Week 2 Flashcards
Describe the unineme hypothesis
DNA of each eukaryotic chromosome consists of one continuous double helical fiber along its entire
• DNA + histones = nucleosomes
• Structural organization: chromosome scaffolds
Describe the experiment showing semiconservative replication of DNA
Chromosome labeled with halogenated thymidine in Metaphase 1
Anaphase separates the chromosome into two chromatids
After replication one strands is labeled with thymidine and newly synthesized strand isn’t
Describe with sister chromatid exchanges are and how they can be detected
- The exchange of segments between sister chromatids represents a deviation from regular semi-conservative pattern of eukaryotic chromosomes
- Involve DNA breakage and reunion
• Taylor et al.(1957) autoradiographic technique – important in understanding the occurrence of SCEs at mitosis
• Led to discovery that tritiated thimidine could be replaced by halogenated thymidine analogs.
– Bromodeoxyuridine (5-bromo-2’-deoxyuridine, BrdU, BUdR, BrdUrd, broxuridine) is a synthetic nucleoside that is an analog of thymidine
• 33258 Hoescht used to detect BrdU-substituting DNA or Giemsa in labs without fluorescent microscopes
What functions does chromatin serve?
• Consists of DNA, protein and RNA • Plays role in: – packaging, – controls replication, – mitosis, – preventing DNA damage – controls gene expression
What is the difference between euchromatin and heterochromatin
Euchromatin
Chromatin or chromosomal regions that are lightly staining and are relatively uncoiled during the interphase portion of the cell cycle.
Contains most the structural genes
Heterochromatin
Chromosome segments that do not undergo despiralization and decondensation at the end of each cell division, remain tighly coiled
What are features associated with transcriptionally active and inactive chromatin
ACTIVE
• CHROMOSOME STRUCTURE – Open extended conformation
• DNA METHYLATION
– relatively unmethylated esp. at
promoter regions
• HISTONE ACETYLATION – Acetylated histones
INACTIVE
• CHROMOSOME STRUCTURE – Highlycondensed
confirmation
• Both Constitutive and Facultative
• DNA METHYLATION
– Methylated incl. promoters
• HISTONE ACETYLATION – Deacetylated histones
What is the mechanism of DNA methylation
• Epigenetic mechanism
– Describes heritable states that do not depend on changes in DNA sequence
- Maintains repression of transcription
- Critically involved in mechanisms operating on some genes to ensure that only 1 of the 2 parentally inherited alleles is expressed (Monoallelic expression)
• 3 major epigenetic mechanisms (Genes regulated/modulated)
– Reversible modification of DNA by the addition or removal of methyl
groups
– Modification of histones by the addition or removal of chemical groups
-RegulationofgeneexpressionbysmallnoncodingRNAmolecules
• Extent of methylation can be tissue specific and can vary from less than 2% to over 7%
What are the targets of DNA methylation
CpG dinucleotides targets for DNA methylation
Repression at methylated CpGsequences in promoter regions mediated by proteins which specifically bind methylated CpG
• 2 proteins ided : Methylated CpG-binding Proteins 1 and 2 (MeCP1 and MeCP2)
• MeCP2 binds histone methyl transferase–H3K9is deacetylated it is methylated.
• Methylated H3K9 is target for heterochromatizing proteins such as HP1 – cause chromatin to condense and become inactive
Methylation occurs in the cytosine of CG doublets in DNA, usually in both strands
• 5-̕mCpG-3̕
• 3̕- GpCm-5̕
• Inverse relationship exists between the degree of methylation and the degree of expression
– Low levels of methylation associated with higher levels of gene expression
• Methylation patterns are tissue specific and once established are heritable for all cells of that tissue
How are methylation patterns conserved
• DNA methylation interacts with histone modification
• Permits stable transmission from a diploid cell to daughter cells of chromatin states that repress expression
• Maintenance methylation is the perpetuation of a preexisting methylation pattern & is carried out by
Dnmt1 methyltransferase
• CpG methylation is perpetuated by a requirement for the specific methyl transferase to recognise a hemi-methylated target sequence
• The sequence CpG has dyad symmetry
• Following methylation of a hemi-methylated target
• The two methylated strands separate at duplication – act as templates for 2 unmethylated daughter strands
• The resulting duplexes provide new hemi-methylated
targets for continuing the same pattern of
methylation
Explain the mutual reinforcement of histone and DNA methylation in an inactive chromatin
- Methylation of DNA attracts proteins that modify associated histone proteins.
- Histone deacetylation and methylation attract proteins that methylate associated DNA
- In addition to the effects shown here some histone methyltransferases can interact directly with DNA methyltransferases
- Endogenous short interfering RNA may be important in forming heterochromatin
Describe the two types of heterochromatin
- Constitutive heterochromatin
- Located at the identical position in homologous chromosomes in all cells as a permanent structural entity
- E.g. Centromeres, telomeres, long arm of Y chromosome,Hetrochromatic knobs B chromosomes
- Facultative heterochromatin
- Varies in its state of condensation in different cell types and developmental stages
- Associated with the switching off of genes
- E.g. Barr body
What are the characteristics of b chromosomes
- Differ morphologically from As
- Display non – Mendelian Inheritance
- No Nucleolus Organizers
- Mitotic and meiotic behaviour
- In high numbers – depress fertility and reduce growth
- Carry no genes with major effect
In many animal and plant species
Describe the structure of B chromosomes
• Smaller than A’s
• Heterochromatic
• Present in excess of the normal 2n chromosome number
• Vary in numbers between cells and tissues
• More tolerant of deletions and other structural
changes
• Do not pair with A chromosomes in Meiosis
• E.g. Allium schoenoprasum (Chives) on next slide
How are B chromosomes transmitted
• Accumulation of B’s from generation to generation
– In higher plants = post – meiotic
– In animals = before or during meiosis
• Search for the cause led to discovery of non – disjunction
- Directednon– disjunction
- Preferential meiotic segregation in egg mother cells
- Preferential fertilization by B- carrying male gametes
When does X inactivation occur
• Early stages of development
• Initiated as cells begin to differentiate - Late blastula stage
• X chromosomes selected for inactivation
– random
• In individuals with an X:autosome translocation
–normal X chromosome is consistently inactivated
• Once a progenitor cell in early embryo has committed to inactivating Xp or Xm chromosome. The inactivation pattern shows clonal inheritance
Describe the mechanism of X inactivation
• The X inactivation center (XIC)
• Controls the initiation and propagation of X-inactivation
• XIC encodes a large noncoding RNA, XIST (X-inactivation-specific transcript)
• XIST is essential for initiating inactivation, NOT required for maintaining the inactivation state
• Another extraordinary gene TSIX
• TSIX has a transcription unit overlapping all of the XIST gene, but on the antisense
strand
• This partner gene is expressed in undifferentiated embryonic stem cells and early embryos
• Tsix deleted from mouse embryogenic stem cells – deletion blocked X-X pairing and resulted in chaotic inactivation – 0,1,2, Barr bodies.
• “Counting” mechanism that designates one X to be inactivated
• Maternal and paternal X chromosomes must 1st pair briefly and align the Xic loci as a mechanism for counting the number of X chromosomes prior to X inactivation.
- XIST shows monoallelic expression
- Uniquely expressed from the inactive X chromosome
- Primary transcript undergoes modification with methylated cap, splicing & polyadenylation to generate a 17kb mature noncoding RNA
- Primary signal for spreading the transcriptionally inactive signal along X chromosome
- Cis-limited spreading of this RNA product acts to coat the inactivated X chromosome over very long distances
- Xist recruits the polycomb repressor complex 2 (PRC2) to the inactivated X, which mediates chromosome wide trimethylation of lysine 27 on histone 3 of nucleosomes - Leading to chromatin condensation and transcriptional silencing.
