Gene Expression & DNA, Chromosomal Mutations and Molecular techniques

Question 1

What is meant by ‘central dogma’?
Why is it important for i) eukaryotes
ii) prokaryotes ?

Answer 1

• it is the process by which the instructions in DNA are converted into functional product i.e the process of transcription (DNA -> RNA* the message), then translation (RNA -> protein).
• eukaryotes= allows for different cell types during development
• prokaryotes= adapting to the environment

Question 2

Outline the process of transcription.

include names of enzymes & where it takes place

Answer 2

• happens in the nucleus
• transcription factors (TFs) are needed for promoters to bind. TFs bind to the TATA box (region on eukaryotic DNA). Only then can RNA polymerase be recruited.
• the DNA helix is unzipped by DNA helicase (by breaking H bonds) to expose the bases that will act as the template (template strand). Transcription starts from 5’ to 3’.
• free mRNA nucleotides align with their complimentary base pairs opposite the template strand.
• the coding strand (non-template, not transcribed, just gives code for RNA, will have same base sequence to RNA, goes 5’ to 3’ same as mRNA being made). The template strand i.e the non-coding, transcribed strand, goes in the opposite direction (3’ to 5’).
• RNA polymerase bonds the RNA nucleotides to form an mRNA polymer chain.
• transcription is terminated, resulting in a pre-mRNA molecule.

Question 3

What post transcriptional modifications take place? Where does this happen? What are they useful for?

Answer 3

• the pre-mRNA leaves the nucleus via nuclear pores & enters the cytoplasm where its introns (non-coding) are spliced and exons bought together by spliceosomes & a protective 5’ UTR cap is added to prevent degradation of mRNA by enzymes in the cytoplasm. A 3’ poly A tail is added (several adenine RNA nucleotides) for protection & regulation.
• matured mRNA will contain an ORF (open reading frame) and 5’ & 3’ UTRs (untranslated regions).

Question 4

Is one gene = one polypeptide?

If not, what is a gene?

Answer 4

• NO, not all genes are expressed as polypeptides, because not all RNA molecules are translated into polypeptides.
• a gene is a functional unit. It can be either a sequence of amino acids in a polypeptide OR a sequence of nucleotides in an untranslated RNA.

Question 5

Give the differences between RNA and DNA.

List the different types of RNA & their properties

Answer 5

• RNA has a -OH group on its C2, DNA has a -H
• RNA has a Uracil instead of Thymine
• RNA is single stranded
• RNA often forms stemloops; complimentary, anti parallel strands w themselves that form a mini helix
• RNA can form complex 3D structures, often w non-standard bases.
  1) rRNA (ribosomal)= more than 80% of all RNA, few kinds but many copies, highly conserved, eukaryotes have 2 small and 2 large, prokaryotes have 1 small and 2 large.
  2) tRNA (transfer)= more than 15% of total RNA, more than 100 kinds, small, clover leaf shape, each is dedicated to 1 of 20 aa’s, specific anticodon sequence.
  3) mRNA (messenger)= less than 2.5% of all RNA, 100 thousands of types, few copies, act as transcripts for proteins.

Question 6

• What polymerase types act on what RNA types?
• Where & in what frequency are RNA polymerase found in eukaryotes & prokaryotes?
• Why is transcription said to be ubiquitous?

Answer 6

1) rRNA= RNA polymerase I
2) mRNA= RNA polymerase II
3) tRNA= RNA polymerase III
- eukaryotes= 3x nucleus, 2x chloroplasts, 1x mitochondria, complex w/ many subunits.
- prokaryotes= 1 copy; RNAP (bacterial RNA polymerase, core enzymes has 5 subunits, holoenzyme has 6 subunits (core + sigma)
- ubiquitous= present, appearing & happening everywhere simultaneously

Question 7

What is the structure of ribosomes in:
a) prokaryotes 
b) eukaryotes 
Give features of the genetic code.
What is the initiation codon & aa?

Answer 7

• prokaryotes= x3 rRNAs, 70S ribosome (lighter)
• eukaryotes= x4 rRNAs, 80S ribosomes (heavier)
• degenerate (different codons can code for the same amino acids), non overlapping, no gaps
• template 5’ to 3’ read through producing N to C polypeptide.
• AUG = Methionine (met)

Question 8

How can tRNA become charged? What is needed for translation to occur? think of the factors required

Answer 8

• when an amino acid binds to tRNA, the tRNA-amino acid complex is said to be charged & the complex can be described as an activated amino acid.
• for translation to happen, we need: mature mRNA, ribosomes, activated tRNAs w aa’s so we can begin to join the aa’s together, initiation factors, elongation factors, release factors & energy i.e ATP.

Question 9

Outline the process of translation.

Answer 9

• initiation= recognition of mRNA cap by tRNA-*Met.
• the mature mRNA attaches AUG codon to the ribosome at the P-site in the cytoplasm (A site is empty & waiting for next tRNA). The ribosome attaches at 3’ end of mRNA @ AUG.
• an activated tRNA with complimentary anticodon sequence to AUG aligns opposite the mRNA, binding to the A site(ACCEPTED), held in place by the ribosome.
• the ribosome translocates along the mRNA molecule from 5’ to 3’ to allow another complimentary tRNA to attach to the next codon on the mRNA.
• the two amino acids attached on the tRNAs will be joined via a peptide bond hydrolysed by peptidyl-transferase. The empty tRNA (now uncharged) leaves the ribosome via the E site.(EXITS)
• This elongation continues (helped by elongation factors) until the ribosome reaches a STOP codon at its A site. Release factors recognise this STOP.
• this causes the ribosome to detach via hydrolysis & the polypeptide has been completed.
• uncharged tRNA is now in P site & the ribosome dissociates into subunits

Question 10

Define mutation & mutagenesis.

Give some exogenous & endogenous causes of mutations.

Answer 10

• mutation= a change in the sequence of nucleotides causing a change in the gene & chromosome. It’s also the process that produces the alteration (is the source of most alleles eg blue eyes are a mutation)
• mutagenesis= process of mutation generation
• exogenous= radiation, UV, carcinogens, mutagenic chemical agents, free radicals
• endogenous= DNA replication errors, free radicals, transposable elements (specific DNA sequences that ‘jump’ randomly from one part of gene to another)

Question 11

There are many types of DNA mutation. Give some examples of spontaneous mutations.
What types of effects can these have on the gene & its expression?

Answer 11

• spontaneous deamination (random hydrolysis of C into U), transposable elements; parts of nucleotide sequence jumps around gene.
• can have no effect (if it’s jumps away from gene), inactivate the gene, re-activate the gene or alter gene expression.
• may or may not cause a phenotypic change. Could be good, bad or neutral. May cause disease.
• drives genomic variation

Question 12

• What is the meaning of SNP? What are the different types?

- give 6 examples of single base/ nucleotide changes.

Answer 12

• SNP= single nucleotide polymorphism: anonymous=no known effect, non-coding=outside a gene, coding=inside a gene.
  1) transition=single base change to the same type of base eg C to T (pyrimidines) or A to G (purines)
  2) transversion= single base change to a different type of base eg A to C (purine to pyrimidine)
  3) missense=single base change that codes for a different amino acid so changes polypeptide coded for.
  4) frameshift=addition or deletion of a nucleotide base (not in x3) that causes the reading frame to shift, affecting every codon after the mutation.
  5) nonsense=mutation coding for an early STOP codon, shortens polypeptide
  6) silent=codes for same amino acid therefore has no effect on polypeptide

Question 13

There are also multi-nucleotide mutations. Give 2 examples

Answer 13

• deletion= deletion of multiple bases from the nucleotide sequence (in in x3s, doesn’t lead to frame shift, if not, frame shift will occur)
• insertion= insertion of multiple bases into the nucleotide sequence.

Question 14

What is fork slippage & what can it cause?

In which disease does this occur?

Answer 14

• it’s a mutation that occurs during DNA replication when the template & newly synthesised strand are mispaired.
• if the newly synthesised strand denatures from the template strand and is complimentary to other stretches of the template strand, it will pair w the wrong sequence leading to repeated sequences (happens due to insertion)
• if the newly synthesised strand loops out, it will be deleted from the sequence.
• Huntington’s disease is caused by CAG repeats.

Question 15

What types of chromosomal abnormalities are there?

What 2 things are important to consider when thinking about their physical characteristics?

Answer 15

1) balanced abnormalities= no genetic info lost & no phenotypic change apparent
2) unbalanced abnormalities= not balanced
- consider; numerical & structural abnormalities

Question 16

The average human will have 23 pairs of chromosomes. Outline some numerical abnormalities of chromosomes. Define mosaicism as one of these.

Answer 16

• polyploidy= gain of haploid set of chromosomes (we normally have 2n so would have 3n which is triploidy), most common cause is x2 sperm fertilising one egg, causes ~15% of miscarriages
• aneuploidy=loss or gain of whole chromosomes (monosomy=1 less, trisomy=1 more), caused by meiotic non disjunction (during anaphase, chromosomes are not pulled to correct poles of cell therefore unequal number in each granddaughter cell), eg Downs is Trisomy 21
• mosaicism=presence of 2 or more cell lines in an individual, can be throughout the body or tissue specific, caused by mitotic non-disjunction

Question 17

Outline some structural abnormalities a chromosome could have.

Answer 17

1) deletions= deletions of portions of genes on the chromosome
2) duplications=duplications of portions of genes on the chromosome
3) substitutions/insertions= addition of genetic material into the chromosome (another chromosome for substitution)
4) inversions= portion of chromosome from 1 flipped and re-inserted into same chromosome.
5) reciprocal translocations=material from one chromosome A is inserted into B and material from B inserted into A i.e swap
6) robertsonian translocation

Question 18

What is cytogenetic testing?

Why do we do it?

Answer 18

• cytogenetic analysis eg karyotyping
• fluorescent in situ hybridisation (FISH)
• microarray
• DNA sequencing
• allows accurate diagnosis and prognosis, better clinical management, assesses for future reproductive risks, prenatal diagnosis

Question 19

What is robertsonian translocation?
Give an explanation using the example of chromosomes 13 and 14 with a health carrier father & healthy mother.
Who is more at risk of carrying the robertsonian chromosome?

Answer 19

• where an individual has 45 chromosomes instead of 46.
• only occurs between the acrocentric chromosomes (13,14,15,21,22)
• 2 acrocentric chromosomes fuse together to form one large chromosome
• when having a child, a robertsonian carrier can have a healthy child- x1 gamete(normal 13+14 and normal 13+14 chromosomes from partner), healthy carrier child- x1 gamete (gives robertsonian ie 13+14 and partner gives 13+14 so health number but carries the chromosome), trisomy child-x2 gametes (robertsonian (13+14) w another 13 plus partner who brings 13+14 so child will have 3 copies of 13 and x2 of 14) or monosomy-x2 gametes (normal 13 and partner gives 13+14 but child lacks another 14).
• women are more at risk of carrying the chromosome

Question 20

What is DNA hybridisation?
What techniques use hybridisation?there are 3
Outline what the uses of & the steps involved in southern blotting.
Give 3 characteristics of probes used in southern blotting.

Answer 20

• dna hybridisation is where double stranded DNA is denatured (heat or high pH) to separate into 2 single strands. The strands are then annealed to probes that are complimentary and are renatured.
• FISH, microarray, southern blotting
• southern blotting=DNA fragments are transferred/blotted from gel to sheet (more solid), a radioactive or fluorescent probe is added w a comp sequence, the fragments are then imaged to locate the DNA fragments to which the probes bound.
• used to analyse/check constructs, to investigate large deletions or duplications, to investigate triplet repeats, nucleotide expansions eg CAG repeats in Huntington’s, to investigate variation eg dna fingerprinting
• probes don’t need to have 100% sequence similarity, don’t need to fully align w target sequence (can still bind w partial overlap) and they don’t affect the position of the target on gel sequence (only bind to target & help them to be located)

Question 21

What are microarray used for, what is it?

What does FISH stand for? What is it?

Answer 21

• allows changes in gene expression to be monitored eg cancer cells vs healthy, 1000s of genes can be analysed at same time, consist of single stranded oligonucleotides w sequences corresponding to coding regions of genome, can use fluorescence to distinguish DNA strands.
• FISH= fluorescent in situ hybridisation.
• labelling chromosomes with complimentary probes that have fluorescence. Allows detection of specific sequences, can dpfing deletions(probe would appear on one chromosome but not the other) or additions(extra probe appears on same chromosome), can view extra chromosomes ie trisomies,

Question 22

What is chromosome painting?

Answer 22

• hybridisation of fluorescently labelled chromosome specific probes. -They bind to the chromosome and allows you to view deletions/additions/translocations of the entire chromosome.
• also allows karyotyping of the entire chromosome set

Question 23

What is PCR? Outline the process.
How can we check PCR has been successful?
What are the uses of PCR?

Answer 23

• polymerase chain reaction, used to hugely amplify target DNA.
• heat DNA to 95C to denature into single strands, cool to 65C so primers can bind to complimentary sequences on single stranded DNA, raise temp to 72C so taq polymerase extends primers allowing synthesis of new DNA (DNA polymerase cannot work w RNA as its template), repeat.
• final PCR product will contain the forward & reverse primers.
• requires pair of primers (forward + reverse that span area to be copied)
• to check it’s worked; check for band of correct size using agarose gel electrophoresis, use positive & negative controls, sequence the PCR product to check for errors.
• useful for amplifying DNA, investigating single base mutations, investigating small deletions or insertions, investigating variation eg DNA profiling, NEW USE=site directed mutagenesis; can make planned mutations in copied DNA.

Question 24

Outline:

• why DNA sequencing is used
• the process of Sanger sequencing

Answer 24

• determines the exact sequence of bases (ATCG) in DNA. Can find location of mutations eg insertions, deletions. Normally used for smaller mutations since sequencing large sections of DNA is timely & costly.
• Sanger sequencing= target DNA has to be amplified first (via PCR), heat is used to denature the DNA to separate strands into a template strand & complimentary strand, a primer is annealed to the 5’ end of the template strand, this primed DNA is equally dispersed among 4 reaction vessels, DNA polymerase & all 4 dNTPs are added to each, a single different ddNTP is added to each vessel, dNTPs anneal and once the ddNTP binds, the sequence is terminated as it lacks a -OH at its 3’ C, DNA fragments of different lengths are formed, then gel electrophoresis, smaller migrated further towards pos charge.

Question 25

• outline what is needed for agarose gel electrophoresis.
• outline the process of gel electrophoresis
• when is this process used?

Answer 25

• 1)gel:a matrix that allows separation of DNA fragments. 2)buffer:allows charge on DNA samples across the gel. 3)power supply:generates charge difference. 4)stain:to identify the strands
• DNA is neg charged & will move towards anode (+) if in electric field, DNA fragments separated on basis of size or shape, DNA is visualised w ethidium bromide.
• AGE is often used in conjunction w DNA fragmentation using restriction endonucleases

Question 26

What does SDS-PAGE stand for?
What type of molecules is this process used for?
Outline the steps in this process.

Answer 26

• sodium dodecyl sulphate (coats protein, removes charge, adds - charge) polyacrylamide gel electrophoresis
• used to separate proteins based on their size
• proteins are charged molecules so will move toward anode or cathode in electrical field, separated on basis of charge, size or shape, requires:gel, buffer, power supply, stain to detect.