Title: InkBiology / Gene regulation, genomics and biotechnology
On every note of this deck, unless a note says otherwise:
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Front: DEFINE
Define: Complementary DNA (cDNA).
Back: DNA made from an mRNA template by reverse transcriptase, and therefore containing no introns.

Front: CHECK YOURSELF
A biotechnology company offers a mail-order test which, from a saliva sample, reports on a microarray of 700 000 sites and returns a list of conditions the customer is 'at increased risk of'. Evaluate this service: explain what the technique can genuinely establish, three limitations of the information it provides, and one argument on each side of the question of whether such tests should be sold directly to the public.
Back: What it can establish is real. Saliva contains cells, so DNA can be extracted and amplified by PCR. Washing the labelled DNA over a microarray whose spots carry known probe sequences shows, by hybridisation, which of those 700 000 variants the customer carries. As a determination of genotype at those sites, the result is likely to be accurate.
The first limitation is that a variant associated with a condition is not a cause of it. Most of the associations reported by these tests come from studies that found a particular variant slightly more often in affected people than in unaffected ones. A relative risk of 1.3 against a background risk of 2 per cent moves the absolute risk to about 2.6 per cent, which is a difference most people would ignore if it were presented that way rather than as 'increased risk'.
The second is coverage of the wrong kind. An array tests the sites that were put on it and no others, so a negative result is not reassurance: a customer may carry a rare pathogenic variant in the very gene being reported on and be told nothing, because that variant was not among the 700 000. Nor does an array detect the insertions, deletions and structural changes that cause a great deal of genetic disease.
The third is that risk depends on far more than genotype, since environment, diet, age, smoking and family history all contribute, and no counselling is attached, so the customer receives a probability with nobody to explain what it means or what to do.
For direct sale, the strongest argument is autonomy: the information is about the customer's own body, adults are entitled to it without a doctor's permission, and some people act on it usefully. Against it, the strongest argument is harm from misunderstanding: a result presented without counselling can prompt unnecessary anxiety, unnecessary surgery or false reassurance, it discloses information about relatives who never consented, and the burden of sorting out the confusion falls on health services that had no part in selling the test.

Front: DEFINE
Define: DNA methylation.
Back: The addition of methyl groups to cytosine bases; heavy methylation of a promoter reduces or prevents transcription of that gene.

Front: SPOT THE ERROR
Say what is wrong with this claim:
A liver cell keeps the genes it needs and throws the rest away.
Back: Nothing is thrown away. A neurone's nucleus holds the insulin gene complete and undamaged, and a β cell holds every gene a neurone uses. What differs is which are transcribed, which is why a nucleus from an adult udder cell produced a whole sheep in 1996.

Front: DEFINE
Define: RNA interference.
Back: The inhibition of gene expression by small RNA molecules binding to complementary mRNA.

Front: DEFINE
Define: Epigenetics.
Back: Heritable changes in gene expression caused by chemical modifications to DNA and to the histones associated with it, without any change to the base sequence.

Front: DEFINE
Define: Proto-oncogene.
Back: A normal gene whose product stimulates cell division in response to a growth signal.

Front: DEFINE
Define: Transgenic organism.
Back: An organism containing DNA transferred into it from another species.

Front: DEFINE
Define: DNA probe.
Back: A short single-stranded length of DNA, labelled radioactively or fluorescently, with a base sequence complementary to a target sequence.

Front: DEFINE
Define: Frame shift.
Back: The displacement of the reading frame caused by insertion or deletion of a number of bases that is not a multiple of three.

Front: CHECK YOURSELF
A company wants E. coli to manufacture a human protein. Describe, in order, how they would obtain the gene, insert it into the bacteria and identify the cells that will produce the protein, giving a reason for each choice you make.
Back: Obtain the gene from mRNA rather than from a chromosome. Choose a human cell type that produces the protein in quantity, extract its mRNA, and use reverse transcriptase to make a single strand of complementary DNA against it; DNA polymerase then builds the second strand. The reason is introns: a bacterium has no spliceosomes, so a gene taken from a chromosome would be transcribed and translated complete with its intron sequence and would yield no functional protein. The gene machine would serve equally well, and would allow the codons to be chosen to suit E. coli.
Cut the cDNA and a plasmid with the same restriction endonuclease, so that both carry complementary sticky ends. Mix them, and the overhangs pair by hydrogen bonding; add DNA ligase to form the phosphodiester bonds that make the join permanent. The plasmid must already carry an origin of replication so that the bacterium copies it, a promoter the bacterium's own RNA polymerase recognises placed in front of the insertion site, and a marker gene.
Transform the bacteria: suspend them in ice-cold calcium chloride and give a brief heat shock, or use electroporation, to make the membranes permeable so that plasmids can enter.
Identify the successful cells with the marker. If the marker is an antibiotic resistance gene, grow the culture on agar containing that antibiotic and only cells carrying a plasmid survive. If the gene was inserted into the middle of a second marker, a fluorescent protein gene say, then among the survivors it is the colonies that do not fluoresce that carry the insert, because the insertion has interrupted that gene. Those colonies are cultured on a large scale in a fermenter and the protein extracted.
A good answer notices what could still go wrong. A bacterium cannot carry out post-translational modification, so a protein needing particular sugars added in a Golgi apparatus has to be made in yeast or in mammalian cell culture instead, however well the transformation worked.

Front: DEFINE
Define: Metastasis.
Back: The spread of cells from a malignant tumour through the blood or lymph to form secondary tumours.

Front: DEFINE
Define: Hybridisation.
Back: The binding of a probe to a complementary single-stranded target sequence by hydrogen bonds between complementary bases.

Front: DEFINE
Define: Mutation.
Back: A change in the base sequence of DNA.

Front: DEFINE
Define: Reverse transcriptase.
Back: An enzyme, obtained from retroviruses, which catalyses the synthesis of DNA from an RNA template.

Front: DEFINE
Define: siRNA.
Back: Small interfering RNA: short double-stranded RNA fragments, one strand of which guides a protein complex to a complementary mRNA so that the mRNA is broken down and cannot be translated.

Front: DEFINE
Define: Histone acetylation.
Back: The addition of acetyl groups to histone tails, which reduces their positive charge, loosens the chromatin and increases transcription.

Front: DEFINE
Define: Promoter.
Back: A region of DNA at the start of a gene to which RNA polymerase and transcription factors bind.

Front: SPOT THE ERROR
Say what is wrong with this claim:
Gene therapy changes your DNA, so any children you have will inherit the change.
Back: Somatic gene therapy alters body cells such as lung epithelium, bone marrow and retina, and does nothing to the cells that become sperm or eggs, so nothing is inherited. Germ line therapy would be inherited, which is why countries that have legislated have prohibited it.

Front: DEFINE
Define: Oestrogen response element.
Back: A specific base sequence near a target gene to which an oestrogen-receptor complex binds, increasing the rate of transcription.

Front: CHECK YOURSELF
A drug inhibits histone deacetylase enzymes in a group of cells. Predict its effect on the transcription of genes in those cells, explain your reasoning in terms of charge, and suggest why such a drug is being investigated as a cancer treatment rather than as a general tonic.
Back: Histone deacetylases remove acetyl groups from histone tails. Inhibiting them means acetyl groups accumulate, because they are still being added and are no longer being taken off.
An acetyl group neutralises the positive charge on a lysine residue in the histone tail. With more acetyl groups, the histones carry less positive charge, so they are attracted less strongly to the negatively charged phosphate groups of the DNA backbone. The chromatin becomes less condensed, promoter regions become accessible to transcription factors and to RNA polymerase, and transcription of the affected genes increases.
The cancer application follows from what has often gone wrong in a tumour cell. Tumour suppressor genes are frequently silenced by heavy methylation of their promoters and deacetylation of the associated histones rather than by any mutation, so the sequence is intact and re-opening the chromatin can restore the protein, and with it the control on division. Several such inhibitors are licensed for particular lymphomas.
It would be a poor general tonic because the effect is indiscriminate: the drug increases acetylation across the genome, so genes that were properly silenced are turned up too, in every cell it reaches. That is where the side effects come from, and it is the general problem with treating a control mechanism rather than a gene.

Front: DEFINE
Define: Transcription factor.
Back: A protein that binds to a specific base sequence in DNA, usually in or near a promoter region, and so increases or decreases the rate of transcription of that gene.

Front: DEFINE
Define: Gene expression.
Back: The process by which the information in a gene is used to produce a functional product, usually a polypeptide.

Front: DEFINE
Define: Control sequence.
Back: A region of DNA that is not translated but determines whether, when and how strongly a gene is transcribed.

Front: DEFINE
Define: DNA microarray.
Back: A plate carrying many different DNA probes at known positions, allowing thousands of sequences to be tested for at once.

Front: DEFINE
Define: Steroid hormone.
Back: A lipid-soluble hormone that crosses the cell-surface membrane by simple diffusion and acts on an intracellular receptor.

Front: DEFINE
Define: Tumour suppressor gene.
Back: A gene whose product slows the cell cycle or triggers apoptosis; loss of both alleles removes that control.

Front: DEFINE
Define: Recombinant DNA.
Back: DNA made by joining together sequences from two different organisms.

Front: SPOT THE ERROR
Say what is wrong with this claim:
Cut the gene out, drop it into a plasmid, mix that with bacteria, and you have bacteria making your protein.
Back: Four things stop it. A restriction enzyme cuts at its recognition sequence, not at the ends of a gene; a chromosomal gene carries introns and a bacterium cannot splice; the cut ends need ligase; and well under one bacterium in a hundred takes up a plasmid.

Front: DEFINE
Define: Silent mutation.
Back: A base substitution that produces a triplet coding for the same amino acid, so the primary structure of the polypeptide is unchanged.

Front: SPOT THE ERROR
Say what is wrong with this claim:
A base substitution that does not change the amino acid is a mutation that never really happened.
Back: Silent describes one triplet in a coding sequence and nothing else. A base change in a promoter alters how much protein is made, and one at an intron boundary ruins the splicing; some β-thalassaemias arise that way with the coding sequence entirely intact.

Front: DEFINE
Define: Oncogene.
Back: A mutated or over-expressed proto-oncogene whose product stimulates division continuously.

Front: CHECK YOURSELF
A gene codes for a protein 480 amino acids long. In one patient a single base is deleted at base 30. In a second, a single base is substituted at base 1437, near the end of the coding sequence. In a third, three bases are deleted at position 900. Predict which of the three is likely to be most severely affected, and explain your reasoning fully.
Back: The patient with the single base deletion at base 30 is likely to be worst affected by a wide margin.
Base 30 is inside the tenth triplet, so nine amino acids are translated normally and everything after that is read in a shifted frame. Because the code is non-overlapping and read in threes from a fixed start, removing one base pulls every subsequent base one place forward, so all of the remaining amino acids are potentially wrong. A shifted frame also throws up stop codons at random, so the polypeptide is usually cut short as well as scrambled. A protein of nine correct amino acids followed by nonsense will not fold into anything, and the cell effectively has no product from that allele.
The substitution at base 1437 falls in the 479th triplet, near the very end of a 480-residue protein, and changes one amino acid. That may do nothing at all: the code is degenerate, so the new triplet may specify the same amino acid, and even if it does not, a single change at the end of a chain rarely disturbs an active site or a binding site. The most likely outcome is a protein that works normally.
The three-base deletion at position 900 removes exactly one amino acid and shifts nothing, because three is a multiple of three. Every triplet after it is read as before. The result is a protein 479 amino acids long with the rest of its sequence intact. It may still be faulty, since the commonest cystic fibrosis allele is precisely this kind of mutation and produces a protein the cell fails to deliver to the membrane, but it is a protein, and it is far closer to normal than a frame-shifted one.
The ranking is therefore: frame shift near the start, worst; three-base deletion in the middle, intermediate; substitution near the end, probably harmless. What decides severity is how much of the protein is affected and where, not how many bases changed.

Front: DEFINE
Define: Epigenome.
Back: The full set of such chemical marks on a cell's DNA and histones at a given time.