Genes and Early Embryology - Week 6 Flashcards
What regions can the fallopian tube be divided into?
The infundibulum, the ampulla, the isthmus and the intersitial (intramural) region.
What happens when the ovary sheds an ovum?
- It sheds it into the abdominal/ peritoneal cavity, and then it’s captured by the fallopian tubes using fimbriae.
- There is continuity in the female with the outside through the vagina, through the uterus, along the fallopian tube, into the abdominal cavity.
- Can be a route for infection to invade the body.
What are the stages of female gametogenesis, oogenesis?
-Before birth, there are structures called oogonia/ oogonium, which sit within ovary and as birth approaches and young childhood, they mature into this primary oocyte, which is surrounded by a single layer of follicular cells, which may be derived from the epithelium of the ovary.
-So you have the primary oocyte surrounded by a single layer of follicular cells, together known as the primary follicle.
-After puberty, each month a number of these primary follicles, 5-12 probably, begin to develop and become growing follicles.
-The surrounding follicular cells multiply quite rapidly and become a several layered surrounding of the oocyte.
-They also become separated from the ovum by an acellular mucopolysaccharide layer, the septum pellucidum.
-Normally all but one of these growing follicles degenerate and form a small scar within the ovary, called the corpus atreticum.
-The follicular cells of the remaining growing follicle secrete a fluid which produces a fluid-filled antrum within the follicle.
-The ovarian non-gamete cells surrounding the follicle also become altered and form a thecal layer around the follicle.
-This can be divided into a vascular theca interna (which produces oestrogen) and a relatively avascular inactive theca externa.
-In the mature or Graafian follicle the majority of the follicular cells form the stratum granulosum (which will produce
progesterone).
-The remainder surround the oocyte as the cumulus oophorus.
-It is now a secondary oocyte.
-The mature follicle ruptures to release the ovum.
-This retains a covering of follicular cells which form the corona radiata.
-The septum pellucidum has expanded to become the zona pellucida.
-The cells of the theca interna and the stratum granulosum enlarge, especially those of the latter, turn yellowish and form the
corpus luteum.
-It secretes large amounts of progesterone, and also oestrogen.
-Prior to ovulation the follicle produced mainly oestrogen.
-If fertilisation doesn’t occur, the corpus luteum has a life of only 12 days after which it degenerates into the corpus albicans.
-The cessation of its hormonal output leads to menstruation.
-If pregnancy occurs, the corpus luteum is sustained by the HCG produced by the conceptus and forms a large corpus luteum
of pregnancy.
-This will eventually form a large corpus albicans. So the normal menstrual cycle is interrupted if fertilisation takes place.
What are the stages of male gametogenesis?
-So load of stem cells which are 46 chromosomes and they undergo mitosis, so still have 46 chromosomes to form the primary spermatocyte.
-Then undergo a meiotic division from their double chromatids and they’re either 23 chromosomes with an X chromosome or 23 chromosomes with a Y chromosome.
-They then undergo a second meiosis so now have a single chromatid from each pair of chromosomes, and they are called spermatids and they have 23 single chromatids which form eventually, as
they mature, spermatozoa, so the spermatozoa will either be 23X or 23Y, and according to which they are when they meet an egg, will either form a male or female baby.
What is the structure of a single sperm?
- The head consists largely of a nucleus, where the chromosomal material is, with a sparse cytoplasmic covering.
- Within that cytoplasmic covering is the acrosomal cap, derived largely from the Golgi Apparatus, that contains enzymes which help in the penetration of the ovum.
- Has a middle piece, about 7 micrometres long, and that is packed with a spiral array of mitochondria surrounding the axial bundle of the sperm, which contains material very similar to the content of a cilium.
- The tail is about 40 micrometres long, and that contains material again similar to a cilium, with a 9+2 microtubule arrangement, and this is what propels sperm forward, it is the motile part.
What does it mean if someone has Kartageners syndrome?
- The sperm lacks the little arms called dynein, which joined the tubules into this array.
- They are all separate, so not propulsive.
- So somebody with this syndrome would be infertile as sperm couldn’t swim far.
What do you end up with at the end of
spermatogenesis?
Single chromatids in each of the sperm.
What do you end up with at the end of
oogenesis?
-In females, egg undergoes mitosis before birth and before birth also produces first meiotic division.
-So the primary oocyte is containing the full complement of chromosomes, and then as it matures, it undergoes the first meiotic division.
-So instead of having two chromosomes in pair, it only has one.
-The other one forms a polar body.
-So it will sit at this stage of development for
a while and then undergo second meiotic division and as it’s released and fertilised, at that point there’ll be a second polar body,
so it’s suspended at this stage, so when it is released from ovary, it is at this stage.
How do spermatogenesis and oogenesis
compare?
-In spermatogenesis, have mitosis followed by meiosis in the testes, but in oogenesis, only have meiosis in the ovaries which
results in haploid ova and a haploid sperm.
-These are produced continuously from puberty in the male, so the stem cells are
retained in the testes, and it’s constantly producing sperm, and that process takes about nine weeks and about 300 million
sperm are in each ejaculate.
-In contrast, in the female, it is discontinuous, all primary oocytes, and there’s about 2 million of them, are present at birth.
-Don’t produce any more eggs beyond birth, there are no stem cells.
-The primary oocytes are suspended part way through meiosis.
-As the five to twelve primary oocytes continue with meiosis each monthly cycle, following puberty there are five to twelve that do this every month, but they don’t actually complete meiosis, they’re still suspended part way through until moment of fertilisation.
-Sperm are motile, eggs aren’t, they rely on movement down fallopian tube being driven
by cilia in tube itself.
-In sperm, there’s very few bits of cytoplasm, mainly of nucleus, so a very low cytoplasmic to nuclear ratio.
-In contrast, in the egg, a lot of cytoplasm, and a high cytoplasmic to nuclear ratio.
-The sperm also requires fluid from other
glands in reproductive system to be added to it prior to ejaculation, so to form seminal fluid from seminal glands and the
prostate.
What happens when sperm are deposited in the vagina?
- Sperm are deposited in the vagina, only about 1% of the sperm deposited penetrates the cervix.
- Several hours later, the sperm have swum up here and got to the isthmus region of the fallopian tube, and when they get here they get less motile.
- They’re waiting for chemoattractants, chemical molecules to attract the sperm that are released from the cumulus cells surrounding the ovum.
- Once they pick up those signals, they become motile again and swim to ampulla region, and that’s usually where fertilisation takes place.
- So egg is picked up by the fimbriae, wafted along the fallopian tube by means of cilia and they meet up normally at the ampulla for fertilisation to take place.
- In that the sperm are waiting for a signal from the ovum, they could wait there a little while if no ovum present, so can have slightly delayed fertilisation.
What is capacitation?
-Sperm requires a process called capacitation, a conditioning of the sperm that is brought about by materials within the
female reproductive tract.
-Once the sperm is capacitated, the acrosomal region, above the nuclear material in the head, loses the glycoprotein coat, so the enzyme sac is much more exposed and available to digest its way into the egg.
What are the stages of fertilisation?
-In fertilisation I, shed secondary oocyte.
-So surrounded by corona radiata cells and surrounded by the zona pellucida.
-The egg was suspended partway through meiosis, so this is half of the genetic material.
-The polar body is the other half, and it’s shed to one side and sits there.
-So it’s sitting there, all the sperm come, they’re attracted to it and eventually one of them will penetrate the zona pellucida and fertilise the egg.
-In fertilisation II the female nucleus completes that second meiotic division, so only when it is fertilised does that happen, and then have two or sometimes three polar bodies, because sometimes the polar body that was here divides as well.
-So sperm fertilises egg and it’s the enzymes in the acrosomal region in the head of the sperm that actually helps that penetration and the nuclear material only is injected into all this cytoplasm to meet with the female nucleus.
-In fertilisation III, fertilised ovum diploid again.
-Once that material has been inserted, the zona pellucida undergoes a reaction called a zona reaction, which makes it almost impossible for further sperm to penetrate, meaning can’t get more than one male nucleus entering the egg.
-At this reaction there are enzymes released by cortical granules which digest sperm receptor proteins ZP2 and ZP3 so they can no longer bind the sperm.
-The ovum shrinks so that there’s a bigger perivitelline space between the zona pellucida and the cytoplasm of the egg itself.
-At this point, all of this cytoplasm has come from the female, whereas the nuclear
material is equal.
-So in consequence all the organelles come from the female and none from the male.
-In fertilisation IV, first thing get is cleavage.
-So the single cell with the two fused nuclei, after about 30 hours will divide into two identical cells called blastomeres formed by normal mitosis.
-The amount of cytoplasm divided between two cells.
-In fertilisation V, get further cleavage, and the cells will continue dividing and what happens is, the zona pellucida is still here, the blastomeres and the amount of cytoplasm get smaller, so a lot of synthesis of DNA to make new nuclei, but virtually no proteins or further cytoplasm made at this stage.
-In fertilisation VI, carry on dividing until get a solid ball of cells called a morula.
-Now the cytoplasmic to nuclear ratio has fallen as all cytoplasm that was in original single cell that filled this cavity has now been divided up amongst these multiple cells through the cell division.
-All through this, zone pellucida is here and intact.
What happens in blastocyst formation, after fertilisation?
-Morula still surrounded by zona pellicuda free within the uterine cavity.
-Now the second week after fertilisation that you start getting more differentiation.
-Still a solid ball of cells.
-First thing that happens is the process of compaction.
-All these cells now instead of being individual, they’re all joined together.
-They formed tight junctions, so the outer layer of cells is now forming tight junctions and at the same time the zona pellucida is
starting to break down, cracks appearing in it.
-So you get compaction and the establishment of tight junctions between surface cells and the zona pellucida starts to split.
-These cells start to organise themselves about five days post fertilisation.
-So all of the outer cells with the tight junctions start pushing some cells towards the middle, these don’t have tight junctions, they’re separate, so you end up with two populations of cells, and a fluid filled space.
-These outer cells are called a trophoblast.
-And the inner cells called inner cell mass.
-These half a dozen cells from the inner cell mass are the ones that will actually form the whole of the embryo.
-The outer cells are mainly involved in forming extra embryonic membranes, membranes outside the embryo and the placenta.
When does implantation begin?
At about six days the blastocyst adheres to the endometrium, usually on the posterior wall of the uterus and nearer the fundus
than the cervix. Implantation begins.
What happens in implantation?
-Blastocyst has outer layer of trophoblast cells, usually made up of approximately 55 cells, and at one end of the ball of cells will
be the inner cell mass, starting off as approximately 5 cells.
-Trophoblast said to be invasive, ingestive and digestive, as can invade epithelium and to some extent digest its way into the wall of the uterus.
-Once it gets there, a decidual reaction occurs in the uterine lining, causing increased secretion from the cells of the uterine wall, and that to some extent nourishes the embryo at this early stage.
-The inner cell mass forms the embryo and some of the membranes that surround it.
-Here the inner cell mass has started to divide into two populations of cells, the blue primitive ectoderm and the yellow primitive endoderm.
-The zona pellucida has disappeared.
-Cells have now pushed their way out to the edges of where the trophoblast is and now have layer of trophoblast cells called the
cytotrophoblast, the inner layer of the trophoblast.
-The trophoblast itself is also differentiating and forming a multicellular layer called syncytiotrophoblast.
-The inner cell mass gives rise to primitive ectoderm, epiblast, which surrounds the amniotic cavity, and primitive endoderm, hypoblast, which surrounds the cavity of the yolk sac.
-Blastocyst will now be two layered.
-By day 10, completely embedded and the epithelial continuity restored.
-Cells start being made in the edges of the conceptus so that the primitive ectoderm and primitive endoderm are pushed away from the trophoblast.
-This is an invasion of the area, and think the cells come from the trophoblast - the cells are termed extra-embryonic mesoderm.
-Purpose of this is to make space within conceptus for embryo to start expanding and growing.
-Within the extra-embryonic mesoderm are cavities.
-The embryo attaches by the trophoblast overlying the inner cell mass.
-Have the conceptus that has now completely invaded the uterine wall.
-Endometrium undergoes decidual reaction.
-In wall of uterus, various cells differentiate so can provide nutrients for growing embryo, so they contain large amounts of glycogen and lipid material and are acting as a gland which is secreting materials to the conceptus.
-The decidual reaction is most intense in regions of implantation, so area immediately surrounding where implantation has taken place.
-Cavity in the extra-embryonic mesoderm expands to completely surround embryo.
-Extra-embryonic mesoderm is left covering the amniotic cavity and yolk sac and lining the trophoblast.
-The cavity is called the chorionic cavity or extra-embryonic coelom.
-Purpose of this cavity is it allows this part of the conceptus to expand and grow.
-Only thing now attaching the embryo to the trophoblast which will form the placenta, is a little strand of extra-embryonic mesoderm, called the connecting stalk.
-So, these two layers of mesoderm are joined by the connecting stalk.
What is the bilaminar disc?
-Where ectoderm and endoderm lie against each other, a flattened bilaminar disc is produced from which will be formed the majority of the fetus.
-The ectoderm of the amnion is continuous with that of the bilaminar disc.
-The endoderm of the yolk sac is
continuous with that of the bilaminar disc.
When does implantation happen?
- In the second week of development, which we call a period of two’s.
- Have two layers of embryo, ectoderm and endoderm, have two cavities, the amniotic sac and the yolk sac, and have two trophoblast derivatives, the cytotrophoblast and the syncytiotrophoblast, the multicellular layer.
What happens in the third week?
Develop three germ layers and three important structures, called the primitive streak, the notochord and the neural tube.
How is the notochord formed?
- Cells from the rostral end of the primitive streak form a midline structure, the notochord, which induces overlying ectoderm to form the neuroectoderm of the neural plate.
- So the primitive streak develops further so at the rostral end, the end nearest the prochordal plate, get an area called a primitive node/ pit.
- From that beneath the surface is fused a rod of condensed mesoderm called the notochord.
- This gives off signals as it grows forwards towards the prochordal plate, telling the area of ectoderm above that it is going to become neural cells, and you end up with an area called neural plate.
What does the neural plate form?
- The neural plate forms neural folds (neuroectoderm) which eventually fuse to form a neural tube - brain and spinal cord.
- It loses contact with the overlying ectoderm.
What are the three major types of RNA in cells?
- Ribosomal RNA, which makes up two thirds of the ribosome and allows the ribosome to act as a catalytic entity.
- Transfer RNA, molecules that deliver amino acids to ribosomes for translation to occur.
- Messenger RNA which is the RNA that will end up encoding protein.
What are all cellular RNAs transcribed from?
DNA templates.
What creates RNA from DNA?
-The production of RNA from DNA is carried out by DNA-dependent RNA polymerases, these multisubunit complexes. -Unlike DNA polymerases which run across all of the genetic material to duplicate it, RNA polymerases are directed to specific genes at specific times the cell requires it.
What does RNA polymerase do?
- They use the DNA template to create RNA.
- In order to create RNA, have to clip together ribonucleotide triphosphates, so the NTPs: ATP, CTP, GTP and UTP.
What are similarities in RNA synthesis between prokaryotes and eukaryotes?
- 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.
What is the difference in protein coding genes in
eukaryotes and prokaryotes?
-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.
“How does RNA polymerase work out which part of the DNA it needs to transcribe?
-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.
Where are promoters found?
-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.
In which direction does RNA synthesis proceed?
In the 5’ to 3’ direction.
What does binding of the RNA polymerase holoenzyme lead to?
-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.
What does it mean that RNA polymerase is processive?
- 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.
How is transcription rapid?
- 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.
How can RNA synthesis be initiated as often as sterically possible?
- 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.
How can transcription be terminated in prokaryotes?
- 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.
What is the rho-independent form of terminator sequence?
- 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.
What is rho-dependent termination?
-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.
What are some of the types of RNA polymerase in eukaryotes?
- 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.
What is alpha-amanitin used for?
- 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.
What does it mean for RNAP II promoters to be more complex and diverse?
-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.
What are enhancers in transcription?
-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.
What are general transcription factors?
-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.
How is Rifamycin B an inhibitor of transcription?
-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.
What happens when transcription successfully
proceeds?
- 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.
What process does RNA undergo to create mRNA?
- 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.
What is co-transcriptional modification?
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.
Why is RNA capped?
- 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.
What is polyadenylation?
- 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.
What is splicing?
- 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.
What is a spliceosome?
- 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.
What is alternative splicing?
- 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.
What are the three main patterns of inheritence?
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.
What is autosomal dominant?
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.
What is autosomal recessive?
- 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.
What is X-linked recessive?
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.
What are some common x-linked recessive disorders?
- 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.
What is Duchenne Muscular Dystrophy?
- 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.”
What are some common autosomal recessive disorders?
- Hereditary haemochromatosis.
- Cystic fibrosis.
- Beta-Thalassaemia.
- Spinal muscular atrophy.
- Many inborn errors of metabolism.
- Some sensorineural deafness.
- 21-hydroxylase deficiency.
What is cystic fibrosis?
-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.
What information is needed to work out the risk of someones child being affected by a condition?
- 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.
What do you do if you don’t know the carrier rate for a recessive condition?
-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.
What is the Hardy Weinburg principle?
-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.
When is the Hardy-Weinburg principle used?
- 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.
How do you use the Hardy-Weinberg principle?
- 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.
How does non-random mating disturb the Hardy-Weinburg principle?
- 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.
How does the mutation-selection equilibrium
disturb the Hardy-Weinburg principle?
-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.
How does selection (heterozygous advantage)
disturb the Hardy-Weinburg principle?
-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.
How does small population size disturb the Hardy-Weinburg principle?
-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.
How does migration (gene flow) disturb the Hardy-Weinburg principle?
Migration and intermarriage can introduce new alleles into a population.
When is the first routine ultrasound?
- 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.
What is a Nucha Translucency (NT) scan?
-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.
What are causes of increased NT?
- Chromosomal - Down syndrome.
- Major congenital heart disease.
- Skeletal dysplasias.
- Diaphragmatic hernia.
What is abnormal MSAFP assay?
- 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.
What are invasive targeted fatal tests?
Chorionic villus sampling (CVS), amniocentesis and cordocentesis (fetal blood sampling) and fetal tissue biopsy which is used
rarely.
What is chorionic villus sampling?
- 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.
What is amniocentesis?
- 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.
What is preimplantation genetic diagnosis?
-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.
What is universal newborn screening?
- 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.
What is polygenic inheritance?
- 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.
What is multifactorial inheritance?
-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.
What are examples of multifactorial conditions?
Autoimmune deficiency, coronary artery disease, late onset forms of diabetes.
Why detect mutations using molecular diagnostics?
-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.
Why is somatic molecular testing used?
-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.
What is an example of using somatic testing in terms of K-ras?
- 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.
What are challenges facing molecular tests?
-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.
