Chapter 20 + 21 - Gene Expression + Recombinant Gene Technology Flashcards
What kind of gene mutations can occur during DNA replication?
Addition - another base added
Deletion - complete removal of a base
Substitution - one base is swapped for another
Inversion - where the sequence of the bases are flipped around
Duplication -
Translocation -
What kind of gene mutations can occur during DNA replication?
Addition - another base added
Deletion - complete removal of a base
Substitution - one base is swapped for another
Inversion - where the sequence of a group of bases is flipped around
Duplication - when one or more bases are repeated after the original base or group of bases
Translocation - when a group of bases leave the chromosome and are added to another chromosome - usally leading to some forms of cancer or reduced fertility
What are potential consequences of these changes?
Many of these mutations cause frame shifts, where the entire sequence after the mutation is altered because they are no longer in the same triplet, so the entire amino acid sequence is different
A substitution could lead to a stop codon being created, so the polypeptide sequence is significantly shorter than it should be
It could change one amino acid, which either could result in a non-functional gene (e.g active site of an enzyme) or have no affect on a protein
Due to the degeneracy of the genetic code, these mutations could lead to the same amino acid being coded for and therefore have absolutely no impact on the polypeptide
What are the 3 major causes of mutations?
Chemical mutations
ionising radiation
random chance
What are the different levels of stem cells, starting from the most potent to the least potent?
Totipotent > pluripotent > multipotent > monopotent
only occur in early mammalian embryos > typically only occur in embryos and can become every type of cell except placenta cells > multipotent are typical in adults and can become any cell from a specific group of cells > can only become one specific type of cell
How can stem cells be made more useful in the treatment of disease?
By the formation of Induced Pluripotent Stem Cells (IPS cells) by using appropriate transcription factors
What is recombinant DNA technology?
The transfer of DNA from one organism or species to another. This works thanks to the universal nature of both DNA and also transcription and translation - allowing the DNA to be translated and therefore expressed in the phenotype of the transgenic (recipient) organism
What are the three types of method to recover DNA fragments?
Reverse transcriptase - where mRNA is converted to complementary DNA (what HIV uses)
Restiction endonucleases - where specific DNA fragments can be cut away from known sequences
The use of the “gene machine”
What is the gene machine?
The gene machine allows scientists to look at the amino acid sequence of desired proteins and “reverse engineer” it, to form the mRNA sequence and then the DNA sequence. This DNA sequence can be fed into a machine which checks for biosafety and then assembles “oligonucleotides” which are bonded together to form the desired DNA sequence
Why is the gene machine so advantageous?
It can be used to great accuracy in a short amount of time (approximately ten days), and any sequence of nucleotides can be formed without the requirement of a pre-existing or known seqeunce. Most notably, the produced sequence is completely free of introns, so is easily translated by prokaryotes
What methods can be used to amplify DNA fragments?
in vivo - use of a vector (done by cells)
in vitro - Polyamerase chain reaction (done in a lab)
How does in vivo cloning work? (The insertion portion)
It is easiest done with restriction endonucleases which leave sticky ends. First, a promoter region and terminater region are added to the DNA fragment and then the same restriction endonuclease is used on the plasmid - the vector. This means that because the sticky ends are palindromic, they will base pair together perfectly, and can then be bonded along the backbone by DNA ligase, resulting in a rembinant DNA sequence
Once you have obtained your recombinant vectors, what needs to be done to transform them?
The cells and plasmids are put in a solution of lots of calcium ions and then heated and cooled several times - which increases permeability of the bacterial membrane allowing the plasmids to enter the cell
What can happen to the plasmids and DNA fragments which doesn’t result in a desired plasmid with the DNA fragment?
The plasmid only has a 1% chance of taking the DNA fragment, this is because:
The plasmid can shut without taking the DNA fragment
The DNA fragment can form its own plasmid
How can you check that the bacteria has both taken the plasmid, and the plasmid has taken the DNA fragment?
The bacteria can be grown on a medium which contains an antiobiotic, and only the cells which have taken up plasmids will have resistance to this DNA therefore they will survive. Then, the use of marker genes can be used to identify genetically modified or cells. Some markers include fluorescence, a producable enzyme with a measurable activity or resistance to another antiobiotic. These markers are typically added to the DNA fragment before uptake by a plasmid
What 4 factors are required for PCR?
The DNA fragment
DNA polymerase, typically taq polymerase because it is heat resistant (comes from bacterial hot springs)
primers
free nucleotides
a thermocycler
What are the 3 heat stages in each PCR cycle, and what happens during them?
In the first stage, the temperature goes to about the 90s so that DNA strands and the hydrogen bonds seperate
The temperature is then reduced to 55 so that the primers can be added, which allows the DNA polymerase to act on the chain and also prevents the two strands coming back together
The temperature then goes to the 70s so the polymerase can form 2 new, seperate double helix strands
this process takes about 2 minutes
What is a transcription factor?
A transcription factor is a protein which is able to bind to a specific section of DNA before a gene and inhibit it or stimulate its transcription
Give a named example of a hormone and how it works on its transcription factor
Oestrogen is lipid soluble and can therefore diffuse directly across the membrane. From there it can bind to the allosteric site of a transcription factor, activating it and causing it to enter the nucleus and activate a protein, leading to its transcription
Define epigenetics:
Define epigenetics:
Epigenetics is heritable changes to DNA without involving changes to the base sequence. These changes arise from factor which inhibit transcription
What is the epigenome?
The epigenome is the genome (the chromosomes of an organism and all of its assosciated proteins) as well as the chemical markers which affect it
What are the chemical tags assosciated with the epigenome and how do they affect transcription?
Increased methylation (addition of methyl groups) leads to higher levels of “supercoiling” preventing access to certain genes
Increased acylation however leads to lower levels of supercoiling, therefore it does not inhibit gene transcription
Why is epigenetics relevant to cancer treatment?
Epigenetics can potentially prevent the transcription of genes which allow the detection and death of cancerous cells, so when they are methylated they are no longer proteins which can be made, meaning there is less chance of the body being able to kill cancerous cells
What can prevent translation in eukaryotic cells?
RNAi, or a RNA interference molecules which can inhibit the production of a protein from mRNA molecules
What are the main characteristics of malignant tumours?
They can grow to large sizes and grow quickly, however they lack capsules and therefore they can grow finger-like projections into the surrounding tissues. They also have larger and denser nuclei due to the large amounts of DNA, and easily become undifferentiated cells. They don’t produce adhesion molecules, meaning they easily dislodge from affected tissue and spread around the body through the blood, known as metastasis. This makes it significantly harder to treat, requiring radiotherapy or chemotherapy, and often reoccur after treatment. They have much more systemic and severe impacts on the body, because they replace healthy tissue with abnormal tissue, leading to fatigue and weight loss
What are the main characteristics of benign tumours?
They are large but grow slowly and remain well differentiated. Their nuclei remain normal looking. They are covered in a capsule so remain fairly solid and well adhesed to their surrounding tissue. Due to their capsule they rarely have major impacts other than disrupting the function of a vital organ, meaning that they have localised affects. They are therefore easy to remove with surgery and don’t often reoccur after removal
What are proto-oncogenes and tumour suppressor genes?
Proto-oncogenes are chemical messengers which signal cells to divide, leading to multiplication to DNA when they attach to a receptor on the cell membrane. Tumour suppresor genes instead can cause apoptosis, correct mistakes in DNA replication and slow down or prevent the division of cells. These two proteins are generally balanced leading to deliberate and controlled cell division
How do epigenetics and mutations affect tumour suppressor and prot-oncogenes?
If tumour suppressor genes become methylated then the proteins are no longer produced so cell divison can’t be regulated as easily. When proto-oncogenes mutate they become oncogenes, and the proteins produced either don’t unbind from the cell meaning it keeps on dividing, or they trigger massive amounts of growth hormone to be produced resulting in uncontrolled and rapid cell divison
What are the links between increased oestrogen concentrations and breast cancer?
After menopause in women, their breasts increase production of oestrogen, potentially causing cancerous cells. When breast cells do become cancerous they release more oestrogen, furthering the development of the cancerous cells
What are sequencing projects?
Projects where the entire genomes of many different organisms are read, including humans
What does determining the genome of simpler organisms allow?
It allows the genes from which certain proteins are produced (proteome-genomic link) - this can have uses in drugs and vaccines
Why is it harder to link the genome and proteome of more complex organisms?
Due to the presence of non-coding DNA
What is a DNA probe and what are the two most common types?
A DNA probe is a single strand of DNA which is complementary to a specific desired allele. They either contain nucleotides made up of 32P which is radioactive and then tested using X-ray or with fluorescent nucleotides which emit light when bound to the DNA sequence
What is DNA hybridisation?
DNA hybridisation is where the DNA double helix of the host is split and mixed with the DNA probe of a complementary allele which binds to the DNA sequence
What do DNA probes and hybridisation allow us to do?
They allow us to test for specific genes and genetic disorders, or genetic resistances to certain drugs for example
What is genetic counselling?
Genetic counselling is testing genes for specific diseases and giving advice on offspring or potential lifestyle choices
What is name of the region between different genes, which differs in different organisms and individuals of the same species?
Variable tandem number repeats (VNTRs), or a repeating base sequence. The chance of two individuals having the same VNTRs is very low
What is genetic fingerprinting? Give 3 uses:
Genetic fingerprinting is identifying the relation of DNA based upon the VNTRs present. It is often used to test for paternity, whether a suspect was present at the scene of a crime, medical diagnosis, and plant or animal breeding (plants can avoid being inbred, or plants and animals can be selected based upon desirable alleles)
Give the stages of genetic fingerprinting through electrophoresis
1) Extraction + PCR
2) Digestion - addition of restriction endonucleases
3) Seperation (electrophoresis)
4) Hybridisation - addition of DNA probes
5) Development - identification of the band placement (either visually with fluorescent probes or with x-ray film for radioactive probes)
