genetics S2 Y1 Flashcards
What is cytogenetics?
Study of chromosomes in health and disease
What is chromatin?
DNA compacted by forming complexes with histones
- What is euchromatin?
- How is it compacted?
- Loosely compacted but dynamic chromatin
- H1 proteins - can dissociate to loosen so RNA pol. can bind
- What is heterochromatin?
- What compacts it?
- Permanently tightly compacted chromatin
- Condenser proteins (recruited in cascade-like mechanism)
What separates euchromatin and heterochromatin?
Barrier proteins - stop condenser proteins compacting euchromatin
Why can barrier proteins sometimes be lost/moved?
Translocation of barrier element that codes for it can be moved
- What does structural heterochromatin contain?
- Where is it?
- Satellite DNA (repetitive DNA sequences)
- Centromeres and telomeres
What are telomeres and what are they for?
Short tandem repeats with a G-rich (overhanging) strand and a C-rich strand that protect ends of chromosomes fusing by capping the ends
- Role of G-rich strand?
- What promotes this?
- Acts as a longer G tail that loops over, displaces some the double stranded parts to create a T-loop
- Telosome-shelterin complex
- 6 steps of cytogenetics?
- Why fixed in metaphase?
- Chromosomes treated with colcemid (arrests cells in metaphase - prevents spindle formation)
- Harvested
- Hypotonic treatment (moves chromosomes to periphery of cell)
- Fixation
- Metaphase spreading
- DNA staining
- Chromosomes treated with colcemid (arrests cells in metaphase - prevents spindle formation)
- So the chromosomes are visible
Why do chromosomes in prometaphase provide more detail than those in metaphase?
Less compact
How are positions on chromosomes defined?
Coordinate system:
- Short p arm, long q arm
- Sub regions via numbers e.g. p22.1
Molecular cytogenetics:
- What is it for?
- Mechanism?
- Higher resolution analysis
- Specific, chemically-synthesised (hybridised) oligonucleotide probe for DNA sequence is labelled with a fluorophore and binds to heat treated chromosome
7 steps of fluorescence in situ hybridisation?
- Colcemid applied (chromosomes fixed in metaphase)
- Cells transferred into hypotonic solution
- Resuspended in methanol/glacial acetic acid fixative
- Cells placed on slide and fixed using formaldehyde
- Heat denatures chromosomal DNA
- Fluorescently labelled probes are hybridised to complimentary sequence
- Counterstain with DNA-binding dye (DAPI - blue)
How does chromosome painting work?
Many probes used to bind along length of chromosome
- What is spectral karotyping?
- How can it identify cancer?
- Entire set of chromosomes analysed by different coloured probes binding to different chromosomes
- Colours of cancerous chromosomes will be different
Sanger sequencing:
- How are DNA clones generated?
- What then happens to clones?
- How is the nucleotide sequence determined?
- Standard PCR reaction
- Polymerase-mediated synthesis step
- Random termination at each nucleotide using dideoxynucleotides/ddNTPs (OH on deoxyribonucleotide replaced with H) that stop any binding to create hundreds of fragments of varying sizes - pieced together to form sequence
3 limitations of Sanger sequencing?
- Necessity of having a clone of the DNA template (to create adequate levels of fluorescence for detection)
- Must have at least some sequence information beforehand
- Short sequencing read length
2 steps of Sanger sequencing?
- Constructing initial framework (contig)
- Sequencing and final assembly of genome
Step 1 of Sanger sequencing:
- What happens to chromosomal DNA?
- How are clones mapped?
- Fragmented into large fragments that are cloned into vectors called YACs
- In terms of original chromosomal location using FISH-type experiments and PCR-based screening for STS (sequence tagged sites)
Step 1 of Sanger sequencing:
- How does a FISH (fluorescence in situ hybridisation) -type experiment work?
- What are STSs and why are they useful?
- What is formed?
- DNA used as a fluorescent probe and matched to region on chromosome
- Sites that have already been sequenced previously - can have primers designed, if one clone gives +PCR result then it will be matched to chromosomal region
- A clone contig - series of overlapping clones that have been mapped for chromosomal location
Step 2 of sanger sequencing:
- Steps? (4)
- Why must the fragments be reordered?
- Random fragmentation - ligated into vectors - make clones of each fragment in bacteria - sanger sequence the clones
- Many overlapping fragments of a single clone are joined to vector molecules, these are then cloned in bacteria and sequenced, then re-constructed using homology
Whole genome shotgun sequencing:
- Who?
- Why was it better than Sanger?
- Celera genomics
- Rapidly reordered fragments (faster)
What is annotation?
Identification of genes present
- What indicated protein-coding genes initially?
- Issue with this?
- An open reading frame with no stop codon in the sequence
- Genes are made up of introns and exons –> introns have stop codons so there are only mini open reading frames
What can inter-species sequence comparison be used for?
If an ORF represents a true protein coding gene
What are mini ORFs associated with?
Evolutionary conserved regions
How are ORFs proven to be representative of protein coding genes?
Using expressed sequence tags (ESTs)
- reverse transcription allows cDNA formation for RNA –> cDNA library cloned –> put into cloning vector to form EST library
What are non-highly conserved regions?
Regions with no protein-coding unit but encode non-coding RNAs
Long non-coding RNAs:
- Main role?
- Conserved?
- Why do they have higher flexibility to sequence changes than protein coding regions?
- Regulating gene transcription by interacting with DNA
- Poorly
- Unlikely to have a negative effect on organism
MicroRNAs:
- How do they regulate translation?
- Conserved?
- Bind to 3’ UTR of mRNAs
- Highly
Transposable elements:
- What are they?
- What can they do?
- Main role?
- Highly repetitive transposon-based genes
- Change position and multiply
- Promote genome evolution by regulating genome complexity
What are DNA transposons?
Sequence is ‘cut and pasted’ from one region to another
What are retro-transposons?
RNA copy inserted (creates 2 copies in genome)
Long interspersed nuclear elements (LINEs):
- What are they?
- Why are they autonomous?
- Type of retro-transposon
- Mini-systems encode enzymes for insertion (e.g. LINE-1 has RNA copy encoding reverse transcriptase and endonuclease to aid with insertion)
How can the LINE-1 gene create a hybrid gene after insertion of the corresponding hybrid cDNA?
LINE-1 in introns has a polyA tail to signal end of transcription but if this messes up, transcription will occur up to the following polyA tail so there will be an exon fused after transcription
What are long terminal repeats (LTRs) associated with?
Endogenous retrovirus (ERV) sequences that derive from non-infectious retroviral sequences with maintained transposon activity
How do short interspersed nuclear elements (SINEs) reinsert themselves?
Hijack LINE-1 mechanisms
Mitochondrial genome:
- Shape?
-Number of genes?
- Circular
- 37
What are siRNAs for?
Regulate gene expression and have a viral defence mechanism
siRNA pathway?
Machinery generates small double-stranded RNAs from viral genomes and then uses them to destroy viral mRNAs
6 steps of detection and degradation of viral mRNA?
- Dicer enzymes produce siRNA from viral dsRNA
- siRNA taken uo by RISC protein complex
- One of the siRNA strands is degraded and the other becomes a guiding strand
- mRNA match found by base pairing
- RISC complex detects match
- RISC complex degrades viral mRNA
Why do small siRNAs need to be in a very precise molecular configuration?
RISC complex is very specific
Why is RNAase not perfectly aligned?
Cuts siRNA so there is an overhang of 2 nucleotides between the two strands - RISC complex only accepts this form of siRNA
Purpose of siRNA guiding strand?
Finds viral RNA
RISC complex:
- What is argonuate?
- Role of N-terminal?
- Role of PIWI protein?
- How is guiding strand locked into RISC complex?
- Major component of RISC complex
- Separates strands
- Breaks down viral mRNA
- 3’ bound to PA2 and 5’ bound to MID
What do microRNAs encode?
MicroRNA-precursor RNAs that are then processed to be shorter by cellular enzymes
Role of DROSHA complex?
Processes pri-miRNA to produce pre-miRNA with an overhang
What happens to pre-miRNA?
Undergoes dicer-mediated cleavage to produce mature miRNA to be recognised by RISC complex
What is the complimentary region between 3’ end of mRNA and 5’ end of miRNA called?
Seed
When is translation inhibited?
If miRNA is partially complimentary to the mRNA strand (if it is a perfect match mRNA degradation occurs)
PIWI-interacting RNAs (piRNAs):
- Role?
- What are they complimentary to?
- Control/suppress bursts of retrotransposon activity by piRNA precursor being processed to form PIWI protein that fragments LINE-1 RNA to piRNA (silencing transposon)
- LINE-1 gene for transposon
How is a gene silenced?
Recognition of LINE-1 RNA by PIWI - DNMT enzyme recruited and methylates DNA (turns off gene transcription)
What biological function can non-coding DNA sequences have?
Transcriptional regulators (promoters, enhancers, silencers, insulators)
How does transcriptional control control lineage differentiation?
Ensures the correct expression of specific genes
Why are somatic cells different to one another in different areas of the body even though they have the same genome?
Specialised by regulated expression of genes
Genes transcribed by the 3 types of RNA polymerase?
- RNA pol. I - 5.8S, 18S, 28S, rRNA genes
- RNA pol. II - all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, most snRNA genes
- RNA pol. III - tRNA genes, 5S RNA genes, some snRNA genes, genes for other small RNAs
3 steps of transcription using RNA pol. II?
- Initiation - recruited to target genes along with general transcription factors and regulatory proteins in a complex called RNA pol. II holoenzyme
- Elongation - RNA pol. II moves stepwise along DNA, unwinds double helix at its active site, complimentary nucleotides are added in a sequential manner using anti-sense DNA strand as a template
- Termination - RNA pol. II stops at end of gene and is released from DNA strand
Promoters:
- What are they?
- Where are they?
- What do they contain?
- DNA sequences that define position where transcription of a gene by RNA pol. II begins
- Upstream of target gene
- DNA sequence motifs bound by GTFs and RNA pol. II in stepwise manner
What do GTFs position?
RNA pol. II to initiate transcription
What are enhancers and silencers needed for?
High levels of accurate transcription and modulation of the rate of promoter transcription
Where are enhancers and silencers?
On the same genes they regulate (cis-regulatory sequences) upstream or downstream of target genes and in introns or exons
- What do transcription factor proteins do?
- What do they also contact with?
- Read sequence of cis regulatory DNA to bind to specific motifs - DNA bound TFs interact with GTFs and RNA pol. II assembled at the promoter
- Intermediary proteins called transcriptional coactivators and corepressors
Role of enhancers?
Promote transcription from the gene promoter by speeding up the rate of RNA pol. II-GTF complex assembly
Role of silencers?
Slows transcription by blocking RNA pol. II-GTF assembly
Insulators:
- Role?
- Where?
- Specific type with essential role?
- Prevent innaporpriate regulation of adjacent genes and control genes/set of genes an enhancer can regulate
- Between enhancer and promoter (blocks enhancer action)
- Act as barrier elements
What does combined activity of multiple cis-regulatory elements generate?
The correct spatial and temporal gene expression patterns
What was the ENCODE project?
Mapped regulatory elements genome-wide using many experimental techniques
Two types of non-coding RNAs that are extensively transcribed but not into proteins?
Housekeeping ncRNAs
Regulatory ncRNAs
Long non-coding RNAs:
- Length?
- 2 ends?
- Difference from mRNA?
- Where?
- > 200 nucleotides
- PolyA tail, 5’ 7-methylguanosine cap
- Fewer exons, shorter transcript length, more tissue restricted expression
- Nucleus or cytoplasm
What are genes made up of?
Interlinked protein and non-coding transcripts
What happens when lncRNAs are coexpressed with protein coding genes?
Have a local transcriptional regulation role
What do most transcriptional enhancers generate?
A lncRNA
What have a new subset of lncRNAs formed?
New class of gene expression regulators that act either directly in the nucleus to regulate transcription OR indirectly in the cytoplasm to post-transcriptionally affect gene expression