MBG block 2 lecture 2.pdf

Lecture Objectives: General/Background 1.Define the following terms: Chromatin: lesser condensed form of DNA, makes up chromosome Chromosome: more fully condensed form of DNA, contains centromere and telomere, Chromatid: One of the two identical halves of a duplicated chromosome, joined at the...

Lecture Objectives: General/Background 1.Define the following terms: Chromatin: lesser condensed form of DNA, makes up chromosome Chromosome: more fully condensed form of DNA, contains centromere and telomere, Chromatid: One of the two identical halves of a duplicated chromosome, joined at the centromere, produced by chromosome replication, separates during cell division to become individual chromosomes Chromosome territories: The distinct regions within the nucleus where individual chromosomes are organized, ensuring that they do not overlap significantly with other chromosomes and can be regulated individually. Chromatin Remodeling Complex: protein aggregates that reorganize the nucleosomes of chromatin, enabling different levels of condensation allowing or restricting access to DNA for transcription, replication, and repair. Histone: A type of protein around which DNA winds to form nucleosomes. Histones help in the organization and compaction of DNA and play a key role in gene regulation. Nucleosome: The basic structural unit of chromatin, consisting of a segment of DNA wrapped around a core of eight histone proteins. Centromere: The region of a chromosome where the two sister chromatids are joined together and where the kinetochore forms, facilitating attachment to spindle fibers during cell division. Kinetochore: A protein complex that assembles on the centromere of a chromosome, serving as the attachment point for spindle microtubules during mitosis and meiosis, ensuring proper chromosome segregation. Neocentromere: An atypical centromere that forms at a location on the chromosome where a centromere did not previously exist, often arising in cases of chromosomal rearrangements. Telomere: The repetitive DNA sequences at the ends of chromosomes that protect them from degradation and prevent fusion with neighboring chromosomes. Telomeres shorten with each cell division. Karyogram: A visual representation of an individual's complete set of chromosomes, organized and displayed in pairs according to size and shape. Karyotype: The full set of chromosomes in a cell or organism, including their number, size, and shape, typically displayed in a karyogram for diagnostic purposes. Autosome: Any chromosome that is not a sex chromosome. Humans have 22 pairs of autosomes and 1 pair of sex chromosomes. Repetitive DNA: DNA sequences that are repeated many times within the genome. These can be short sequences, like satellite DNA, or longer ones, like transposable elements. Pseudoautosomal Regions: Regions of the sex chromosomes (X and Y) that are homologous and undergo recombination during meiosis, similar to autosomes. Banding/Banding Pattern: The pattern of light and dark bands on chromosomes when stained, used to identify chromosome structure, detect abnormalities, and map genes. Euchromatin: The less condensed form of chromatin that is transcriptionally active, allowing access to DNA for gene expression. Heterochromatin: The tightly packed form of chromatin that is generally transcriptionally inactive and often associated with structural and regulatory roles. Locus (Loci): The specific physical location of a gene or other significant sequence on a linear chromosome. Allele: One of two or more alternative forms of a gene that arise by mutation and are found at the same locus on homologous chromosomes. Different alleles can result in different traits or functions. Chromosomes Structure and Function 2.Explain the relationship between types of DNA sequences and the structures formed in chromosomes and identify the expected chromosome structure formed from a sequence of DNA or histone variant incorporated at a given chromosome position 1. Types of DNA Sequences and Chromosome Structures: Unique DNA Sequences: ○ These are single-copy sequences that typically contain genes and regulatory elements. Represented only once in a haploid set of chromosomes ○ They form euchromatin, the less condensed and transcriptionally active regions of the chromosome. Euchromatin has a loosely packed structure, allowing for gene expression. Repetitive DNA Sequences: ○ Satellite DNA: Clustered repeated sequences found mainly in centromeric and telomeric regions. forms heterochromatin, which is densely packed and transcriptionally inactive. It provides structural stability to chromosomes and plays a role in chromosome segregation during cell division. ○ Interspersed repeated sequences SHORT Alu family, ~10% of genome Regions rich in GC content (G-light bands) LONG Long Interspersed Nuclear Element (LINE) L1 sequences, ~20% of genome Regions rich in AT content (G-dark bands) Chromosome territories ○ Chromatin fibers in nucleus of nondividing cell form discrete chromosome territories Chromosome territories are correlated with gene density ○ Gene-Rich chromosome domains located interior of nucleus Gene-rich DNA Sequences:  ○ sequences form euchromatin structures, where genes are actively transcribed. ○ Gene regulatory elements like promoters and enhancers can be found within these regions chromatin here is more loosely packed, promoting accessibility to transcription factors. The chromatin structure allows for the efficient condensation of the long DNA molecules into the nucleus while regulating access to genetic information for processes like transcription, replication, and repair. 1. Histones: The Protein Core of Chromatin Histones are small, positively charged proteins that bind to negatively charged DNA, helping to package it. There are five main types of histones: Core Histones (H2A, H2B, H3, H4): Histone octamer, make up core particle ○ These histones form the structural core around which DNA is wrapped. Linker Histone (H1): This histone binds to the core particle and to the linker DNA ○ stabilizes the higher-order structure of chromatin. 2. Nucleosomes: The Basic Repeating Unit of Chromatin The nucleosome is the basic structural unit of chromatin, composed of DNA wrapped around a histone core. Structure of a Nucleosome: ○ The core of the nucleosome consists of an octamer of histones, made up of two copies each of the core histones: H2A, H2B, H3, and H4. ○ About 147 base pairs of DNA are wrapped around this histone octamer, making approximately 1.7 turns. ○ The linker DNA (20-80 base pairs) connects adjacent nucleosomes. The linker histone H1 binds to the linker DNA, aiding in the compaction of nucleosomes into higher-order structures. 3. Chromatin Structure and Compaction Chromatin can exist in various states of compaction, largely determined by the arrangement of nucleosomes and histone modifications. Beads on a String (10 nm Fiber): ○ This is the most relaxed form of chromatin, where nucleosomes are connected by linker DNA, resembling a string of beads. The DNA is accessible for transcription and other processes. 30 nm Fiber: ○ In this more compact structure, nucleosomes fold into a helical or zigzag structure, which compacts the chromatin further. This is mediated by the presence of histone H1, which helps to pull nucleosomes closer together. Higher-Order Structures: ○ Chromatin fibers can be further folded and compacted into loops, domains, and eventually into the highly condensed structures visible during mitosis and meiosis. In these states, chromatin is tightly packed and less accessible for transcription, forming what is known as heterochromatin. 3.Summarize the processes of chromosome condensation with particular emphasis on the components involved in the generation of fully condensed chromosomes and Assess the consequences for changes in protein availability or incorporation in terms of chromosome structure or condensation level Condensin - Provides force to get passed random looping phase and continue to dense higher order structure Cohesin - Separating SMC1 and SMC3 1. Chromatin formed by the winding of DNA around histone proteins 2. Structure comprising DNA loop linked by condensin (SMC2, SMC4) 3. Structure comprising several units of condensins 4. Sister chromatid, a structure comprising many densely condensed units  5. Structure comprising two sister chromatids with DNA sequences linked by cohesion (SMC3, SMC1) 6. Condensed chromosome of two sister chromatids produced from the replicated DNA sequences connected at centromere 4.Summarize the function, location, gene content, and importance of the following chromosome structures: centromere, kinetochore, telomeres, and NORs Centromere - a region of highly specialized chromatin that is largely heterochromatic and contains lots of alpha satellite DNA - Centromere Proteins: there are more than 20 now known - primary function: - be the foundation for the formation of the Kinetochore - essential for chromosome segregation Kinetochore - Protein complex essential for proper chromosome segregation during mitosis - Massive complex requires several proteins Nucleolar Organizer Regions (NORs) - Located on the satellite stalks of acrocentric chromosomes - Location of nucleoli formation in interphase - Site of ribosomal RNA genes and production of rRNA 5.Explain the role of the centromere and kinetochore in chromosome segregation with an emphasis on components required for function and the role of centromere positioning to chromosome segregation 6. Connect the kinetochore, the processes of chromosome condensation, and centromere positioning with cellular regulation, kinase activity, and the Anaphase Promoting Complex from block 1 1. The Centromere: Role in Chromosome Segregation: The centromere is the specialized chromosomal region where sister chromatids are held together and where the kinetochore forms. Its primary role in chromosome segregation is to anchor the kinetochores, allowing the attachment of spindle fibers and proper chromosome alignment on the metaphase plate. Components Required for Function: ○ Alpha-Satellite DNA: In humans, centromeres are rich in repetitive alpha-satellite DNA sequences, which act as a foundation for kinetochore assembly. However, the DNA sequence alone is not sufficient for centromere function. ○ CENP-A: A variant of histone H3, called CENP-A, replaces standard H3 in centromeric nucleosomes. CENP-A is critical because it marks the centromere and serves as a platform for kinetochore assembly. ○ Centromeric Chromatin: Centromeric chromatin is highly specialized and organized to attract kinetochore proteins. The presence of CENP-A recruits additional centromere proteins, such as CENP-B and CENP-C, which help stabilize the centromere.  Centromere Positioning: The position of the centromere divides the chromosome into two arms: a short arm (p)and a long arm (q). Based on centromere positioning, chromosomes are classified as: ○ Metacentric: located in middle of chromosome ○ Submetacentric: located closer to one end of chromosome ○ Acrocentric: located near one end of chromosome ○ Telocentric: located at the telomere The centromere serves as the anchor point for the kinetochore and ensures that sister chromatids are pulled apart at the correct time and in the right direction. Mispositioning or malformation of the centromere can lead to improper segregation, which can cause aneuploidy or cell death. 2. The Kinetochore: Role in Chromosome Segregation: ○ The kinetochore is a multi-protein complex that assembles on the centromere ○ Its role is to mediate the attachment of chromosomes to the microtubules of the mitotic spindle, allowing the chromosomes to be properly aligned and segregated to daughter cells during cell division. ○ Aurora B kinase and CDK 1 activity help to detach and reattach to the correct spindles in meiosis 1 and cell division Components Required for Function: The kinetochore has two main parts: ○ Inner Kinetochore: This part binds directly to the centromeric chromatin and interacts with CENP-A, CENP-C, and other centromere-specific proteins. ○ Outer Kinetochore: This part attaches to microtubules and is involved in chromosome movement. Key proteins include: Ndc80 Complex: Connects microtubules to the kinetochore, anchoring the spindle fibers to the chromosome. Spindle Assembly Checkpoint Proteins: Proteins like Mad2 and BubR1 ensure that each kinetochore is properly attached to spindle microtubules before the cell proceeds with chromosome segregation.  Dynein and Kinesin Motors: These motor proteins are essential for the movement of chromosomes along microtubules during anaphase. Kinetochore-Microtubule Attachment: Each kinetochore attaches to microtubules, which emanate from opposite spindle poles. ○ During metaphase, kinetochores pull chromosomes to the center of the cell, aligning them on the metaphase plate. ○ Once proper alignment is confirmed, the kinetochores facilitate the separation of sister chromatids by pulling them to opposite poles during anaphase. Tension-Sensing Mechanism: The kinetochores create tension by pulling on the spindle microtubules. ○ This tension is crucial because it signals that the kinetochores are properly attached to microtubules from opposite poles. ○ If tension is not sensed, the spindle assembly checkpoint delays progression to anaphase until proper attachments are established. This ensures the accurate distribution of chromosomes to daughter cells. 3. The Role of Centromere Positioning in Segregation: The correct positioning of the centromere is essential for the balanced segregation of chromosomes. ○ The centromere’s central or near-central positioning in most chromosomes ensures that the two sister chromatids are distributed equally between daughter cells. Metacentric and submetacentric centromeres typically promote balanced segregation, as they divide the chromosome into roughly equal halves. ○ Acrocentric centromeres, with their very short p-arms, may be more prone to missegregation if centromeric function is impaired. Centromere positioning also determines the attachment of spindle fibers, as kinetochores form at the centromere and bind to microtubules. ○ Accurate attachment and tension at the centromere-kinetochore interface are critical for proper chromosomal segregation. Summary of the Centromere and Kinetochore in Chromosome Segregation: Centromeres serve as the foundation for the kinetochore assembly and ensure proper chromosomal alignment and segregation by providing a structural platform. Kinetochores are the actual attachment points for spindle microtubules, pulling chromosomes to opposite poles during cell division. Proper centromere positioning is critical for ensuring balanced chromosome segregation, with the formation of kinetochores at these regions guaranteeing the correct attachment to spindle fibers and successful chromatid separation. 7.Assess the consequences for the chromosome or cell particularly in terms of chromosome integrity or DNA repair induction following changes in length, availability or activity of telomere/telomerase associated proteins, activity or function of telomerase Telomeres - Repetitive sequences of DNA at the ends of the chromosomes that assist with protecting the chromosome from genetic loss (TTAGGG) - Telomerase maintains telomere length by two components - Reverse transcriptase protein subunit (hTERT) - RNA component (hTR or hTERC) - Telomere length represents age - Long telomeres are capable of protecting ends of chromosomes via capping - Short telomeres can no longer provide caps, and will lead to further loss of DNA and appear broken - Loss capping = reduced chromosome integrity = G1 senescence The telomere cap forms when telomeres with their associated proteins loop into themselves - A 3’ overhang called the G-strand Overhang folds back and invades the dsDNA forming T and D loops - The looped “cap” protects the 3’ overhang Associated proteins assist with this: - TRF 1 = telomere repeat binding factor 1 - Complex with it = Length regulation - TRF 2 = telomere repeat binding factor 2 - Complex with it = Protective end cap Sacrificial Buffer for Protection of Genetic Material - Can be maintained or restored via an enzyme called telomerase Stem Cells - Need to be maintained throughout the entire life cycle - Telomerase maintains telomere length TERT = telomerase reverse transcriptase TERC (or hTR) = human telomerase template RNA - Loss of this causes disorders due to its importance in maintaining stem cells - Bone marrow failure, pulmonary fibrosis, etc. 8.Evaluate the process of telomerase-mediated elongation of telomeres and Explain the role of telomere associated proteins in telomere maintenance and/or telomerase activity Nomenclature Section: Introductory level ONLY; no complex or difficult designations/rearrangements should be expected 9.Explain how chromosome abnormalities are named and identify the proper designation for a given change ◦Be able to identify the specific change from a summary karyotype ◦Be able to write the summary karyotype for a given specific change (or normal human karyotype) or identify the specific change (or normal expectation) from the karyotype summary provided On which type of chromosome would you expect to find a nucleolar organizing region (NOR)? (LO4) - Acrocentric What is considered the typical, healthy male karyotype if no chromosome abnormalities are observed (a.k.a. traditional/normal male karyotype)? (LO1 and LO8) - 46,XY Female? - 46,XX What would you expect to see if (LO3): - There was a mutation in condensin that prevented its incorporation on chromosome 3? - Wouldn't full condense, stall as coiled 30nm fibers, but not higher ordered structures - prevented its breakdown? - Inability to return to a normal arrangement, lose genetic activity of entire chromosome - Histone variants typically associated with a centromere were incorporated into a region near the telomere for a metacentric chromosome? - Formation of a neocentromere closer to the telomere - Potential for a dicentric chromosome What would you expect to see if (LO6/LO7): - Telomeres became critically short? - Cellular senescence (G0 from G1) - Telomerase became inactive in adult stem cells? - Shortening telomeres even in stem cells