Seminario 9 Genoma y Mecanismos de estabilidad y variabilidad génica - Sebastian Giusti

Introduction to the Human Genome

Overview of Seminar Topics

• The seminar will cover two main topics: characteristics of the human genome and mechanisms determining sequence stability and variability, focusing on mutation generation.

Defining the Human Genome

• The human genome encompasses not only coding genes but also non-coding sequences that do not contain genetic information.

• Each cell contains a copy of the nuclear genome and multiple copies of smaller mitochondrial genomes, with today's focus solely on the nuclear genome.

Characteristics of the Human Genome

Diploidy in Human Cells

• A key feature of the human genome is its diploid nature, meaning each cell nucleus contains two complete sets of genetic information from maternal and paternal origins.

• The fusion of these parental genomes forms the zygote's genome, which is present in all mitotically derived cells.

Chromosomal Structure

• The karyotype technique allows visualization of chromosomes at maximum condensation, showing homologous pairs originating from different parents (e.g., chromosome 5 from mother and father).

• Humans have 22 types of autosomal chromosomes plus two sex chromosomes (X and Y).

Size and Composition of the Human Genome

Measuring Genetic Length

• The smallest unit for measuring nucleic acid length is a base pair; each human chromosome consists of millions of base pairs. A complete diploid set comprises approximately 3.2 billion base pairs (3,000 million).

The Human Genome Project

• Initiated in 1990, this global research consortium aimed to decode all human chromosome sequences using Sanger sequencing methods over about 10 to 13 years at a cost of $3 billion. Samples were collected from several individuals for analysis.

Findings from the Human Genome Project

Gene Count Surprises

• Initial estimates suggested around 100,000 genes in humans; however, it was found there are approximately 20,000 protein-coding genes identified post-project completion. This contradicted earlier assumptions about gene quantity.

Coding vs Non-Coding Genes

Genomic Insights: Understanding Our DNA

Overview of Gene Coding and Non-Coding Regions

• The human genome contains approximately 23,000 genes, which is fewer than previously speculated. A small fraction of the genome encodes information for protein translation.

• Only about 2% of our genome consists of strictly coding information; the remaining sequences are non-coding regions with various functions.

• Approximately 50% of the genome comprises repetitive sequences, while the other half includes exonic information and regulatory sequences like enhancers and silencers.

Classification of Repetitive Sequences

• Repetitive sequences in our genome can be classified into two main categories: tandem repeats and dispersed repeats.

• Tandem repeats can be further sub-classified into satellite, minisatellite, and microsatellite types based on their length and arrangement.

Functional Aspects of Tandem Repeats

• Satellite repeats are longer and play structural roles in centromeres, aiding chromosome separation during cell division.

• Minisatellites serve a similar structural function in telomeres but are shorter than satellite repeats.

• Microsatellites are short repetitive sequences that currently lack known endogenous functions but exhibit high polymorphism among individuals.

Applications in Forensic Genetics

• Due to their high variability, microsatellites are utilized in forensic genetics to establish biological relationships between individuals, such as parent-child connections.

Diploid Nature of Repetitions

• Microsatellites follow a diploid organization; both homologous chromosomes carry similar types but potentially different alleles (e.g., differing numbers of repetitions).

Origin of Dispersed Repeats

Transposons and Retrotransposons in the Human Genome

Overview of Transposons

• Transposons are DNA sequences that can move within the genome, including a category known as retrotransposons, which utilize RNA intermediates for their movement.

• An example of DNA transposons is the TC1 or mariner family, which encodes an enzyme called transposase necessary for their mobility.

Mechanism of Action

• Transposase recognizes specific ends of transposable elements and facilitates their movement through a "cut and paste" mechanism, cutting double-stranded DNA to relocate segments within the genome.

• This process is termed conservative transposition; it does not increase copy numbers unless certain conditions allow for duplication during cell division.

Evolutionary Insights

• Over millions of years, mutations have rendered many transposons inactive by disrupting their ability to produce functional transposase or recognizing sequences.

• The remnants of these once-active elements serve as evolutionary vestiges in our genome, challenging the notion that all genomic elements are functional.

Retrotransposons: Characteristics and Functionality

• Retrotransposons resemble retroviruses and require RNA intermediates for mobility. They integrate into host genomes using reverse transcriptase enzymes derived from viral mechanisms.

• Integrated retroviral genomes can generate multiple copies through transcription; some may undergo further reverse transcription to insert into new genomic locations.

Active Elements in Modern Genomes

• Some families of retrotransposons still possess active members capable of mobility today. For instance, Long Interspersed Nuclear Elements (LINEs) encode reverse transcriptases and endonucleases necessary for replication.

• LINE activity was prevalent in ancestral lineages, resulting in over half a million copies present in modern human genomes.

Additional Mobile Genetic Elements

• Short Interspersed Nuclear Elements (SINEs), while not coding for reverse transcriptases themselves, exploit LINE machinery to replicate within the genome.

Genetic Information and Its Complexity

Overview of Genetic Makeup

• Genetic information is inherited from both maternal and paternal origins, represented in homologous chromosome pairs found in our cells.

• Only about 2% of the genome consists of strictly coding regions that contribute to functional gene products, while half relates to genes through regulatory sequences or non-coding RNAs.

Repetitive Sequences in the Genome

• The other half of the genome comprises repetitive sequences, including tandem repeats like satellites and microsatellites, which have structural roles in chromosomes.

• A significant portion of repetitive sequences arises from mobile genetic elements, considered evolutionary vestiges without specific functions.

Variability Among Human Genomes

• Post-Human Genome Project research focused on analyzing genomic variability among individuals and populations worldwide.

• A study sequenced over 2,500 individuals, revealing that humans share approximately 99.9% of their genomic sequences despite a large total number of base pairs.

Mechanisms Behind Genetic Variation

• The small percentage difference between genomes translates to roughly 4 to 5 million variable sites when comparing two human genomes.

• This leads to inquiries about the mechanisms responsible for these variations, introducing the concept of mutations as permanent changes in DNA sequence.

Types and Effects of Mutations

• Mutations can be classified into micromutations (affecting few base pairs) and macromutations (involving large chromosomal segments).

• Distinction is made between somatic mutations (not heritable) and germline mutations (heritable), emphasizing only germline mutations can be passed to future generations.

Understanding Premutational Changes

• The class will explore how mutations arise from premutational changes—initial alterations that may be corrected by DNA repair mechanisms before becoming fixed mutations.

• These premutational changes can occur spontaneously or be induced by mutagenic agents but are not yet classified as mutations until they evade repair during DNA replication.

Repair Mechanisms and Mutation Fixation

• Various DNA repair mechanisms exist but are not infallible; unrepaired premutational changes can become permanent upon subsequent DNA replication.

Spontaneous Premutational Changes in DNA Replication

Mechanisms of Premutational Changes

• The discussion begins with the consequences of changes fixed during DNA replication, highlighting spontaneous premutational changes associated with the process.

• DNA polymerase generates a new strand using a template strand; however, geometric changes in the double helix may lead to the incorporation of non-complementary nucleotides, resulting in premutational changes.

Tautomerization and Its Effects

• Tautomeric forms of nucleotides are introduced; these are structural isomers that differ in arrangement but have the same chemical composition.

• The predominant forms of adenine and cytosine are amino types, while guanine and thymine predominantly exist as keto forms. However, they can spontaneously transform into rarer tautomeric forms through proton and electron shifts.

Equilibrium Between Forms

• The equilibrium between predominant and rare tautomeric forms can be understood temporally (most time spent in predominant form) or population-wise (majority in predominant form at any moment).

• Rare tautomeric forms exhibit different pairing patterns than their predominant counterparts; for instance, rare thymine can pair with guanine instead of adenine.

Consequences of Tautomeric Incorporation

• During DNA replication, if a rare tautomer is incorporated (e.g., tautomérical cytosine with adenine), it may disrupt base pairing when reverting to its predominant form.

• This disruption leads to what is known as mispairing. If DNA polymerase remains at this site, it may activate proofreading mechanisms to correct this error.

Repair Mechanisms for Mismatches

• If mispairing occurs after additional nucleotides have been added by polymerase, another repair system called REMA detects mismatches based on helical distortions.

• REMA consists of enzymes that identify which strand is newly synthesized versus the template strand to ensure accurate repair without introducing mutations.

Understanding DNA Mutations and Repair Mechanisms

Mechanism of Mutation Fixation

• The process begins with the polymerization of guanine using cytosine as a template, leading to a permanent difference in the resulting strands compared to those generated from the complementary strand.

• Spontaneous premutational changes occur due to biomolecule degradation within cells, such as depurination and deamination of nucleotides.

Types of Chemical Changes

• Deamination can transform natural DNA bases into non-standard ones; for instance, deaminated cytosine becomes uracil, while adenine turns into hypoxanthine.

• These spontaneous chemical changes may evade repair systems if not detected promptly, potentially leading to fixed mutations during replication cycles.

DNA Repair Systems

• The REBA repair system identifies unnatural bases like uracil through specific enzymes that signal their presence for correction.

• If the REBA system fails to correct certain deaminations before replication occurs, these errors can become permanent mutations in subsequent generations.

Epigenetic Influences on Mutation Rates

• Certain biochemical contexts hinder effective correction; for example, methylation of cytosines near gene promoters can mask potential mutations from detection by repair systems.

• Methylated CpG islands are particularly prone to mutations since deamination leads to undetectable nucleotide changes.

Consequences of Mutations

• The impact of a mutation is influenced by its genomic location; coding regions are less common but critical for functional outcomes.

• Mutations can be classified based on their effects on polypeptides: synonymous (no change), nonsynonymous (amino acid change), or nonsense (premature stop codon).

Types of Mutations Explained

• Synonymous mutations do not alter amino acids due to genetic code redundancy.

• Nonsynonymous mutations result in different amino acids being incorporated into proteins, potentially altering function.

Mutations During DNA Replication

Types of Mutations

• Mutations during replication can include not only substitutions but also insertions and deletions of nucleotides, which occur due to a phenomenon called slippage.

Mechanism of Slippage

• The process involves DNA polymerase detaching from the template strand and reattaching, potentially leading to loops in repetitive sequences that result in additional copied regions.

Consequences of Insertion Errors

• If the loop created during replication is not repaired, it may lead to an insertion in the newly synthesized strand compared to the parental strand.

Repair Mechanisms for Insertions and Deletions

• Two main repair mechanisms exist: non-homologous end joining (prone to errors) and homologous recombination (more accurate), which uses a homologous sequence as a template for repair.

Impact of External Factors on Mutations

Consequences of Mutations in Coding Regions

Impact of Insertions and Deletions

• Mutations such as insertions or deletions in coding regions can lead to various consequences depending on the specific site and number of nucleotides involved.

• A single nucleotide insertion or deletion may cause a frameshift mutation, altering the reading order of codons and changing the amino acid sequence in proteins synthesized from that region.

Effects of Nucleotide Changes

• An insertion of three nucleotides (in-frame) between established codons may have a lesser impact, resulting only in the addition of an interstitial amino acid within the protein's primary structure.

• Mutations can also occur in non-coding gene regions, affecting splicing sequences that remove introns. This could lead to entire exons being removed alongside adjacent introns.

Consequences on Gene Expression

• Mutations affecting regulatory elements like promoters or enhancers can influence gene expression, even if they do not occur within strictly coding regions.

• Studies estimate that approximately three mutations accumulate per cell division due to unrepaired premutational changes, leading to an increase in mutations with age.

Mutation Accumulation Across Generations

• Between generations, studies show 100 to 200 new mutations accumulate, indicating ongoing genetic change within human populations.

• While many mutations are deleterious and linked to diseases, they also serve as a source for genetic variability essential for evolution.

Understanding Functional Impacts of Mutations

• The effects of mutations on gene functions must be analyzed case by case; predicting outcomes from substitutions or insertions is complex and often requires experimental validation.

• Classifying mutations involves considering factors like their occurrence in germline cells which may allow transmission to future generations.

Summary of Mutation Processes

• The process generating permanent changes in DNA sequences involves three steps: initial premutational damage, potential recognition by repair mechanisms, and fixation during replication if unrepaired.

Mutations and Their Classifications

Types of Mutations

• The discussion begins with the classification of mutations based on their size, distinguishing between point mutations and variable-length mutations.

• Modifications are categorized into substitutions, insertions, and other types of lesions that can occur during fertilization events.

Future Topics on Mutations

• Upcoming classes will cover triplet expansion mutations, which are a specific type of genetic mutation.

Functional Consequences of Mutations

• The impact of mutations may be functional or non-functional depending on whether they affect gene products; this leads to classifications as silent mutations or those resulting in gain or loss of function.