Genes and Early Embryology - Week 6 Flashcards

Question

What are similarities in RNA synthesis between prokaryotes and eukaryotes?

Answer 1

- Unlike DNA replication where you will see both strands of chromosome being completely copied, with RNA synthesis, it's initiated at DNA specific sites, so getting small single stranded portions of nucleic acid which are complementary to small specific sites within the DNA. - No primer needed unlike replication, and the template is fully conserved. - In transcription, would find only going to be transcribing one particular strand of the DNA to create your RNA. - In the vicinity of the RNA strand being formed, have melting of DNA helix, an unbinding of the helix in this vicinity - it forms a transcriptional bubble. - This will move with the RNA polymerase as it moves down its nucleic acid template. - The template strand of DNA is known as the antisense/ noncoding strand. - The sense or coding strand has the same sequence as RNA. - Can have RNA being produced from both strands in a particular region of DNA.

Answer 2

-Genes that encode proteins are called structural genes. -If you look at how genes are operated and lie within the genome of prokaryotes vs eukaryotes, notice some difference. -Eukaryotes tend to have genes transcribed individually, so will have own control systems mandating their operation. -But in prokaryotes, will find structural genes creating proteins involved in very similar procedures, will find those genes will be in a tandem layout, so are often side by side to one another. -They will be transcribed together, can be controlled by one set of regulatory sequences upstream of what’s referred to as the operon, so this grouping of genes. -Means can turn everything on and off quickly, meaning processes can be better controlled for a more rapid response, that’s what’s shown in bacteria. -In eukaryotes, gene will have its own initiation and termination sequences or regulatory sequences.

Answer 3

-RNA polymerase binds to initiation site through base sequences known as promoters. -These are sequences of DNA that promote gene expression. -In prokaryotes, only have one RNA polymerase which will bind to all of the genes. -They've got different promoters and only want certain genes turned on at certain times, so the RNA polymerase found in prokaryotes has a little sigma factor - so prokaryotes recognised by RNA polymerase sigma factor.

Answer 4

-Promoters about 40 base pairs on the 5’ side of the Transcriptional Start Site. -That is the position at which start to produce RNA. -It will lie on the upstream side of the TSS. -The TTS is called +1. -The nucleotide before that in the DNA, that is not part of the RNA, that’s given the acronym of -1. -There is no zero in this naming scheme. -Can then number base pairs accordingly. -The 14 base pairs upstream of the TSS will be part of the promoter. -These promoters often have conserved sequences, probably the bit the RNA polymerase latches onto.

Answer 5

In the 5' to 3' direction.

Answer 6

-Melting of the DNA forming the transcription bubble. -Allows complementary RNA strand synthesis. -The bubble will travel with the RNA polymerase as it moves - their form of action is processive.

Answer 7

- Tends to form multiple catalytic actions in one go and don’t dissociate after each action, so don’t dissociate after each ribonucleotide triphosphate is added onto the nucleic acid chain. - It will actually add a large number of them before the RNA polymerase dissociates from the template, and this is necessary because especially when you’re dealing with eukaryotes, some of the genes can be thousands of base pairs long and if your enzyme had to dissociate after adding every NTP to the end of the RNA it would take a long time. - Experiments have suggested in E. coli that there’s going to be up to 1900 base pairs incorporated before an enzyme dissociates.

Answer 8

- The processive enzymes work particularly quickly, they can add up to fifty ribonucleotide triphosphates per second. - Have an error frequency of 1 per 10^4.

Answer 9

- RNA synthesis can be initiated as often as sterically possible meaning when an RNA polymerase has moved out the way from the promoter and there’s enough space, another RNA polymerase complex can form and start transcription. - With prokaryotes as don’t have a nuclear envelope, the ribosomes can start their job as soon as the RNA starts to emerge from the RNA polymerase and it’s sterically feasible. - So, only in prokaryotes can protein synthesis begin before RNA is completely synthesised.

Answer 10

- Different ways of turning off transcription depending on whether it’s a eukaryote or prokaryote. - In prokaryotes, there are sites within the DNA called the termination sites that can contain palindromic sequences. - Those can be a marker for the RNA polymerase complex to stop its work. - There are two different types of terminator sequences. - There is the rho-independent form and the rho-dependent form.

Answer 11

- The rho-independent form are sections of the DNA, the intrinsic terminators, where they’ll form a self-complementary hairpin. - The RNA will fold back on itself causing the RNA polymerase to sense it and it pauses in its job. - That permits reannealing of the DNA and dissociation of the RNA from the RNA polymerase.

Answer 12

-Rho-dependent requires action of a protein, a helicase by the name of Rho factor. -This helicase moves along the RNA until it encounters a paused RNA polymerase. -The RNA polymerase has paused because it has encountered a particular sequence in the vicinity of the termination site. -As soon as the helicase catches up with it, it hydrolyses ATP and then will cause a rewinding of the helix, a release of the RNA and a release of the RNA polymerase. -Once RNA polymerase has dissociated form the RNA it can go back and start process again.

Answer 13

- Got type I in nucleolus, which will make ribosomal RNAs, so will transcribe the ribosomal RNA genes. - There’s type III in nucleoplasm, they’ll transcribe some rRNA but also tRNAs as well. - Then there’s type II in nucleoplasm, the RNA polymerase that creates the mRNA that encodes protein. - So essentially it will be transcribing your structural genes.

Answer 14

- Alpha-amanitin is a toxin that can be used to tell the difference between RNA polymerases in an experimental fashion. - So type I is insensitive to it, type II is strongly inhibited by it, and type III is inhibited by a high concentration.

Answer 15

-Promoters for RNA polymerase II are particularly complex and diverse. -Noticed in experiments that there are particular parts of these promoters often conserved across the genes that require RNA polymerase II to do its job and we refer to these as core promoter regions. -Some of these regions are well known and conserved across many of the genes and the ‘TATA’ box is one of those showing this. -These core promoter regions will allow a certain set level of transcription to go ahead, a basal level.

Answer 16

-There are other sequences often found that can encourage transcription and they will aid the promoter sequences in their job and these are given the term of enhancers. -Enhancer sequences don’t have to be upstream of the transcriptional start site, can be downstream, because what they’re relying on is the fact that DNA does not lie in a linear orientation, it can be curled up on itself. -So will find there are proteins that will bind the enhancer region and due to the way DNA lies in 3D space, it will actually be in close spatial proximity to the core promoter regions where they can bind the RNA polymerase and in this case encourage transcription. -So these are known as enhancer sequences. -Can also get silencers. -So rather than interact with RNA polymerase, enhancers are recognised by transcription factors. -They can stimulate RNA polymerase II binding and mediate selective gene expression in eukaryotes.

Answer 17

-To recognise these diverse promoters, will find there are a range of protein interactors that help the RNA polymerase find what it needs to bind to. -In RNA polymerase II transcription, general transcription factors are required. -If look at RNA polymerase II find general transcription factors that help form the preinitiation complex on the core promoter. -They’ve got the acronym of TFII and a letter after them, A, B, E, F or H or D. TFIID is also known as the ‘TATA binding protein’, that’ll help locate for example the ‘TATA’ box that the RNA polymerase II can bind to. -That’s a preinitiation complex. -Once that’s been formed and transcription starts, then enter the elongation mode and will have other transcription factors involved. -General transcription factors will allow a slow level of transcription. -Will often need gene specific transcription factors in the local vicinity of the gene in order to really encourage transcription.

Answer 18

-Rifamycin B is a compound and antibiotic derived naturally and produced by Streptomyces bacteria. -Will encounter as an antibiotic in the form of rifampicin, which is a synthetic derivative. - It inhibits prokaryotic but not eukaryotic transcription, which is good when trying to heal patients. -Rifampicin prevents further chain elongation, so they end up locking the RNA polymerase onto the promoter on the early stages of the gene, and then nothing else can get past it. -No other RNA polymerases can get past it and that blocks any further attempts at initiating transcription.

Answer 19

- With a prokaryote, often find the RNAs that are produced are complete, they are exactly what you need to create proteins. - Only modified to small extent. - But eukaryotes, undergo a lot of processing in order to get in the condition they will need to produce a protein.

Answer 20

- First transcript produced from transcription is known as hnRNA, heterogenous RNA, and that can undergo a range of different processing procedures. - Can have a cap added on the 5’ end, may end up being cleaved, may have a large section of the tail or 3’ end tail cleaved before having a process called polyadenylation occur. - Get your runs of adenosines added to the end of the transcript. - Can then undergo process of splicing, by which introns, these areas of the RNA that won’t end up coding for parts of the protein, will be removed. - Splicing will join the exons, the expressed sections, of the protein.

Answer 21

Many of these processing procedures can take place while the RNA is being synthesised, so can be known as co-transcriptional modifications of the transcript.

Answer 22

- Could aid in the location of the start codon, that AUG within eukaryotic translation. - Could also permit promoter escape by the RNA polymerase, so find need to have successful capping of the RNA to permit the polymerase to move away from the promoter. - Could be involved in preventing mRNA from being degraded by enzymes, hence permitting the ribosome to go about its job.

Answer 23

- Eukaryotes don’t have very specific termination sites, at least not the transcription termination sites would see in bacteria. - Part of reason for that is because have polyadenylation occurring. - This means many adenosines. - What you have is a string of up to 250 adenosines added to your mRNA in the process of its maturation. - The act of adding this tail ends up helping the RNA polymerase to terminate transcription and dissociate from the mRNA so it can then carry on its job.

Answer 24

- The big difference between eukaryotic and prokaryotic RNA processing will be with splicing. - Splicing means joining exons together. - Introns weren’t discovered until sequencing came about. - There are some interesting distributions of introns with organisms. - Yeast for example, a unicellular eukaryote, may have just 239 introns spread throughout entire genome. - Humans, however, can have up to 50 per gene, or more. - Many introns will retain their positions within the gene.

Answer 25

- A spliceosome is a complex of proteins and small nuclear RNAs, snRNAs. - These snRNAs and proteins will form small ribonucleoproteins, snRNP. - They’re going to end up binding different parts of the intron, either the 5’ border region or the branch point and then they’re going to assemble the spliceosome complex on the intron and essentially allow it to fold, forming a lariat. - If knock the branch point out, can’t form the lariat and this is deleterious. - If mutate any of the conserved sequences within the intron, is deleterious. - If end up affecting the sequences of the snRNAs or the proteins that make up the spliceosome, is deleterious. - So have a source for mutation here. - So why is that this splicing process has become embedded within eukaryotic organisms? - This is because of alternative splicing.

Answer 26

- Got gene, and when splice RNA, can have multiple versions of mRNA being produced if you’re looking at protein coding gene for example. - Means when have to translate it, can have variations on your same protein. - So, the coding potential of your genome expands, because can have protein a for example where have all of the exons from your gene expressed and incorporated and translated into your protein. - Or you could lose an exon, hence giving different capabilities. - Expands proteins within a cell.

Answer 27

Autosomal dominant where heterozygotes with one copy of the abnormal gene are affected, autosomal recessive where homozygotes with two copies of the abnormal gene are affected, and X-linked recessive where males with one copy of the X chromosome are affected.

Answer 28

Have a fault on one copy, on one of the alleles of your pair, and it is a strong gene change and it shows itself even though you’ve got a second standard copy.

Answer 29

- In recessive inheritance, the gene change is such that you can cope with having one working copy and so can be a carrier and have one gene change, but as long as your other copy is standard, you will be fine. - To have a child that has a recessive condition or develop one as an adult have to have a change on both copies of the gene so both have to be faulty.

Answer 30

Last inheritance pattern is x linked and the most common form is x linked recessive, and in that the changes on the x chromosome, and so, males because they only have one x chromosome will tend to run into trouble, and women because they have 2 x chromosomes, if they have a second standard copy will usually be okay or might have some mild features.

Answer 31

- Duchenne Muscular Dystrophy. - Becker Muscular Dystrophy. - Haemophilia. - Red-Green Colour Blindness. - G6PD deficiency. - One form of hereditary motor and sensory neuropathy called Charcot-Marie-Tooth Disease. - Retinitis Pigmentosa.

Answer 32

- Most common and severe form of muscular dystrophy. - Presentation in boys usually between 3-5 years. - About one third have mild to moderate learning difficulties. - Waddling gait and positive Gower sign. - Difficulty running and climbing stairs. - Gradual deterioration, leading to loss of mobility and wheelchair bound. - Progressive muscle weakness, leading to cardiorespiratory failure. - Get pseudohypertrophy of calf muscles and proximal muscle weakness. - Difficulty rising from the floor."

Answer 33

- Hereditary haemochromatosis. - Cystic fibrosis. - Beta-Thalassaemia. - Spinal muscular atrophy. - Many inborn errors of metabolism. - Some sensorineural deafness. - 21-hydroxylase deficiency.

Answer 34

-Population incidence of 1 in 2000. -Carrier frequency is 1 in 25. -Autosomal recessive inheritance. -Disease characterised by progressive lung disease, pancreatic dysfunction and elevated sweat electrolytes.

Answer 35

- A risk figure for being a carrier for both parents. - So for cystic fibrosis for example, 1/25 is the population carrier rate, so this is the chance for someone with no history of the disease in their family.

Answer 36

-You have to calculate it using the Hardy-Weinburg principle. -The Hardy-Weinburg principle allows the calculation of carrier rates once the incidence of a condition is known as long as the gene frequency is in equilibrium. -So as long as you know how common the condition is, can use that to work back and work out what the carrier rate must be from the general population, and as long as those genes are in equilibrium and the incidence of that condition is staying pretty much stable all the time.

Answer 37

-For this principle to apply, need a big population that's randomly mating so the relative proportions of different genotypes remains constant. -This only holds true if there are no outside influences, e.g. selective or assortive mating. -If you have two alleles for an autosomal condition: A and a, have frequency p and q and p+q=1. -Put in a punnet square as shows all the possible combinations could have if people had either one of those two gametes. -So if you're AA, that's p^2, if you're Aa, that's 2pq and if you're aa, you're q^2. -p^2 are homozygous affected, so people who have two normal copies of the gene, 2pq are carriers, so have one faulty copy, and q^2 are the affected, as have two copies of the abnormal gene. -As the generations continue, the relative proportions remain the same.

Answer 38

- Don't use for autosomal dominant conditions as usually straightforward to see who is a gene carrier and who isn't. - For autosomal and X linked recessive conditions, however, the carrier rate is not obvious - don't use so much for x linked however.

Answer 39

- e.g. PKU has an incidence of 1 in 10,000 live births. - Means q^2 is 1/10,000, so q is 1/100. - Can then work out p by using p+q=1, which means p=99/100. - 2pq then equals 1/50. - Can then work backwards to double check, 1/50 x 1/50 x 1/4 = 1/10,000.

Answer 40

- Hardy-Weinburg principle doesn't work if mating is assortive or there is consanguinity. - Assortive mating is the tendency to choose a mate with similar characteristics, e.g. height or IQ. - Consanguinity is relationships between close relatives that can lead to an increased carrier risk within a family.

Answer 41

-New mutations are arising all the time. -Different genes have different new mutation rates according to their size and structure, e.g. in Duchenne Muscular Dystrophy the new mutation rates are high. -Usually this is balanced by loss of alleles due to reduced reproductive fitness in affected individuals. -An alteration in this balance will affect the equilibrium.

Answer 42

-For some autosomal recessive conditions, carriers seem to have a reproductive advantage. -This has lead to certain genes being very common in a particular population. e.g. sickle cell carriers are resistant to falciparum malaria. -Infected cells probably sickle as a result leading to their preferential removal from circulation.

Answer 43

-This is the founder effect. -One allele can be transmitted to a large proportion of children purely by chance leading to an increased incidence of a certain condition in a population.

Answer 44

Migration and intermarriage can introduce new alleles into a population.

Answer 45

- Earliest scan offered at eight weeks. - Gives limited view of baby. - Can give dates of pregnancy, essential for other screening tests. - Can show whether it's a single or multiple pregnancy.

Answer 46

-Performed between 10-14 weeks gestation. -Thickness of NT, back of the babies neck, measured by ultrasound scan in relation to crown-rump length. -Other abnormalities may be detected.

Answer 47

- Chromosomal - Down syndrome. - Major congenital heart disease. - Skeletal dysplasias. - Diaphragmatic hernia.

Answer 48

- Check for chemical called AFP - increased levels means certain structural problems with way baby is forming. - Reduced MSAFP levels (<0.5MoM) can mean Down Syndrome. - Elevated MSAFP levels can mean neural tube defects, anterior abdominal wall defects, missed or threatened miscarriage, intra-uterine growth retardation, multiple pregnancy and congenital nephrotic syndrome.

Answer 49

Chorionic villus sampling (CVS), amniocentesis and cordocentesis (fetal blood sampling) and fetal tissue biopsy which is used rarely.

Answer 50

- Usually between 11-13 weeks. - Usually transabdominal approach and use needle to take sample of placenta. - The placenta comes from the same first cell the baby comes from. - So good yield of fetal DNA for molecular tests. - 1% miscarriage risk.

Answer 51

- Usually 15-16 weeks gestation. - Put needle in tummy and take sample of fluid which will have some of babies skin samples in and can use those skin cells to do a genetic test of baby. - Low yield of fetal DNA. - 1% chance of miscarriage.

Answer 52

-PGD is the process of testing embryos produced by IVF for inherited disorders, so that embryos that are free of the disorder to be replaced. -Suitable for couples at substantial risk of transmitting a serious genetic condition to their children, such as chromosome translocations, x-linked disorders and some single gene disorders. -Not available for all conditions. -Requires considerable preparation - clinical, counselling, laboratory. -Stimulate ovulation, oocyte retrieval, fertilisation and culture and then blastocyst biopsy on D5. -Success rate is 35%. -Criteria are it should be an important public health problem (i.e. common enough), there should be a treatment or intervention available, facilities should be available to make a firm diagnosis, the test should be acceptable, sensible and specific, and participation should be voluntary after full information and counselling.

Answer 53

- The newborn blood spot screening programme. - Currently all babies have a series of blood spots taken at seven days of age onto a Guthrie or Scriver card to screen for phenylketonuria and congential hypothyroidism. - CF, sickle cell anaemia and MCADD are now screened for nationally on the same sample.

Answer 54

- Polygenic inheritance is controlled by many genes with small additive effects. - Genes at many different loci contribute to the phenotype. - No one gene is dominant or recessive to another - there is a cumulative effect. - Remember, in locus heterogeneity, more than one gene leads to the same phenotype.

Answer 55

-In reality, our environment often has an influence, to a greater or lesser extent on our phenotype, e.g. height. -Multifactorial inheritance is where a condition is caused by the interaction of multiple genes and the environment. -The sum of environmental influences and genetic predisposition gives a person liability to be affected. -If the threshold is exceeded the condition results. -The liability-threshold model explains many observations seen in this situation but remains a theory. -The worse the genetic makeup is, the more it pushes the curve over to be affected, as does a worse environment.

Answer 56

Autoimmune deficiency, coronary artery disease, late onset forms of diabetes.

Answer 57

-Gene mutations make a significant contribution to human disease. -Inherited mutations are called 'germline' mutations and are present in every cell. -Acquired mutations are somatic and are present in the diseased tissue. -Detection of both germ-line and somatic mutations is important in clinical management.

Answer 58

-Somatic testing is usually done in tumours. -Information from somatic testing can be used to make a diagnosis and help to classify tumours - some tumours now depend on molecular diagnostics to confirm the actual diagnosis of the tumour. -Somatic testing can also give information about outcomes, so prognostic indicators. -May also give prediction of tumour response to chemotherapy. -This is different to pharmacogenetics because the we’re talking about presence of a mutation in the tumour which will dictate whether that tumour cell will respond to a chemotherapy or not. -That will be called predictive testing.

Answer 59

- Know K-ras mutation is something that prevents response to cetuximab. - If patients have wild type K-ras gene (wild type is non- mutant) then if give cetuximab, their outcome is better than if don’t receive. - But conversely if they have a mutation in the K-ras gene, this is in a tumour cell, then if there is a somatic mutation in the tumour, the cetuximab will just be ineffective, and so in the cases where there’s a somatic K-ras mutation in the tumour, giving cetuximab would have no benefit, would be expensive and would have the side effects as well. - So doing somatic testing for K-ras mutations in colorectal tumours is obligatory to decide whether to give cetuximab.

Answer 60

-There are lots of different types of mutations which occur, ranging from single base mutations up to changes in large chromosomal fragments. -So the variety of mutations which occur is highly variable, which is a challenge. -Different types of mutation require different types of test, so test would use to look at a single base mutation would be different to one would use for chromosomal translocation. -If different types of mutation in the same gene give the same effect, multiple tests may be required. -Some genes have regions which show a high frequency of mutations. -These are hotspots, so a region where lots of mutations are occurring. -So allows us to, rather than testing the whole gene, to just test where the hotspot is. -In other genes, multiple different mutations within the same gene may give the same phenotype, allele heterogeneity. -Means are looking for different types of mutation rather than specific types of mutation, which is a challenge. -Different mutations within the same gene may give different phenotype. -May find mutations in different genes in a pathway may give same syndrome, locus heterogeneity.

Answer 61

-BRCA1, which has evolved in breast cancer and ovarian cancer, show allelic heterogeneity so mutations may occur anywhere along the whole of the gene. -So whole gene then needs to be screened for the presence of the mutation.

Answer 62

-This is an example of mutation in the same gene producing different phenotypes. -Duchenne's and Becker's arise because deletions occur in the Dystrophin gene. -Shows how different mutations in the same gene cause different phenotypes. -DMD due to frameshift mutations causing truncated protein and total loss of function. -BMD due to in-frame deletions causing partial loss of function. -In both diseases 60% mutations are deletions of whole exons in two hotspots. -They are both X-linked recessive progressive muscle wasting diseases. -First symptoms from ages 2 to 5 years in DMD with loss of ambulation by 12 in DMD. -Death in DMD secondary to cardiac dysfunction or respiratory complications, in third decade. -BMD individuals may survive to old age.

Answer 63

-May have one phenotype, but may need to do a wide variety of testing for it. -Syndromes often result from malfunction of physiological pathways, and if there are a lot of genes within that pathway, would have to test for each one, e.g. many genes responsible for hearing loss. -Some functions depend on multimeric complexes. -Loss of any member may disrupt the whole complex, e.g. mismatch repair. -Locus heterogeneity increases the number of tests required to screen for the syndrome.

Answer 64

-Imprinting is about silencing using methylation of DNA. -All genes are represented twice - we have 23 pairs of chromosomes and the genes are duplicated on each one. -In small minority of genes, have imprinting, where only one of the alleles from the parental chromosome is expressed. -This silencing is done by epigenetic modification, which is adding methyl residues onto cytosines. -Usually find it's consistent, but can have some variations. e.g. Prader-Willi syndrome and Angelman syndrome. -Find the syndromes caused by deletion of the same region, but depending on whether it’s the paternal allele or the maternal allele, that’s what dictates what syndrome you get. -The relevant genes are located on 15q but whether you lose the paternal or maternal allele will dictate what syndrome you get. -Then becomes important to know which allele has been lost. -Similar events occur in tumours at tumour suppressor loci .

Answer 65

- Size analysis to detect expansion mutations, PCR for X-linked deletions in males, and PCR to detect common point mutations, which would be called ARMS PCR, which looks for specific point mutations. - Can also do screening of PCR products by heteroduplex analysis in order to identify where mutations occur. - Can do sequencing. - Can do MLPA when looking for deletions over a large area. - Can use PCR for microsatellites to track unknown mutations in families.

Answer 66

Genetic tests are performed on DNA derived from lymphocytes from blood, mouthwash cells or buccal scrapes from babies, and chorionic villi, such as from the placenta, or can do a placental tap if looking for embryonic defects.

Answer 67

-PCR is used to amplify a specific region of interest, such as a hotspot in a gene. -It represents a form of in-vitro DNA replication. -It requires tiny quantities of starting material and produces huge amounts of target product. -The PCR product can then be analysed using a variety of assays.

Answer 68

-PCR product can be evaluated for size by electrophoresis. -So DNA is negatively charged, and if move it across a charged gradient, then different size fragments will move at different speeds, and will basically resolve into different sizes. -By doing electrophoresis, can do it in a gel or in a capillary tube, and will allow identification of expansion mutations such as Huntingdon’s disease. -Will allow polymorphic microsatellite markers to be followed for linkage analysis, so wherever you have a variation in size you can use PCR.

Answer 69

-Expansion mutations are a special form of mutation involving expansion of triplet repeat sequences, so it’s a codon which is just amplified repeatedly. -Expansions may be within coding or non-coding regions. -May exert an effect by turning off the gene, such as Fragile X syndrome. -Or may cause changes in protein processing, such as a myotonic dystrophy. -Or may produce non- functional proteins, like Huntingdon’s chorea.

Answer 70

-With the expansion mutation, can get anticipation, meaning with the successive generations, the age of onset of the disease gets lower, or becomes more severe, or more penetrant. -Reason for that is have these expansions, and if carry on expanding then they have a more severe effect. -So it’s due to the progressive increase in the size of repeats. -There are thresholds above which allele causes disease, e.g. over 40 repeats In Huntingdon’s disease, will then result in a phenotype, whereas under 50 probably won’t as there’s still a lot of residual function there. - Over 200 repeats in Fragile X is the number that then causes a problem.

Answer 71

-If there is a polymorphic marker near to a mutant allele it can be used to track the mutation. -There has to be polymorphism, so has to be a variation in size. -Is possible you know there’s a mutation there, but don’t know what the mutation is or you don’t want to sequence for it every time. -But adjacent to a mutation is a specific polymorphic marker, so it’s a marker of a specific size indicating it’s linked to the mutation. -So if see a marker of that size and it says there’s a mutation there, but if see a marker of a different size, indicates don’t have mutant allele. -Can use this then for tracing. -Thus rather than sequence for the mutation everytime, the presence of the polymorphic marker is used to indicate the presence of the mutant allele. -If the specific mutation is unknown, there may be no choice other than to track the polymorphic marker.

Answer 72

-Can analyse presence or absence of a product, so is a slightly different way of testing. -So showing that presence or absence of a deletion. -Not actually looking at sequence as such, just looking at deletion or loss of sequence. -Can use multiplex PCR with several primers to amplify several regions all in one go that can then be analysed.

Answer 73

-In MPLA, design PCR primers which have common sequences. -They're designed to hybridise onto different exons and once they've been ligated to the DNA, only need one primer pair to amplify the PCR rather than in previous example and had six different exons, so six different primer pairs. -If do MPLA can test a large number of different things using just one primer pair after ligated different probes on. -Smaller peak indicates there’s a deletion occurring. -As it’s not completely gone, that represents one of the alleles, and the bit that’s missing here represents deletion of the other alleles.

Answer 74

- Can be used to detect imprinted or epigenetically silenced alleles. - To do with the fact that when the DNA has become methylated with methyl residues, it changes the physical properties of the cytosines. - What that means is that you expose the DNA to bisulphites, then the methylated cytosines are resistant to the effects of bisulphites, whereas the non-methylated cytosines undergo a change converting the cytosines to thymine, so the sequence actually changes. - By then designing methylation specific primers, can test for the presence or absence of a PCR product because your primers are methylation specific so only amplify when there is a methylated DNA there. - If not methylated, due to bisulphite response, the sequence will change and PCRs won’t respond. - Can do the bisulphite conversion to cause the non-methylated primers to change, and that then means by using methylation specific PCR can then test for differential modification in the DNA. - And so we know which of the alleles was meant to be silenced or imprinted by methylation, so can design maternal specific primers or paternal specific primers for same sequence. - Depending on which one disappears, will then be indicative of where the deletions have occurred.

Answer 75

- If want to sequence PCR products, is a trick of doing mutation scanning. - Purpose of that is for example if looking for a large area, so have multiple PCR products, but know mutation will only occur in one of those areas. - So rather than sequencing everything which will be expensive, only sequence PCR product containing mutation. - In cases of allelic heterogeneity, this is where lots of different variants anywhere along the allele may cause the phenotype, may have to test whole gene. - If test each one by sequencing is expensive, but can reduce costs by testing the whole allele using a variety of different PCR products. - Then you screen those PCR products for presence of a mutation. - So use these physical characteristics of that gene to screen for the presence of mutation, and you can use things like heteroduplex formation single strand conformation polymorphism, and protein truncation test.

Answer 76

-Dideoxy/ Sanger sequencing is the most commonly used method. -Newer methods include pyrosequencing. -Whilst all mutations should be validated by sequencing, it is not always easy and it is expensive. -Thus, in many contexts, it is better to use scanning methods before deciding when to sequence. -Pyrosequencing is comparitavely new and slightly more sensitive than Sanger sequencing but it has some shortcomings.

Answer 77

Molecular tests will give information on predisposition - risk of developing disease, diagnosis - for both inherited and acquired diseases, prognosis - mainly in cancer, responsiveness to therapy - predictive testing/ patient stratification, metabolism of therapuetic drugs - pharmacogenetic testing, and therapeutic targets - pharmacotherapeutic testing.

Answer 78

-The process during which embryonic cells specialise and diverse tissue structures arise, each with specific functions in the body. -In very early embryonic development, the embryo doesn't possess these varied cells. -Differentiation of cells during embryogenesis is the key to cell, tissue, organ and organism identity.

Answer 79

-The zygote divides into multiple cells in a process called cleavage, triggering the beginning of embryonic differentiation. -This zygotic division produces blastomeres which later make up the hollow sphere known as the blastocyst. -Cells migrate within the blastocyst to locations that will later define the structure of the embryo and consequent organism. - In the process, called gastrulation, three germ layers arise, endoderm, mesoderm and ectoderm. -These are then committed cells, so endodermal cells are committed to form cells that endoderm form.

Answer 80

The trilaminar disc, so the three germ layers.

Answer 81

-The ectoderm forms the surface of the body, particularly the epidermis of the skin, but also hair, nails, mammary glands and the anterior pituitary. -Ectoderm also differentiates into neuroectoderm, which forms the neural tube which forms the CNS, retina and posterior pituitary. -Neuroectoderm also forms the neural crest, which forms the ganglia, adrenal medulla, connective tissue and parts of the heart.

Answer 82

- Lateral mesoderm forms connective tissue and muscle of viscera, serous membranes, heart, blood, spleen and adrenal cortex. - Intermediate mesoderm forms urinogenital system, ducts and accessory glands. - Head mesoderm forms the skull, connective tissue and dentin. - Paraxial mesoderm forms muscles of the head, trunk, and limbs, the skeleton, dermis and connective tissue.

Answer 83

Endoderm forms the epithelia of internal surfaces, e.g. respiratory system, alimentary system and urinary system.

Answer 84

-Cells continue to differentiate from what were initially the same types of pluripotent embryonic stem cells. 0Can differentiate into many different things according to which genes are expressed, controlled by transcription factors that actually switch genes on and off, and these cells have an inherent ability to control to some extent what they become. -And if we take those cells, we can actually give them various signals, and we know that different signals cause those embryonic cells to become different things. -So end up with whole population of different cell types within the body and that allows for process division of labour, so different cells specialise to do different things within the body.

Answer 85

Differentiation of cells is brought about by both internal cellular factors as well as extracellular factors.

Answer 86

Regulative development involves the interaction of adjacent cells, within embryonic morphogenic fields, giving flexibility to differentiation.

Answer 87

-They are unspecialised and can divide repeatedly over long functions. -Under certain conditions, they can be induced to differentiate into cells with special functions, like muscle cells and neurones, and hence could be important in treatment of diseases.

Answer 88

- Embryonic stem cells are studied quite a bit, in vitro or in culture. - Where there obtained from, is to isolate the inner cell mass of an embryo and then can put them on special culture conditions and the cells will perpetuate as stem cells under the right conditions, dividing by mitosis. - What differentiation takes place, can be triggered by adding various factors to these cells in cultures.

Answer 89

-Once embryo is forming, all the cell divisions will be by mitosis, and at these early stages, we have cell division taking place, so cleavage, and this cell division is a single cell dividing into two identical cells, dividing into more identical cells. -So there’s no differentiation, all these cells are identical. -Can also have cell division taking place later on during development, where you get cell division into two identical cells, and both of them will then differentiate into something.

Answer 90

- In many cases, get cell division and differentiation into two identical cells, one of which will carry on in that way, so no differentiation and will remain as a stem cell. - Other one may differentiate into a particular type of cell. - So, in skin, at bottom of skin have basal cells which are the stem cells and they divide and only some of them are committed to differentiating and others just remain as stem cells. - So in skin as they divide and specialise, start making keratin and eventually will form the keratinised layer of the skin.

Answer 91

Can give rise to all cell types found in the adult organism, plus extraembryonic cells, e.g. zygote.

Answer 92

- Can give rise to all three of the germ layers, but not extraembryonic, e.g. cells of the inner cell mass of the blastocyst - from where embryonic cells are derived. - As development proceeds, stem cells may lose their pluripotency.

Answer 93

-Restricted in their ability to form different cell types and therefore are multipotent, not pluripotent. -Scientists are finding methods to cicumvent this disadvantage. -Disadvantages of the approach include the slow rates of cell division and their scarcity.

Answer 94

- Mature (adult) stem cells may replace tissue that is damaged by disease or injury. - Can replace neurones damaged by spinal cord injury, stroke, Alzheimer's disease, Parkinson's disease or other neurological problems. - Can produce insulin that could treat people with diabetes. - Produce heart muscle cells that could repair damage after a heart attack. - Potentially replace virtually any tissue or organ that is injured or diseased.

Answer 95

Derived from the inner cell mass of the embryo - pluripotent - can form virtually any cell or tissue type.

Answer 96

Diabetes, Alzheimer's and Parkinson's disease, anaemias, and spinal cord injuries.

Answer 97

-Embryonic stem cells may be obtained from embryoes after IVF, a process called reproductive cloning. -May cause immune rejection, but could be modified to circumvent this problem. -Ethical considerations, as the cells are derived from viable embryos.

Answer 98

-Nuclei from adult cells and introduce them into enucleated oocytes. -Oocytes are stimulated to differentiate into blastocysts, and embryonic stem cells are harvested. -As the cells are derived from the host, they are compatible genetically, and as fertilisation is not involved, the technique is less controversial.

Answer 99

- The exact number of abnormal zygotes formed is unknown. - Usually lost within 2 to 3 weeks of fertilisation, before the woman realises she is pregnant, and therefore not detected. - Estimates are that as many as 50% of pregnancies end in spontaneous abortion and half of these losses are as a result of chromosomal abnormalities. - Abortions are a natural means of screening embryos for defects. - They reduce the incidence of congenital malformations. - Without, approximately 12% instead of 2-3% of infants would have birth defects. - With the use of a combination of IVF and polymerase chain reaction, molecular screening of embryos for genetic defects is being conducted. - Single blastomeres from early stage embryos can be removed, and their DNA can be amplified for analysis.

Answer 100

- Gene regulation is critical for determining cell type, whether something becomes a hepatocyte or a fibroblast, or a neurone etc. - Also determines function of the proteins that are produced in those genes.

Answer 101

-Ribosomal DNA is the genes within the nucleolus of the nucleus of a eukaryotic cell that are going to express the ribosomal RNA species, which become part of the ribosomes, which are then critical for translating the mRNA from a protein coding gene. -Have a random protein coding gene, may have four exons for example, as well as introns. -If going to be expressed, an RNA polymerase has to transcribe it starting with initiation point, initiation transcription, to migrate from one end of gene down the other end where it terminates and produces an mRNA species. -Transcription will do something like this, as the RNA polymerase migrates down from the promoter to the transcriptional termination point, it’s producing a transcript, and that transcript has portions that represent the exons and introns. -So have transcription, and that’s a critical point for regulation. -It makes sense to regulate gene expression primarily at transcription because you don’t waste materials, metabolites etc, by doing regulation later on, can control it at the level of production of the transcript at the first point. -Not to say don’t see regulation elsewhere.

Answer 102

-Proteins come together with this transcript, to process it etc. -During its transcription, as a co-transcriptional process, the RNA gets a cap on its 5’ end, what will become an mRNA gets a 5’ cap in eukaryotic cells. -Essential for translation. -Also during cleavage of the initial transcript, get a polyadenylation event which is a post transcriptional event. -So get this thing called a poly(a) tail on the 3’ end of the message. -So now have what would be described as a hnRNA or heteronuclear RNA, and that still has to go through some processing to become an mRNA to be translated as still has the introns in it. -At this point could be degraded, which would control gene expression, as the potential message has now been degraded. -Or we can do a message. -Splicing of that would give a mature mRNA where these intron regions have been spliced out. -They are non-protein coding so don’t want them present in an mRNA that’s going to encode a protein. -And there’s regulation potentially at splicing events. -So can get alternative splicing, so may get a splicing even where one exon is present in one form of an mRNA that’s come from the same transcript as another, where that exon has been spliced out as an alternative splicing endpoint. -So can get regulation of gene expression by this differential splicing. -All of this regulation here would be called posttranscriptional processing, so capping, polyadenylation, degradation and splicing events.

Answer 103

-If going to translate this mRNA, need some ribosomes, need ribosomal subunits, and they come from nucleolar rDNA, where they transcribed to the ribosomal RNAs. -They assemble with various proteins that make up the ribosomes to give us the functional ribosomes, and then we’ve got to, we’re still in the nucleus at this point, so we have to get these building blocks out into the cytosol, so there’s a transport step, so the messages, the ribosomal subunits have to be transported to nucleopores within the nuclear membrane, into the cytosol where can be translated. -Some genes, the regulation can be controlled at this level of transport, particularly the mRNA, out of the nucleus, so another step. -So now we’re at the point where the message has been exported to the cytosol, and have the ribosomal building blocks which assemble into ribosomes, so there’s a bit more regulation can get here. -Once get into cytosol, message can be degraded, another potential regulatory step, negatively regulated. -Something people didn’t originally recognise is this message can be sequestered in an inactive form, so it can become associated with certain cytosolic proteins and form into inactive bodies within the cytosol, where the message isn’t actively translated. -The reason that’s useful for a cell is you’ve now got a pool of silent mRNAs. -So if you want a very fast switch on of the expression of a protein, if you’ve got a pool of inactive RNA that you can rapidly activate, you’ve short circuited the transcription, capping, polyadenylation, splicing steps, and you’ve got a very rapidly available pool of RNA to increase the level of protein. -So that’s a useful regulatory step, to sequester this and make it available later. -So the mRNA availability is something that’s regulated. -The ribosomes then come in, the ribosomal subunits can assemble at the cap generally, and then they scan down the mRNA looking for a start codon to start translating to produce protein. -So protein produced by the ribosomes will fold before it becomes functional, and again another regulatory possibility, degradation of the protein before it becomes fully functional. -These regulatory steps further down, particularly protein stability for example, are critically important to many cellular processes. -So, a lot of proteins need post-translational modification and processing. -Maybe you’ve got a proprotein, this proteolytic event, that clips off a bit that allows the mature protein, so chymotrypsinogen, becoming chymotrypsin etc. -Maybe carbohydrate modification. -And then have protein becoming fully functional, so these proteins may be compartmentalised, may be secreted, and then you get to function.

Answer 104

- Transcriptional regulation is paramount, largely for reasons to do with resource. - Critical for most eukaryotic genes. - Only transcriptional control prevents the formation of any unwanted intermediates etc. - Anything down stream from that, possible you can get intermediates that may be partially functional, or interfere in ways cell doesn’t want. - Only stopping transcription will prevent any of that. - So it’s important.

Answer 105

- There are two core mechanisms. - Binding of sequence specific transcription factors to DNA, so protein factors that bind to the gene, either at the promoter region or elsewhere to regulate that gene expression. - Mustn’t forget about the control of the availability of the gene and its promoter, how the DNA is packaged into chromatin. - Because if it’s tightly packaged, it’s inaccessible to the RNA polymerase, its clearly not going to be expressed, and if it’s open, and the RNA polymerase can get in and recognise promoter, it’s going to be expressed in all likely hood. - So these two essential parts of transcriptional regulation.

Answer 106

-Most eukaryotic genes controlled by complex cis-acting sequences within their promoters. -Cis-acting means on same bit of DNA, they’re adjacent to the transcriptional start site, so acting in close ranges, they’re what would be described as proximal to the transcriptional start site, as opposed to distal which is a long way away but is still influencing. -So most genes in eukaryotes are controlled by these elements to a large extent with additional input from distal or other inputs. -So a lot of them are proximal, and a lot of what we understand about transcriptional regulation comes from proximal elements and the binding of classic transcription factors to these regions near to the transcription start site.

Answer 107

-Discrete cis-acting sequences are recognised by transcription factors, and then they influence the expression of genes. -So many of these transcription factors contain elements described as DNA binding domains, so the protein component of these transcription factor elements that will recognise specific DNA sequences, so called consensus sequences, and they also have transactivation domains, so these are domains that once it’s bound to the DNA in the right place, there’s another part of the protein that for example interacts with and binds to the RNA polymerase and tells the RNA polymerase to go, and it goes off and starts transcribing. -So consist of typically bits that bind to DNA and these transactivation domains. -Can also get transcription factors that are negative regulators of transcription that have DNA binding domains and repressor domains that then switch off the transcription as well.

Answer 108

-Transcription factors bind to DNA, they’ve got to recognise discrete DNA patterns. -Those patterns are typically sequences of about 6-8 bases pairs they bind to. -So the protein sequence within the transcription factors can recognise specific sequences. -When people first realised 6-8 bases were the kind of consensus, there became a question that say for six bases, that’s something that occurs not unfrequently in a genome, so going to stand the chance of finding that quite regularly on a random basis. -So that would seem to be to frequent for tight control of genes. -It turns out most transcription factors act as dimers. -So you’ve got dimerization, two parts of a transcription factor to produce a dimer. -Each half of the dimer then recognises one copy of these consensus sequences. -That gives more specificity, now seeing 12-16 base pairs because you’ve got this dimer that’s each binding to the same consensus, and that the chances of finding that consensus in duplicate in close proximity on the DNA is quite rare, so got increased specificity.

Answer 109

- DNA contains two grooves, major and minor groove, and it’s the information that the transcription factors can see in these grooves that’s recognised by the transcription factors. - Most transcription factors bind in the major groove, there’s more information available there, evolutionarily it’s easier to get higher specificity there. - Those that bind in the minor groove tend to have a weird property, they often cause bending of the DNA so they are what we describe as architectural transcription factors, those that bind to the minor groove, they cause disruption of the overall structure and bending of the DNA, and that can be important for the regulation of the gene. - Major groove is bigger, more molecular features available, it’s better for specificity.

Answer 110

- Most transcription factors bind as dimers to bind to DNA, giving increased specificity. As they’re dimeric as well, you can get transcription factors functionally that are homodimers, so both subunits are the same. - Or heterodimers, where two different protein components make up the functional transcription factor. - The ability to form homo- or heterodimers will change the binding specificity of the DNA. - They will change the options and the complexities within which we can regulate genes as have more variety for producing transcription factors that are able to bind to different DNA sequences.

Answer 111

-Transcription factors fall into general families, general transcription factors and transcription regulators. -General factors find associated with most promoters, so more associated with general transcription rather than regulation. -Then have the regulators which are the additional factors that come in and control your on and off primarily and response to the extracellular signals.

Answer 112

-Typical eukaryotic gene, typical in the sense it has a TATA based promoter, a TATA box. -Have a TATA box based promoter that binds the polymerase, it binds the general transcription factors in the very immediate region next to the polymerase. -Got downstream gene that’s going to be transcribed, and then various other features, so you’ve got features some of which are relatively near, so proximal to the promoter which bind specific gene regulatory proteins. -Got some that can be a distance away up stream of the promoter. -Got some that can be even further away, and then some that can be downstream as well. -Can be 100,000 base pairs between the transcriptional start site and some of the regulatory elements. -Get some kind of DNA looping. -Thought that way you get interactions is through the fact the intervening DNA is looped out, and then the distal regions interact somehow with the promoter. -Now become apparent there’s a large multisubunit protein complex called the mediator which is often what facilitates this interaction. -So this mediator complex can interact both with the proximal promoter region through protein-protein interactions, but can also interact with the distal elements, which themselves have transcription factors bound to them and mediate this looping out and then drive the activation of gene expression. -So this would appear to be what is the accepted model now for the way these distal elements function.

Answer 113

-Many transcription factors don’t actually bind DNA. -Some of these are critical, they function in groups. -A lot of transcription factors recruit other essential regulators, and you would describe these regulators as transcription factors as well, so called co-activators and co-repressors. -These are protein components that come in and act in concert with the DNA binding transcription factors to fine tune the regulation of gene expression, either to activate it or repress it. -Many of these activators and repressors are enzymes, so they have enzymatic function. -They’re able to methylate or phosphorylate other protein components, and that’s the way they regulate gene expression. -A lot of the function, as DNA is packaged and condensed into chromatin, a lot of the co- activators and repressors re functioning because of the packaging of chromatin and making it ether available for transcription or packaging it up so it’s not available. -So, a lot of these activators and repressors modify local chromatin structure and do so in a way that either opens up the chromatin and activates the gene, or closes the chromatin and activates the gene, or closes the chromatin and shuts it down.

Answer 114

-Chromatin formation requires DNA interaction with a set of proteins called histones. -These are small basic proteins; they carry a lot of lysine residues with the C termini of the histone proteins. -That’s good for DNA interaction, as lysine is net positive charge at neutral pH. -So, if you got a protein with a lot of lysines in it, will carry a lot of net positive charge, and DNA is a polyanion it has a phosphate backbone, negatively charged so get really high affinity interaction between these histones and DNA.

Answer 115

-What you end up with is an interaction between the core histone complex, so there are eight histones, two copies each of four histone proteins, H2A, H2B, H3 and H4, two copies of each forming this structure in the middle, around which is wrapped the DNA. -Wraps round twice round each structure to give a nucleosome, this is the lowest level of the higher order DNA structure you see in a eukaryotic cell. -Tight association with the histones, because of the negative phosphate backbone of the DNA. -If look at it from the other plane, can see each nucleosome is encompassed by two coils of the DNA wrapping around this, this histone octamer, producing each nucleosome and these nucleosomes are strung together like beads on a string on the DNA. -These beads assemble into a superstructure called a solenoid, which is packed more tightly together. -Interaction all relies on positively charged lysine residues and negatively charged DNA.

Answer 116

-Transcription co-activators open chromatin structure and therefore activate gene expression, many of those are histone acetyltransferases. -What they do is they’re enzymatic, they’re recruited into the promoter structure, and when they’re bound there, they then acetylate histones on those lysine residues. -If remove positive charge on the histones by acetylation, the interaction between the positively charged histones and negatively charged DNA becomes weaker, and that chromatin structure starts to open up. -So histone acetyltransferases are important activators. -But there’s other proteins involved, e.g. other proteins that are chaperones, that bind histones and remove them from the chromatin, and therefore that region of DNA is opened up, and if there’s a tata box as part of the promoter there, you now have an open region of chromatin that can recruit the polymerase and drive gene expression.

Answer 117

-When look at whole process, it’s a cascade of different enzymes involved. -Get successive modifications, e.g. one of the first events to bind a gene activator protein, which then recruits a histone acetyltransferase due to protein-protein interactions between the two. -That histone acetyltransferase that acetylates for example histone three on its lysine nine position, which then causes recruitment of a histone kinase which does phosphorylation’s on serines, which then recruits a modelling complex for example, which then opens the chromatin to allow transcriptional activation, so it’s a cascade of different modifications. -Sometimes called these modifications of histones to activate gene transcription or to shut it down, the histone code. -So you get acetylation, phosphorylation, other modifications as well, so this is an epigenetic code, that it can be read by different proteins that then open the chromatin for transcription.

Answer 118

-Co-repressors do the opposite, they close the chromatin down. -Example would be histone deacetylases, they take the acetyl group back off the lysines, the lysines become positively charged, they interact more tightly with the DNA and start to close the chromatin structure up. -Can also get factors that bind to over lapping sequences for those on the DNA that bind the activators. -So, if you’ve got a repressor protein that binds to a side that overlaps the binding site for the activator, then those two binding events are mutually exclusive. -Maybe under certain circumstances, the affinity for the repressor is higher than for the activator on the DNA and therefore the activator is displaced and you get a closing down of transcription. - You get binding site repressors which interact with the transactivation domains of activators, therefore mask the activation surface, and they can now no longer interact with the polymerase and switch on transcription. - Then in these looped out type examples, got binding site for oppressors that interact with the same surface on the RNA polymerase as the activator, so there’s a competition between the binding of the repressor and the activator. -If repressor wins, shut down transcription. -Also got repressors that are histone methyltransferases, so methylate lysines that the acetyltransferases acetylate, but the chemistry is different here. -If look at a primary amino group on a lysine, if acetylate can’t acquire a positive charge in neutral pH. -If you methylate it, that promotes formation of a positive charge on methylated nitrogen on side chain of lysine. -Will facilitate a stronger interaction between the DNA and histones. -So histone methyltransferases function normally to shut down transcription by facilitating tighter interaction between histones and DNA and closing up chromatin structure.

Answer 119

-There’s a group of transcription factors, called the hox genes, in humans. -They all encode transcription factors that control brain and neuronal development. -Hard to study in humans. -Mouse have the same homologous sets of genes, and if you look at where those genes are expressed during mouse developed, they’re expressed along the developing spinal cord, in Hox zones. -They control all sorts of neurological development. -Each Hox protein activates a battery of other genes. -Defects in these genes result in congenital malformation in humans, so HoxA1, defects in that produces deafness, facial defects etc.

Answer 120

- Pax transcription factors control development of neural crest cell migration, so these are in a developing embryo. - The developing neural crest that becomes the spinal cord, some of the cells migrate away from there during development into other parts of the developing embryo to become eye, ear etc.

Answer 121

-p53 is a transcription factor. -It is mutated in up to 50% of human cancers. -One of the reasons for that is it activates multiple genes downstream of itself to cause cell cycle arrest, to allow DNA repair, DNA damage is detected, and also if that damage can’t be repaired to trigger apoptosis, this programmed cell death to cause removal of the damaged cell from the overall organism.

Answer 122

- Myc is a master regulator, it’s critical for cell cycle entry and growth, so is often over expressed or mutated to produce a constitutively active transcription factor in many cancers, as its progrowth and will drive growth when it shouldn’t. - A hotspot in many human cancers.

Answer 123

-RNA stability illustrated by iron and regulation of iron storage. -In order to get iron in the cell, need the transferrin receptor to be produced, so if a cell is low in iron starvation, it needs to make the transferrin receptor, and in the transferrin receptor RNA, in the 3’ translated region of the RNA, there’s a secondary structure stem loop which binds this enzyme cytosolic aconitase, stabilising the RNA, allowing the transferrin RNA to be translated and therefore the receptors go to the cell surface to allow the cell to bring transferrin into the cell carrying the iron. -If there’s a lot of iron in the cell, some of that free iron binds to the cytosolic aconitase, causes a changing shape of that aconitase, it can no longer bind to this 3’ UTR stem loop structure. -By coming off of that, that opens up access to a cleavage point via an endonuclease, a ribonuclease, so this protein removed as it changes shape binding to iron, it loses affinity for this structure, that leads to endonulceolytic cleavage and that stimulates breakdown of RNA and therefore no transferrin receptor made as have plenty of iron in cell so don’t need any more of it. -Ferritin mRNA, has a UTR with a 5’ stem loop. If the cell is starved of iron it doesn’t need to make the storage protein ferritin, so cytosolic aconitase binds to this, preventing ribosome getting through and translating it, as don’t need it. In excess iron, it binds to the aconitase, it changes shapes, loses affinity for stem loop, stem loop now no longer stable enough to prevent ribosome reading through and translating the ferritin storage protein.

Answer 124

-Human genome now known to produce thousands of non-coding RNAs. -Many of these are miRNAs, microRNAs. -They’re small microRNAs, single stranded RNAs, they associate with a number of proteins to produce the risk complex or the RNA induced silencing complex, so miRNAs generally are a way of shutting down the expression of a gene at the RNA level. -So once this particular RNA is associated with the risk complex, it can then go and search out specific RNAs, using this RNA as a template to then degrade or silence particular RNAs. -So get a double stranded RNA. -It produces interfering RNA which is homologous to the RNA we’re trying to target, shut down. So this interfering RNA is used as a template to bind to by complementary base pairing to the target RNA, and that target RNA is either destroyed or translationally repressed. -So you get this risk complex, it carries a guide strand. -This guide strand is complementary to the target -mRNA we want to shut down. -If it is an extensive match by homology, you get splicing, get endonucleases which break this target RNA down, which is then degraded. -Can also have a less extensive match between the RNA target, get translational repression, so the sequestering of the RNA into inactive granules within the cell, and they’re not translated. -So microRNAs have become massively important in translational regulation.

Answer 125

- Chromosomes are the structures that carry the genetic material. - They consist of DNA carried on a protein skeleton. - They are localised in the nuclei of almost every cell in the body. - One exception is red blood cells, which don't have a nucleus, so no chromosomes. -For most of the cell cycle the chromosomes are decondensed and cannot be visualised.

Answer 126

-For actively dividing cells, the cell cycle lasts approximately 24 hours. -However, there are a large number of cell types that aren't going through the cell cycle and aren't spontaneously dividing - they are in the stage referred to as G0, and have to be stimulated by use of a mitogen to enter the cell cycle in vitro. -There is usually a 24 hour lag before cells start cycling after exposure to a mitogen.

Answer 127

-One example of this is most of the cells within the blood. -Blood cells serve a function within the blood, they all have different jobs to do, but none of them are actually dividing and generating new cells within the blood, most of that cell manufacture happens in things like bone marrow. -So will not usually find cells spontaneously dividing within the blood. -Generally speaking, don’t have spontaneously dividing cells in the blood, and have to use a mitogen to persuade those cells to go into cell division.

Answer 128

- Need a fresh sample, and have to be able to persuade it to divide. - If cells aren’t dividing, can’t see the chromosomes.

Answer 129

- Blood samples generally don’t have actively dividing cells, so have to persuade the cells to divide with use of mitogen, most commonly used is PHA, and that mitogen is specific to the T cells. - So when look at cytogenetic preparations from blood, mostly looking at chromosomes from T cells.

Answer 130

-Can prepare chromosome preparation from prenatal samples for prenatal diagnosis purposes. -One sample type we get is the amniotic fluid, the fluid surrounding the baby in the womb, and within that fluid will be some cells from the skin or the bladder surfaces of the fetus. -Those cells are not spontaneously dividing, they’re inactive , so put them into a situation where provide them with substrate to grow on, so grown within a sterile vessel which has a flat surface, provide them with a medium with all the growth factors they need for growth, and as they find themselves in that situation, they will start to divide and become active. -The chorionic villus which is part of the placenta can also be sampled. -It’s a part of the placenta that grows very rapidly during fetal development, so you have very actively dividing cells within the chorionic villus, especially within the first and second trimester, placenta growing much more quickly than the fetus for part of that time, so have very rapid turnover of cells.

Answer 131

-Look at chromosome preparations in cancer. -One of the reasons why cytogenetics is useful in cancer, is the hallmark of cancer is you have cells actively dividing in an uncontrolled manner. -So by trying to collect the cells that are most actively dividing in the tumour, probably looking at the most significant tumour cells. -So for patients with leukaemia, receive bone marrow samples. -The cells which are actively dividing will overwhelmingly be from the tumour clone. -Also occasionally receive blood samples from a leukaemia patient if you’ve got a very high turnover of tumour within bone marrow, those cells will spill over into blood, so will often find have actively dividing cells within blood of leukaemia patient. -Also receive lymph node biopsies from patients with the lymphoma. -Can also receive fresh tumour biopsies from a whole variety of different solid tumour, idea being if cells are actively dividing and we can culture them, and we can look at the chromosomes, we will be looking at the specific cells which are the core of the tumour.

Answer 132

- Metacentric, submetacentric and acrocentric. - In metacentric chromosomes, the centromere is in the middle. - May be large, or small. - Each half of the metaphase chromosome is a chromatid. - In submetacentric chromosomes, the centromere is towards one end. - Short arms (p) uppermost. - Long arms (q) below. - In acrocentric chromosomes, centromere very close to one end. - May have satellites separated from the small short arms by a secondary constriction or satellite stalk.

Answer 133

-The X is considerably larger, about same size as middle row of chromosomes. -Y chromosomes are one of the smallest chromosomes, can slightly vary in size between the individuals but is about the size of chromosome 22. -Issue here in that women have two X chromosomes each with a full complement of genes, and males only have one X chromosome, so only one copy of all those genes, and then the Y chromosome is smaller and has a smaller complement of genes. -Problem in that females carry two copies of the genes on the X chromosome, males only carry one copy of those genes, so would logically be a massive dosage difference between a male and a female. -Was a problem and finally realised, in women there’s a process called X inactivation and all women switch off one copy of their X chromosomes for most of the time, which means the dosage for all the genes carried on the X is the same for men and women. -This is dosage disequilibrium.

Answer 134

When we say males have a wide chromosome and females don’t, what the main difference is, is that males have this sex determining region gene, SRY, and that’s what makes the difference in gender development in the early fetus’, the presence or absence of that sex determining gene.

Answer 135

- X-inactivation isn’t there right from the beginning, it doesn’t happen until the 5000 cell stage, so it occurs two weeks post fertilisation. - At that point, it occurs randomly, so in every single cell, one or the other of the X chromosomes will switch off. - Once the X inactivation has happened in that cell, when that cell then divides and produces more and more daughter cells, the daughter cells carry the same pattern of X-inactivation as that first progenitor cell at the point of X-inactivation.

Answer 136

Can have numerical changes, uniparental disomy where the parental origin of the chromosome or part of a chromosome becomes important, and structural chromosome abnormalities.

Answer 137

Changes in the copy number of the chromosomes, can have trisomy’s, monosomy which is a loss of a chromosome, so go down to one chromosome instead of a pair, mosaicism, and polyploidy.

Answer 138

-Translocations where material from two different chromosomes exchange. -There are inversions, which are rearrangements within a single chromosome where the chromosome has two breakpoints in it and the central part of it is inverted. -Deletion, which is chopping out a section of chromosome. -Duplication, which is doubling up a section of a chromosome and then inserting it next to the original copy so you get a repeat within the chromosomes. -Chromosomes rarely form a ring, where in effect you have to chop off the ends of the chromosomes and join the cut ends together to form a ring structure with a centromere within it. -An isochromosome is a chromosome which consist of the same arm twice, so is like a mirror image around the centromere, so you can have an isochromosome for the long arm of the X chromosome, which will exist of two X q’s joined at the centromere and loss of the X p. -Can have a marker chromosome which means there’s an extra bit and don’t know what it is. -Fragile sites exist, things that appear like the end of a chromosome consistently appears to be falling off. -Can also have gaps or breaks.

Answer 139

-Down Syndrome is the most common genetic cause of mental retardation. -One in 650 births. -94% caused by a trisomy of chromosome 21, so an extra copy of chromosome 21. So karyotype is written as 47, XX, +21. -Majority of Down’s arise through non-disjunction in the maternal meiotic first division, so it’s an error that arises at the point in meiosis where the eggs are being formed. - Of the 94% which have trisomy 21, there are a small number who don’t have that extra copy of chromosome 21. -4% of these have unbalanced ‘Robertsonian translocation’ so a translocation between two chromosomes, one of which is 21, and is inherited in an unbalanced form, so in effect that child has affectively got three copies of 21, but it doesn’t appear as an additional chromosome, it’s actually attached to a different chromosome. 2% are mosaic, so a post-zygotic, mitotic non-disjunction event can give rise to a cell early in embryogenesis with an extra copy of 21, and then all the daughter cells from that first cell will have three copies of 21, but there will also be some normal cells present.

Answer 140

-There are only two other trisomy’s that are viable in live born humans in non-mosaic form other than Down Syndrome. -One of them is Edward’s syndrome. -Rarer, birth rate of 3 in 10,000. -Features are growth retardation, a prominent occiput (back of head), small mouth, clenched hands, overlapping fingers and prominent heels (rocker bottom feet). -85% have congenital heart disease and 30% have renal anomalies. -50% die by two months of age. -Also called trisomy 18, there are three copies of chromosome 18.

Answer 141

-Third live-born human trisomy. -Rare, effects 2 in 10,000. -Features are scalp defects in 75%, hypotelorism (narrow between eyes), polydactyly (extra digits) in 78%, and cleft lip and palate. 70% have brain malformation, some have congenital heart disease, 30-60% have renal anomalies, and males have undescended testes. -69% die by six months. -Has three copies of chromosome 13.

Answer 142

-Live-born monosomy, when have a single copy of a chromosome. -Not viable In humans for any of the autosomes, the only viable monosomy is for the sex chromosome, and so this is when only have a single sex chromosome, and that sex chromosome has to be an X, can’t survive with only a Y chromosome, but can exist with only an X chromosome. -Probably because in females we are inactivating one of the X chromosomes anyway. -Affects 1 in 10,000 females. -Features are short stature, webbed neck, lymphoedema of hands and feet, low posterior hairline, wide carrying angle at elbows and small nails. -60% have renal anomalies, 15% have coarctation of aorta, some have bicuspid aortic valve and some have gonadal dysgenesis.

Answer 143

-Caused by deletion within the proximal long arm of chromosome 15, but was realised early on patients always had a deletion of one copy of chromosome 15, and was more recently realised it was always the paternal chromosome that had the deletion. -So need a deletion of the paternal copy of the critical region on chromosome 15, or can have two apparently normal copies of chromosome 15, but both of those copies have come from the mother and you have no paternal copy of chromosome 15. -Extremely floppy in early infancy. -Developed marked obesity through over-eating. -Mild to moderate learning difficulties.

Answer 144

-Associated with a deletion of the proximal long arm of chromosome 15. -It’s the same critical region as for Prader Willi syndrome. -But for Angelman syndrome it’s the maternal copy of chromosome 15, from which the deletion occurs. -Or have no maternal chromosome 15 at all and have two copies of the paternal chromosome 15. -Inappropriate laughter, convulsions, poor coordination (ataxia) and learning difficulties.

Answer 145

-(1) First of all they have to bind mRNA, the RNA transcribed from protein encoding genes. - Ribosomes must bind mRNA such that codons can be read. -(2) The ribosome must be large as it has to interact with the tRNA molecules, that set of molecules that help interpret the codon and match codons to different amino acids. -(3)Need a set of non-ribosomal protein factors involved as there are complex procedures, so you need initiation, elongation and termination of polypeptide chains to have extra regulators that can help control the job. -So in that case the ribosome needs to be capable of interacting with these extra factors. -(4)Need a stable structure in which to catalyse peptide bond formation. -The bigger structure you have, the more effective a catalytic moiety is. - (5) Ribosomes have to be complex enough or have to have capabilities that will permit them to move down an mRNA or to allow an mRNA to move through them. -Must mean there is a sequential display of your codons to the required tRNAs at specific times. -So, added complexity there.

Answer 146

-Can gauge ribosomal size based upon their ability to be centrifuged, so their sedimentation rates. -This roughly correlates with size. -If look at prokaryotic ribosome, there are two subunits. -If you were to sediment this ribosome in one go, it comes out at 70S. -Now if you are to break apart the subunits or ribosome, will find two subunits. -There is the small subunit within the prokaryotic ribosome, and then the large subunit. -If you centrifuge those, have different rates. -Small 30s ribosomal subunit and the large 50s ribosomal subunit. -As this isn’t a linear function, 50 and 30 doesn’t add up to 70, but 80. -These ribosomes are collections of rRNA and protein. - If look at small subunit, there’s just one rRNA in prokaryotes and this is the 16S ribosomal RNA, and there’s a mix of 21 proteins helping the 16S do its particular job. -In the large subunit of the prokaryotic ribosome, have two rRNAs. -Have the 5S and 23S rRNA, and then 31 proteins there.

Answer 147

The ribosome, be that of a eukaryotic or prokaryotic cell, is the place where a peptide bond is formed and there is no other place for the formation of that peptide bond, and remembering proteins are the agents of action in both, then find ribosomes are very numerous in both types.

Answer 148

-Find ribosomal proteins, if to look at structure of a ribosome, they’re often on the outsides of the ribosome. -Proteins act as a scaffold. -If tried to locate site of the peptidyl transferase reaction, so the catalytic site of the peptide bond being formed, its 18 angstroms away from a protein. -So it turns out the ribosome is actually a ribozyme, it’s an example of catalytic RNA. -So the ribosome is an example of a ribozyme, a catalytic bit of RNA.

Answer 149

-Ribosomes are large cellular moieties, because of complexity of job. -Have to interact with the tRNA, that molecule that helps the process of translation actually function. -If were to look at a ribosome, and its ultrastructure, would note that there were three parts to the ribosome, especially within the large subunit, where would find certain tRNAs fitted, so sites where they were present during the translation process. -Ribosomes have three tRNA binding sites, one of which is called the A site or the aminoacyl site, this will accommodate the incoming aminoacyl-tRNA. -Also has P site, the peptidyl site, where have tRNA attached momentarily to the extending polypeptide chain, so the growing protein. -Then have E site, the exit site, which accommodates the tRNA, without amino acid, that is leaving. -All three tRNAs have anticodons bound to 30s subunit, and the rest of the tRNA bound to the 50s subunit.

Answer 150

-tRNAs on the A site and P site interact closely with mRNA via base pairing. -Acceptor ends, the ends with the amino acids, are close together. -That permits the peptidyl transferase reaction to occur, so the formation of the peptide bond, which will then allow a growing polypeptide to emerge. -Benefits of having a complex tertiary structure within tRNA, it’s tight and bound, so not delaying with a flapping clover leaf structure, it’s all been wrapped up on itself, such that you can have a close interaction of acceptor stem.

Answer 151

- Eukaryotic ribosomes are 40% bigger than bacterial version, they are 80s ribosomes. - Small subunit is 40s and contains 33 polypeptides and 18s rRNA. - Large subunit is 60S and contains 49 polypeptides and 28S, 5.8S and 5S rRNA.

Answer 152

-As run through process of translation, need helpers, some of which are catalytic, some which stabilise what is happening, to help ribosome do its job. -May be more of these helpers involved in eukaryotic than prokaryotic translation. -Apart from having extra helpers, need sources of energy. - So will have nucleotide triphosphates, either ATP or GTP being required. - For example, GTP will need to help bind the aminoacyl-tRNA to the A site within ribosome, and also need a GTP to allow either the RNA to move through the ribosome in on codon, or for ribosome to move along the mRNA depending on how look at it.

Answer 153

-Translating a three-letter code, the codon, into 20 amino acids. -The mRNA will be read in a 5’ to 3’ direction. -mRNA is synthesised in a 5’ to 3’ direction by transcription. -Means in prokaryotes where don’t have a nuclear envelope, as soon as an mRNA is produced, a ribosome can start interpreting it and creating a polypeptide very rapidly. -The three stages of translation are initiation, elongation and termination.

Answer 154

-Will have an amino acid that arrives in the A-site of ribosome, the one that’s going to contain the new amino acid that’s going to be added onto the polypeptide chain. - The tRNA found within the p-site is the one containing the currently growing polypeptide. - So, the embryonic protein as it were, the still elongating protein. - So what happens is going to have a peptidyl transferase reaction where will find the elongating chain of the polypeptide would actually form a peptide bond with the new amino acid in the A-site, and momentarily would find that the entire polypeptide would suddenly exist on the tRNA found within A-site. - Then the process of translocation occurs, and the mRNA or the ribosome moves a codon and suddenly the tRNA that was once in you’re A-site that contained the extending protein is now in the P-site. -The empty t-RNA that was once in the p-site but has been used up or at least no longer contains the elongating protein, that’s now in the E-site of the ribosome. -That means now the cycle can begin again. -It will carry on until encounter a nonsense codon.

Answer 155

-Due to the length of mRNA, they’re often quite long, and the ribosome doesn’t span the entirety of the mRNA, it is possible that as the mRNAs start to move through the ribosome/ the ribosome moves along the mRNA, another ribosome can get it on the act. -When have multiple ribosomes translating message from one length of mRNA, can refer to them as polysome complex. -In some cases, may find prokaryotic mRNA can encode more than one protein, sometimes depending which reading frame you’re in, can have overlapping genes. -Can also have multiple genes in same reading frame in same bit of mRNA. -Sometimes prokaryotic mRNAs referred to as polycistronic, so have more than one coding region, so one single mRNA can code for more than one protein. -These different coding regions can have own initiation and termination codons, meaning ribosomes will bind to different part. - Eukaryotic mRNA is simpler, generally would only have one coding region, it’s only going to give one protein, so can refer to as monocistronic.

Answer 156

-Where get an assembly of the different components found within the translation system. -It’s the process which has to take place in order to permit the system to be set up so the peptide bond can be catalysed by the ribozyme. -Going to need a ribosome, whether in prokaryotic or eukaryotic system, going to need two subunits that have come together in a particular spatial location for the ribosome to do its job, will need the mRNA which is going to be translated, will need an aminoacyl-tRNA specified by the first codon, also need a source of energy - GTP. -In prokaryotes need some extra factors to help, and in this particular part of the process, those are going to be given the acronyms of IF in prokaryotes for initiation factors, so can have three of those IFs, one, two and three. -In eukaryotes, initiation is more complex, have more initiation factors involved.

Answer 157

-First going to need an alignment of mRNA with ribosome, in a way the start codon, AUG, can get recognised. -Prokaryotes and eukaryotes do this slightly different. -With prokaryotes, if look at 30S ribosomal subunits, see a 16S ribosomal RNA, and towards its 3’ site, will have a particular sequence, which will base pair with a sequence about 10 bases up from your start codon in mRNA, and this particular reverse complementary base pairing, allows mRNA to be located such that AUG will be under the P-site, or within the vicinity of the P-site of the ribosome. -It’s a way of locking in mRNA in the correct position. -This particular sequence is highly conserved between prokaryotes and is called the Shine-Dalgarno sequence, and would be upstream, so towards 5’ end of mRNA. -In very close proximity to start codon, AUG. -That will mean that ribosome will select the correct start codon for particular peptide of choice. -Can have mRNAs in prokaryotes that can be polycistronic, so may be multiple start codons and they’ll all have Shine-Dalgarno sequences and other sequences that contribute to how well the ribosomes bind. - In eukaryotes, they’re monocistronic, so only have one start codon. -The cap structure find on mRNA, will have a role in locating this. - So, ribosome will detect that, and it scans down from that cap towards the AUG. - The Kozak sequence can also be involved in finding AUG. -The first tRNA then binds to mRNA. The first tRNA is an initiator tRNA, it’s going to bind the start AUG codon that’s been designated to the ribosome, in the case of prokaryotes by the Shine-Dalgarno sequence, and the case of eukaryotes, the scanning process. - Special as it will be the only tRNA that binds the ribosome at the P-site, all the other following tRNAs, will access the ribosome via the A-site, and will complementary base pair at that position. -Also, in the case of prokaryotes, the amino acid attached which is a methionine, is different to normal methionine’s, it’s been n-formulated, and that means it can only act as the n-terminal part of the protein as it has an extra amide bond in it. -This tRNA is spotted by a form of initiation factor called IF-2, that’ll do its job of collecting the formulated methionine tRNA and bringing it to the P-site of the ribosome. - Once that is there, have mRNA, tRNA with methionine on bound to AUG, will suddenly have the large subunit then join the complex and complete it ready for the next stage.

Answer 158

-Addition of amino acids to the carboxyl end of the growing chain. -Have elongation factors involved and also tRNAs as specified by the mRNA because will just be clipping together those that complementary base pair with the mRNA. -Going to be finding the polypeptide is going to be expanding on its C-terminus region, and as it does that, it will be moving as a polypeptide, it’s going to slowly be pushed through the ejection channel and through the ribosome. - The aminoacyl-tRNAs due for connection to the growing polypeptide chain will be brought to the ribosome by elongation factor 2. -It does this by hydrolysis GTP. -However, that is continually replenished by another type of elongation factor called ET-S that will swap out the hydrolysed or the guanosine diphosphate and replace it by guanosine triphosphate, hence allowing EF-2 to continually do its job. -That’s like the third stage of the process. -Then find have peptidyl transferase reaction, the creation of the peptide bond, which is going to take place between the acceptor stems of tRNA. -This activity, this is catalysed by the 23S ribosomal RNA of your large subunit. -So step four going to have peptide bond created. -Then get translocation step, facilitated by elongation factor G which will hydrolyse GTP in order to permit this movement. -Empty tRNA in P-site acts like a marker that will allow the ribosome to progress by three bases, so it will shuffle along by three bases during translocation process, and hence the growing peptide chain which is found in the tRNA which was once in you’re A-site, now ends up in the P-site. -Once in this particular position, steps three, four and five can carry on again.

Answer 159

-At some point the elongation phase has done its job and will have a protein that’s capable of performing the function that it’s evolved for. -So protein synthesis process has to draw to a close. -Couple of codons that no tRNA will bind to called nonsense codons, termination codons. -So one of these will move into view and occupy the A-site of ribosome. -Once it does that, there’s no tRNA to complementary base pair with it, will find extra factors required for translational process will get involved. -These particular factors at this stage are known as release factors, RF. -Some of them, RF-1 and RF-2, will recognise the different types of termination codon present within mRNA. -They’ll bind to those codons. -Once it does that will find that peptide linkage between tRNA and the polypeptide chain will get trimmed, will release protein. -Another release factor gets involved, RF-3. -This hydrolyses GTP and breaks the whole ribosomal complex apart. -Once its does, that ribosome can go back to beginning of mRNA and start process again. -Many of these mRNAs will have multiple ribosomes working alongside them in one particular go, in a polysome, so many release factors involved etc, to produce large numbers of proteins as required by cell.

Answer 160

The majority of antibiotics block translation, possibly due to the complex of the translational machinery.

Answer 161

-Translation occurs multiple times a second in a cell with many players involved. -Number of compounds that do a good job in playing around with protein synthesis. -Well known example of an antibiotic that effects protein synthesis is streptomycin. -Has a range of effects depending on strengths of the antibiotic provided. -So at low streptomycin concentrations, will find mRNA gets misread by the ribosome, essentially will find for example a pyrimidine may be mistaken in the first and second codon positions. -If however you up the amount of streptomycin, rather than having inaccurate elongation, will just stop initiation dead.

Answer 162

-Have 2 germ layers, the epiblast or primitive ectoderm, and the hypoblast or primitive endoderm. - Have two trophoblast layers, the cytotrophoblast which is cellular, and the syncytiotrophoblast which is multinucleate. -The two cavities, the amniotic and chorionic cavity, also known as the extraembryonic coelom. -Two membranes, the amnion, which consists of the amniotic epithelium plus the extraembryonic mesoderm, and the chorion which consists of the trophoblast, particularly the cytotrophoblast, and the extraembryonic mesoderm.

Answer 163

-When placental villi first form they are called primary stem villi have a core of cytotrophoblast covered by a layer of syncytiotrophoblast.

Answer 164

Secondary villi have developed a core of extra-embryonic mesoderm inside the two trophoblast layers.

Answer 165

- Tertiary villi have developed fetal blood vesels within the mesodermal core. - So now have possibility of transport but still quite a barrier between fetal blood vessels and what would be outside is one of the lacunae’s filled with maternal blood. - Anything outside has a long way to travel, has to get through syncytiotrophoblast, cytotrophoblast, mesoderm, endothelium of blood vessel to get there, so any transport via this route at this stage would be very minimal.

Answer 166

-Discoidal (15-25cm diameter) and weighs 450-600g. - Maternal aspect divided into 15-20 cotyledons. -Maternal blood enters cotyledons via 80-100 spiral arteries, and leaves via tributaries of uterine veins. -The maternal (intervillous) blood space surrounds villous trees. - Fetal blood (50ml) lies in vessels located within the villi and separated physically from maternal blood. -The intervascular barrier between maternal and fetal blood is made mainly of syncytiotrophoblast and vascular endothelium. -It has a large surface area, 10-15m^2, but is thin, average thickness about 4 micrometres.

Answer 167

-Placental abruption happens fairly late in pregnancy, where placenta peels away from uterine wall either partially or completely. -At that point growing baby will be short of nutrients and more particularly short of oxygen. -As it’s quite a traumatic peeling away, mother can be bleeding profusely from spiral arteries, and usually will trigger a premature birth, but if not, baby would need to be induced or delivered by caesarean. -Quite often leads to still birth if no intervention. -Happens in 1 in 100 pregnancies.

Answer 168

-Placenta previa is when the placenta is very low lying, so implantation has taken place very low down in uterus, and placenta grows so it covers all or part of the opening of the cervix. -Means baby can’t be born through cervix without placenta going first. -And because of the connections of the blood vessels it can cause severe bleeding when birth takes place as baby being expelled through mass of maternal and fetal blood vessels. -Can also cause premature birth. -1 in 200 pregnancies.

Answer 169

- Placenta accreta is the rarest, 1 in 2500 deliveries. - Placenta implants very deeply and firmly into uterine wall, may even go through uterine muscle or even through entire thickness of the uterus to emerge into the peritoneal cavity. - At birth, amount of bleeding excessive and mother can bleed out if this isn’t detected at the time. - Relatively well diagnosed condition and can be managed.

Answer 170

-Can have errors in polymerisation, maybe DNA polymerase slips, or incorporates the wrong base isomer. -The base isomer, the nitrogenous bases have different arrangements in 3D space, they exist in equilibrium with one another, there’s what’s known as the -enol form, the amino form, and the keto form. -It’s the keto form want in DNA and permits Watson and Crick base pairing. -Sometimes the other arrangements in 3D space end up being incorporated in DNA. -Given a phrase “tautomerism’. -So sometimes tautomerism can occur by accident, corrupting the quality of DNA have. -DNA can also be altered chemically by reactants present within the cells themselves, and also agents that occur in the environment.

Answer 171

-If mutations occur, either due to errors in polymerisation or due to problems in cell, can get a change in nucleotide sequence. -That can mean a change which ends up being inherited by other copies of the DNA or by the progeny of the organism, which can be a problem. -So the alteration of the nucleotide sequence of DNA, either by insertion, deletion, or maybe gross rearrangement. -Other terms include lesion, which means injury/ corruption. -Genetic lesion – areas of altered nucleotide sequence. -In many cases, will find mutations go unnoticed, or will just impact somatic cells, so the different cells that aren’t involved in progression of DNA to progeny. -So may not necessarily be noticed. -In some cases, somatic cells will form cancers, so will undergo metastatic change and then will notice these mutations. -If these mutations occur within the germline cell, so cells that lead to sperm or eggs, they can be transmitted to progeny.

Answer 172

-Environmental agents can be of a variety of different natures. -Quite a large number of them, a severe risk is posed by electromagnetic radiation, hence part of the light spectrum, so UV light between 200-300nm, for example, is problematic. -Other forms of radiation with an even higher energy quanta associated, one of those would be the X-rays and gamma rays. -Those are problematic when interacting with DNA.

Answer 173

"-Can encourage pyrimidines to form a cyclobutyl ring arrangement, causing them to dimerise, so have a pyrimidine dimer, a thymine dimer. -These dimers get in way of enzymes that are performing, so replication or transcription, so lead to problems with expression, especially when dealing with genes.

Answer 174

-Ionising radiation can cause problems by direct impact, the photons will interact with backbone of DNA and lead to its shearing, so it will break, and double stranded breaks in DNA is a problem, normally a lethal problem, end up leading to necrosis. -Ionising radiation can also cause reactive oxygen species to be created by aqueous environment – the cell contains a significant quantity of water, and these reactive oxygen species can go and impact DNA and effect its structure.

Answer 175

-Chemical mutagens can cause insertions or deletions of nucleotides, others can perform a point mutation. -There are two classes of point mutation – will have transitions, where purines change identity to another form of purine, or a pyrimidine changes to another pyrimidine. -In other cases can perform transversions, meaning they alter the structure of a pyrimidine to make it a purine, or vice versa. - Problematic as if change identity of bases within a particular position of DNA, means when comes to replication, suddenly will have a progeny or a daughter duplex being formed where at that position have wrong base pair occurring.

Answer 176

-These mutations often arise due to treatment with intercalating agents. -For example, actinomycin-D likes to intercalate and wedge itself within base pairs, leading to a distortion, basically doubles distance between the two and then the polymerase thinks there should be a base there and DNA replication then inserts or deletes a base. -Ethidium bromide is another mutagen.

Answer 177

- A polycyclic aromatic hydrocarbon encountered in cigarette smoke. - Actually formed by incomplete combustion of vegetative matter. - Benzo[a]pyrene gets acted upon by some of the enzymes present within cells. - Its attempt at detoxification makes it worse. - Binds to guanines, then means guanine is misread as a thymine and get a point mutation. - Benzo[a]pyrene is a cause of chimney sweep cancer, a cancer found on genitals of chimneys sweeps, many male, because of their poor hygiene.

Answer 178

-Genes will no longer be referred to as wild type. -Wild type is a term used in molecular biology, referring to the allele, the nucleotide sequence which is most common or occurs at the highest frequency within the population. -So wild type is the most common allele. -Anything that diverges from wild type can be called a mutation. -When dealing with a protein coding gene, a structural gene, can lead to an impact on the codon, which can have varying effects. -Sometimes it’s a silent mutation, sometimes will find the mutation changes just one position of the codon, but thanks to the redundancy found within the genetic code, the same amino acid is present. - Other times can have a missense mutation where will have a change in the codon that leads to an amino acid that has different biochemical properties. -Then impacts protein function. -In other times, can have a termination/ nonsense codon and will stop things dead.

Answer 179

- If have insertions or deletions, as long as not dealing with a bunch of three insertion or deletions, will have a frameshift mutation, which is very problematic – depends upon where mutations occur within gene sequence. - If occurs right at end and only affects like two amino acids, may not have a problem. - But if it starts right at beginning of gene, may have a problem. - With regards to missense mutations, may not have a problem if that mutations happens to be in say a surface loop of a protein, but if it’s in the active site of an enzyme, will have a problem. - So all depends on where mutation takes place within gene and protein

Answer 180

-Sometimes will find there are mutations that effect downstream genes, so may occur in one gene but this will impact expression of other genes. - In bacteria, this is understood in the terms of operons, the aggregations of genes that may all be turned on or off in one go. - Refer to these types of changes as polar mutations.

Answer 181

-Mutations don’t have to just be small numbers of nucleotides, can be gross chromosomal changes. - For example, can find parts of the chromosomes that have been duplicated, and can then have recombination occur, which can lead to deletion of sequences between, so if have two repeats, due to active recombination, the bit of spanning DNA between them ends up being cut out. -There are evolutionary reasons for having recombination but can lead to disease in some cases. -Example is Charcot-Marie-tooth syndrome which shows between 5 and 15 years of age, where develop physical weakness and difficulty walking. -Get a 1.4 mega base interval that is deleted on chromosome 17, so a huge number of nucleotides cut out, which impacts on a number of processes.

Answer 182

- Not all DNA alterations are noticeable as have redundancy in genetic code and we are diploid organisms, so will often have alleles, or sister chromosomes that can potentially compensate. - Not every part of a chromosome is expressed, not all of it is genes, there’s non-coding DNA. - So not everything will lead to a significant problem. - Sometimes will get a mutation that leads to a metastatic event, so when get cells growing out of control and spreading, which is cancer.

Answer 183

- Examples of enzymes that try to reverse damage directly and do good job of breaking up pyrimidine dimers caused by UV light. - So will require light energy and transfer it to the dimer, get excited and split the dimer. - They are found in prokaryotes and eukaryotes but not humans.

Answer 184

-Not all damaged bases can be directly repaired by enzymes. -They must be removed and replaced by base excision repair. -Glycosylases cleave the glycosidic bond of corresponding altered nucleotide. -Leaves a deoxyribose residue with no attached base, leaving an apurinic or apyrimidinic site, which is bad. -Enzymes can get stuck to them and ribose can linearise and stick to other cellular components, so cell needs to sort this out. -The deoxyribose residue is cleaved on one side, 5', by apyrimidinic endonuclease. -The deoxyribose and adjacent nucleotides are removed by deoxyribose phosphatelyase. -Gaps are then filled by DNA polymerase and ligase. -AP sites in mammalian DNA are highly cytotoxic and irreversibly trap mammalian topoisomerase I. -Ribose at AP site lacks glycosidic bond, so can linearise and then cross-link to other cellular compoents. -So, remain tightly bound to glycosylase.

Answer 185

- Both base excision repair and nucleotide excision repair act when lesion affects one strand. - However, DNA is susceptible to double strand breaks. - This can be lethal to a cell. - Common, 5-10% of dividing cells will have undergone double stranded breakage of a chromosome, so important have a mechanism. - Two types of repair mechanism, non-homologous end-joining (NHEJ), and recombination repair. - Need these as double stranded breaks, the end of DNA are capable of binding to other types of cellular components, potentially other parts of nucleus or organelles, which can cause problems. - That’s why have mechanisms.

Answer 186

- Will have protein called Ku, it acts as the broken DNA sensor of cell. - Will non-specifically bind the major and minor groups of DNA and will slide up and down the DNA as required and one will go on one part of the broken helix, and one will go on the other. - It will interact with a range of proteins that will permit resecting, so a bit of trimming back of DNA, and then filling sections in and ligation. - Sometimes will lead to scars in genome.

Answer 187

-In homologous end-joining, where have an arrangement called a Holliday junction, and due to action of Rad51, ends of helix which has been sheared can invade the sister helix, the sister chromosome, and due to formation of two of these Holliday junctions, they end up swapping over bits of DNA and then using polymerases to fill in the joints. -That can totally restore the nucleotide sequence. -So another way by which nucleotide sequences can be repaired.

Genes and Early Embryology - Week 6 Flashcards

(211 cards)