unit 8 Flashcards
What are mutations?
Any change to a nucleotide base.
What are mutations usually caused by?
errors during DNA replication.
What are the 6 types of mutations?
*Substitution
*Deletion
*Addition
*Duplication
*Inversion
*Translocation
What can a mutation to a polypeptide that makes up an enzyme cause?
It may change the shape of the enzymes active site. This may stop substrates from being able to bind to the active site meaning the enzyme cannot catalase its reaction.
Do some mutations increases the risk of developing a cancer?
Yes, BRAC1 can increase the chances of developing breast cancer.
Can some mutations cause genetic disorders?
Yes, e.g. cystic fibrosis. If a gamete containing a mutation for a certain type of cancer or genetic disorder is fertilised, the mutation will be present in the foetus. These are hereditary mutations.
Why do not all mutations lead to a change in the amino acid sequence?
Degenerate code
What is a frameshift mutation?
Additions, duplications and deletions almost always cause a change to the amino acid sequence of a polypeptide. It causes a change in the number of bases in the genetic code causing a ‘shift’ in the triplets being read called a frameshift. The entire gene is read in a different way. The earlier on the gene the mutation is the bigger the change.
What increases the rate of mutations?
Mutagenic agents
What are examples of mutagenic agents?
Radiation such as UV or ionising, some chemicals e.g. benzene and some viruses e.g. Human papillomaviruses (HPV).
What ways do mutagenic agents increase the rate of mutations?
*Acting as a base
*Altering bases
*Changing the structure of DNA
Mutagenic agents acting as bases-
chemicals called analogues can substitute for a base during replication changing the base sequence eg 5-bromouracil can substitute for thymine and pair with guanine instead of adenine causing a substitution mutation.
Mutagenic agents altering bases
some chemicals cans delete or alter bases Eg alkylating agents can add an alkyl group to guanine so that it pairs with thymine instead of cytosine.
Mutagenic agents changing the structure of DNA
some types of radiation can change the structure of DNA e.g. UV radiation can cause thymine bases to join up together.
What are acquired mutations?
Mutations which occur after fertilisation.
What happens if these acquired mutations occur in the genes which control mitosis?
It leads to uncontrollable cell division and will result in a tumour forming.
What is a tumour?
A mass of abnormal cells. Two types-
Benign and Malignant.
What is a benign tumour?
These are not cancerous. They usually grow slowly and are often covered in fibrous tissue which stops them invading other tissues. They are often harmless unless they cause blockages, put pressure on other organs or are difficult to remove e.g. in the brain. Some benign tumours can become malignant.
What is a malignant tumour?
These are cancerous. They grow rapidly and to a large size. Cell nucleus is larger and darker due to an abundance of DNA. Cells become unspecialised. Form secondary tumours. Effects such as weight loss and fatigue. Freq occur again after treatment.
In what ways do tumours differ from cells which are normal?
- The nucleus is larger and darker and can have more than one nucleus
- Have an irregular shape
- They don’t produce all the proteins to function properly
- They have different antigens on their surface
- They don’t respond to growth regulating processes
What are the two types of genes which control cell division?
Tumour suppressor genes (TSG) and Proto-oncogenes
What other factors can cause the growth of tumours?
Oestrogen and Abnormal methylation of DNA
What are tumour suppressor genes?
These genes slow cell division, repair mistakes in DNA and tell cells when to die by producing proteins which either stop cell division or cause the cell to self destruct (apoptosis).
If a mutation occurs to the TSG, it is inactivated so stops inhibiting cell division and cells can rapidly divide. Those TSG’S that are inactivated that survive can clone themselves and form tumours.
What are proto-oncogenes?
These genes stimulate a cel to divide when growth factors attach to a protein receptors on its cell surface membrane.
If a mutation occurs then the cell can overreact and the cells will divide uncontrollably.
A mutated proto-oncogene is called an oncogene.
If a proto oncogene mutates to an oncogene it can become permananently activated for what two reasons?
-oncogene may code for a growth factor that is then produced in excessive amounts, again stimululating excessive cell division.
-Receptor protein on the cell surface membrane can be permanently activated so so division is switched on even in absence of growth factors
Abnormal Methylation of TSGs
Methylation means adding a methyl group to something. When methylation occurs normally to DNA it can control if a gene is transcribed (copied to mRNA) and translated (made into a protein).
Hypermethylation is when methylation occurs too much= TSGs being inactivated.
hypomethylation is when methylation occurs too little (normally in oncogenes)=inactiavted
Oestrogen
Some women produce more oestrogen than others. This can cause menstruation to begin younger and menopause to begin later. There is a theory that exposure to oestrogen for a long period of time can increase the risk of breast cancer but researchers are not completely sure why. Transdermal HRT has no extra risk associated. Some theories include:
*Oestrogen causes proto-oncogenes to mutate into oncogenes in breast tissue
What does it mean by some cancer’s being polygenetic?
That they are triggered by many environmental factors which means it is difficult to know which factor has the greatest effect.
Ways of preventing cancer?
Screening such as a masectomy to get breasts removed if a women has the BRAC1 gene
How can you treat skin cancer?
If it is due to BRAF protooncogene it can be treated with a drug which inhibits the enzyme produced by the mutated gene to stop the cell expressing.
What are examples of future stem cell therapies?
For spinal cord injuries the damaged nervous tissue can be replaced.
To replace bladders damaged by disease.
For respiratory and heart diseases.
What are the ethical issues surrounding embryonic stem cells being used?
They have a right of life.
Embryo not used in IVF would be destroyed anyway.
Embryo can become every type of cell.
What are the ethical issues surrounding adult stem cells being used?
Doesn’t destroy an embryo
Only become a limited no of cells
What are the ethical issues surrounding stem cells from unfertilised eggs which have been induced to divide?
No right to life as not an embryo
Would not produce a foetus if implanted in the womb.
What are the benefits of stem cell therapy?
Can improve the quality of life for ppl
Expensive
Using patients own cells to grow organs and tissues which would not be rejected.
What is bone marrow used for?
Make blood cells. Transplanting stem cells which is found in bone marrow from a healthy patient to a patient producing abnormal stem cells can produce new healthy stem cells.
Used to treat leukemia, lymphoma cancer, sickle cell anaemia etc
What are stem cells?
Undifferentiated cells which can differentiate into specialised cells.
What are sources of stem cells?
Embryonic stem cells-can differentiate into any cells
Umbilical cord stem cells- taken from umbilical cord blood straight from birth
Placental stem cells- only specialise into some cells
Adult stem cells- found in foetus to adult. They are specific to a particular tissue or organ within which they produce the cells to maintain and repair tissue.
What are forms of stem cells?
Totipotent stem cells which can differentiate to any type of body cell including placenta. Only in mammals in the first few cellular divisions of embryo.
Pluripotent stem cells-are after 1st division of embryo. Form any body cell excluding placenta.
Multipotent- found in adults and can differentiate to limited no of specialised cells eg adult and umbilical cord blood.
Unipotent- only differentiate to one type of cell. Derived from multi potent stem cells and are made in adult tissue
What are induced pluripotent stem cells?
Where scientists obtain stem cells from embryos and some adult tissues and also creating stem cells in the lab. Process involves reprogramming these stem cells to express transcription factors which express genes associated w pluripotent cells.
How to induce pluripotent stem cells?
Induce the stem cells with a modified virus. The virus has genes coding for transcription factors in it’s DNA. When the virus enters and infects adult cells the viral DNA is passed into adult cells DNA and these cells will produce transcription factors.
What type of stem cells form from adult mammals from 8 weeks onwards?
Multipotent and unipotent
What are gene switches?
Where stem cells differentiate in their development and they only transcribe and translate part of their DNA. All stem cells produce the same genetic info but not all will express the same genes. Different conditions will cause some genes to be on or off.
What is specialisation like in most animal stem cells?
In most, specialisation is irreversible. For example, red blood cells loose their nucleus when they differentiate so cannot differentiate again. Adult stem cells replicate by mitosis to replace any which have been specialised.
What are cardiomyocytes?
Unipotent heart muscle cells. Old or damaged cardiomyocytes can be replaced by small no of unipotent cardiomyocyte cells in the heart.
What are transcription factors?
They are proteins which control transcription by binding to DNA. Most of the time they are inactive in cytoplasm as they have inhibitor attached.
What happens to transcription factors in eukaryotes?
They move from the cytoplasm to the nucleus by diffusion. In the nucleus they bind to specific DNA base sites called promoters. These are specific base sequences at the beginning of the gene and are many bases long. When transcription factor binds, transcription begins. mRNA produced and info is translated to polypeptide. If a gene is switched off the site of transcription factor that binds to DNA is not active.
What are activators?
A type of transcription factor which stimulates or increases the rate of transcription. Helps RNA polymerase bind to promoter region of target gene.
What are repressors?
A type of transcription factors which inhibit or decrease the rate of transcription. Bind to promoter region of target gene preventing RNA polymerase from binding and stopping transcription.
What is oestrogen?
A steroid hormone which can switch a gene on and start transcription
It binds to an oestrogen receptor and forms oestrogen-oestrogen receptor complex.
This changes the tertiary structure of DNA binding site
Inhibitor released and transcription factor can bind to promoter sequence.
What is RNA interference (RNAi) in eukaryotes?
A small double stranded RNA molecule which stops mRNA from being translated from proteins (gene silencing)
What is siRNA?
double stranded, taken up by cells and enters the cells through vectors
What is miRNA?
Single stranded and made inside cell. Within introns of larger RNA.
where is siRNA found?
lower animal and plant kingdoms
where is miRNA found?
all animals and plants
How does siRNA bind?
Perfectly to its mRNA target in animals.
How does miRNA bind?
It binds imperfectly to its mRNA target so can inhibit translation of many different mRNA sequences
What happens to miRNA and siRNA in the cytoplasm?
They are both processed by Dicer before forming part of protein complex called RISC. They emerge from longer RNA precursors.
SiRNA
Once RNA is transcribed it leaves the nucleus to the cytoplasm. The double stranded siRNA unwinds. 1 strand is selected and 1 strand is degraded. The selected strand binds to mRNA as its base sequence is complementary to mRNA’s base sequence.
The proteins associated with siRNA cut mRNA into pieces meaning it can no longer be translated. Therefore the pieces are degraded.
MiRNA in mammals
MiRNA is not fully complementary to mRNA as it targets more then 1 RNA molecule.
When first transcribed it is a long folded strand. It is processed to a double then single strand. 1 of the strands binds to mRNA which blocks the translation of mRNA. Gets moved into the processing body (stored or degraded).
What is epigenetics?
A study of how environmental cues can cause heritable changes phenotype without inheriting the DNA genotype. Affects how genotype is read. Could be used to cure cancer.
What does epigenetic gene control determine in eukaryotes?
Whether a gene is switched on or off. It works through the attachment or removal of epigenetic tags to or from DNA or histones. These tags form the epigenome which determines the shape of the DNA- histone complex. It does not alter DNA but decided how easily enzymes can transcribe DNA.
Why is the epigenome flexible?
As tags respond to environmental cues.
What is the epigenome?
Accumulation of tags during our lifetime
What usually happens to epigenetic markers between generations?
Most are removed between generations in the early foetus but some escape the removal and are passed onto offspring. The expression of genes can be affected by environmental changes which affected their grandparents.
How do acetyl groups act as epigenetic markers?
By them being added or removed from histones.
When histones are acetylated the chromatin becomes less condensed and can be transcribed which switches the gene on.
When and acetyl group is removed (deacetylation) from a histone it makes the chromatin more condensed and cannot be transcribe, which switches the gene off.
What is methylation?
Methyl is an epigenetic marker. A methyl group attaches to where a C + G base are next to eachother. Increased methylation changes the DNA structure so transcriptional enzymes cannot interact with the gene so it is switched off.
It does this by-
1. Preventing transcription factors binding to DNA
2. Attaching proteins that condense the DNA histone complex making the DNA inaccessible.
Is there methylation in normal cells?
The promoter regions in normal cells have no methylation meaning they are switched on and DNA can be transcribed.
What happens when a tumour suppressor gene is methylated and promoter genes are inactivated?
DNA can’t be transcribed and the tumour suppressor gene is inactivated which occurs in the early stages of cancer.
What can changing the epigenome cause?
Genes which repair DNA and prevent cancers being switched off.
Increased methylation of protective genes being switched off means damaged DNA cannot be repaired which leads to cancer (which is why some cancers are inherited).
What is epigenetic therapy?
Treatments involve drugs which inhibit the enzymes involved in DNA methylation or histone acetylation.
Drugs which inhibits enzymes which cause DNA methylation can reactivate genes which are switched off.
So when treating cancer genes must be specifically targeted as to not switch on or off genes which are correctly read and create a secondary cancer.
What is the genome?
All DNA of an organism. It includes all genes that carry genetic info for making all proteins required by an organism, a complete map of all the genetic material in an organism.
What is the human genome project?
The sequence is not from 1 person but it is a composite from several individuals. To ensure anonymity of the DNA donors most blood samples were collected then used and no names attached.
What is whole genome shotgun sequencing?
It involves cutting DNA into smaller sections with overlapping ends.
Computer programs then use the overlapping sections to assemble the entire genome.
These are continuously updated and has led to sequencing being extremely quick.
Why is it easy to see the proteome of organisms of bacteria such as bacteria?
They do not have many introns
What is the proteome?
All proteins the genome codes for
What is recombinant DNA?
DNA from 2 diff organisms that have been combined.
These organisms are known as transgenic or a gentically modified organism.
What is the 1st step to make recombinant DNA?
Make DNA fragments.
What does recombinant technology involve?
Transferring DNA from 1 organism to another, even if it is a different species.
This can be done as genetic material is universal, as the same base codes make the same amino acids and all proteins and organisms.
Process of making DNA fragments
MRNA is isolated and combined with reverse transcriptase enzyme and nucleotides two create a single strand of complimentary DNA. DNA polymerase is used to convert the single strand to a double strand which contains the desired code for the gene
Some DNA have palindromic sequences of nucleotides which means parallel base pairs read the same in opposite directions.
Restriction endonucleases bind to a specific restriction site. They separate the two DNA strands at the specific base seq by cutting the sugar-phosphate backbone in an uneven way to give sticky ends which results in one strand being longer than the other.
What do sticky ends allow?
Make it easier to insert the desired gene into another organisms DNA as they can easily form hydrogen bonds with the complementary base sequences on other pieces of DNA that have been cut w the same restriction enzyme.
What is the process of the gene machine?
- The sequence is designed
- First nucleotide is fixed with some form of support eg a bead.
- Nucleotides are added 1 by 1 with a protecting group which ensures that nucleotides are joined at the correct place without unwanted branching.
- Oligoucletides (short sections of DNA) are made and are freed of support and protecting groups. They can be joined together to form longer sections of DNA.
-DNA fragments can be made from scratch with out a DNA template.
What is in vivo cloning?
Where copies of genes are made within a living organism.
What is the process of in vivo cloning?
- Insert DNA fragment into a vector
-vector DNA is cut open using the same restriction endonucleases for DNA fragmentation, this ensures sticky ends will be complementary.
-vector DNA and fragment DNA are mixed w DNA ligase which joins the ends together through ligation.
-this creates recombinant DNA (new combo of bases) - Vector (w/recombinant DNA) transfers the DNA into host cells.
-If a plasmid is used, change in temperature and certain chemicals will encourage the cell to take it in.
-If a bacteriophage is used, they inject the DNA into host and target DNA integrates into bacterial DNA.
-Host cells taking up the vectors with the gene are transformed.
3.Identify transformed cells
-Marker cells inserted into vector with the DNA fragments
-Marker gene codes for antibacterial resistance or will make the transformed genes fluorescence under UV light.
-Only those who are resistant to the specific antibacteria can survive and replicate, so those who grow are transformed.
What does the vector need if you want the host cell to produce a particular protein?
Specific promoter and terminator sequences. The sequences can either be in vector DNA or added w the DNA fragments.
What is in vitro cloning?
Copies of genes are made outside a living organism using the Polymerase Chain Reaction.
What is the process of in vitro cloning?
- Create a mixture containing the DNA fragment, free nucleotides and DNA polymerase and primers (short pieces complimentary to bases at the start).
- DNA mixture heated at 95 degrees celsius to break hydrogen bonds. It is then cooled (50-66 degrees) for primers to bind to the strands.
- Heated to 72 degrees for DNA polymerase to work. It lines up with free nucleotides along each template fragment strand to create complementary strands.
- 4 strands of the DNA fragment are created are created in 1 cycle which then starts again. Polymerase chain reaction doubles the number of strands in each cycle.
What is genetic engineering?
Where organisms can be transformed using recombinant DNA technology, gene fragments.
How do you transfer plants?
The cell is treated with enzymes which dissolve the cell wall. The new gene is inserted and the cell is then placed in a broth of plant hormones and nutrients that induce it to regenerate. It first re-forms a cell wall. If the correct promotor gene is added, then the plant will produce the protein. This can develop plants with added nutrients or resistance to pests.
How do you transform animals?
The desirable gene can be added into an egg cell or into an early animal embryo. When you genetically modify an egg, you are altering the genes of the germ cells, which then mature into genetically modified eggs and sperm. Germline editing is gene editing of only reproductive cells, which include sperm and egg
Benefits and risks to medicine using recombinant DNA technology?
+Many new drugs and vaccines are produced using recombinant DNA
New drugs can be made cheaply, quickly and in large quantities
+Higher yield and more nutritious leading to less famine.
-Companies who have the patient on certain drugs may limit the use of them by charging high prices
-Could be used to make ‘designer babies’
Benefits and risks to industry using recombinant DNA technology?
+Industry uses enzymes for many reasons such as in food production and cleaning. The enzymes can be made from recombinant DNA making them cheaply and in large quantities
-Inaccurate labelling means people may not have a choice about eating GM food
-Larger companies who control the GM technology may become more powerful, forcing smaller business out
Benefits and risks to agriculture using recombinant DNA technology?
+Can grow in more extreme climates
Increase shelf life
+Crops produce herbicides and pesticides resistance
-Breed with wild plants to create ‘superweeds’
-Seeds may blow into nearby farms – if organic, they will contaminate and lose money
What is gene therapy?
altering defective alleles to treat genetic disorders and cancer.
What does the type of gene therapy used depend on?
whether the disorder is caused by a mutated dominant allele or 2 mutated recessive alleles.
What do both types of gene therapy involve?
Inserting a DNA fragment into a persons DNA. This involves a vector to get the DNA fragment into the cell. These can be viruses, plasmids or liposomes.
What are liposomes?
small artificial vesicles of spherical shape that can be created from cholesterol and natural non-toxic phospholipids. Due to their size and hydrophobic and hydrophilic character liposomes are promising systems for drug delivery.
What can you do if the faulty DNA is caused by a dominant allele?
You can ‘silence’ it by inserting a DNA fragment into the middle of it to prevent it from working.
What can you do if the faulty DNA is caused by 2 recessive alleles?
You can add a dominant gene to one of the chromosomes which will ‘supplement’ it.
What are the two types of gene therapy?
Type 1 – Somatic therapy, this alters the faulty alleles which are affected. It does not affect the sex cells, so the disorder can still be inherited e.g. cystic fibrosis.
Type 2 – Germ line therapy, this involves altering the alleles in the sex cells. This means all the cells will be altered and the individual and their offspring will not suffer the disease. This currently illegal.
What are the ethical issues surrounding gene therapy?
- Who decides which traits are normal and which constitute a disability or disorder?Will the high costs of gene therapy make it available only to the wealthy?
*Could the widespread use of gene therapy make society less accepting of people who are different?
*Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?
What are DNA probes?
Short strands of DNA. They have a specific base sequence which is complementary to the base sequence of the target allele e.g. the allele of the disorder. The DNA probe will hybridise (bind) to the target allele if it is present in the sample of DNA.
The marker will help to show where the probe has bound.
What can DNA probes be used for?
They can be used to locate specific alleles on genes. This is then used to screen for inherited conditions, drug responses and health risks.
How are DNA probes made?
You first need to sequence the allele you want to screen for and then use PCR to produce multiple complimentary copies of the allele - these are the probes.
What do DNA probes contain so they can be detected?
A marker. The most common markers are radioactive detected using x-ray film or a fluorescent marker detected with UV light.
What is the step-by-step process of Locating Alleles Using DNA Probes?
Step 1 – The DNA sample is digested into fragments using restriction enzymes. It is then separated using gel electrophoresis.
Step 2 – The DNA fragments are then transferred to a nylon membrane and incubated with the fluorescent marker. If the allele is present the DNA probe will hybridise to it.
Step 3 – UV light is then shone on the nylon membrane. If the allele is present then the probe will have bound to the DNA fragment, and will fluoresce.
What is the function of a microarray?
It can screen for lots of different genes at the same time.
What happens to the microarray?
A sample of fluorescently labelled DNA is washed over the microarray. Any DNA sequences which match the probes will attach to the array. This means lots of different mutated genes can be screened for at the same time. The array is washed to remove DNA which has not attached and put under a UV light. Any spot which glows means that the allele is present.
What is genetic counselling?
used to give advise to people and their families about the options for genes to be screened e.g. a history of family cancers, and to offer advise upon test results.
It can also advise if someone is a carrier for a mutant allele, what type of mutation it is and what treatments are an option.
If test results are positive for a mutated allele, then the counselling advises on the treatments or preventative treatments available.
What are personalised medicines?
tailored to a persons DNA. Doctors can predict the drugs you will respond best to and prescribe those. This means recovery time should be quicker and have a lesser impact on your life.
What is variable no of tandem repeats?
The number of base sequences, which don’t code for proteins (introns), but repeat over and over.
The number of repeats differs from person to person and the number of bases repeated also varies. GTGA might be repeated twice in one person (8 nucleotides) and 10 times in another person (40 nucleotides) .
What is genetic fingerprinting?
NNTR’s can occur anywhere in your genome. Because of the differences between peoples genomes, they can be compared between individuals called genetic fingerprinting. The probability of 2 people having the same genetic fingerprint is very low.
What is the process of making genetic fingerprints?
.A sample of DNA e.g. saliva is taken, it is cut into fragments (restriction enzymes), and PCR is used to make many copies of the areas of the DNA with VTNRs.
2.Primers attached to the sticky ends at either side of the repeats and the whole repeat is amplified.
3.Different primers are used for each repeated base sequence.
4.A fluorescent tag is attached usually to the primer to be easily viewed under UV.
5.You finish with fragments of different lengths which correspond to the number of repeats at a specific location. E.g. at one location the repeat might be 50 nucleotides for one person and another person 80 nucleotides.
How to separate DNA fragments of different lengths?
Gel electrophesis: The fragments are placed into a well of agarose gel and covered in a buffer solution which conducts electricity. An electric current is passed through the gel.
DNA is negatively charged and so will move towards the positive electrode. Shorter, lighter fragments will move further and longer heavier fragments wont move so far.
The segments separate according to fragment length and form bands.
Analysis of gel electrophesis
After a certain amount of time the electricity is switched off and the gel is viewed under UV light. The DNA fragments are seen as bands.
One of the wells might have had a DNA ladder added to it. This contains DNA fragments of known lengths which allows you to calculate the length of the fragments which appear.
Two or more individuals bands can be compared. If they have the same bands, then they have the same length of VNTRs at the same location, so it’s a match.
