Genetics Flashcards
5 features of genetic information that have to be true for it to be able to work?
Stable for storage over time
Be able to faithfully replicated (semi conservative replication)
Be able to control expression of traits ( encode the sequence of proteins)
Be able to change in a controlled way
But also available for direct cellular processes
DNA has the structure of? Therefore leading to the creation of?
Double helix
Minor grooves and major grooves
What is a nucleotide?
Polymer made up of the repeating units of a sugar a base and a phosphate
Phosphate binds to sugar, which binds to base
Sugar + base =?
Nucleoside
Deoxyribose + adenine =?
Deoxyadenosine
Deoxyribose + guanine?
guanosine
Deoxyribose + cytosine?
cytidine
Deoxyribose + thymine?
thymidine
How do you know which end is the 5’ end and which is the 3’ end?
The 5’ end which have carbon 5 (the chimney) bonded to a phosphate group
The end will have an OH group on carbon 3
What is the directionality of a DNA strand?
5’ to 3’
DNA strands are anti parallel in a double stranded structure, what does this mean?
They are going in opposite directions (still going 5’ to 3’)
What holds DNA strands together?
Hydrogen bonds between complementary base pairs
Adenine to thymine
Cytosine to guanine
What’s a 2 carbon ring base called and what are the examples?
Purine
Adenine and Guanine
What’s a single carbon ring called, and what are the examples?
Pyrimidine
Cytosine and thymine
A purine will always bind to a?
Pyrimidine
How many hydrogen bonds between Adenine and thymine?
2
How many hydrogen bonds between Guanine and cytosine?
3
Why is the minor and major groove created?
Base pairs are planar
Stack?
Hydrophobic interactions
What type of double helix is DNA?
Right handed, (put left thumb up, direction of fingers shows the right hand direction)
Genetic information is carried in?
The sequence of bases in DNA = digital code
How does the structure of DNA provide a mechanism for heredity?
You can control the expression of traits (encode the sequence of proteins)
Be able to change in a controlled way
What is the genome?
All the DNA in the nucleus of a cell (ie. the complete set of genes of an organsism)
What’s special about mitochondria and chloroplasts in terms of DNA?
They have their own small circular genomes
Nuclear genetic material is distributed among discrete units called?
Chromosomes
How are nucleosomes formed?
DNA wraps twice around 8 core histone proteins to form nucleosomes
How are chromatin fibres formed?
Histon H1 brings the nucleosomes together
How is chromatin fibre folded into loops (part of the chromosome)?
Chromatin fibres are further condensed by scaffold proteins
What is the net result of creating chromatin fibre which then forms the chromosome?
Each DNA molecule has been packaged into a mitotic chromosome that is 10,000 fold shorter than it’s fully extended length
2 types of chromatin?
euchromatin
heterochromatin
Features of euchromatin?
Less compact DNA form
Contains genes that are frequently expressed
Features of heterochromatin?
More compact
Contains DNA that is not transcribed
What are nucleoli?
Regions where chromosomes carrying rRNA cluster, rRNAs are synthesised and ribsomes are assembled
Chromosomes organise themselves into discrete patches called?
Chromosome territories
Where are active genes normally located?
In the euchromatic region of the chromosome, and near the chromosomes territory boundary
What does transcription do very basically?
Prodcues an RNA molecule that is complementary to one strand of DNA
DIfferences in RNA compared to DNA?
RNA has the sugar ribose instead of deoxyribose
Has the base uracil instead of thymine
What do differences in RNA mean?
RNA is More chemically reactive than DNA as RIbose has 2’ OH group (so less stable so only used for short term storage of information, whereas DNA is long term)
RNA is more prone to mutate than DNA, as cytosine deamination to uracil can’t be detected and repaired in RNA
RNA is single stranded and not double, meaning there is no backup of genetic information (but it can still base pair with itself)
What enzyme helps synthesise RNA and describe its structure?
RNA polymerase, Has area where the short region of DNA has been separated, and a ribonucleoside triphosphate uptake channel allows the ribonucleosides to come in
What direction is RNA synthesised?
5’ to 3’ direction (so DNA will be read 3’ to 5’)
How is energy provided for the formation of a phosphodiester bond when synthesising RNA?
Breakage of phosphoanhydride bond of NTP (the phosphate chain in the nucleoside triphosphate, 2 are lost and the other binds to the sugar backbone with the base attached)
What does mRNA do?
Messenger RNA
Code for proteins
What does rRNA do?
Ribosomal RNA
Forms the basic structure of the ribosome and catalyze protein synthesis
What do tRNA’s do?
Transfer RNA, central to protein synthesis as are adaptors between mRNA and amino acids
What is the transcriptome?
Set of all RNA molecules
Varies on the cell (different genomes)
What is the central dogma?
Information flow from DNA to RNA via transcription then from RNA to Protein via translation
How is mRNA processed before it leaves the nucleus?
The 5’ end of the mRNA gets “capped” with an atypical nucleotide
The 3’ end of the mRNA gets a tail of poly-A nucleotides
Introns (non coding sequences) have to be removed from the mRNA
One way in which a bacterial gene is different to a eukaryotic gene?
In a bacterial gene all the gene is a protein coding region (exon)
In a eukaryotic gene there are protein coding regions (exons), but also non-coding regions (introns)
Describe briefly the splicing process that occurs in pre mRNA to remove introns?
3’ clevage and addition of a poly(A) tail
Intron excision occurs by snRNA’s that bind to proteins to form snRNP’s, which form the core of the splicesosome
The splicesosome causes the intron to bend and one side which break and bind to itself forming a intron lariat
Exon ligation then occurs to create the mRNA reading for translation
What can alternative splicing lead to?
More than one gene being expressed from one gene
Describe mRNA’s keaving the nucleus?
RNA binding proteins mark a mature mRNA for export from the nucleus
Leaves through nuclear pore
What determines how much protein is translated by a gene relating to the mRNA?
The stability of the mRNA, as it’s being degraded by the cell
So more stable it is the more times it can be translated
How many types of amino acids?
20
How do we know that genetic code isn’t read overlapping?
Single base mutations only affect one amino acid
What is proflavin?
Planar molecule that intercalates between base pairs in DNA
Causes frameshift mutations (insertion of an extra base, or deletion of a base)
What is the genetic code of mRNA read in?
Sets of 3 nucleotides called codons
Some amino acids have more than one tRNA, meaning they are?
degenerate
The genetic code is universal, meaning?
It’s found in all organisms, however there are variations in mitochondria
Very bsaically what do mRNA, tRNA and rRNA do in translation?
mRNA carries the genetic information
tRNA deciphers the codons of the mRNA
rRNA makes up the ribosome
Structure of tRNA?
Anticodon loop which binds to mRNA, (bases will be opposites)
Acceptor stem which binds to correct amino acid, via a high energy ester bond. This is initiated by specific enzymes
Structure of rRNA?
Made up of a large subunit and a small subunit
4 RNA molecules and lots of protein molecules
Has 3 binding sites
A-site = aminoacyl-tRNA site
P-site = peptidyl-tRNA site
E-Site = exit site
What are the 3 steps in translation called?
Initiation
Elongation
Termination
What are the steps of initiation?
Translation initiates at the start codon (AUG)
The start codon is bound by the initiator tRNA (will always be UAC, which is methinonine
Binding of the initiator tRNA to the small subunit allows the large subunit to bind and the ribosome to assemble
P-site is filled with initiator tRNA but the A-site is empty
The tRNA with an anticodon that corresponds to the codon in the A-site binds
The amino acid on the tRNA in the P-site is transferred onto the amino acid on the tRNA in the A-site
Steps of elongation?
The now empty tRNA moves to the exit site and is released, so now there is a peptidyl tRNA in the A site
The ribosome slides down the mRNA so that the tRNA with the growing peptide is in the P-site, leaving the A site free to bind to another tRNA
The growing peptide chain is transferred to the aminoacyl tRNA, converting it to a peptidyl tRNA
This cycle continues
Describe the termination step?
When the stop codon is reached, translation will stop
There are no tRNAs with anticodons that bind to Stop codons. Instead stop codons are bound by termination factors
Translation ends with the mRNA and ribosomal subunits diassembling and the release of the newly synthesised protein
What direction is polypeptide synthesis?
N to C (amino-terminal to carboxy terminal)
Why is the ribosome a ribozyme?
It catalyses the formation of a peptide bond
What are telomores?
Highly repetitive DNA that allows the ends of chromosomes to be replicated, also protects the ends of chromosome being mistaken as broken
What are centromers?
Repetitive DNA which forms the spindle attachment site in mitosis
WHat is the origin of replication?
Special sequence where duplication of the DNA begins, each chromosome will have many origins
What occurs in the S phase?
DNA replication
What occurs in the M phase?
Mitosis
WHat do you need for DNA synthesis?
Enzyme DNA polymerase
DNTP’s
Single stranded template DNA
Primer 3’-OH (allows replication to start)
Results in DNA synthesis 5’ to 3’ direction
What’s more accurrate DNA polymerase or RNA polmerase?
DNA polymerase
2 terms of DNA polymerase uses to be accurate?
Accurate polymerization 5’ to 3’
Accurate exonucleolytic proofreading 3’ to 5’
What direction is DNA replication?
Bidriectional- moves away from the origins of replication
Do genome sizes vary?
Yes largely
Features of a bacterial genome?
Small compact and usually circular
Features of genetic material?
Single circular double stranded DNA molecule which is a chromosome or nucleoid
No histone proteins
Associated with Mg2+ and polyamines
May also contains smallers circular DNA’s called plasmids
What happens at the replication fork?
Both strands are copied
In a 5’ to 3’ direction
(one continuous (leading strand) and one discontinues (lagging strand))
As DNA replication can only go 5’ to 3’ direction how is it replicated in the other direction (in the lagging strand)?
Discontinous
Primase synthesises short RNA Primers copied from DNA
DNA polymerase elongates RNA primers with new DNA to from okazaki fragments
Nucleases remove RNA at 5’ end of neighbouring fragment and DNA polymerase fills the gap
DNA ligase connects adjacent Okazaki fragments
What do helicase enzymes do?
Unwinds double stranded DNA, using ATP
What do single-stranded binding proteins do?
Prevent base pairing until DNA polymerase arrives
What is primase?
A specialised RNA polymerase
Why is DNA replication fast?
DNA polymerase is binded to a sliding clamp which encircles the double stranded DNA
Sliding clamp is loaded around DNA by a clamp loader
What is the replisome?
large protein complex that carries out DNA replication
In eukaryotes, what do histone chaperones do?
Load histones on to newly synthesised DNA
What do topoisomerases do?
Helicase unwinding of DNA causes supercoiling (“twists”) ahead of the replication fork, which need to be unwound
So they unwind these twists
What does mismatch repair protein MutS do?
Detects incorrect base pairing in newly synthesised DNA
What is associated with mutations in mismatch repair proteins?
Predisposition to cancers
Overall what contributes to very low error rate in DNA synthesis?
5’ - 3’ polymerization errors = 1 in 10^5
3’ - 5’ exonucleolytic proofreading 1 in 10^2
Strand-directed mismatch repair 1 in 10^2
Combined 1 in 10^9
What do you require for PCR?
Taq DNA polymerase dNTPs Template DNA Primer Buffer (Mg2+)
Steps of PCR?
High temperature 95 degrees -Denaturation
Double DNA strand melts open as hydrogen bonds break
Low temperature 45 degrees - annealing
Primase binds to DNA polymerase attaches and starts copying DNA
Intermediate temperature - Extension
Optimum temperature for polymerase and extension of fragment
What part of DNA is amplified during PCR?
Only DNA between primers
What are primers?
Short roughly 20 bp long single stranded DNA, marker for DNA polymerase to bind on to
Chemically synthesised
In gel electrophoresis of DNA moving to the positive electrode, what factors affect it’s movement?
Shape and size
Smaller fragments move faster
What are the limitations of PCR?
Sequence inforamtion is required to design 2 primers
Limit on length of amplified fragment
Very sensitive to reaction conditions
Tiny amounts of contaminating DNA will also be amplified
What’s a gene libary?
Collection of recombinant clones
Can screen for clones containing gene of interest
Describe transgenics?
Genes between species
Genetic code is universal, so expression is possible
In eukaryotes what are the 3 enzymes that transcribe different sets of genes?
RNA polymerase I
RNA polymerase II
RNA polymerase III
What is required for RNA polymerase to bind to the RNA strand?
A transciption factor binded to the control region
What are Cis-acting regulatory regions?
DNA sequences recognised by proteins
What are the 2 types of Cis-acting regulatory genes and features of them?
The promoter:
Very close to the protein coding region, and includes the initiation begins and a “TATA” box ( sequence that allows transcription factors to bind and therefore RNA polymerase II)
Basal factors including RNA pol II bind here
The enhancer (Upstream activating sequences)
Regulatory sites that can be distant from the promoter
Activators and repressors bind here
What are trans-acting proteins?
Transcription factors, bind to the promoter and enhancer to control transcription of the gene
Describe the process of RNA polymerase binding to the RNA strand?
TBP binds to the TATA box
TBP recruits TFIID complex and TFIIB to the promoter
RNA pol II and further transcription factors bind to the promoter to give the basal transcription complex
Describe the gal4 system in yeast?
Gal4 is bound to enhancer region, but Gal80 is also bound to it, meaning there is no transcription of Gal1
However if galactose is present it binds to Gal80, removing it from Gal4 so RNA pol II can bind and Gal1 can be transcribed
How can chromatins stop transcription?
By condensing
Describe the 4 ways in which DNA can be made accessible to transcription factors (uncondensed)?
Chromatin remodelling complex binds:
1. Remodelling the nucleosomes
- Histone chaperone removes histones
- Histone chaperone replaces histones with other histones
Histone modifying enzyme binds:
- Specific pattern of histone modification
Where can core Histones be genetically modified by covalent addition of functional groups?
On their N-terminal tails
Differences in bacterial gene expression compared to eukaryotes?
No nuclear membrane
One cytoplasmic compartment
No histones
Coupled transcription and translation
Features of an operon in bacteria?
Has one promoter
All genes in an operon are transcribed together
The mRNA is translated to give separate proteins
Describe the lac operon in bacteria?
Has a promoter region, lacZ, lacY and lacA which make mRNA which then makes B-galactosidase, B-galactoside permease, B-galactoside transacetylase
Which results in lactose metabolism
When no lactose present, there is no transcription as lac repressor is bound
When lactose is present, it binds to lac repressor removing it so proteins are created
What are polycistronic transcripts?
Transcripts that able to produce multiple proteins from one mRNA transcript
How can single pre-mRNAs produce mutiple products?
Alternative splicing
How is alternativ splicing regulated?
By activators and repressors
What about mRNA’s determines how much protein is translated in the cell?
The stability of the mRNA
What does injecting double strands of RNA do?
Reduces the expression of specific genes, by reducing the levels of mRNA
It does the by the clevage of target RNA
Translational repression and eventual destruction of target RNA
Formation of heterochromatin on DNA from which target RNA is being transcribed
How are eukaryotic genomes arranged as?
Linear chromosomes
What are transposons (jumping genes)?
Can replicate and insert into other parts of the genome
Some transpose via mRNA
Transposons 2 categories?
DNA transposons (cut and paste) Retrotransposons (Copy and paste, using RNA polymerase, and reverse transcriptase)
What are LINEs?
Long interspersed nuclear elements
What are SINEs?
Short interspersed nuclear elements
Most mobile elements in bacteria are?
DNA transposons
Most mobile elements in eukaryotes are?
Retrotransposons
Process of Retrotransposons transpose?
Transcription of retrotransposon to form mRNA
Which is translated to form a reverse transcriptase
Then there is reverse transcription of RNA to DNA via the reverse transcriptase
Synthesis of second double DNA strand
Insertion of retrotransposon make into genome
Features of Eukaryotic genomes in relation to their variability?
Highly variable in size
Much of the variability is attributable to repeated sequences and other non coding DNA
Transposons can insert copies elsewhere in the genome
Retrotransposons transpose via RNA
Features of Sanger sequencing, Next generation sequencing (NGS), and single molecule sequencing
Sanger sequencing - Accurate but low throughput
NGS - Extremely high throughput, but difficult to assemble repeated sequences
Single molecule sequencing - Error rate, high cost, long sequences can be done
(nanopore can be used in field)
What are polymorphisms?
2 or more alleles of a gene, common in a population
caused by different base order
Why does most genetic variation genetic variation have no phenotypic effect?
Very small amount of genome codes for proteins
What do wild type genes produce?
Functional proteins
What do mutant genes produce and what does this infer?
Often non functional proteins, therefore in diploids most mutations are recessive
Also means mutations are lethal in haploid or hynozygous diploid
Some mis-sense mutations are lethal
Example of a conditional lethal mutation?
Temperature sensitive
As protein only functional at certain temperatures
Features of mitosis?
Somatic cells
Produces genetically identical daughter cells
2n = 2n + 2n
Happens to all the chromosomes not just one
Features of meiosis?
Haploid gamete production
2n = n x 4
Genetically different daughter cells
Happens to call chromosomes not just one
Where does homologous recombination occur?
Prophase of meiosis 1
Homologous chromosomes pair
Crossing over occurs
What are animals sterile sometimes?
Don’t have homologous chromosomes
What occurs in recombination?
Start with 2 double strands of DNA which are homologous (1 from mother, 1 from farther)
Double stranded break occurs
MRX performs 5’ to 3’ resection (removes some of strand)
Dmcl and Rad51 strand invasion causes 2 holiday junctions
Which can then be cleaved to produce non cross over products or cross over products
What can homologous recombination cause In meiosis?
Generates cross overs- creates holliday junctions which are then broken in a certain way to create crossing over potentially
Effects of base substitutions in coding regions?
Sileent - Base changed will still code for the same amino acid
Neutral - amino acid that is now coded for is very similar to the original - protein not affected
Mis-sense - new amino acid coded for does affect the overall protein created
Nonsense - if the base change causes a stop codon to form - protein will be incomplete
What’s a frameshift mutation?
When you insert or delete a base and codons are read completely differently
What are rare alleles termed as and what are normal alleles termed as?
Mutations
Wild type
2 common single gene mutations?
Sickle cell amenia
Cystic fibrosis
4 mechanisms cells have to prevent mutation?
Proofreading DNA polymerase
Postreplication mismatch repair
DNA repair by homologous recombination
Cell cycle checkpoints
2 environmental factors that can cause mutations?
Radiation
Chemical mutagens
2 types of mutations and which is more common?
Transition - Purine is replaced by a purine, or pyrimidine is replaced by a pyrimidine
Transversion - Purine is replaced by pyrimidine or visa versa
Transistions are more common as transversions are normally detected as there is a larer change in shape
3 examples of chemical mutagens?
Intercalating agents: (planar molecules)
Insert between base pairs resulting in frameshift mutations
Base analogues
Incorporated into DNA in place of normal base ( as chemically similar), mis-pair as they hydogen bond to different things so synthesised strand will be different, resulting in a base substitution
Base modifying agents
Covalently alter a base causing it to mispair
Resulting in a base substitution
Features of when homologous recombination repairing double strand breaks?
Break is accurately repaired
Universal process
Non-reciprocal
Local loss of heterozygosity (A to a)
Features of homologous recombination generating cross overs?
In eukaryotes occurs in meiosis
Breakage and joining of DNA
Reciprocal
Genetic rearrangement
Holliday junctions are generated (from D loops)
Cut horizontally = chromosomes without crossover
Cut vertically = chromosomes with crossover
