Chapter 7: Genes, Chromatin & Chromosomes
The Gene: The Physical and Functional Unit of Heredity
Definition of a Gene by Cell Biologists:
Consists of the entire DNA sequence necessary for the production of a functional protein or RNA molecule, marking it as the fundamental unit of inheritance.
Types of RNAs included:
Messenger RNAs (mRNAs) that act as templates for protein synthesis.
Ribosomal RNA (rRNA), Transfer RNA (tRNA), and other smaller functional RNAs (e.g., snRNAs, snoRNAs, microRNAs) that perform diverse cellular functions without being translated into protein.
Includes both introns (non-coding intervening sequences) and exons (coding sequences) as integral components, which are both transcribed into pre-mRNA.
Contains non-coding transcriptional control regions crucial for regulating gene expression.
Example: enhancer sequences that can significantly boost transcription rates. These enhancers could lie some distance away from the promoter, either upstream (5') or downstream (3', or even within introns), and exert their effects through DNA looping mechanisms.
Fundamental Differences between Prokaryotic and Eukaryotic Genes
Prokaryotic Polycistronic Transcription Unit:
Features a simple transcription unit, often organized into operons (e.g., the trp operon), leading to the production of polycistronic mRNAs. This means a single mRNA molecule can encode multiple different proteins, allowing for efficient co-regulation of functionally related genes.
Eukaryotic Simple Transcription Unit:
Demonstrates a more complex structure compared to prokaryotes, characterized by introns, exons, and distinct control regions. Each eukaryotic gene typically produces a monocistronic mRNA, encoding only one protein. This complexity allows for sophisticated regulation and processing.
A schematic example illustrating this complexity is often displayed in Fig. 7-3a, highlighting multiple regulatory elements and post-transcriptional processing steps.
Components of Eukaryotic Genes
Gene Structure:
Upstream enhancers can significantly influence transcription initiation by binding specific activator proteins, promoting the recruitment of the transcription machinery.
Promoter sequences (e.g., TATA box, initiator sequences) are necessary for the accurate initiation of gene expression, serving as the binding site for RNA polymerase and general transcription factors.
Pre-messenger RNA (pre-mRNA), also known as heterogeneous nuclear RNA (hnRNA), comprises the initial RNA transcript that contains both introns and exons.
Intronic enhancers and downstream enhancers can also play a role in regulating transcription, adding further layers of control.
Processing of pre-mRNA is a crucial step that leads to mature mRNA:
Adding a 5' 7-methylguanosine cap: This cap is essential for protecting the mRNA from degradation, facilitating its export from the nucleus, and promoting ribosome binding for translation.
Adding a 3' poly(A) tail: Consisting of 50-250 adenine nucleotides, this tail also contributes to mRNA stability, nuclear export, and translational efficiency.
Splicing: Removal of introns and ligation of exons to form the continuous coding sequence.
Introns
Introns:
Extremely rare in prokaryotes, reflecting their compact genome organization and selection pressure for rapid gene expression.
Yeast possess introns, but their prevalence is not as high as in higher eukaryotes, where they are almost ubiquitous.
Most animal and plant genes are characterized by the presence of multiple introns, which can be much longer than the exons themselves.
Alternative Splicing:
A significant post-transcriptional process where a single pre-mRNA can be spliced in different ways to yield different (yet related) mature mRNA products. This allows a single gene to encode multiple protein isoforms, vastly increasing the protein diversity from a limited number of genes.
Over 90% of human genes are estimated to undergo alternative splicing, showcasing its profound versatility and importance in eukaryotic gene regulation and cellular complexity.
Alternative Splicing of Complex Eukaryotic Genes
Complex transcription units may lead to the production of multiple related proteins from a single gene locus.
Different internal and terminal splicing variations can result in diverse mRNA products. This includes mutually exclusive exons, skipped exons, alternative 5' or 3' splice sites, leading to functionally distinct protein isoforms.
Multiple mRNAs can share common exons while presenting unique sections, highlighting the intricate regulation of gene expression at the post-transcriptional level.
Examples include:
Splicing variations resulting from exon duplication, where duplicated exons can be alternatively included or excluded.
Alternate polyadenylation site usage, which defines different 3' ends of mRNAs and can affect mRNA stability or the protein's C-terminus.
Alternate promoter use, which can lead to transcripts with different 5' ends and potentially different N-terminal protein sequences.
Genome Organization
Approximately an 80 kbp region of the human genome largely consists of non-functional DNA, emphasizing that the majority of the genome is not protein-coding.
Pseudogenes:
Non-functional relatives of known genes that have lost their protein-coding ability due to mutations (e.g., stop codons, frameshifts, regulatory element deletions). They can arise from gene duplication followed by inactivation or from retrotransposition of mRNA.
Examples include ¥ß2 and ¥ß1 (gamma-globin pseudogenes) that are considered non-coding and illustrate gene loss of function.
Alu Sequences:
Short (approximately 300 bp) interspersed nuclear elements (SINEs) that are highly abundant in the human genome, making up over 10% of our DNA. They are retrotransposons that move via an RNA intermediate and reverse transcriptase. Alu sequences can contribute to genomic rearrangements and diseases.
Human ẞ-globin Gene Cluster (Chromosome 11):
Illustrates a classic example of a gene family with developmental regulation. It shows the organization of linked globin genes (e.g., ε, Gγ, Aγ, δ, ß) and pseudogenes (¥ß1) within a confined genomic region, reflecting their evolutionary relationship and coordinated expression during embryonic, fetal, and adult stages, as depicted in Fig. 7-4.
The C-value Paradox
The C-value refers to the total amount of DNA in a haploid genome (e.g., in picograms, pg, or number of base pairs, bp).
The paradox implies discrepancies in the amount of DNA per genome across various organisms that do not correlate with perceived organismal complexity. For example, some simple organisms have larger genomes than more complex ones.
Genome Sizes (C-values):
Yeast: 0.015 pg/haploid genome (12 Mbp)
Fruit fly: 0.15 pg/haploid genome (140 Mbp)
Chicken: 1.3 pg/haploid genome (1.2 Gbp)
Human: 3.2 pg/haploid genome (3.2 Gbp)
Notably, certain amphibians (e.g., salamanders) and plants (e.g., onions, wheat) possess significantly larger haploid genomes than humans, further complicating the relationships between DNA quantity, gene number, and organismal complexity.
Most of the excess DNA noted in larger genomes often represents repetitive sequences, rather than a proportional increase in the number of protein-coding genes.
Eukaryotic Genome Organization: Major Classes of Nuclear Eukaryotic DNA
Eukaryotic DNA can be broadly classified based on its repetition frequency and organization:
Unique (Single-Copy) DNA: Comprising genes that are present in one or a few copies per haploid genome. This class includes most protein-coding genes and untranslated regions.
Moderately Repetitive DNA: Includes sequences repeated from a few hundred to tens of thousands of times. This class contains functional genes (e.g., rRNA and tRNA genes), transposable elements (e.g., LINEs, SINEs like Alu), and some short tandem repeats.
Highly Repetitive DNA: Consists of sequences repeated tens of thousands to millions of times, often arranged in tandem arrays. This includes satellite DNA (found around centromeres and telomeres) and minisatellites/microsatellites (used in DNA fingerprinting).
Table 7-1 (not provided in the prompt but inferred from context) typically summarizes detailed proportions of these major DNA classes in humans, highlighting their characteristics, copy numbers, and functional significance (or lack thereof).
Solitary Genes
Known as single copy genes (or unique sequence genes):
Represent approximately 25-50% of haploid genes in mammals, depending on how they are defined.
These genes generally encode proteins that are required in specific amounts or at specific times, thus not needing multiple copies.
Example: The chicken lysozyme gene, spanning 50-60 kb with several introns, is an example of a solitary gene. Despite its large size due to significant intronic regions, it yields a single type of functional mRNA, demonstrating the unique structure of such genes where non-coding sequences vastly outnumber coding ones.
Gene Families
Gene families:
Define sets of genes that encode functionally related, homologous proteins or RNAs. These genes have typically evolved from a common ancestral gene through gene duplication events.
Can arise from gene duplication processes, primarily through mechanisms like unequal crossing over during meiosis or retrotransposition.
Illustrated are gene duplication events where one gene copy can diverge over evolutionary time due to mutations, leading to either subfunctionalization (sharing ancestral functions) or neofunctionalization (acquiring new functions). This divergence and expansion into gene families, such as the B-globin gene family, allow for the development of diversified functions or tissue-specific expression patterns.
Heavily Used Repetitive Genes
Encode nearly identical proteins or RNAs crucial for cellular efficiency, as these products are required in very high कॉपी numbers.
Examples consist of genes required for fundamental cellular processes, particularly during active cell division and functional RNA synthesis:
Pre-ribosomal RNA (rRNA) genes: These genes are typically found in large tandem arrays within the nucleolus, facilitating the high-volume production of the structural and catalytic components of ribosomes.
Genes encoding tRNAs: Also found in multiple copies, reflecting the high demand for tRNAs during protein synthesis.
Renaturation Kinetics
Describing the processes involved in genomic DNA denaturation (melting into single strands, typically by heat) and subsequent re-annealing (re-association of complementary strands) over time. This technique, often analyzed through C_0t curves, highlights different classes of DNA based on their repeat levels, as repetitive sequences re-anneal faster than unique sequences.
Highly Repetitive DNA:
Accounts for 10-15% of the mammalian genome. These sequences have very high copy numbers and re-anneal most rapidly. They are often found in tandem arrays (e.g., satellite DNA) and contribute to structural functions like centromeres and telomeres, generally not encoding proteins.
Middle Repetitious DNA:
Constitutes about 25-40% of the genome. These sequences have intermediate copy numbers (hundreds to thousands) and re-anneal at an intermediate rate. This class includes transfer RNA and ribosomal RNA genes, histone genes, and transposable elements (LINEs, SINEs).
Single Copy DNA:
Represents roughly 50-60% of the mammalian genome and re-anneals most slowly due to its low copy number. This class includes most protein-coding genes. However, less than 5% of this single-copy DNA actually codes for proteins or functional RNA sequences, with the rest consisting of introns, regulatory regions, and intergenic spacers.
Mobile DNA Elements
Defined as transposable elements (also known as "jumping genes") that are segments of DNA capable of moving from one location to another within the genome. They create mutations, diversify genetic material, and can significantly shape genome evolution and structure.
First identified by Barbara McClintock in the 1940s from her work on maize, demonstrating their ability to cause changes in phenotype.
Transposons and retro-transposons are the primary classes of these mobile elements, differing in their mechanism of movement.
Transposons vs Retro-transposons
Differences in their mechanism of movement and structure:
Transposons (DNA Transposons):
Involve a "cut and paste" mechanism, where the DNA segment is excised from its original location and inserted elsewhere. This process typically utilizes a transposase enzyme encoded by the transposon itself, acting directly on DNA intermediates.
Retro-transposons:
Utilize an RNA intermediate for their transposition, a "copy and paste" mechanism. The retro-transposon DNA is first transcribed into an RNA molecule, which is then reverse-transcribed back into DNA by a reverse transcriptase (often encoded by the retro-transposon). This new DNA copy is then inserted into a new genomic location, leaving the original copy intact. They are broadly classified into LTR (Long Terminal Repeat) retro-transposons and non-LTR retro-transposons (like LINEs and SINEs).
The Role of Centromeres and Telomeres
Centromeres are specialized constricted regions on chromosomes that are key for proper mitotic and meiotic segregation during cell division. They serve as the attachment site for the kinetochore, a protein complex that links the chromosome to spindle microtubules, ensuring accurate distribution of sister chromatids to daughter cells.
Telomeres play a vital role in chromosome stability, preventing the ends of linear chromosomes from being degraded by nucleases or fusing with other chromosomes. They also address the "end replication problem," ensuring complete replication of chromosome ends.
Telomeres and Telomerase
Telomeres:
Consist of tandem arrays of short, highly repetitive DNA sequences (e.g., 5'-TTAGGG-3' repeats in humans), which prevent chromosome degradation and maintain genomic integrity.
Require maintenance by the enzyme telomerase during replication in germline cells, embryonic stem cells, and many cancer cells.
Telomerase: A ribonucleoprotein reverse transcriptase enzyme that synthesizes telomeric DNA repeats using an intrinsic RNA template, thereby extending the 3' end of the chromosome. This counteracts the progressive shortening of telomeres that occurs with each round of DNA replication in most somatic cells due to the inability of DNA polymerase to fully replicate the very ends of linear chromosomes.
Understanding telomere structure and its function leads to crucial insights into mechanisms of aging, as telomere shortening in somatic cells is linked to cellular senescence and organismal aging, and cancer biology, particularly observing telomerase reactivation in malignant growths, which allows cancer cells to evade growth limits and achieve immortal proliferation.
Histone Modification and Chromatin Structure
The dynamic nature of chromatin, its ability to switch between condensed (heterochromatin) and open (euchromatin) states, is largely controlled by various post-translational modifications of histones, influencing gene expression.
Histone Acetylation:
The addition of acetyl groups to lysine residues on histone tails, catalyzed by histone acetyltransferases (HATs), is generally associated with gene activation. Acetylation neutralizes the positive charge of lysine, reducing the affinity between histones and the negatively charged DNA, thereby relaxing chromatin structure and making DNA more accessible to transcription factors.
Histone Methylation:
The addition of methyl groups to lysine or arginine residues on histone tails, catalyzed by histone methyltransferases (HMTs), can have context-dependent effects. It is generally associated with transcriptional silencing when occurring on specific residues (e.g., H3K9me3, H3K27me3), by creating binding sites for repressive protein complexes (like HP1 in heterochromatin). However, methylation on other residues (e.g., H3K4me3) is associated with gene activation.
X-Chromosome Inactivation
X-inactivation is a dosage compensation mechanism in female mammals where one of the two X chromosomes is randomly chosen and epigenetically silenced early in development to equalize the gene expression levels between males (XY) and females (XX). This process leads to mosaicism in females, where different cells express genes from either the paternal or maternal X chromosome.
The inactivation process involves turning one copy of the X chromosome into a condensed, transcriptionally inactive structure called a Barr body (a form of facultative heterochromatin).
It is primarily regulated by the Xist RNA (X-inactive specific transcript), a long non-coding RNA that coats the entire future inactive X chromosome (Xi). This coating then recruits various chromatin-modifying enzymes, leading to specific histone modifications (e.g., deacetylation, methylation of H3K27) and DNA methylation that subsequently silence most of the genes on Xi.