chromosomes and cytogenetics

chromatin

• In chromosomes, DNA is compacted by forming complexes with histone proteins 

• Most condense form 

• Can exist at euchromatin or heterochromatin 

• Occurs during metaphase 

• Regulated by interaction with histone proteins 

  • Wraps around twice 

  • Forms a nucleosome 

  • Can form a 30 nm fibre 

  • 30 nm fibre loops around a protein scaffold 

euchromatin

• Most of the genome is in the form of 'euchromatin' 

• The degree of compaction varies and can be very dynamic 

  • Can regulate association with H1, which in turn regulates rate of gene transcription 

• Contains all of the genes 

• Forms 90% of chromosomes 

heterochromatin

• A smaller proportion of human chromosomes is in the form of heterochromatin, where the chromatin is more permanently compacted 

• Forms via condenser proteins 

• Not dynamic (cannot reverse compactness) 

• Barrier elements recruit barrier proteins 

  • Prevent heterochromatin spilling into euchromatin 

  • Can be lost following a chromosomal translocation 

spilling of heterochromatin

• If heterochromatin spills = position effect variegation 

• Shuts off all the genes in the euchromatin 

• Heterochromatin has a tendency to spread (e.g. into neighbouring euchromatic regions) 

  • Molecular mechanisms exist to prevent this 

structural heterochromatin

• Structural heterochromatin usually contains repetitive DNA sequences known as satellite DNA 

• It is found in structural elements such as centromeres and importantly in telomeres 

  • Prevent fusion of chromosomes 

• Telomeric DNA sequences are composed of long arrays (10-15 kilobases) of short tandem repeats 

  • G rich strand = longer, resulting in an overhang called the G-tail (around 30 repeats) 

  • C rich strand = shorter 

G-tail

• Protects chromosome ends 

  • Prevents chromosome ends from being recognized as DNA double-strand breaks 

  • Avoids activation of DNA damage response pathways 

• Enables telomere capping 

  • G-tail folds back to form a T-loop 

  • The T-loop physically hides the chromosome end 

• Recruits shelterin complex 

  • Binds telomere-specific proteins (e.g. TRF1, TRF2, POT1) 

  • Shelterin stabilizes telomere structure and maintains heterochromatin 

• Prevents end-to-end fusions 

  • Blocks non-homologous end joining (NHEJ) at chromosome ends 

• Maintains genomic stability 

  • Ensures proper chromosome maintenance across cell divisions 

• Facilitates telomere replication 

  • Provides a substrate for telomerase to extend telomeres 

function of structural heterochromatin

• Maintains chromosome structural integrity 

  • Provides mechanical stability to chromosomes 

• Located at repetitive DNA regions 

  • Found at centromeres, telomeres, and other repeat-rich regions

• Ensures proper chromosome segregation 

  • Centromeric heterochromatin is essential for kinetochore formation 

  • Prevents mis-segregation during mitosis and meiosis 

• Suppresses recombination 

  • Prevents homologous recombination between repetitive sequences 

  • Reduces chromosomal rearrangements 

• Silences repetitive DNA 

  • Transcriptionally inactive 

  • Prevents expression of transposons and satellite DNA 

• Protects genome stability 

  • Limits insertional mutagenesis by mobile elements 

• Maintains nuclear organization 

  • Anchors chromatin to the nuclear periphery 

  • Contributes to higher-order chromatin architecture 

• Defines chromosomal domains 

  • Acts as a boundary separating active euchromatin regions 

• Epigenetically maintained 

  • Characterized by DNA methylation 

  • Enriched in H3K9me3 and HP1 proteins 

• Evolutionarily conserved 

  • Present in most eukaryotes with similar structural roles 

cytogenetics

• Chromosomes from cell preparations can be 'spread' on a microscopic slide for analysis 

  • Want to collect them in metaphase 

  • As they are more condensed 

  • Can be distinguished from each other 

  • Chromosomes in prometaphase are less compacted and thus reveal more detail 

    • Can find structural features 

hypotonic treatment

• Cells are placed in a hypotonic solution (e.g. 0.075 M KCl) 

• Water enters cells by osmosis 

• Cells swell due to increased internal pressure 

• Nuclear membrane weakens and ruptures 

• Chromosomes spread apart within the swollen cell 

• Cell is then fixed (e.g. methanol:acetic acid) to preserve chromosome structure 

• Chromosomes are dropped onto a slide for staining and analysis 

• Stains create a banding pattern 

  • Can distinguish one chromosome from another 

  • Using karyotyping 

molecular cytogenetics

• Higher resolution analysis (e.g. detection of specific DNA sequences within chromosomes) 

• Achieved by designing and chemically synthesising an 'oligonucleotide probe' directed against a target DNA sequence 

  • Labelled with a fluorophore 

how does FISH (fluorescence in situ hybridisation) work

• Purpose: 

  • Detects and localizes specific DNA (or RNA) sequences on chromosomes or in cells 

• Starting material: 

  • Metaphase chromosomes or interphase nuclei fixed on a slide 

• Denaturation: 

  • Chromosomal DNA is heated or chemically treated 

  • Double-stranded DNA separates into single strands 

• Probe preparation: 

  • Short, single-stranded DNA oligonucleotide probes 

  • Labelled with a fluorescent dye 

• Hybridization: 

  • Probe is applied to the slide 

  • Incubation allows probe to find and bind its complementary sequence 

• Washing: 

  • Removes unbound or weakly bound probes 

  • Ensures specific binding 

• Detection: 

  • Slide viewed under a fluorescence microscope 

  • Bound probes appear as bright fluorescent signals 

how does the oligonucleotide probe attach

• Base pairing 

  • Probe binds by complementary base pairing 

  • A–T and G–C hydrogen bonds form between probe and target DNA 

• Hybridization conditions 

  • Temperature, salt concentration, and time control stringency 

  • High stringency → only perfect matches bind 

  • Low stringency → partial mismatches may bind 

• No covalent bonds 

  • Attachment is non-covalent (hydrogen bonds only) 

  • Binding is reversible under denaturing conditions 

• Probe length matters 

  • Short oligos → high specificity 

  • Longer probes → stronger binding, lower specificity 

chromosome painting

• Uses a large cocktail of probes that will bind to many different locations along the length of a single type of chromosome 

• Spectral karyotyping (SKY) can allow for the entire set of chromosomes to be analysed