Genetics Exam 2 Study Guide: DNA Technology, Transcription & Translation, and Cell Division

DNA Technology

Restriction Digests and Agarose Gel Electrophoresis

Restriction enzymes cut DNA at specific sequences, producing fragments that can be separated by agarose gel electrophoresis. The resulting pattern of bands can be interpreted using a DNA ladder, which provides size references.

• Restriction Enzymes: Proteins that recognize and cut specific DNA sequences, often producing 'sticky' or 'blunt' ends.

• Agarose Gel Electrophoresis: Technique to separate DNA fragments by size; smaller fragments migrate further.

• DNA Ladder: A set of DNA fragments of known sizes used as a reference.

• Sticky Ends: Overhanging single-stranded DNA produced by certain restriction enzymes, facilitating ligation.

• Ligation: The process of joining DNA fragments, often using DNA ligase.

• Interpreting Gels: Compare sample bands to the ladder to estimate fragment sizes.

Example: EcoRI cuts at GAATTC, producing sticky ends. If two DNA fragments have compatible sticky ends, they can be ligated together.

Cloning Vectors and Plasmid Features

Plasmids are circular DNA molecules used as vectors to clone foreign DNA. Essential features include:

• Origin of Replication (ori): Allows plasmid replication in host cells.

• Selectable Markers: Genes (e.g., AmpR for ampicillin resistance) that allow identification of cells containing the plasmid.

• Multiple Cloning Site (MCS): Region with several restriction sites for inserting foreign DNA.

• Reporter Genes: Such as LacZ, which enables blue/white screening for recombinant clones.

Example: Cells with plasmids containing AmpR survive on ampicillin plates; insertion into LacZ disrupts β-galactosidase, producing white colonies.

Restriction Mapping and Site Identification

• Restriction sites can be identified by analyzing DNA sequences for enzyme recognition motifs.

• Compatible sticky ends from different enzymes can sometimes be ligated if their overhangs match.

Transcription and Translation

The Central Dogma

The central dogma of molecular biology describes the flow of genetic information: DNA → RNA → Protein.

• Transcription: Synthesis of RNA from a DNA template.

• Translation: Synthesis of protein from an mRNA template.

Equation:

$\text{DNA} \xrightarrow{\text{transcription}} \text{RNA} \xrightarrow{\text{translation}} \text{Protein}$

DNA vs. RNA

• Sugar: DNA contains deoxyribose; RNA contains ribose (with an extra –OH group at the 2' position).

• Strandedness: DNA is usually double-stranded; RNA is single-stranded.

• Nitrogenous Bases: DNA uses A, T, C, G; RNA uses A, U, C, G.

• Stability: DNA is more stable; RNA is more prone to degradation.

• Secondary Structure: RNA can form complex secondary structures (e.g., tRNA, rRNA).

Types of RNA and Their Functions

• mRNA (messenger RNA): Encodes protein sequences.

• tRNA (transfer RNA): Brings amino acids to the ribosome during translation.

• rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

• snRNA (small nuclear RNA): Involved in splicing of pre-mRNA.

• miRNA/siRNA: Regulate gene expression post-transcriptionally.

Gene Structure

• Promoter: DNA sequence where RNA polymerase binds to initiate transcription (e.g., Pribnow box in prokaryotes, TATA box in eukaryotes).

• 5' UTR and 3' UTR: Untranslated regions flanking the coding sequence, important for regulation.

• Exons: Coding sequences retained in mature mRNA.

• Introns: Non-coding sequences removed during splicing.

Transcription Initiation and Termination

• Prokaryotes: Use a single RNA polymerase; sigma factor recognizes promoter; termination can be Rho-dependent or Rho-independent.

• Rho-independent termination: Formation of a GC-rich hairpin in RNA followed by a poly-U tract causes dissociation.

• Rho-dependent termination: Rho protein binds to RNA and causes dissociation of the transcription complex.

• Eukaryotes: Multiple RNA polymerases; complex promoter elements (TATA box, GC box); transcription and translation are separated by the nuclear envelope.

mRNA Processing (Eukaryotes)

• 5' Capping: Addition of a methylated guanine cap.

• Splicing: Removal of introns by the spliceosome.

• 3' Polyadenylation: Addition of a poly(A) tail.

• Alternative Splicing: Allows production of multiple proteins from one gene.

Translation: Genetic Code and Mechanisms

• Codon: Three-nucleotide sequence in mRNA specifying an amino acid.

• Sense Codon: Codes for an amino acid.

• Nonsense Codon: Stop codon (UAA, UAG, UGA).

• Initiator Codon: Usually AUG (methionine).

• Reading Frame: The way nucleotides are grouped into codons.

• Wobble: Flexibility in base pairing at the third codon position.

• Charged tRNA: tRNA with an attached amino acid, catalyzed by aminoacyl tRNA synthetase.

Five Main Features of the Genetic Code:

• Triplet code

• Non-overlapping

• Degenerate (redundant)

• Unambiguous

• Nearly universal

Translation Initiation, Elongation, and Termination

• Prokaryotes: Shine-Dalgarno sequence aligns mRNA with the ribosome; 70S initiation complex forms.

• Eukaryotes: Kozak sequence surrounds the start codon; 80S initiation complex forms.

• Initiation Factors: Proteins that assist in ribosome assembly on mRNA.

• Elongation: Amino acids are added to the growing chain at the ribosome's A, P, and E sites; peptidyl transferase catalyzes peptide bond formation.

• Termination: Release factors recognize stop codons and release the polypeptide.

Directionality: Polypeptides are synthesized from the N-terminus to the C-terminus.

Mutations Affecting Protein Coding

• Silent (Synonymous) Mutation: No change in amino acid sequence.

• Missense (Nonsynonymous) Mutation: Changes one amino acid.

• Nonsense Mutation: Introduces a premature stop codon.

• Frameshift Mutation: Insertion or deletion alters the reading frame.

Translational Start and Stop Codons

• Start Codon: Usually AUG (methionine).

• Stop Codons: UAA, UAG, UGA.

• Translation of a sequence involves identifying the start codon and reading in triplets until a stop codon is reached.

Posttranslational Modifications

• Phosphorylation: Addition of phosphate groups to proteins.

• Glycosylation: Addition of carbohydrate groups.

Cell Division: Mitosis and Meiosis

Chromosome Structure and Terminology

• Chromatid: One of two identical halves of a replicated chromosome.

• Kinetochore: Protein structure on the centromere where spindle fibers attach.

• Cohesion: Protein complex holding sister chromatids together.

• Separase: Enzyme that cleaves cohesion during anaphase.

• Dyad: Pair of sister chromatids.

• Tetrad: Pair of homologous chromosomes (four chromatids) during meiosis I.

• Synapsis: Pairing of homologous chromosomes during prophase I of meiosis.

• Synaptonemal Complex: Protein structure facilitating synapsis and crossing-over.

• Chiasma: Site of crossing-over between homologous chromosomes.

Cell Cycle and Chromosome Condensation

• Chromosomes are uncondensed during interphase and condensed during mitosis/meiosis.

• Stages of the cell cycle: G1, S, G2, M (mitosis/meiosis).

Homologous Chromosomes vs. Sister Chromatids

• Homologous Chromosomes: Chromosomes with the same genes but possibly different alleles; one from each parent.

• Sister Chromatids: Identical copies formed by DNA replication, joined at the centromere.

• Non-homologous Chromosomes: Chromosomes that do not share the same genes.

Stages of Mitosis and Meiosis

• Mitosis: Prophase, Metaphase, Anaphase, Telophase; produces two identical diploid cells.

• Meiosis: Meiosis I (reductional division) and Meiosis II (equational division); produces four non-identical haploid cells.

Tracking Chromosome and DNA Content

• Number of chromosomes, chromatids, and DNA molecules changes during cell division; important to track through each stage.

Haploid vs. Diploid

• Haploid (n): One set of chromosomes (gametes).

• Diploid (2n): Two sets of chromosomes (somatic cells).

End Products of Mitosis and Meiosis

• Mitosis: Two genetically identical diploid cells.

• Meiosis: Four genetically diverse haploid cells.

Reduction Division and Genetic Diversity

• Reduction Division: Occurs in Meiosis I, reducing chromosome number by half.

• Genetic Diversity: Increased by crossing-over (recombination) and independent assortment.

Calculating Genetic Combinations

• The number of possible combinations of maternal and paternal chromosomes in gametes is $2^n$, where n is the haploid number.

Equation:

$\text{Number of combinations} = 2^n$

Key Terms Table

The following table summarizes some of the key terms and their definitions:

Term	Definition
Recombinant DNA	DNA molecules formed by laboratory methods of genetic recombination
Cloning Vector	DNA molecule used to carry foreign genetic material into another cell
Promoter	DNA sequence where RNA polymerase binds to initiate transcription
Exon	Coding region of a gene
Intron	Non-coding region removed during RNA processing
tRNA	Transfer RNA; brings amino acids to the ribosome
Mutation	Change in DNA sequence
Chiasma	Site of crossing-over between homologous chromosomes
Haploid	Cell with one set of chromosomes
Diploid	Cell with two sets of chromosomes

Additional info: Some explanations and examples were expanded for clarity and completeness based on standard genetics curricula.