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Overview of Genetics

• Genetics: The study of heredity and variation, fundamental to understanding trait inheritance.
• Gene: The unit of heredity encoding specific traits through a functional product like a polypeptide

Fields of Genetics

• Broadly split into transmission genetics, molecular genetics, and population genetics.

Transmission Genetics

• Concentrates on trait inheritance from parents to offspring.
• Gregor Mendel's work in the 1860s is the foundation, introducing the law of segregation and independent assortment.
• Principles explain how genes are passed as discrete units.
• Analyze inheritance patterns through genetic crosses over generations.

Key Concepts in Transmission Genetics

• Law of Segregation: Each individual has two alleles per gene, separating during gamete formation
• Law of Independent Assortment: Genes for different traits are inherited independently.

Molecular Genetics

• Explores biochemical mechanisms underlying genetic material, specifically DNA structure, function, and regulation.
• Field analyzes DNA, RNA, and proteins, to understand how genes are organized, expressed, and regulated at the molecular level.

Fundamental Processes

• Gene Expression: It is aTwo-step process where DNA is transcribed into RNA and then translated into proteins.
• These proteins are crucial for cellular function and phenotypic traits.

Population Genetics

• Focuses on the genetic composition of populations and its variation across time and space.
• It connects Mendelian genetics and Darwinian evolution.
• Studies: Gene pools and evolutionary forces like mutation, selection, genetic drift, and gene flow.

Key Focus Areas

• Genetic Variation: It is the Differences in inherited traits among individuals, from mutations, chromosome structure changes, or chromosome number variations.
• Role in Evolution: Genetic variation provides the raw material for evolutionary change, enabling adaptation.

Genetic Variation and Interaction of Genes

• Variations in genes and alleles lead to different traits, influenced by several factors.
• Gene Mutations: Small sequence changes create different alleles.
• Chromosomal Alterations: Segments may be lost or duplicated, impacting function/expression.
• Chromosome Number Variations: Changes resulting to phenotypic changes.

Genes and Environment Interplay

• Traits are influenced by both genes and environment.
• Phenylketonuria (PKU) illustrates this.
• Individuals with PKU can't metabolize phenylalanine, so dietary control can mitigate this.

Genetic Material Requirements

• Must contain essential information for creating an organism.
• Information must be transmissible from parent to offspring.
• Replication: Genetic material must replicate for transmission during cell division.
• Variation: Genetic material must be capable of variation for diversity within a species.

Griffith's Transformation Experiment

• Frederick Griffith studied Streptococcus pneumoniae, which comes in smooth and rough strains
• S Strain: Secretes a protective polysaccharide capsule, producing smooth colonies.
• R Strain: Lacks said capsule and therefore produces rough colonies.
• Heat-killed S cells transform live R cells into virulent S cells in mice, indicating a transferable "transforming principle".

Avery, MacLeod, and McCarty's Experiment

• DNA identified as the transforming principle.
• DNA extracts from S cells transform R cells into S cells.
• DNase eliminated transformation; RNase and protease don't.

Hershey-Chase Experiment

• Bacteriophage T2 used to show DNA is genetic material.
• Labeled phage DNA with radioactive phosphorus-32 and protein with radioactive sulfur-35.
• Radioactive phosphorus-32 carrying DNA entered bacterial cells, confirming DNA carries genetic information.

RNA as Viral Genetic Material

• Gierer and Schramm isolated RNA from tobacco mosaic virus (TMV).
• Purified RNA could infect plants, confirming RNA as the genetic material in RNA viruses.

DNA Structure

• Two strands twisted into a double helix.
• Nucleotides: Structural components that consist of a phosphate group, deoxyribose sugar, and a nitrogenous base

Structural Features

• Double helix is stabilized by hydrogen bonding between complementary bases (A-T and G-C).
• Base stacking interactions among planar surfaces of bases also stabilize the double helix.

Chargaff's Rules

• Adenine (A) equals Thymine (T), and Guanine (G) equals Cytosine (C)
• Rules provided key evidence for the base-pairing mechanism in DNA structure.

The Double Helix Model by Watson and Crick

• Rosalind Franklin's X-ray diffraction and Chargaff's rules helped to formulate this.
• Antiparallel Orientation: Strands run in opposite directions (5' to 3' and 3' to 5').
• Right-handed helix: Helix spirals clockwise away from the observer.

Alternative DNA Structures

• B-DNA: The common physiological form
• A-DNA: A right-handed occurring under low humidity
• Z-DNA: A left-handed helix that may be involved in transcription and recombination.

RNA Structure

• Typically single-stranded, though can form short double-stranded regions through complementary base pairing.
• RNA uses uracil (U) instead of thymine (T).
• RNA contains ribose sugar instead of deoxyribose.

Base Stacking in DNA

• Refers to non-covalent interactions between nitrogenous bases.
• Stabilizes the double helix structure by minimizing bases exposure to water.

Significance of Griffith's Transformation Principle

• Revealed genetic material could transfer between bacteria.
• Showed that DNA carries genetic information

Antiparallel Strands of DNA and its Contribution

• Strands run in opposite directions, permitting complementary base pairing.
• Arrangement stabilizes the double helix structure.
• Enables proper replication, transcription, and accurate genetic information transfer.

Importance of DNA Directionality (5' to 3')

• Determines how DNA is replicated and transcribed.
• DNA polymerase can add nucleotides in this direction.
• Ensures proper base pairing and genetic information flow.

Structural Features of the DNA Double Helix

• 2 strands twisted into a helix.
• Sugar-phosphate backbone
• Nitrogenous bases (A, T, C, G) paired in the center.
• Strands are antiparallel.
• Complementary base pairing (A with T, C with G).
• Major and minor grooves along the helix.

Transformation in Griffith's Experiments

• Uptake of genetic material causes a change in phenotype.

Hydrophobic Interactions and DNA Stability

• Hydrophobic interactions stabilize by causing hydrophobic bases to stack inside, away from water.
• Stacking minimizes exposure to the aqueous environment.

Key Difference Between A-DNA and Z-DNA

• Primary difference is that A-DNA is a right-handed helix, and Z-DNA is a left-handed helix.

Significance of Major and Minor Grooves

• They serve as binding sites for proteins that regulate gene expression.

Role of DNase in Bacterial Transformation

• Used to degrade DNA.

DNA Replication

• Complementarity of DNA strands: Base pairing follows the AT/GC rule.
• Parental Strands: Original DNA Strands
• Daughter Strands: Newly synthesized strands from parental templates.

DNA Replication Models

• Conservative Model: Parental strands remain together with newly synthesized DNA after replication.
• Semiconservative Model: Each daughter DNA has one parental and one new strand.
• Dispersive Model: Parental and new DNA are interspersed in both strands.

Bacterial DNA Replication

• Meselson and Stahl Experiment: Validates correct DNA replication model.
• Used E. coli cultures in mediums with different nitrogen isotopes (15N and 14N).
• DNA via centrifugation showed the semiconservative model.

Replication Initiation

• Origin of Replication (oriC): Contains AT-rich regions, DnaA boxes, and GATC methylation sites.
• DnaA Proteins: Bind to DnaA boxes, initiating unwinding.
• DNA Helicase: Unwinds DNA creating supercoiling, which DNA gyrase (topoisomerase II) alleviates.

Elongation

• DNA Primase: Synthesizes short RNA primers.
• DNA Polymerase III: Attaches nucleotides to the 3' end of the RNA primer. Higher processivity due to the Beta (β) subunit (clamp protein), allowing DNA to synthesize rapidly.

Leading and Lagging Strand Synthesis

• Leading Strand: Continuous synthesis towards the replication fork.
• Lagging Strand:
  • Synthesized in Okazaki fragments away from the fork.
  • RNA primers are replaced by DNA by DNA Polymerase I.
  • DNA ligase connects DNA fragments.

Termination

• Ter Sequences: Terminate replication, facilitated by Tus proteins.
• Topoisomerases: Resolve intertwined DNA (catenanes) by cutting and rejoining strands.

Proofreading and Fidelity

• High fidelity mechanisms include:
• Instability of Mismatched Pairs: Reduces errors due to inherent instability.
• Active Site Configuration: Prevents errors via correct nucleotide matching ability.
• Proofreading Activity: DNA polymerase 3' to 5' exonuclease activity corrects mismatches.

Eukaryotic DNA Replication

• Large Linear Chromosomes: Require multiple origins of replication for efficient synthesis.
• Origins of replication resemble bacterial systems, esp. in yeast (e.g., ARS elements).

Initiation and Assembly

• Pre Replication Complex (preRC):Assembled at G1 phase, involves ORC (Origin Recognition Complex) and MCM helicase, activating upon phosphorylation.

Elongation

• DNA Polymerases α, δ, ε: involved in DNA synthesis and there is transition from Pol α to Pol δ/ε for elongation.
• Involved in DNA synthesis.
• Polymerase Switch: Transition from DNA Pol a synthesizing an RNA-DNA hybrid primer to DNA Pol δ/ε taking over for elongation .
• PCNA: Similar to bacterial β subunit, increasing processivity as a clamp.
• Replication Machine (Replisome): Facilitates DNA strand copying via proteins with polymerases.

Telomeres and Termination

• Telomeres: They protect ends of chromosomes, prevent degradation and fusion.
• Replication issues at 3' ends, solved by telomerase adding repetitive sequences.
• Telomeres shorten with age/cellular senescence; cancer cells increase telomerase activity to prevent this.

Effects of Mutations on DNA Replication

• Loss of polymerase III 3'→5' exonuclease activity: Increased error rate in replication.
• Loss of polymerase I 5'→3' exonuclease activity: RNA primers remain in lagging strand thus preventing completion of DNA synthesis and ligation.

Telomerase Effects

• Telomerase RNA loss: Telomerase can't extend ends of chromosomes, leading to telomere shortening with each division.

Okazaki Fragment

• DNA synthesis happens in 5'→3' direction, with the right Okazaki fragment made first.
• The right RNA primer is removed first since primer removal proceeds in the same 5'→3' direction as DNA synthesis
• Polymerase I fills in the primer gap as long as there is an adjacent DNA strand, the middle Okazaki fragment does not need to be synthesized firsthand
• DNA ligase joins the left Okazaki fragment with synthesized DNA at the left.

Stages of Transcription

• Introduction:
  • Transcription is the process where genetic information is copied from DNA to RNA using RNA polymerase.
• Transcription occurs in three main stages:
  • RNA polymerase binds to a DNA sequence called the promoter, signaling the start of transcription at the initiation phase
  • RNA polymerase synthesizes an RNA molecule as it moves along the DNA strand during the elongation phase
• RNA polymerase releases the newly formed RNA molecule after reaching a termination or stopping sequence

Initiation

• The promoter region contains critical sequence elements, such as the Pribnow box in bacteria
• These sequences help transcription factors and RNA polymerase for initiation of transcription.

Elongation

• RNA polymerase synthesizes the RNA strand in a 5' to 3' direction, reading the template DNA strand in a 3' to 5' direction

Termination

• In bacteria, there are two mechanisms: rho-dependent and rho-independent termination
• Specific signals such as RNA strands are used within the processes to release the RNA polymerases and newly synthesized RNA strand.

Promoter Structure in Bacteria

• Promoters have conserved base sequences like -10 and -35, which sigma factors recognize.
• Such holoenzymes carry transcription initiation through base-pairing.sigma factors, and

Promoter Structure in Eukaryotes

• Eukaryotes: These have a core promoter that contains the TATA box which has a transcriptional start site
• Multiple transcription factors and mediators are required to make transcription more efficient

Eukaryotic RNA Polymerases

• RNA Polymerase I transcribes ribosomal RNA (rRNA) genes
• RNA Polymerase II transcribes protein-coding genes (mRNAs) and several snRNA.
• RNA Polymerase III transcribes transfer RNA (tRNA) gene as well as the 5S rRNA genes.

RNA Modification

• RNA Stability modifications affect the stability, transport and translation of RNA while pre-mRNA is being converted to mature ones.
• 5' Capping: Most mature mRNAs gain a 7-methyl guanosine cap during transcription by polymerase II.
• The result is that the mRNA produced will be highly stable for the cells to transport to the cytoplasm as part of the translation initiation instructions.
• Splicing: RNA splicing will target non-coding region, namely the introns, within the pre-mRNA
• Through a process with spliceosomes or self-splicing mechanisms, which involves the RNA itself functioning as a ribozyme, the remaining mRNA will be stabilized further for efficient translation.
• 3' Polyadenylation: addition of a polyA tail happens after transcription to optimize mRNA function such as stability efficiency

Alternative Splicing:

• Different exons can also be spliced together in various combinations, leading to the production of proteins with different functions from the same gene precursor. Hence alternative splicing can be a mechanism for a single gene to produce multiple proteins.

Regulatory Elements

• Enhancers: activator proteins affect transcription by promoting DNA sequencing to increase rates of same

• Silencer: when repressor proteins affect DNA sequencing as bounded to this elements

• The DNA strands follows rules where (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G). Template strand (3' to 5'): 3'-CCGTACGTAATGCCGTAGTGTGATCCCTAG-5'

• Coding strand (5' to 3'): 5'-GGCATGCATTACGGCATCACACTAGGGATC-3'

• Promoter Location: Located on the 3' end of the template strand.

Bacterial consensus sequences

• The -10 region (Pribnow box or TATAAT) aids in DNA unwinding.
• The -35 area is recognized through RNA polymerases by encoding its promoter.

-dependent Termination vs. p-Independent Termination

• P-Dependent: requires the recognition of rho (p) to protein for termination of transcription.
• P-Independent: includes U-rich sequence that follows after the recognition of GC-rich hairpin.

Mutations Affecting Transcriptional Termination

• Mutations will affect the rates of transcription.
• In the absence of transcription there potentially, it starts leaking and begins entering non-coding/downstream portions.
• Mutations could potentially hinder the formation of U-rich portion of the mRNA.

Key Promoter Elements in Eukaryotic Genes

• The first TATA box is attached for transcription initiation starting at -25 to -30 through TATA-binding protein (TBP).
• BRE(TFIIB Recognition Element, -35 bp): will bind to DNA, enhancing polymerase
• Inr(Initiator, +1 bp): the starting signal for transcription.
• DPE(Downstream Promoter Element, +28 to +32 bp): functions well with Inr in order to facilitate promoter recognition

RNA transcription: process

• The promoter is scanned upon Rna Polymerase's binding to promoters of holoenzymes.
• Specific bases trigger RNA Polymerase.
• polymerase's attachment of dna ensures appropriate preparation of gene expression.
• RNA polymerase synthesizes an RNA transcript during elongation.
• Strand templates without using encoding strands are made. t from DNA is replaced with U in the newly synthesized strand

What Happens During Termination?

• Transcription stops and the RNA/DNA detaches as Polymerase releases (which is independent regarding rho).

Enhancers and Silencers:

• Regulatory elements: Binding sites for transcription factors that regulate gene expression.
• Enhancers: DNA sequences that increase transcription when bound by activator proteins promoting binding of RNA to polymerase while the silencer decreases rates for transcription binding polymerases where both of these occur at the promoter
• The open complex ensures the proper transcription rates by initiation of the phase

Transcription Regulation Factors

• Transcription factors: the domains enable activation binding of proteins allowing influences for expressional/inhibition within the genes/gene expression.
  • Cis-acting elements: the elements are DNA regions with genes to affect transcription rates (TATA box, etc)
  • Trans elements are regulatory ones.

Impacts of Mutations in the Promoter Region

• Mutations in the promoter region hinder RNA polymerase binding, eventually decreasing transcription and genetic expression overall.

Genetic basis for Protein Synthesis

• Genetic material encodes production of proteins for cellular structure and function.

Genes

• Structural Genes: Encode polypeptides and are transcribed into messenger RNA (mRNA)
• Archibald Garrod: Demonstrated the relationship between genes/protein production studying alkaptonuria establishing inborn errors of metabolism .

Process and Components

• A process involving ribosomes, tRNA and a genetic code where ribosomes act as the production factories made proteins and RNA strands with specific structures that include codon/anti-codon zones, thus amino acids will be encoded in Triplet to which the beginning signals occur at AUG (while UAA;UAG + UGA is where it stops).
• The genetic code is inherently degenerate, meaning that there exist overlapping or multiple sets or sequences of codons/triplets.

Ribosome Structure

Functional Sites: - Three sites - Peptidyl (P) site, binding transfer site. Aminoacyl (A) site, for receiving coded RNA for cellular/organelle

Stages of Translation

• Formation of activation complexes by the aid of initiation factor proteins to recognize target codons that bind to mRNA.
• For eukaryotes, this means attaching tRNA to the ribosomal subunit for the start codon.

Elongation

• Ribosomes travels across mRNA by deciphering the sequence and binding acids to a growing strand along the A site, where it travels and leaves.

Termination

• The process ceases when the RNA sequence reaches a "stop" command or set of bases.
• This then tells proteins to dissociate, thus breaking their chains. Bacteria has 3RFs whereas eukaryotes have two (more simplistic structures as opposed those in organelles or cytoplasm)

Dr.Biliran Questions

• A sequence with a start sequence to then read the codons will be translated into the amino strand (Met-Gly-Asn, etc).
• The Shine-Dalgarno is found in RNA which attaches and reads mRNA: located a few spaces up on mRNA and has certain sequences for the 30S site which starts binding/translation.

Translation in Ribosomes

• Ribosomes identify the codon upon binding to the 5’ site. In bacterial regions, the mRNA and 16S base pairings will initiate/ elongate termination.
• Codons are located on the anticodon side or areas.

Lactose Permease

• Has 417 acids where codons are numbered beginning at the start.
• Codon sequence that are for amino acid are read from 5'->3' mRNA which encode 417 base proteins needs at least 1251 bases (and those will contain untranslated regions). Both will travel via protein chain through tRNA sequences that are linked 3-CUU-5, etc (the codon matches via tRNA to amino acid). Synthetases: attach proteins/acids to enhance translation

Polysome and polyribosome

• Where multiple strands can become mRNA.

Gene Mutations Definition

• These are heritable changes in genetic material which may contribute disorders.

Mutation Types:

• Chromosome Mutations: affects genome/genes with changes in composition Genome Mutations: affects genome at the variations in numbers Single-Gene: mutations happening at smaller scale within genes

Point Mutations

• Involutive when a single base sequence is modified with substitutions, which are swapped with another base one (either transversions or translations). Mutations are often an outcome of:
• Base pair additions
• Removal sequences along the downstream

Protein Polypeptide Effects

Gene mutations which occurs in the coding sections has an impact to protein production

Types of mutations

• Silent: substitutions/alterations to not involve changes during codon sequences Missense: Changes in acids but may retain a small similarity/function
• Incomplete: the acids have early termination which prevents all the expression
• Frameshift: reading is disrupted upon additions/removal of protein sequences/acids

Mutation Origins

• Occur spontaneously or upon mutagen influences Process:
• Check plate for natural vs rates exposed to specific mutagen