic cells.
Eukaryotic gene expression is regulated at multiple levels:
1. Chromatin accessibility (epigenetic level)
2. Transcriptional control (e.g., transcription factors, enhancers)
3. RNA processing (splicing, capping, polyadenylation)
4. mRNA transport (nucleus to cytoplasm)
5. mRNA stability (lifespan of mRNA)
6. Translation control (initiation and inhibition)
7. Post-translational modification (phosphorylation, cleavage)
8. Protein degradation (via proteasomes)
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2. Define epigenetic inheritance and describe the mechanism involving chromatin modification.
Epigenetic inheritance refers to heritable changes in gene expression that do not involve changes in the DNA sequence. Mechanisms include:
* Histone modifications (e.g., acetylation opens chromatin, methylation can repress or activate)
* DNA methylation (typically represses transcription)
* These modifications influence chromatin structure, making genes more or less accessible for transcription.
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3. How do transcription factors contribute to gene expression?
Transcription factors are proteins that:
* Bind to specific DNA sequences (promoters or enhancers)
* Help or hinder RNA polymerase binding and initiation
* Can act as activators (enhance transcription) or repressors
They ensure that specific genes are turned on in the right cell at the right time.
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4. How is gene activation regulated in two different cells (liver and lens cells) having identical DNA?
Though liver and lens cells have identical genomes, they express different genes due to:
* Different combinations of transcription factors
* Epigenetic differences (methylation patterns, histone marks)
This results in differential gene expression, giving each cell type its unique function.
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5. How does alternative RNA splicing contribute to the regulation of gene expression?
Alternative splicing allows a single gene to produce multiple mRNA variants, leading to different proteins. This:
* Increases protein diversity
* Enables tissue-specific expression of protein isoforms
* Plays roles in development and adaptation
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6. What determines the lifespan of mRNA and proteins?
* mRNA lifespan is influenced by:
* Sequences in the 3’ UTR
* Poly-A tail length
* Binding of regulatory proteins or microRNAs
* Protein lifespan is controlled by:
* Specific degradation signals (like ubiquitin tags)
* Targeting to the proteasome for degradation
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7. How do non-coding RNAs control gene expression?
Non-coding RNAs (ncRNAs) regulate gene expression at multiple levels:
* MicroRNAs (miRNAs): Bind complementary mRNA sequences to block translation or trigger degradation
* siRNAs: Similar to miRNAs, used in RNA interference
* lncRNAs: Involved in chromatin remodeling, transcriptional regulation, or splicing
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8. How do cytoplasmic determinants in the egg contribute to differential gene expression during development?
Cytoplasmic determinants are mRNAs and proteins unevenly distributed in the egg cytoplasm. After fertilization:
* As cells divide, they inherit different determinants
* This leads to differential gene expression, driving cell fate specification
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9. How does MyoD act as a master regulator of gene expression?
MyoD is a transcription factor that activates muscle-specific genes. It:
* Binds DNA and promotes transcription of genes needed for muscle differentiation
* Can convert non-muscle cells (like fibroblasts) into muscle-like cells
* Controls a regulatory cascade leading to a muscle cell identity
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10. Describe the process of pattern formation in the fruit fly.
Pattern formation involves setting up the body plan of the embryo. In Drosophila:
1. Maternal effect genes (like bicoid) establish axes.
2. Gap genes divide the embryo into broad regions.
3. Pair-rule genes refine these into segments.
4. Segment polarity genes define anterior/posterior orientation in segments.
5. Homeotic (Hox) genes assign identities to each segment.
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11. How does the bicoid gene contribute to axis establishment in the fruit fly?
bicoid is a maternal effect gene:
* Its mRNA is localized at the anterior end of the egg.
* After fertilization, it’s translated into Bicoid protein, forming a gradient.
* Cells "read" the gradient to determine their position along the anterior-posterior axis.
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12. What genes are associated with cancer and how do mutations in these genes contribute to cancer?
Key gene types involved in cancer:
* Oncogenes: Mutated proto-oncogenes that promote uncontrolled cell division (e.g., ras)
* Tumor suppressor genes: Normally inhibit growth or promote DNA repair (e.g., p53, BRCA1/2)
Mutations can lead to:
* Uncontrolled proliferation
* Avoidance of apoptosis
* Genomic instability
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13. How do multiple mutations contribute to colorectal cancer?
Colorectal cancer is a multi-step process:
1. Loss of APC tumor suppressor – early polyp formation
2. Activation of ras oncogene – promotes proliferation
3. Loss of DCC and p53 – leads to malignancy and metastasis
Each mutation adds to the breakdown of cellular control, highlighting the multi-hit model of cancer.
Biotechnology
1. Describe the process of cloning a gene using bacteria.
Gene cloning using bacteria involves:
1. Isolating the gene of interest from donor DNA.
2. Inserting it into a plasmid vector using restriction enzymes and DNA ligase.
3. Transforming the plasmid into bacteria (usually E. coli).
4. Selecting transformed colonies using antibiotic resistance or marker genes.
5. Growing bacteria to replicate and express the gene or produce the protein.
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2. How do restriction enzymes contribute to DNA cloning?
Restriction enzymes (endonucleases) cut DNA at specific sequences, producing:
* Sticky ends (overhanging single strands) or
* Blunt ends (straight cuts)
These cuts allow foreign DNA to be inserted into plasmids with matching ends, creating recombinant DNA.
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3. How are genomic and complementary DNA (cDNA) libraries established in bacteria?
* Genomic library: DNA is extracted, cut with restriction enzymes, and cloned into vectors. It includes introns, exons, and regulatory regions.
* cDNA library: Made by reverse-transcribing mRNA into cDNA using reverse transcriptase, then cloning it. It only contains expressed genes (no introns).
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4. How are genomic and cDNA libraries screened for a gene of interest?
* Hybridization: A labeled DNA or RNA probe complementary to the target gene is used to detect matching sequences.
* PCR-based screening: Specific primers amplify only the gene of interest from library clones.
* Antibody screening (for cDNA): If the gene expresses a protein, antibodies can detect it.
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5. Describe the steps involved with Southern blotting analysis.
Southern blotting detects specific DNA sequences:
1. Digest DNA with restriction enzymes.
2. Separate fragments by gel electrophoresis.
3. Transfer DNA from gel to a membrane (blotting).
4. Hybridize with a labeled probe complementary to the target sequence.
5. Detect the probe, revealing bands corresponding to the gene.
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6. How can RFLP be utilized to compare two different DNA molecules?
Restriction Fragment Length Polymorphism (RFLP) involves:
* Digesting DNA with restriction enzymes.
* Running fragments on a gel and probing with a specific DNA sequence.
Differences in fragment lengths reflect mutations or polymorphisms, useful in genetic mapping, forensics, or paternity testing.
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7. What factors do biologists consider when deciding the appropriate expression system?
Factors include:
* Host organism (bacteria, yeast, insect, mammalian cells)
* Post-translational modifications (e.g., glycosylation in eukaryotes)
* Protein folding and solubility
* Speed and cost of expression
* Safety and scalability
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8. How does PCR amplify minute quantities of DNA?
Polymerase Chain Reaction (PCR):
1. Denaturation: DNA is heated to separate strands.
2. Annealing: Primers bind to flanking sequences.
3. Extension: Taq polymerase adds nucleotides to extend primers.
This cycle repeats (~30–40 times), doubling the DNA each cycle → exponential amplification.
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9. Describe the steps involving DNA sequencing with dideoxyribonucleotides.
Sanger sequencing (chain-termination method):
1. Set up four reactions (or a single mix) with DNA polymerase, template, primer, dNTPs, and labeled ddNTPs.
2. ddNTPs terminate DNA synthesis at specific bases.
3. Run fragments on a gel or capillary system.
4. Read sequence from fragment lengths and labels.
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10. What is the purpose of Northern blot analysis?
Northern blotting detects specific RNA sequences:
* Assess gene expression levels
* Determine mRNA size
* Compare expression across tissues or conditions
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11. How does reverse-transcriptase PCR amplify target mRNAs?
RT-PCR steps:
1. Use reverse transcriptase to convert mRNA → cDNA.
2. Amplify cDNA with PCR using gene-specific primers.
Used to quantify gene expression or detect viruses like HIV.
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12. What is the purpose of DNA microarray analysis?
DNA microarrays:
* Contain thousands of DNA probes fixed on a chip.
* Hybridize with labeled cDNA from samples.
* Measure gene expression patterns across the genome.
Used for:
* Disease profiling
* Identifying differentially expressed genes
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13. How can scientists determine the function of a gene, and what methodologies can they employ to knockdown gene expression?
Gene function determination methods:
* Gene knockout (CRISPR, homologous recombination)
* RNA interference (RNAi) using siRNA or shRNA to knock down mRNA
* Overexpression or reporter assays
* CRISPR interference (CRISPRi) for gene repression
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14. How can SNPs be used to pinpoint the location of disease-causing genes?
Single Nucleotide Polymorphisms (SNPs) are:
* Common genetic variants
* Mapped across genomes
Researchers use linkage analysis or genome-wide association studies (GWAS) to correlate SNP patterns with disease traits, identifying candidate gene loci.
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15. Describe the methodology involving the cloning of animals.
Animal cloning (e.g., somatic cell nuclear transfer):
1. Remove nucleus from an egg cell.
2. Insert nucleus from a somatic (body) cell.
3. Stimulate the egg to divide and develop.
4. Implant embryo into a surrogate mother.
This produced Dolly the sheep.
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16. What may have contributed to the early death of Dolly, the first cloned mammal, and what may have been the mechanism involved?
Dolly died early (age 6) likely due to:
* Shortened telomeres: Her cells came from an adult, already biologically "aged."
* Epigenetic abnormalities: Imperfect reprogramming may have led to developmental defects.
* Increased susceptibility to disease: Possibly from immune or organ function issues.
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17. How can stem cells be used to treat disease?
Stem cells can:
* Differentiate into specialized cell types.
* Replace damaged or diseased tissues (e.g., neurons in Parkinson’s, beta cells in diabetes).
* Be used in regenerative medicine and gene therapy.
iPS cells (induced pluripotent stem cells) offer personalized, ethical options.
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18. How can therapeutic genes be placed within target cells?
Gene delivery methods:
* Viral vectors (retrovirus, adenovirus): Efficient, but may integrate into host DNA.
* Non-viral methods: Liposomes, electroporation, nanoparticles.
* CRISPR-based systems: Direct gene editing at specific loci.
Goal: Correct genetic defects or add beneficial genes (e.g., for sickle cell anemia, cystic fibrosis).