Unit 4 Human Biology Flashcards
Restriction Enzymes
Because DNA molecules are very long, often consisting of millions of base pairs, restriction enzymes cut the DNA at specific points to make smaller fragments known as restriction fragments.
- The fragments can be separated using gel electrophoresis & recombinant DNA.
- Particular repeat sequences can be cut out by restriction enzymes:
— Short tandem repeats (STRs).
— Restriction fragment length polymorphism (RFLPs).
— Variable number tandem repeats (VNTRs).
STRs, RFLPs, VNTRs
- ≥90% of DNA does not carry nucleotide triplets that code for proteins.
- Some of the non coding regions (introns) consist of repeated sequences of nucleotides.
- The number of repetitions in any one section of DNA varies from one individual to the next.
Short Tandem Repeats
- Occurs wen a pattern of two or more nucleotides is repeated & the repeated sequences are adjacent to each other.
- Pattern can range in length from 2 to 10 base pairs.
- Typically non-coding intron region.
- Count of repeats of a specific STR at a given locus can create unique genetic profile.
- Currently over 100 000 published STR sequences in human genome.
- Prevalent method for determining genetic profiles in forensic cases.
- Analysis is performed by extracting nuclear DNA from cells of interest.
- DNA is amplified using PCR.
- Tested by gel electrophoresis or capillary electrophoresis.
- Applications: forensics (crime, mass disaster, paternity testing, military DNA ‘dog tag’, convicted criminal DNA databases), bone marrow transplant follow-up (important for establishing graft rejection & disease relapse).
Variable Number Tandem Repeats
- These can be found on many chromosomes & often show variation in length - the repeat number may vary from 1 - 30 repeats of 8 - 50 base pairs.
- Each variant acts as an inherited allele allowing use for identification.
- These repeat regions are usually bounded by specific restriction enzyme sites.
Restriction Fragment Length Polymorphism
- Variation in the DNA sequence of a genome detected by breaking DNA into pieces with restriction enzymes.
- Analyse fragments by gel electrophoresis.
- Important tool in genome mapping, localisation of genetic disease genes, determination of risk for a disease, genetic fingerprinting & paternity testing.
- Applications: agriculture (direct method for selecting desirable genes such as disease resistance), forensics, genetic mapping (determine disease status of an individual (eg. Huntington’s disease, cholera), cystic fibrosis, sickle cell anaemia), genetic counselling.
STR
Short tandem repeat
RFLP
Restriction fragment length polymorphism
VNTR
Variable number tandem repeats
Steps in DNA Identification
- Isolate DNA & make copes (PCR).
- Cut the DNA into shorter fragments that contain STRs (using restriction enzymes).
- Sort the DNA by size (gel electrophoresis).
- Compare samples to identify a person.
PCR
- Polymerase Chain Reaction
- Can produce many copes of a specific target segment of DNA.
- Three step cycle - denaturing (heating), annealing (cooling), extension (replication) - that brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
- Can copy different lengths of DNA, it does not have to copy the whole length of a DNA molecule:
→ One gene
→ Several genes
→ Lots of genes - Artificial process which imitates natural DNA replication.
Reagents Needed for PCR
- DNA sample which is wanted to be replicated.
- Taq DNA Polymerase - enzyme that works at high temperatures.
- Nucleotides (called dNTPs).
- Pair of primers
→ One primer binds to the 5’ end of one of the DNA strands.
→ The other primer binds to the 3’ end of the other anti-parallel DNA strand.
→ Delineate the region of DNA that is to be amplified.
PCR Steps
- Denaturing: heated to 92ºC briefly to separate strands by breaking H-bonds.
- Annealing: cooled to 55ºC to allow primers to form H-bond with ends of target sequence.
- Extension: heated to 72ºC, DNA polymerase adds nucleotide bases from the 3’ end of each primer.
Gel Electrophoresis
- The different sized fragments are separated by a process called gel electrophoresis.
- The separation takes place in a sheet of a firm but jelly-like substance (a ‘gel’).
- Samples of the DNA extracts are placed in shallow cavities (‘wells’) cut into one end of the gel (in the negative end).
- A voltage is applied to opposite ends of the gel.
- DNA has a negative charge & moves slowly towards the positive end.
- The shorter fragments travel through the gel faster than the longer fragments, creating ‘lanes’.
Genetic Fingerprinting
- DNA analysis can be sued for catching criminals, establishing parentage, finding how closely organisms are related & many other applications.
- The pattern of bands in a gel electrophoresis is known as a genetic fingerprint or a ‘genetic profile’.
- If a genetic fingerprint in a sample of blood or other tissue at the scene of a crime matches the genetic fingerprint of a suspect, this can be used as evidence.
- A DNA sample can be obtained from the suspect using blood, check epithelial cells taken from the mouth lining of even the cells clinging to the root of a hair.
DNA Sequencing
- Modified nucleotides called dideoxyribonucleotides (ddNTPs) attach to synthesised DNA strands of different lengths.
- Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment.
- The DNA sequence can be read from the resulting spectrogram.
- Can only sequence 750 base pairs at a time.
- Genome must be broken up into sections.
- Done a number of times with overlapping pieces.
- Overlapping sections analysed & put back together.
Analysing Gene Expression
- Nucleic acid probes can hybridise with mRNAs transcribed from a gene.
- Probes can be used o identify where or when a gene is transcribed in an organism.
Studying Expression of Single Genes
- Changes in the expression of a gene during embryonic development can be tested using:
→ Northern blotting.
→ Reverse transcriptase-polymerase chain reaction. - Both methods are used to compare mRNA from different developmental stages.
Northern Blotting
Combines gel electrophoresis of mRNA, followed by hybridisation with a probe on a membrane.
Reverse Transcriptase-Polymerase Chain Reaction
- Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest.
- The products are run on a gel & the mRNA of interest identified.
- Quicker & more sensitive.
Recombinant DNA
DNA produced by combining DNA from different sources.
Vectors
In DNA cloning, the plasmid/chromosome used to carry the cloned DNA segment to a desired location (frequently a virus or liposome is used).
Restriction Enzyme
Enzyme that cuts DNA at a specific sequence of nucleotides.
Gel Electrophoresis
Procedure used to separate & analyse DNA fragments by placing a mixture of DNA fragments at one end of a porous gel & applying an electrical voltage to the gel.
PCR Definition
Technique that allows molecular biologists to make many copes of a particular gene.
Plasmid
A circular DNA molecule found in bacteria.
Cloning
Process of making a genetically identical organism.
Transgenic Organism
An organism that contains genes from another organism.
Recombinant DNA Process
- Plasmid isolated with RE.
- DNA isolated with RE.
- Gene inserted into plasmid.
- Plasmid put back into bacterial cell.
- Cell multiplies with gene of interest.
- Copies made of gene, cell & protein.
Cutting & Sticking
- Recombinant DNA made by cutting DNA & sticking it together. REs from bacteria cut DNA at specific points.
- REs cut DNA at the restriction site on the DNA.
- REs cut leaves some exposed single strand bases known as a ‘sticky’ end.
- Recombinant DNA made by cutting DNA & sticking it back together.
- Separate fragments of DNA joined into plasmid by enzyme DNA ligase.
- Both pieces of DNA but must have been cut by the same RE so that the sticky ends are complementary.
- The DNA is now called recombinant DNA.
Bacterial Cells & Plasmids
- Plasmids cut with restriction enzyme used to isolate the chosen gene.
- Complimentary sticky ends formed.
- Plasmid & gene mixed & they combine.
- Plasmid then seals & forms recombinant plasmid with help of ligase enzyme.
- Plasmids mixed with bacterial cells which take up plasmid.
- Less than 1% of bacteria take up the plasmid & they are now known as transformed bacteria/transgenic bacteria (changed genes).
Examining the Colonies
Three types of colony form:
- Bacteria that did not take up the plasmid.
- Bacteria containing a plasmid that did not seal in a copy of the DNA.
- Bacteria containing the new recombinant plasmid known as transformed bacteria.
Identifying Transformed Bacteria
- Original plasmids have antibiotic resistance gene (used as a genetic marker) for the antibiotics ampicillin & tetracycline.
- Plasmids cut in the middle of the tetracycline gene & insulin gene inserted means there is no longer any resistance to tetracycline.
- Bacteria then grown on agar plates.
- Then transferred onto plates treated with ampicillin to see if they have the plasmid & will grow.
- Then some transferred onto plates treated with tetracycline to see if they still grow or not. If they do not, the contain the insulin gene.
- This is called replica plating.
Medical Applications of Recombinant DNA
- Production of pharmaceuticals for treatment of diseases (eg. human insulin, interferons).
- Production of pharmaceuticals for disease prevention (eg. vaccine (hepatitis B vaccine)).
Examples of Genetic Engineering
- If a person has T1 diabetes & cannot produce insulin, it is possible through genetic engineering to add the insulin gene to the body, so the person can produce insulin.
- The first human protein made commercially suing engineered bacteria was human insulin, but many other hormones & proteins are now being produced. In addition, many recombinant vaccines have been produced.
- Many human proteins that were formerly extremely expensive to produce because they were found in human tissues only in small amounts can now be made in large amounts from cloned genes.
- Many recombinant vaccines have been produced. These include live recombinant, vector, subunit & DNA vaccines.
Gene Therapy
- Viruses are often sued because they have the ability to enter a cell’s DNA.
- The virus particles are modified so that they cannot cause a disease. Then a DNA fragment containing a replacement gene is spliced to the viral DNA.
Somatic Cell Gene Therapy
- Certain genes switched on & off in differentiated cells.
- Augmentation: adding functioning genes into the relevant specialised cells meaning that the protein can be made & the cell will function normally.
- Killing specific cells: making cancer cell express genes producing proteins that make their cells vulnerable to attack by the immune system for targeted cancer treatments.
- Artificially replace the disease-causing gene with a normal allele.
- The normal allele can be carried by a virus vector to the target tissues (eg. treatment for cystic fibrosis).
- The patient is the infected with T he modified virus particles, which should carry the gene into cells to correct the genetic defects.
- Unfortunately, these experiments have not been very successful.
- Gene therapy remains a high-risk, experimental procedure.
Germline Gene Therapy
- Stem cells that could become any type of cell or a new human being.
- Germline therapy is changing the genes of the first few cels meaning that the organisms are different.
- Somatic cell therapy is changing genes on part of the body, however the disease can still be inherited as the gene still exists in every body cell & gametes.
- Germline gene therapy is altering the organisms genes before development into a foetus, however this is illegal in humans due to:
- Potential for creation of new diseases.
- Human evolution would be interfered with.
- Too many ethical & moral issues with changing a human genome.
Issues: Somatic Gene Therapy
- Getting genes into target cells is difficult.
- Treatment if short-lived & has to be repeated.
- Difficult to get the gene functioning in the genome.
- Only the actual patient is affected.
Issues: Germ Line Gene Therapy
- More straightforward to deliver gene into cell.
- All subsequent cells will have the functioning gene.
- Considered unethical.
- Genetic manipulation passed on to children - could be a good or bad thing.
Cloning
- A member of a population of genetically identical cells produced from a single cell.
- Researchers hope that cloning will enable them to make copies of transgenic animals to help save endangered species.
Clonal Propogation
- A source of tissue or organ for transplantation.
- Avoids all problems of immunoincompatibility.
Sickle-Cell Anaemia: Identifying
Replaces GAG with GTG at the MstII site.
Transforming Bacteria
- When organisms contain genes from another species, they are called transgenic.
- Transgenic bacteria now produce important substances useful for health & industry.
- These transformed bacteria produce proteins cheaply, quickly & abundantly.
- Eg: human insulin, clotting factor for people with haemophilia.
Pharming
- Animals used to create/reproduce a human gene which can be isolated and used for human use.
- Transgenic sheep used to carry a gene for a human blood protein, which they secrete in their milk. This protein inhibits an enzyme that contributes to lung damage in patients with cystic fibrosis, emphysema & other chronic respiratory diseases.
- Golden rice: transgenic rice produces beta carotene (used in human bodies to make Vit. A) that gives the rice its golden colour & their increased nutritional value. Vit. A deficiency can lead to blindness & infection.
Xenotransplantation
- 60% transplant patients die on transplant list.
- We can now consider xenotransplantation - transplantation from other species.
- Pigs have been genetically engineered so they do not produce an enzyme involved in organ rejection.
Xenotransplantation Problems
- Different organ size.
- Pigs only live to 15 years, so organs may age quickly.
- Pigs’ body temperature is 39ºC, whereas ours is 37ºC.
- Animal welfare groups think killing animals for organisms is wrong.
- Certain religious groups cannot eat pork, so could not have transplants.
- Possible disease transfer between animals & humans.
Genetic Engineering: Risks & Ethical Concerns
- Risk to researcher - using crippled strains.
- Possible ecological damage from pollen transfer between GM & wild crops - superweeds.
- Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars.
Human Genome Project: Risks & Ethical Concerns
- How can we be sure that a transferred gene makes the appropriate amount of a protein & is expressed at the right time/place.
- What do we reserve gene therapy for - serious diseases, athletic enhancement, appearance, intelligence?
- Reduced genetic diversity.
Summary of Applications of DNA Technology
- Medicine: bacteria produce human proteins.
- To identify people.
- To diagnose genetic diseases.
- To find & identify all of the human genes.
- Gene therapy.
- Genetically engineered goods.
- Agriculture: bacteria that decompose nitrogen faster, disease & pest resistant plants, bigger, sweeter, more nutritious plants, bigger, leaner animals.
- Industry: bacteria used to break down pollutants.
Mutations: Genetics Overview
- DNA = deoxyribonucleic acid.
- DNA carries the instructions for making all the structures & materials the body needs to function - in particular, proteins.
- A chromosome consists of segments of DNA known as genes.
- Genes contain the instructions for the construction of a particular protein.
- It is estimated that there are about 20 000 - 25 000 genes in the human genome (about 3 bn base pairs).
- The sequence of bases in a gene is a code instructing the cell how to construct a particular protein.
Modes of Inheritance
- Dominant alleles cover or override the effect of an alternative form of a gene (allele).
- Capital used to indicate the allele.
- Recessive alleles are not expressed unless there are two, one on each homologous chromosome.
- Use a lower case letter to indicate the allele.
- Homozygous (purebred): the individual has the same alleles for a particular allele pair (tt or TT).
- Heterozygous (hybrid): the individual has different alleles for an allele pair (Tt).
- Genotype is the genes an individual has (TT/Tt/tt).
- Phenotype is the observed physical expression of the allele of the observed characteristic.
Co-Dominant Inheritance
- Where two alleles are equally dominant.
- A third phenotype is produced.
- Eg. sickle cell anaemia.
Sickle Cell Anaemia
- Haemoglobin S is produced in large amounts.
- This causes RBCs to become distorted in shape, reducing their oxygen carrying capacity & decreasing their ability to travel through capillaries.
- This results in capillary blockage & damage to the area.
- RBCs with haemoglobin S have an average life of 16 days.
- Results in anaemia.
Sex-Linked Inheritance
- The allele for a specific trait is carried on the X-chromosome.
- Males cannot be carriers of the trait.
- These traits show up more frequently in the male population.
Multi-Allelic Inheritance
- Instead of two alleles for a trait, there are ≥3.
- Human blood types are multi-allelic & co-dominant.
Polygenic Inheritance
- Polygenic traits are determined by more than one pair of genes.
- Polygenic phenotypes exhibit continuous variation, since each different gene permutation results in just a small phenotypic change.
- Many medical conditions such as autism, cancer & T2 diabetes are polygenic.
- Often continuous traits.
- Responsible for many phenotypic traits.
- Eg. skin pigmentation, height, intelligence, stature.
- These traits all results from the interaction of the genes with environmental factors.
- Many traits such as height, shape, weight, colour & metabolic rate are governed by the cumulative effects of many genes.
Gene Expression & Environment (Skin Pigmentation)
- Results from the pigment, melanin, which occurs mainly in the skin & hair follicles.
- Melanin is produced in organelles called melanosomes, which occur in melanocyte cells in the basal layer of the epidermis.
- Melanosomes are transferred to keratinocytes (the principal cell type in the epidermis).
Skin Colour
Depends on:
- Rate of melanin synthesis.
- Relative amounts of brown-black pigments & red-yellow pigments.
- Number & size of melanosomes.
- Rate of transfer of melanosomes from the melanocytes to the keratinocytes.
Tanning Reaction
- Ultra violet (UV) radiation in sunlight can cause severe damage to the DNA in the skin & underlying tissues.
- Melanin acts like a natural sunscreen & protects the tissues from UV damage.
- When exposed to UV, melanocytes are simulated to produce more melanin. This is often referred to as the tanning reaction.
Regulation of Pigmentation
- The regulation of pigmentation is very complex - more than 120 genes have been identified.
- When keratinocytes are exposed to the sun’s rays, they produce melanocyte stimulating hormone (MSH).
- MSH stimulates receptors on the surface of melanocytes, causing them to produce more melanin.
Chromatin
- The DNA associated with proteins (histone S) that form chromosomes.
- It appears as dark-staining, granular material in the cell nucleus.
Epigenetics
- Epi = on/over.
- Epigenetics = on/over the genetic code.
- Inheritable by dividing cells.
- The changes in gene expression that do not involve changes in the cell’s DNA but can be passed on from generation to generation.
- Epigenetic events explain the observation that cells with the same genotype can have more than one phenotype.
- Epigenetic events generally act like an on/off switch, either silencing or activating a specific gene affecting a protein production.
Histones
- It is estimated that every cell nucleus contains 2-3m of DNA.
- To prevent the very fine strands of DNA becoming tangled, the filaments are coiled around special proteins called histones.
- As well as organising DNA, histones also affect gene expression.
Epigenetic Mechanisms
- Knowledge of epigenetic mechanisms is still developing.
- DNA methylation, histone modification & prion proteins are all known to have important epigenetic effects.
- Epigenetic processes accumulate during a person’s life.
- Epigenetic processes are believed to be important in the development of cancer & in the differentiation of embryonic tissues.
DNA Methylation
Methyl tags attached to DNA bases repress gene expression (‘silence’ genes).
Histone Modification
Molecules attached to the histone tail alters the activity of the DNA wrapped around them.
Changes to Epigenome
→ Nutrition
- Shortages or excesses of food during adolescence leads to risk of obesity, diabetes & early puberty.
- During times of famine, the P-generation may have an epigenetic shift where their needs for certain nutrients are increased.
- If this is passed to their offspring, in time of surplus, the F1 may become overly nourished & develop health risks.
- This also occurs if the P generation is overly-nourished & their offspring then live in famine. They would be less likely to handle smaller portions.
→ Parenting
- Mother rats that infrequently groom & nurse their pups rear anxious offspring.
- This alters genes controlling the production of stress hormone.
- Nature’s way of preparing young for a potentially dangerous environment.
- Fathers affect epigenome as well.
- Crop fungicide vinclozolin can cause susceptibility to cancer & kidney defects.
- Mice fed with cocaine passed on memory failure to three generations.
Cancer Epigenetics
- Originally thought that methylation in cancer was an epiphenomenon, meaning that it was random & arose after the cancer did, not playing a role in the formation of cancerous cells.
- Differed from general genetics:
— Reversible
— Position effects (acts over larger distances than intended).
— High frequencies of mutations. - Gene silencing: essentially turning on & off genes.
- Hypermethylation leads to silencing of tumour suppressor genes.
Jumping Genes
- Transposons (also known as ‘jumping genes’ or transposable genetic elements) are discrete pieces of DNA that can move around to different sites along a chromosome.
- A large proportion of the human genome consists of transposons.
Transposing Mechanisms
- Several types of transposons have been discovered.
- Some use a direct ‘cut & paste’ mechanism (top diagram).
- Others are first coped to RNA & then back to DNA before being inserted into a new location (reverse transcription or retrotransposon mechanism) (lower diagram).
- Transposons can act as mutagens - they can alter gene expression. They can often activate or disable a gene but sometimes have no observable effect.
- Some transposons resemble retroviruses (viruses such as those responsible for AIDS & certain cancers) & may even be derived from them.
Prions
- Infectious protein.
- Normal body proteins that get converted into an alternate configuration by contact with other prion proteins.
- They have no DNA or RNA.
- The main protein involved in human & mammalian prion diseases is called ‘PrP’.
Prion Diseases
- Prions form insoluble deposits in the brain.
- Causes neurons to rapidly degenerate.
- Mad cow disease (eg. bovine spongiform encephalitis: BSE)
- People for New Guinea used to suffer from kuru, which they got from eating the brains of their enemies.
Allele Frequency: Gene Pool
A gene pool is the sum of total genes, with all their variations, possessed by a particular species in a particular place at a particular time.
Changes in Gene Pool
The allele frequency in a population’s gene pool can be affected by evolutionary mechanisms, such as natural selection, or by change occurrences, such as founder effect & random genetic drift (RGD).
Mutations
- A permanent structural alteration in an organism’s DNA that has never been seen previously in ancestors.
- An process by which the base pair sequence of a DNA molecule is altered.
- An important source of genetic variation.
- In most cases, DNA changes either have no effect or cause harm, but occasionally can improve an organism’s chance of surviving.
- Mutations in reproductive cells can be passed on to an organism’s descendants.
- Mutation rate: the number of mutations occurring or estimated to occur per generation or per nucleotide pair.
- Somatic mutation: mutation occurs but is not passed on to the next generation.
- Germ line mutation: mutation occurs in gametes & is passed to the next generation, in both its somatic & germ line cells.
- Without mutations, there would be no new alleles, new genes or evolution.
- Three main types: deletion (section of chromosome is deleted), duplication (section of chromosome is duplicated) or inversion (section of chromosome is inserted upside-down).
- Many human conditions result from genetic mutations.
- Some, such as sickle cell trait, are beneficial in some situations, whereas others, such as cancer, can be lethal.
- Natural selection determines which mutations remain in a gene pool & which ones are eliminated.
Natural Selection (Gene Pools)
- Determines which mutations remain in a gene pool & which ones are eliminated.
- Provides a ‘plausible’ explanation for evolution.
- Scientific theory proposed by Charles Darwin & Alfred Wallace that organisms best adapted to their environment tend to survive & out-multiply those that are less well adapted.
- The allele frequency is the relative frequency of a particular allele in a population. This ranges from 0 to 100%.
- NS affects the frequency of the alleles in a population’s gene pool. The frequency of alleles determining favourable features increases & the frequency of the alleles determining unfavourable features decreases.
Natural Selection (Gene Pools)
- Determines which mutations remain in a gene pool & which ones are eliminated.
- Provides a ‘plausible’ explanation for evolution.
- Scientific theory proposed by Charles Darwin & Alfred Wallace that organisms best adapted to their environment tend to survive & out-multiply those that are less well adapted.
- The allele frequency is the relative frequency of a particular allele in a population. This ranges from 0 to 100%.
- NS affects the frequency of the alleles in a population’s gene pool. The frequency of alleles determining favourable features increases & the frequency of the alleles determining unfavourable features decreases.
Population Size: Large
- Individuals make only a small contribution to the gene pool.
- Evolution occurs slowly.
- Most changes are adaptive.
- NS is the main driving force.
Population Size: Small
- Individuals make up a relatively large contribution to the gene pool.
- Evolution can occur rapidly.
- Many changes are non-adaptive.
- Many changes are due to chance events.
Artificial Selection
- The selective breeding of domesticated plants & animals for a particular trait by man.
- Results with variety within a species.
Terms: Macroevolution
The origin of taxonomic groups higher than the species level.
Terms: Microevolution
A change in a population’s gene pool over successive generations.