3.8 The control of the gene expression Flashcards
What is a gene mutation?
Change in the base sequences of DNA
When can gene mutations arise?
can arise spontaneously during DNA replication, during interphase of the cell cycle
What is an mutagenic agent?
Things that increase the rate of gene mutation
What are some examples of mutagenic agent?
- Ionising radiation
- Carcinogens
- Some viruses
Name the types of gene mutations
Substitution, Addition, Deletion, Inversion, Duplication and Translocation
Describe the effect on amino acid sequence in the encoded
polypeptide when Substitution happens
- No change due to degenerate nature of genetic code
- 1 tripelt code change -> 1 amino acid change
Describe the effect on amino acid sequence in the encoded
polypeptide when Addition happens
- Frameshift; triplets / codons change
downstream of mutation → amino acid
sequence changes
OR - If multiple of 3 bases added – no frameshift, but
extra triplets / codons → extra amino acids
Describe the effect on amino acid sequence in the encoded
polypeptide when deletion happens
- Frameshift; triplets / codons change
downstream of mutation → amino acid
sequence changes
OR - If multiple of 3 bases lost – no frameshift, but
missing triplets / codons → missing amino acids
Describe the effect on amino acid sequence in the encoded
polypeptide when inversion happens
- No frameshift because number of bases stays
the same - Triplets / codons in inverted region change →
sequence of amino acids encoded by inverted
region change
Describe the effect on amino acid sequence in the encoded
polypeptide when Duplication happens
- Frameshift; triplets / codons change
downstream of mutation → amino acid
sequence changes
OR - If multiple of 3 bases added – no frameshift, but
extra triplets / codons → extra amino acids
Describe the effect on amino acid sequence in the encoded
polypeptide when Translocation happens
- Significant impact on gene expression and
amino acid sequences at original and new
location
Why may some mutations not affect the order of amino acids in a polypeptide?
- new codon might still code for the same amino acid
- due to degenerate nature of the genetic code
- also, some gene mutations occur in the introns and therefore won’t affect amino acid sequences
Explain ‘frameshift’
- occurs when gene mutations change the number of nucleotides by any number not divisible by 3
- codons downstream from the mutation change
- sequence of amino acids encoded changes accordingly and the effects on the encoded polypeptide is significant
What is a stop codon?
Codons that terminate translation
What can a mutation to the stop codon cause?
Premature stop codon
- result in production of shorter and often non-functional polypeptide
How can mutations lead to the production of a non-functional protien/enzyme?
- Change in base / triplet sequence of DNA / gene
- Changes sequence of codons on mRNA
- Changes sequence of amino acids in primary structure of polypeptide
- Changes position of hydrogen / ionic / disulphide bonds in protein tertiary structure
- Changes tertiary structure / shape of protein and in the case of enzymes, the active site
will change shape - In the case of enzymes, the substrate will be unable to bind to active site and form an
enzyme-substrate complex
What is a stem cell?
Unspecialised cell
What are stems cells capable of?
- Self renewal
- Specialisation/Differentiation
How does stem cell specialisation occur generally?
- Stimulus e.g. chemical
- Causes selective activation of genes – some genes activated while others inactivated
- mRNA only transcribed from active genes → translated on ribosomes = proteins
- These proteins modify cell permanently and determine cell structure / function
Name and describe the types of stem cells
Totipotent cells
* Occur for a limited time in early mammalian embryos
* Can divide and differentiate into every cell type in body
Pluripotent cells
* Found in embryos
* Can divide and differentiate into most cell types (every cell type in body but not the cells of the placenta)
Multipotent cells
* Found in mature mammals
* Can divide and differentiate into a limited number of cell types
Unipotent cells
* Found in mature mammals
* Can divide and differentiate into just one cell type
What is an example of a multipotent cell?
- cells in the bone marrow
What is an example of a unipotient cell?
Cardiomyocytes ( cardiac muscle cells)
What is an example of a unipotient cell?
Cardiomyocytes ( cardiac muscle cells)
How are Induced Pluripotent stem cells produced?
- Produced from adult somatic cells (non-pluripotent cells or fibroblasts)
- Specific protein transcription factors associated with pluripotency put into cells, causing the cell to express genes associated with pluripotency (reprogrammed)
- Cells cultured
- = Induced pluripotent stem cells
Why would IPS cells be used over embryonic cells?
✓ No immune rejection as can be made using patient’s own cells
✓ Overcome some ethical issues with using embryonic stem cells e.g. no
destruction of embryo and adult can give permission
What are the for’s for stem cell treatment?
- embryo are incapable of feeling pain as they are not equivalent to a human being
- embryos would otherwise be destroyed in clincs
- Duty to apply knowlege to relive human suffereing
- Bone Marrow, less likely to be rejected if it comes from patients own body
What are the arguments opposing the use of stem cells in treating human disorders?
Use of embryonic stem cells:
* Embryo is a potential human; should be given rights
Scientific:
* Induced pluripotent stem cells – cannot yet reliably reprogramme stem cells
* Could begin to multiply out of control, and cause tumours
- Can be expensive?
What are transcription factors and how do they work?
- Protiens
- Move from cytoplasm to nucleus
- Bind to DNA at a specific DNA base
sequence on a promotor region - Stimulate (‘activator’) or inhibit
(‘repressor’) transcription ( of target
gene(s) by helping or preventing
RNA polymerase binding
What is Oestrogen?
A steroid hormone initiating transcription
Why can Oestrogen diffuse across the phospholipid bilayer?
Lipid -soluble
Describe the role of oestrogen in initiating transcription
- Oestrogen diffuses across the phospholipid bilayer
- In cytoplasm, oestrogen binds to a
receptor of an inactive
transcription factor, forming a
oestrogen-oestrogen receptor complex - Inactive transcription factor
changes shape, resulting in active
transcription factor - Diffuses from cytoplasm into
nucleus and binds to specific DNA
base sequence on a promotor
region - Stimulates transcription of genes
by helping RNA polymerase to bind
What is the genome?
The complete set of genes in a cell
What is recombinant DNA Technology?
The transfer of DNA fragments from one organism or species, to another
Why can transferred DNA be transcribed/translated into proteins of a transgenic organism?
Genetic code is universal
- Transcription and translation mechanisms also are
Name the three methods fragments of DNA can be produced by
- Conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
- Using restriction enzymes to cut a fragment containing the desired gene from DNA
- Creating the gene in a ‘gene machine’
Describe the conversion of mRNA to complementary DNA (cDNA), using reverse
transcriptase
- mRNA isolated from a cell that readily synthesises the protein coded for by the desired gene
- Mix mRNA with DNA nucleotides and reverse transcriptase
- reverse transcriptase uses mRNA as a template to synthesise a single strand of cDNA
- DNA polymerase forms second strand of DNA (= double stranded = gene) using cDNA as
a template
Describe using restriction enzymes to cut a fragment containing the desired gene from
DNA
- Different restriction endonucleases cut DNA at
specific sequences of bases called a ‘recognition
sequence’
* Shape of recognition site complementary
to active site - Some restriction enzymes cut in a staggered
fashion = ‘sticky ends’ formed
Describe creating the gene in a ‘gene machine’
- Synthesises fragments of DNA from scratch without the need for a pre-existing DNA
template
Why is gene machine better than other methods of making DNA fragments
- DNA fragments produced quickly / accurately
- Free of introns → can be transcribed by a prokaryote who can’t remove introns
Advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut gene from DNA
- More mRNA in cell than DNA → easily extracted
- Introns removed by splicing (in eukaryotes) whereas DNA contains introns
- Bacteria can’t remove introns
Define epigenetics
Heritable changes in gene function without changes to the base sequencs of DNA, caused by changes in the environment
What can epigentic changes cause?
can inhibit transcription
Describe the effect of increased methylation
- methyl groups added to cytosine bases
- nucleosomes pack tightly as dna wrapped around histones tighter
- prevents transcription factors binding –> genes are not transcribed as RNA polymerase unable to bind
- irreversible
Describe the effect of decreased acetylation of associated histones
increases positive charge of the histones
bind DNA more tightly
prevents transcription factors binding
genes not transcribed
rna polymerase cannot bind
but is reversible
How can hypomethylation cause the production of oncogenes?
proto-oncogens can be hypomethylated
increases transcription of proteins that activate cell division
lead to tumour formation (increased cell division)
proto-oncogenes now act like oncogenes
What is an oncogene?
mutated proto-oncogenes that become overreactive. This stimulates cells to divide uncontrollably resulting in a tumour.
Define the term ‘tumour suppressor’?
normal genes that code for proteins that regulate the cell cycle
What is RNA interference?
RNA molecules inhibit translation of mRNA produced by transcription (encoded protein is not produced)
What are the two types of RNAi?
siRNA
miRNA
Describe differences between siRNA and miRNA
micro-rna: not fully complementary to target mrna; binds to more than one molecule; associates w/ proteins + binds to targed mrna in cytoplasm; blocks translation of mrna site; mrna then stored or hydrolysed
small interfering rna: associates w/ several proteins; unwinds single strand + binds to target dna; base sequencse of sirna fully complementary to base sequences in sections of target mnra; proteins cute the mrna into fragments; no longer translatable so hydrolysed in the body
What is cancer?
uncontrolled cell division
What can tumours be classified as?
Benign
Malignant
Give characteristics of benign tumours
- Grow slowly
- Well differentiated
- Normal nuclei
- Well defined borders
- Can be removed by surgery
What are promotor regions?
DNA sequences that tell RNA polymerase when to start producing mRNA
What are terminator regions?
Regions that tell RNA polymerase to stop
Describe the stages of the culture of transformed host cells as an in vivo method to amplify DNA fragments
- Promotor and terminator regions are added to fragments of DNA
- Vector transports DNA into host cell e.g. plasmids or bacteriophages
- Vector DNA and DNA fragment cut using same restriction enzyme
- Vector DNA and DNA fragment have complementary sticky ends → complementary base pair
- DNA ligase forms phosphodiester bond between adjacent nucleotides on sticky ends
- Marker genes, inserted into vectors at same time as target gene, are added in order to
identify which cells have the desired gene
Describe how restriction endonucleaes and ligases are used to insert fragments of DNA into vectors
- Vector transports DNA into host cell e.g. plasmids or bacteriophages
- Vector DNA and DNA fragment cut using same restriction enzyme
- Vector DNA and DNA fragment have complementary sticky ends →
complementary base pair - DNA ligase forms phosphodiester bond between adjacent nucleotides on sticky ends
Describe the difference between a plasmid vector + Bacetriophage vector during the transformation of a host cell
Plasmid vector:
- host cells have to be persuaded to take in plasmid vector + its DNA
Becteriophage vector:
- infects host bacterium by injecting it’s DNA into it, then the phage DNA with target genes intergrates into bacterial DNA
Why are marker genes used?
to detect genetically modified cells as not call cells will take up the vector and be transfomed
Gene markers can be: ?
- resistance to an antibiotic
- fluorescent protein
- enzyme whose action can be identified
What does PCR stand for?
Polymerase chain reaction
What is PCR?
The amplification of specific DNA fragments where in every cycle, DNA doubles
What is in the reaction mixture?
DNA fragments
DNA polymerase
primers
nucleotides
Describe and explain how the polymerase chain reaction (PCR) is used to amplify a
DNA fragment.
- (Requires DNA fragment) DNA polymerase,
(DNA) nucleotides and primers; - Heat to 95 °C to break hydrogen bonds (and
separate strands); - Reduce temperature so primers bind to
DNA/strands; - Increase temperature, DNA polymerase joins adjacent
nucleotides forming phosphodiester bonds (and repeat method);
Why during PCR, the number of DNA molecules being produced plateu?
Plateaus as no more nucleotides/primers
What are primers
short length of DNA
Single stranded
with specific base sequence/complementary base sequence
Why are primers needed?
to mark the beginning sequence for DNA polymerase to start DNA copying
Prevent 2 seperate strands from re-joining
State the three main stages of PCR
- Seperation of the DNA strand
- Addition
- Synthesis of DNA
What is a DNA Probe?
short single stranded section of DNA that has specific base sequence of DNA, so it binds to complementary genes
What are the two ways DNA probes can be labelled?
- radioactively
- fluorescently labelled
Describe how a DNA probe can be identified using fluroscence
- mutated allele/gene has specific base sequence
- Make complementary base sequence to mutaed gene in the form of DNA probe
- then labell with fluroscence
- Heat the donors DNA to break hydrogen bonds and form seperate strands
- Then if mutaed allele present in donors dna, the dna probe will form base pair to complementary bases
- Wash the plate to remove any unattached DNA probes
- Postive result will show fluroscence
What is gel electrophoresis used for?
used to seperate dna fragments according to their size
How does gel electrophoresis seperate the proteins?
- Depends on size/mass of protein
- Depends on charge of the protien
What are VNTRs?
repetitive sequences in non-coding bases (introns) of DNA
What are the 5 stages of genetic fingerprinting?
- Extraction
- Digestion
- Seperation
- Hybridisation
- Development
