Genomes and Gene Technologies

Genomes and Gene Technologies

outline the steps involved in sequencing the genome of an organism

Genome – all the genetic information within an organism OR all the genetic information within an individual.

Genome sequencing – the technique used to give the base sequence of DNA of a particular organism.

Genomes are mapped to identify which part of the genome they have come from.
Samples of the genome are sheared (mechanically broken) into smaller sections of around 100,000 base pairs.
These sections are placed into separate bacterial artificial chromosomes (BACs) and transferred to coli cells. As these cells grow in culture, many clones of the sections are produced – clone libraries.
PCR: DNA is extracted from BACs and replicated. Different restriction enzymes are used to cut the DNA into smaller overlapping fragments.
Electrophoresis: The fragments are separated into size order. Computer programmes are used to reassemble the full BAC sequence by analysing the overlaps shown by the fragment sequences.

Automated DNA Sequencing Based on Interrupted PCR and Electrophoresis:

Sequencing DNA fragments was initially slow using radioactively labelled nucleotides. The development of automated sequencing has led to a rapid increase in the number of organism genomes sequenced and published in recent years.

The primer joins (anneals) at the 5’ end, allowing DNA polymerase to attach.
DNA polymerase adds free nucleotides by complementary base pairing the strand extends (PCR).
If a modified nucleotide is added, the DNA polymerase enzyme is ‘thrown off’ and replication stops at that point. The nucleotide is modified by adding different coloured fluorescent markers to different bases.
This process is repeated and many different sized DNA strands are created due to the random joining of modified nucleotides. Some may only have one additional nucleotide added to the primer, others may have more.
The different sized DNA strands run through the machine (electrophoresis) sorting them from smallest length first to the longest A laser reads the colour sequence of the modified nucleotide on the end of each DNA strand, displaying the sequence of bases formed. Complementary base pairing of this sequence will tell you the base sequence on the original DNA strand.

outline how gene sequencing allows for genome-wide comparisons between individuals and between species

Genomics – the study of the whole set of genetic information in the form of the DNA base sequences that occur in the cells of organisms of a particular species.

Comparative gene mapping – knowing the sequence of bases in a gene of one organism and being able to compare genes for the same (or similar) proteins across a range of organisms.

The DNA of all organisms contains sections known as genes which code for the production of polypeptides and proteins. However, this coding DNA is only 1.5% of the genome of humans. Much DNA is non-coding DNA and has been referred to as junk DNA, which is misleading as this non-coding DNA carries out a number of regulatory functions. Genomics is seeking to map the whole genome of an increasing number of organisms. Comparing genes and regulatory sequences of different organisms will help us to understand the role of genetic information in a range of areas including health, behaviour and evolutionary relationships between organisms.

Comparative gene mapping has a wide range of applications:

The identification of genes for proteins found in all/many living organisms shows the importance of these in life.
Comparing DNA/genes of different species shows evolutionary relationships. The more DNA sequences organisms share, the more closely related they are likely to be.
Modelling the effects of changes to DNA/genes can be carried out, e.g. testing the effects of mutations on genes obtained from yeast that are also found in the human genome.
Comparing genomes from pathogenic and similar but non-pathogenic organisms can be used to identify the genes that are most important in causing the disease, leading to the identification of targets for developing more effective drug treatments and vaccines.
The DNA of individuals can be analysed – reveal mutant alleles, or the presence of alleles associated with increased risk of particular diseases, e.g. heart attack or cancer.
define the term recombinant DNA

Recombinant DNA – the resulting DNA formed from DNA fragments from different organisms joined using restriction enzymes and ligase enzymes.

explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product

The two main reasons for carrying out genetic engineering are:

Improving a feature of the recipient organism – e.g. herbicide resistance, growth-controlling genes.
Engineering organisms that can synthesise useful products – e.g. hormone genes (insulin, growth hormones), Golden Rice^TM (beta-carotene production) and pharmaceutical chemicals (inserting genes into sheep so that they produce chemical products that can be easily collected from their milk.

There are 4 steps of genetic engineering:

Obtain the required gene.

mRNA is used as a template strand to make a copy of the gene using reverse transcriptase.
Use an automated polynucleotide sequencer to synthesise the gene.
Use a DNA probe to locate the gene on DNA fragments and cut using restriction enzymes.

Place a copy of a gene in a vector.

Gene is sealed into a bacterial plasmid using DNA ligase, or the gene is sealed into virus genomes or yeast cell chromosomes.

The vector carries the gene to the recipient cell.

The gene, one packaged in a vector, can form quite a large molecule that does not easily cross the membrane to enter the recipient cell. Methods used to get the vector into the recipient cell include:

Electroporation – high-voltage pulse disrupts the membrane.
Microinjection – DNA injected using a micropipette into the host cell’s nucleus.
Viral Transfer – uses the virus’ mechanism for infecting cells by inserting DNA directly.
Ti Plasmids (vectors) inserted into soil bacterium Agrobacterium tumefaciens – plants are infected with bacterium, inserting the DNA plasmid into the plant’s genome.
Liposomes (DNA wrapped in lipid molecules) – fat-soluble and can cross the lipid membrane by diffusion.

The recipient cell expresses the gene through protein synthesis.

describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes

Enzymes known as restriction enzymes (restriction endonucleases) are used to cut through DNA at specific points. A particular restriction enzyme will cut DNA wherever a specific base sequence occurs called the restriction site (usually less than 10 base pairs long). The enzyme catalyses the hydrolysis reaction that breaks the sugar-phosphate backbones of the DNA double helix. This gives a ‘staggered cut’, which leaves some exposed bases known as sticky ends.

outline how DNA fragments can be separated by size using electrophoresis

Electrophoresis – used to separate DNA fragments based on their size. This method relies on the substances within the mixture having a charge. When a current is applied, charged molecules are attracted to the oppositely charged electrode. The smallest molecules travel fastest through the stationary phase (a gel-based medium) and in a fixed period of time will travel furthest, so the molecules separate out by size.

describe how DNA probes can be used to identify fragments containing specific sequences

DNA probe – a short single-stranded piece of DNA (around 50-80 nucleotides long) that is complementary to a section of the DNA being investigated.

The probe is labelled one of two ways by attaching to the phosphate on nucleotides:

Radioactive marker – the location can be revealed by exposure to photographic film.
Fluorescent marker – emits a colour on exposure to UV light.

Different bases will have different coloured markers so that they can be distinguished.

outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments

The polymerase chain reaction is basically artificial DNA replication. It can be carried out on tiny samples of DNA in order to generate multiple copies of the sample. The differences between PCR and natural DNA replication include it can only replicate relatively short sequences of DNA (not entire chromosomes), the addition of a primer molecules (to start the process) and PCR involves a cycle of heating and cooling to separate and bind strands.

Primers – short, single stranded sequences of known bases (around 10-20 bases in length), that are the ‘starting point’ for DNA polymerase to bind – DNA polymerase enzymes cannot bind directly to single-stranded DNA.

The DNA polymerase enzyme is described as ‘thermophilic’ because it is not denatured by the extreme temperatures used in the process. The enzyme is derived from a thermophilic bacterium, Thermus aquaticus (Taq), which grows in hot springs at a temperature of 90^oC.

explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase

To join isolated fragments of DNA, an enzyme known as DNA ligase catalyses the condensation reaction that joins the sugar-phosphate backbones of the DNA double helix together. In order to join together DNA fragments from different sources, both fragments need to have originally been cut with the same restriction enzyme. This means that the sticky ends are complementary allowing the bases to pair up and form hydrogen bonds. DNA ligase then seals the sugar-phosphate backbone.

state other vectors into which fragments on DNA may be incorporated

Vectors carry the desired gene to the recipient cell. The most common vectors are bacterial plasmids sealed using DNA ligase. Others include virus genomes and yeast cell chromosomes. Vectors often have to contain regulatory sequences of DNA to ensure that the inserted gene is transcribed in the host cell.

explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product

Plasmid – a small circular piece of DNA. Plasmids are found in many types of bacteria and are separate from the main bacterial chromosome. Recombinant plasmids form from the joining of a plasmid and the desired gene using restriction enzymes and DNA ligase. However, it is important to remember that many cut plasmids will, in the presence of ligase enzymes, simply reseal to reform the original plasmid.

Large quantities of the plasmid are mixed with bacterial cells, some of which will take up the recombinant plasmid. The addition of calcium salts and ‘heat shock’ (temperature is lowered to around freezing, then quickly raised to 40^oC) increases the rate at which plasmids are taken up by bacterial cells. Even so, the process is very inefficient – less than a quarter of 1% of bacterial cells take up a plasmid.

Transformed bacteria – bacterial cells that take up the plasmid.

Transgenic organism – an organism that contains DNA that has been added to its cells as a result from genetic engineering.

Conjugation:

Conjugation is the process in bacteria where genetic material is exchanged. In this process, copies of plasmid DNA are passed between bacteria, sometimes even of different species.

A conjugation tube forms between a donor and a recipient. An enzyme makes a nick in the plasmid.
Plasmid DNA replication The free DNA strands starts moving through the tube.
In the recipient cell, replication starts on the transferred DNA.
The cells move apart and the plasmid in each forms a circle.

Evidence for Conjugation:

R-strain of Pneumococcus = mouse lives.
S-strain of Pneumococcus = mouse dies.
It was found that only mice infected with the S-strain were killed by a protein that was toxic – S-strain must have the instructions to make the protein but the R-strain does not.
Heat-treated S-strain of Pneumococcus = mouse lives – no protein synthesis.
R-strain and heat-treated S-strain of pneumococcus = mouse dies.

The R-strain was capable of taking up DNA from their surroundings (DNA from S-strain), so synthesis of the toxic protein occurred and killed the mouse.
describe the advantages to microorganisms of the capacity to take up plasmid DNA from the environment

The advantage to the bacteria of conjugation is that it may contribute to genetic variation. Bacteria can gain useful characteristics, so they’re more likely to have an advantage over other microorganisms, which increases their chance of survival. Plasmids may contain:

Genes that code for resistance to antibiotics, e.g. genes for enzymes that break down antibiotics.
Genes that help microorganisms invade hosts, e.g. genes for enzymes that break down host tissues.
Genes that mean microorganisms can use different nutrients, e.g. genes for enzymes that break down sugars not normally used.
outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid

Plasmids are carry ampicillin and tetracycline resistant genes are chosen as they are resistant to the antibiotics ampicillin and tetracycline. The resistance genes are known as genetic markers.
The plasmids are cut by a restriction enzyme that has its target site in the middle of the tetracycline resistance gene. The gene for tetracycline resistance is broken up and does not work, however, the gene for ampicillin resistance still works.
Replica Plating:

The plasmids are mixed with bacteria, some of which will take up the plasmids. The bacteria are grown on standard nutrient agar, so all bacterial cells grow to form colonies.
Some cells from the colonies are transferred onto agar that has been made with ampicillin – only those that have taken up a plasmid will grow.
Some cells from the colonies are transferred onto agar that has been made with tetracycline – only those that have taken up a plasmid that does not have the insulin gene will grow.

By keeping a track of which colonies are which, we know that any bacteria that grow on the ampicillin agar, but not on the tetracycline agar, must have taken up the plasmid with the insulin These bacteria can then be grown on a large scale to harvest insulin.

outline the process involved in the genetic engineering of bacteria to produce human insulin

People who cannot produce the hormone insulin suffer from type I diabetes mellitus. Until the early 1980s, insulin was extracted from the pancreatic tissues of slaughtered pigs, which is not identical to human insulin, is less effective and very expensive to produce, since only a very small amount of insulin is present in pancreatic tissue.

mRNA is extracted from the β-cells of the pancreas.
Reverse transcriptase is used to synthesise a complementary DNA strand.
DNA polymerase and DNA nucleotides produce a copy of the original gene (cDNA gene) in a PCR machine.
Plasmid is extracted from coli and cut open with restriction enzymes at the restriction site.
The plasmid is mixed with the cDNA genes and some plasmids take up the gene. DNA ligase then seals up the plasmids creating a recombinant plasmid.
The plasmids are mixed with bacteria, some of which take up the recombinant plasmids.
The bacteria are grown on agar plates, where each bacterial cell grows to produce a colony.

outline the process involved in the genetic engineering of ‘Golden Rice^TM’

Golden Rice^TMis a type of genetically engineered rice produced to reduce vitamin A deficiency. Vitamin A (retinol) in the diet only comes from animal sources. In vegetarians and those without access to meat, vitamin A is derived from the intake of beta-carotene (precursor), which is converted to active vitamin A in the gut.

Rice plants (Oryza sativa) contain the gene that code for the production of beta-carotene. Unfortunately, in the plant of the plant that is eaten – the endosperm (grain) – the genes for beta-carotene production are switched off. It was found that insertion of two genes into the rice genome was needed in order for the metabolic pathway to create beta-carotene to be activated in the endosperm cells.

The genes that give Golden Rice^TM its ability to make beta-carotene in the endosperm are obtained from daffodil plants (Phytoene synthetase) and from the soil bacterium Erwinia uredovora (Crt 1 enzyme) using restriction enzymes.
A plasmid is removed from the Agrobacterium tumefaciens bacterium and cut open with the same restriction enzymes. The genes that code for the enzymes Phytoene synthetase and Crt 1 enzyme are inserted into the plasmid using ligase enzymes.
The recombinant plasmid is put back into the bacterium. Rice plant cells are incubated with the transformed tumefaciens bacteria, which infect the rice plant cells.
tumefaciens bacteria inserts the genes into the plant cells’ DNA, creating transformed rice plant cells.

outline how animals can be genetically engineered for xenotransplantation

Xenotransplantation – transplantation of cell tissues or organs between animals of different species.

Allotransplantation – transplantation of cells tissue or organs between animals of the same species.

Engineered Pigs as Organ Donors:

α-1,3-transferase is the enzyme a key trigger in transplant rejection in humans. In 2003, it was reported that pigs engineered to lack the enzyme α-1,3-transferase had been successfully developed. In 2006, scientists reported that engineering of the human nucleotidase enzyme (E5’N) into pig cells in culture reduced the active of a number of immune cell activities involved in xenotransplant rejection. These developments have encouraged xenotransplantation between pigs and humans.

explain the term gene therapy

Gene Therapy – adding new alleles to the DNA of cells to treat genetic disorders – we are only able to treat recessive disorders.

In simple terms, if the working copy of a gene is placed into cells that contain only the dysfunctional copies of that gene, then transcription of the added working copy will mean that the individual may no longer have the symptoms associated with the genetic disorder.

explain the difference between somatic cell gene therapy and germ line cell gene therapy

Somatic Cell Gene Therapy:

This method uses fully differentiated cells (adult, fully-grown, developed cells found in the body) with specific genes switched on or off to suit their requirements.

Augmentation (adding genes) – applies to conditions that are caused by inheritance of faulty alleles and leads to the loss of a functional gene product. Engineering a functional copy of the gene into relevant specialised cells means that the polypeptide is synthesised and the cells can be function normally. However, this can only happen with recessive diseases.
Killing Specific Cells – e.g. cancers, genetic techniques can be used to make cancerous cells express genes to produce proteins (such as cell surface antigens) that make the cells vulnerable to attack by the immune system could lead to targeted cancer treatments.

Germline Cell Gene Therapy:

This method uses undifferentiated, totipotent cells (zygote, early embryonic cells, sperm, egg cells). Cells of an early embryo are stem cells – it can divide and specialise to become any cell type within the body. Engineering a gene into these stem cells or sperm, eggs or zygotes means that as the organism grows every cell contains a copy of the engineered gene. This gene can then function within any cell where that gene is required. Although wildly employed in experimental animals, germline gene therapy in humans is illegal and ethically unacceptable.

discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms