Week 3 2024 Chromosome Structure and Morphology.pdf

Chromosome Structure & Morphology [email protected] Chromosome Structure Revision Metaphase chromosome (1400nm) The looped domains of DNA coil to give rise to highly compacted chromosomes There is a central scaffold formed of acidic histone proteins to which the chromatin fibre is attached a...

Chromosome Structure & Morphology [email protected] Chromosome Structure Revision Metaphase chromosome (1400nm) The looped domains of DNA coil to give rise to highly compacted chromosomes There is a central scaffold formed of acidic histone proteins to which the chromatin fibre is attached at repeated sequences so that loops of fibre (Laemmli loops) radiate out from the scaffold to form the chromatid (600 nm) Types of DNA Unique sequence (single copy) Highly repetitive (>105 copies) – Tandem Sequences – Interspersed Middle repetitive sequences (102 to 104 copies) Unique sequence copy DNA Most common class of DNA comprising 70-75% of the genome The nucleotide sequences that are represented only once (or a few times) in a haploid set Genes that code for proteins are single copy DNA Highly Repetitive DNA DNA sequences found at >105 copies remaining 25% of the genome (includes middle repetitive DNA) Classified according to the number of repeats and whether the repeats are tandem or interspersed among unique sequence Highly Repetitive DNA Tandem Repetitive – Termed satellite DNA because it separates from the main band DNA by density gradient centrifugation because of the differing CG content % – Categorised based on the length of sequences that make up the tandem array and the total length of the array Alpha satellite (repeat of ~170bp sequence monomer organised in a tandem array of up to a million bp in total length) located in and around the centromere Mini satellite (repeats that are 20-70bp in length, with a total length of a few thousand bp) extremely polymorphic due to variation in repeats. Located primarily around the sub-telomeric regions Micro satellite (repeat units of 2, 3 or 4bp and total length of a few hundred bp) widespread occurrence Interspersed Repetitive DNA – Found throughout the genome and make up 1/3 of total repetitive DNA – Classified as either short or long Short interspersed elements (SINES) range in size form 90 to 500 bp » Have a high G-C content and are found in the G-light bands Long interspersed elements (LINES) range in size form 1.5 to 5 kb » Have a high A-T content and are found in the G-dark bands An example of SINES are the Alu elements (~300bp long) found in large numbers (>1 million copies) within the human genome. Human karyotype: Alu elements (green) and counterstained with TOPRO-3 (red). Image from: - Bolzer et al., (2005) Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes. PLoS Biol 3(5): e157 DOI: 10.1371/journal.pbio.0030157 7a Middle repetitive sequences – DNA sequences found with 102-104 copies – Genes for 18S and 28S ribosomal RNA – Several hundred copies of these genes are tandemly arranged on the short arms of acrocentric chromosomes Chromatin Is complex material, intricate in its organisation It is composed of DNA, proteins, RNA and polysaccharides – Euchromatin: loosely packed within the nucleus – Heterochromatin: tightly packed/condensed within the nucleus Constitutive Facultative Image: Atlas of Plant and Animal Histology https://mmegias.webs.uvigo.es/02-english/5-celulas/4-cromatina.php Euchromatin Unique sequence DNA Contains the coding DNA (Genes) Is loosely organised, extended and uncoiled Contains active, early replicating genes and stains lightly with conventional G banding Heterochromatin Contains highly repetitive DNA Contains few active genes Replicates late during the synthesis (S) phases of mitosis Is highly contracted Located at the centromeres Constitutive Heterochromatin Consists of simple repeats of nitrogenous bases that are located around the centromeres of all chromosomes and distal end of the Y chromosome There are no transcribed genes Variations have no effect on phenotype Chromosomes 1, 9, 16 and Y have variably sized constitutive heterochromatic regions Function is the regulation of crossing over, the exchange of genes from one sister chromatid to the other during cell division Constitutive Heterochromatin C-banded chromosomes Picture provided by Western Diagnostic Pathology Facultative Heterochromatin One X chromosome of every female cell is randomly inactivated (Lyonisation) This inactivated X is condensed during interphase and replicates late during the synthesis (S) stage of the cell cycle – This inactive X can often appear slightly smaller than the active X in the metaphase. Chromatid One of a pair of metaphase chromosome strands Each chromatid contains the same genetic material During the cell cycle the chromosomes replicate and two sister chromatids form At metaphase each chromosome can be seen as 2 chromatids joined together Telomere Human cells in culture divide a limited number of times, then stop dividing. Normally, cells hit their limit after about 50-70 cell divisions. RNA-protein enzyme telomerase which rebuilds telomeres by the addition of guanine-rich repetitive sequences. Active telomerase is a factor in allowing cells to keep on dividing - they stop dividing when the telomeres become critically short thereby activating DNA damage signalling which results in cells death. (Approx. 50 nucleotides are lost after each cell cycle). Telomerase rebuilds the chromosomal telomeres by repetitively adding the DNA sequence 5`-TTAGGG-3` https://somaticmovementcenter.com/wp-content/uploads/2019/12/dna-telomere-diagram.jpg Centromere A constriction visible on metaphase chromosomes where the 2 sister chromatids are joined together Is a specialised region of DNA at which the kinetochore forms and the spindle apparatus attaches during cell division Mitotic spindle fibres separate the sister chromatids to move to opposite poles of the dividing cell Attachment point into which the microtubules insert is called the kinetochore Centromeric heterochromatin contains highly repetitive microsatellite repeat DNA NOR Nucleolar Organising Regions Are located on the satellite stalks on the short arms of the acrocentric chromosomes (13, 14, 15, 21, 22) They contain multiple clustered copies of genes for 18S and 28S rRNA and are the site of synthesis of ribosomal material in the interphase nucleus All may not be active Active NORs stain darkly with silver nitrate Fluorescent probes also available for the rRNA. NOR Image shows eight of the ten acrocentric chromosomes with the active NORs (stalks) stained. Image courtesy of Western Diagnostic Pathology Gross Structure: Acrocentrics Satellite (s) Satellite stalks (stk) Proximal Short arm (p) Centromere (cen) Long arm (q) NOR Satellite association Image courtesy of Western Diagnostic Pathology Landmarks and Regions Defined as consistent and distinct morphological features important in identifying chromosomes – Telomeres – Centromeres – Prominent bands As an area of the chromosome lying between two adjacent landmarks Band A part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or lighter with one or more banding techniques Chromosome Structure and Identification Chromosomes are arranged in a karyogram based on centromere position, band pattern and length of the chromosome. The smaller arm of the chromosome is designated as being the “p” arm and the long arm is the “q” arm. They are ordered from largest to smallest in ascending order ie 1 to 22 Karyotype – the number of chromosomes, sex chromosomes, then abnormalities are listed in numerical order. For example 47,XY,t(3;17)(q12;p13),+18 Chromosome Structure and Identification Metacentric - centromere near the middle. The p and q arms are of similar size Sub-metacentric – centromere is closer to one end one end, giving a smaller p arm and a larger q arm Acrocentric – centromere at top. Acrocentric chromosomes have very small p arms and often have satellites (NOR region) on the end Telocentric - have the centromere at the end with no p arm (not present in the human karyotype). Chromosome Structure Image: https://ib.bioninja.com.au/standard-level/topic-3-genetics/32chromosomes/chromosome-types.html Groups on solid stain Chromosomes are distinguished on the basis of their – Size – Centromere position – Banding pattern Group A (1-3) Large metacentric Group B (4-5) Large submetacentric Group C (6-12 & X) Medium sized sub-metacentric Group D (13-15) Medium sized acrocentric with satellites Group E (16-18) Short metacentric (16) and sub-metacentric Group F (19-20) Short metacentric Group G (21-22 &Y) Short acrocentric with satellites – (Y has no satellites) G Banding Generally the preferred method for the routine staining of chromosomes – Simple – Slides can be kept for a long time without deterioration Usual method to obtain G-banding involve treating the slides with a protease such as trypsin – Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. Banding pattern obtained are thought to reflect both structural and functional composition of the chromosome G Banding G-Dark bands G-Light bands Replicate mid to late in S phase Replicate early in S phase Condense early in mitosis Condense later in mitosis Gene poor tissue specific genes Gene rich housekeeping genes A-T rich DNA C-G rich DNA Enriched in LINES Enriched in SINES (long interspersed elements) (short interspersed elements) Localised at nuclear periphery Located in interior of nucleus G-banded chromosomes Image courtesy of Western Diagnostic Pathology G banding ISCN International System for Cytogenomic Nomenclature Provides a universally understandable description of a karyotype and the aberrations of chromosomes First established in 1960 and has been updated regularly to include new molecular cytogenetic techniques including FISH, Microarray/arrayCGH, region specific assays and sequencing Guidelines for reporting Constitutional and Malignant karyotypes. List of cytogenetic terms and abbreviations. Ideograms for each chromosome at varying resolutions. Chromosome ideograms Banding Resolution “Quality” score given as “bands per haploid set” (bphs) Cytogenetics generally analyse from 400bphs in a prenatal or oncology prep., to up to 550bphs in a routine peripheral blood prep. And up to 800bphs when looking for a specific microdeletion /duplication. Constitutional or Acquired? Cytogenetic changes may be constitutional or acquired Constitutional changes are present from early fetal development, either at the zygote stage or shortly afterwards in embryogenesis Acquired changes occur during the life of a person eg leukaemia or solid tumour growth Cytogenetic Changes Numerical/Aneuploid: where the number of chromosomes varies from 46 Structural: where the structure of one or more of the chromosomes has altered. These changes include translocations(t), inversions (inv), duplications (dup), insertions (ins), rings (r) and markers (mar). Significance of Changes Clinical symptoms are accompanied by cytogenetic alteration/s. Some changes are population “variants”, occurring in the general population without adverse effect. They are discovered incidentally. Cytogenetic changes can also be observed in mosaic form Mosaics Cytogenetic changes can also be observed in mosaic form There may be two or more cell lines The % of different cell lines may vary between different tissues Some mosaic cell lines can only be detected in specific tissues