Recombination and Gene Mapping

Recombination of Linked Genes

• Genes/alleles tend to be inherited together more often than expected due to linkage
• Crossing over during prophase 1 of meiosis 1 leads to recombination between nonsister chromatids

Estimating Linkage and Gene Distances

• Perform a test cross by crossing a heterozygous organism with a homozygous recessive one
• Identify parental and recombinant offspring to calculate recombination frequency

Test-Crosses

• Test crosses determine if two or more traits are linked

Phenotypic and Genotypic Ratios in Test-Cross

• An expected phenotypic ratio of 1:1:1:1 occurs for two unlinked traits (dihybrid cross)

Two-Point Crosses

• A ratio of 1:1:1:1 occurs when a heterozygous organism is crossed with a homozygous recessive organism (test cross)
• A ratio of 9:3:3:1 occurs if both parents are heterozygous

Three-Point Crosses

• A heterozygote with a triple recessive produces 8 phenotypes with a 1:1:1:1:1:1:1:1 ratio

Chi-Squared Tests and Gene Linkage

• Chi-squared tests compare the null hypothesis of no linkage
• χ² ≤ Critical Value means the null hypothesis fails to be rejected and the genes assort independently
• χ² > Critical Value means the null hypothesis is rejected, and there is evidence of gene linkage.

Recombination Map

• The largest number is the most common and represents the parental type
• The lowest number is the rarest and represents double recombinants
• The intermediate numbers are intermediate recombinants

Recombination Frequency (RF)

• Recombination frequency (RF) can be estimated from a recombination map by looking at the distances between genes, measured in map units (mu, cM)

Mapping Units

• A mapping unit measures distance between genes on a chromosome
• 1 map unit (m.u.) = 1 centimorgan (cM)

Relationship Between Map Units and Recombination Frequency

• 1 map unit (m.u.) = 1% recombination frequency (RF)
• Genes with a 10% recombination frequency are 10 map units (10 cM) apart on the chromosome

Recombination Frequency and Physical Distance

• Recombination frequency roughly correlates with the physical distance between genes
• Close genes have a low recombination frequency and are more likely to be inherited together
• Distant genes have a higher recombination frequency because there are more crossovers between them.

Interference

• I = 1 means complete interference (no double crossovers occur)
• I = 0 means no interference (crossovers occur independently)
• I < 0 means negative interference (crossovers promote additional crossovers)

Chromosome Map

• Chromosome maps are diagrams showing the arrangement of genes and their relative positions on a chromosome, based on genetic data.

Locus

• The locus is the specific physical location of a gene or genetic marker on a chromosome

Recombination Map

• A recombination map is a chromosome map showing the relative distances between genes based on recombination frequency
• One map unit (centimorgan, cM) equals a 1% recombination frequency

Linked Traits

• "Linked" means traits are linked together but separate

Coefficient of Coincidence (C.O.C.)

• C.O.C. is the ratio of observed double crossovers to expected double crossovers (C.O.C.=Expected Double CrossoversObserved Double Crossovers)
• Cis conformation is when both dominant alleles are on the same chromosome/homolog (AB/ab or ++/ab, heterozygote)
• Trans conformation is when dominant alleles are on different chromosomes/homologs (Ab/aB or +b/a+)
• Crossing over is the exchange of genetic material between homologous chromosomes during prophase I of meiosis
• A crossover product results from a crossing-over event, leading to chromosomes with a mix of genetic material from both parents
• Interference is calculated using the formula I=1−C.O.C.

Linkage Map

• A linkage map is a genetic map that shows the relative positions of genes on a chromosome according to recombination frequencies
• 1 m.u. = 1 cM, equaling the distance between two genes for every 1 in 100 recombinant offspring
• Recombination frequency correlates to physical distance between two genes/loci on a chromosome
• Recombinant frequency of less than 0.50 indicates genes are linked
• The centimorgan (cM) is a unit of measurement to express the distance between genes on a chromosome (1 cM represents a 1% recombination frequency)

Griffith's Experiment

• In Griffith's experiment, mice were injected with different strains of S. pneumoniae (Smooth (S) = deadly, Rough (R) = harmless).
• The reasoning was to test if a substance could transfer genetic information.
• Mice died when injected with a mix of heat-killed S strain and live R strain
• This led to the discovery of transformation, where a substance from the dead S strain transformed the R strain into a virulent form

Avery, Macleod, and McCarty Experiment

• This used enzymes to selectively destroy DNA, RNA, and proteins in heat-killed S strain before mixing with R strain in order to identify which molecule was responsible for transformation
• Transformation only failed when DNA was destroyed
• This concluded that DNA is the genetic material

Hershey-Chase Experiment

• DNA was labeled with radioactive phosphorus (³²P) and proteins with radioactive sulfur (³⁵S) in bacteriophages and then allowed to infect bacteria
• The reasoning was to determine whether DNA or protein carried genetic information
• Only ³²P was found inside bacterial cells, thus confirming that DNA, not protein, is the genetic material

Meselson-Stahl Experiment

• E. coli was grown in heavy nitrogen (¹⁵N) and then switched to light nitrogen (¹⁴N) and used density gradient centrifugation to track DNA replication
• This determined if DNA replication followed the conservative, semi-conservative, or dispersive model
• After one replication, DNA was intermediate (one heavy and one light strand).
• After two replications, there were both intermediate and light DNA
• The experiment supported the semi-conservative model of DNA replication, where each new DNA molecule has one original and one new strand

Nucleotides

• Nucleotides are the building block of DNA
• The components are a phosphate group, deoxyribose sugar, and nitrogenous base (A, T, C, or G)

DNA Structure

• DNA is structured as a double helix with two strands running antiparallel (5’ to 3’ and 3’ to 5’)
• Bases pair via hydrogen bonds (A-T with 2 hydrogen bonds, and C-G with 3 hydrogen bonds)
• The sugar-phosphate backbone is formed by phosphodiester bonds

Purines and Pyrimidines

• Purines (2 rings) are Adenine (A) and Guanine (G)
• Pyrimidines (1 ring) are Cytosine (C) and Thymine (T)

Chargaff's Rules

• A = T and C = G
• Total purines = Total pyrimidines
• Complementary base pairing in DNA is maintained

Watson & Crick's DNA Model

• DNA is a right-handed double helix
• The strands are antiparallel
• Complementary base pairs form via hydrogen bonds

Semiconservative Replication

• Each daughter DNA molecule has one original strand and one new strand

Conservative Replication

• The entire parental DNA remains intact, producing one all-new DNA molecule

Dispersive Replication

• Parental DNA is fragmented and reassembled with new DNA

Template and New Strand Synthesis

• The template strand is read 3' to 5'
• The new strand is built 5' to 3'
• DNA Polymerase can only add nucleotides to the 3' end of the growing strand

DNA Polymerase III

• DNA Polymerase III is the main enzyme for DNA synthesis.
• It adds nucleotides in the 5' to 3' direction and has proofreading ability to fix errors

Sliding Clamp

• The sliding clamp holds DNA Polymerase III onto the DNA strand, ensuring it remains attached for efficient replication

DNA Polymerase I

• DNA Polymerase I removes RNA primers from Okazaki fragments on the lagging strand and replaces primers with DNA nucleotides

Leading and Lagging Strand Synthesis

• The leading strand is synthesized continuously in the 5' to 3' direction, following the replication fork
• The lagging strand is synthesized discontinuously in the 5' to 3' direction, moving away from the fork and forming Okazaki fragments

Okazaki Fragments

• Okazaki fragments are short DNA segments formed on the lagging strand, synthesized in the opposite direction of the replication fork
• DNA ligase seals these fragments together

Primase

• Primase synthesizes a short RNA primer to provide a starting point for DNA Polymerase III

DNA Polymerase III

• DNA Polymerase III adds nucleotides to the 3’ end of the primer, matching complementary bases (A-T, C-G)

DNA Polymerase I

• Replaces RNA primers with DNA nucleotides

Ligase

• Ligase seals the gaps between Okazaki fragments by forming phosphodiester bonds

Proofreading

• DNA Polymerase III checks and corrects errors

Telomerase

• Telomerase extends repetitive sequences at chromosome ends to prevent loss of genetic material

Nitrogenous Bases

• Adenine: A purine nitrogenous base in DNA that pairs with thymine (T) using two hydrogen bonds
• Cytosine: A pyrimidine nitrogenous base in DNA that pairs with guanine (G) using three hydrogen bonds

Definition

• Antiparallel: DNA strands run in opposite directions, one strand is 5' to 3', and the other is 3' to 5'
• Base: A nitrogen-containing molecule (adenine, thymine, cytosine, or guanine) that forms the rungs of the DNA double helix
• Conservative replication: A disproven model of DNA replication where the parent DNA remains intact and an entirely new copy is made
• Daughter Molecule: The newly synthesized DNA molecule that results from DNA replication

Sugars

• Deoxyribose: A five-carbon sugar in DNA nucleotides that lacks one oxygen atom compared to ribose in RNA
• Ribose: The sugar in RNA, containing one more oxygen than deoxyribose

Replication

• Dispersive replication: A disproven model of DNA replication where parental and new DNA fragments are interspersed within both strands
• Origin of replication: A specific DNA sequence where replication begins, containing DNAa boxes in prokaryotic cells.
• DNAa Box: A specific sequence in the origin of replication (oriC) in prokaryotic DNA where DNAa proteins bind to initiate replication
• DNAa proteins: Proteins that bind to DNAa boxes to begin the unwinding of the DNA double helix at the origin of replication

Enzymes

• DNA gyrase: A type of topoisomerase enzyme that relieves tension caused by supercoiling ahead of the replication fork
• Helicase: An enzyme that unwinds the DNA double helix at the replication fork by breaking hydrogen bonds between complementary bases
• DNA ligase: An enzyme that seals gaps between Okazaki fragments on the lagging strand by forming phosphodiester bonds
• Endonuclease: An enzyme that cuts RNA or DNA internally.
• Exonuclease: An enzyme that removes nucleotides from the ends of RNA or DNA strands
• DNA polymerase I (DNA pol I): Replaces RNA primers with DNA nucleotides on the lagging strand and has exonuclease activity for error correction
• DNA polymerase III (DNA pol III): The primary enzyme responsible for synthesizing new DNA strands by adding nucleotides in the 5' to 3' direction
• Primase: An enzyme that synthesizes short RNA primers, providing a starting point for DNA polymerase to begin DNA synthesis.
• Topoisomerase: An enzyme that relieves the supercoiling tension in DNA ahead of the replication fork by cutting and rejoining DNA strands
• poly(A) polymerase (PAP): An enzyme that adds the poly(A) tail to the 3' end of eukaryotic mRNA.
• B clamp (sliding clamp): A protein that encircles DNA and anchors DNA polymerase to the template strand, increasing its efficiency during replication

Strands

• Lagging strand: The DNA strand synthesized discontinuously in the 3' to 5' direction using Okazaki fragments
• Leading strand: The DNA strand synthesized continuously in the 5’ to 3’ direction, following the replication fork
• Non-template strand (coding strand): The DNA strand that has the same sequence as the mRNA (except with thymine instead of uracil).
• Template strand (noncoding strand): The DNA strand that serves as the template for RNA synthesis

Other Factors

• Single-strand DNA-binding (SSB) proteins: Proteins that bind to separated DNA strands to prevent them from reannealing or being degraded
• Telomere: The protective, repetitive DNA sequences at the ends of linear chromosomes that prevent degradation during replication
• Poly(A) tail: A string of adenine (A) nucleotides added to the 3' end of a eukaryotic mRNA molecule. Typically 50-250 A’s long. Added by the enzyme poly(A) polymerase (PAP).
• Semiconservative replication: The correct model of DNA replication where each daughter DNA molecule consists of one original (parental) strand and one newly synthesized strand
• Promoter: A DNA sequence that signals RNA polymerase where to begin transcription
• Uracil (U): A pyrimidine nitrogenous base found in RNA and replaces thymine (T) in DNA
• Ribosomal RNA (rRNA): Structural and functional components of ribosomes
• Transfer RNA (tRNA): Carries amino acids to the ribosome during protein synthesis
• Messenger RNA(mRNA): Carries genetic information from DNA to the ribosome for protein synthesis

RNA vs DNA

• RNA: Single-stranded, contains ribose, has uracil (U) instead of thymine (T)
• DNA: Double-stranded, contains deoxyribose, uses thymine (T)

Gene Features

• RNA features include Exons, Introns, Untranslated Regions (UTRs), Open Reading Frame (ORF), Upstream, Downstream

UTRs

• Untranslated Regions (UTRs): Regions at both ends of mRNA (5’ and 3’) that regulate translation

Exons vs Introns

• Exons: Coding regions of a gene
• Introns: Non-coding regions removed during splicing

Open Reading Frame (ORF)

• The sequence between the start and stop codon that codes for a protein

DNA Sequences

• Open Reading Frame (ORF): The sequence between the start and stop codon that codes for a protein
• Upstream: Sequence before the transcription start site
• Downstream: Sequence after the transcription start site
• Promoter Sites: DNA sequences where RNA polymerase binds to initiate transcription

Transcription in Prokaryotes vs Eukaryotes

• Prokaryotes transcription occurs with a -10 and -35 region for RNA polymerase binding
• Eukaryotes transcription occurs with a TATA box utilizing general transcription factors (GTFs)

Termination in Prokaryotes

• Factor-Independent Termination: Hairpin loop followed by a string of uracils
• Rho-Dependent Termination: Rho protein chases RNA polymerase and dislodges it

Termination in Eukaryotes

• Allosteric Termination Model: Conformational change in RNA polymerase
• Torpedo Termination Model: Exonuclease degrades RNA, pushing off polymerase

RNA Decay

• Prokaryotes: RNA degrades quickly, helping regulate gene expression
• Eukaryotes: mRNA decays via deadenylation (removal of poly(A) tail) or decapping.

RNA Polymerases

• RNA Polymerase I: Transcribes rRNA (except 5S rRNA), initiates when it binds to rDNA promoter with transcription factors, and terminates with a termination sequence and proteins
• RNA Polymerase II: Transcribes mRNA, snRNA, and miRNA, initiates when it binds to the TATA box with general transcription factors (GTFs), and Terminated when it follows the torpedo or allosteric model
• RNA Polymerase III: Transcribes tRNA, 5S rRNA, and other small RNAs, recognizes internal promoter sequences to initiates, and the termination process is similar to prokaryotic factor-independent termination

RNA Processing

• 5’ Capping: A 7-methylguanosine (m7G) cap is added to the 5’ end for protection and translation initiation
• Polyadenylation: A poly(A) tail is added to the 3’ end by poly(A) polymerase (PAP) for stability
• Splicing: Introns are removed, and exons are joined using a spliceosome

Splicing

• Splicing is the removal of non-coding introns from pre-mRNA to create mature mRNA.
• Benefits include increased genetic diversity through alternative splicing, allowing multiple proteins from one gene, and regulating gene expression

Alternative Splicing

• It can generate different mRNA variants from a single gene
• Types include exon skipping, mutually exclusive exons, intron retention, and alternative 5’ or 3’ splice sites

Eukaryotic RNA Decay

• The purpose of eukaryotic RNA decay is to regulates gene expression and remove defective mRNAs
• Pathways include Deadenylation-Dependent Decay, Decapping Pathway and Endonucleolytic Pathway.

Other Descriptions

• 3′ untranslated region (3′ UTR): Region before the coding sequence. Important for ribosome binding and translation regulation
• 5′ untranslated region (5′ UTR): Region after the coding sequence and contains signals for mRNA stability, localization, and translation regulation
• Alternative splicing: A process that produces different mRNA molecules from the same gene by including or excluding specific exons
• Constitutive exon: An exon that is always included in the final mRNA after splicing
• Decay: The breakdown of RNA molecules to regulate gene expression and remove defective RNA
• Elongation: The stage of transcription where RNA polymerase adds nucleotides to the growing RNA strand

Enzymes

• Exonuclease: An enzyme that removes nucleotides from the ends of RNA or DNA strands
• Helicase: An enzyme that unwinds the DNA helix during replication or transcription
• General transcription factor (GTF): Proteins that help RNA polymerase bind to the promoter and initiate transcription in eukaryotes
• Endonuclease: An enzyme that cuts RNA or DNA internally
• General transcription factor (GTF): Proteins that help RNA polymerase bind to the promoter and initiate transcription in eukaryotes.
• RNA polymerase: The enzyme that synthesizes RNA from a DNA template.
• Primase: An enzyme that synthesizes short RNA primers, providing a starting point for DNA polymerase to begin DNA synthesis RNA editing: Post-transcriptional modifications where RNA nucleotides are added, deleted, or changed

Termination

• Factor-independent termination: A transcription termination mechanism in prokaryotes where a hairpin loop followed by uracils causes RNA polymerase to detach
• Rho-dependent termination: A prokaryotic transcription termination method using the Rho protein to dislodge RNA polymerase

Strands

• Intron: A non-coding region in a gene that is removed during RNA splicing
• Exon: A coding region in a gene that remains in the mature mRNA after splicing
• Mutually exclusive intron: Occurs when only one of two possible introns is retained in the mRNA, never both
• Facultative intron: An intron that is sometimes retained or removed depending on the cell’s needs

Other Descriptions

• Ribose: The sugar in RNA, containing one more oxygen than deoxyribose
• Transcript: The RNA molecule produced during transcription, mRNA
• Half-life: The time it takes for half of an RNA molecule to degrade
• RNApolymerase core enzyme: The essential enzyme complex that carries out transcription without additional factors.
• TATA box: A promoter DNA sequence in eukaryotes where transcription factors bind to initiate transcription.
• 7-methylguanosine (m7G): A modified guanine nucleotide added to the 5' end of eukaryotic mRNA. Forms the 5' cap.