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Posts Tagged ‘genetics

a DNA dialogue 5: a first look at DNA replication

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schematic of ‘replisome’ structures involved in DNA replication

 

Jacinta: So let’s scratch some more of the surface of the subject of DNA and genetics. A useful datum to remember, the human genome consists of more than 3 billion DNA bases. We were talking last time about pyrimidines and purines, and base pairs. Let’s talk now about how DNA unzips.

Canto: Well the base pairs are connected by hydrogen bonds, and the two DNA strands, the backbones of the molecule, run in opposite, or anti-parallel, directions, from the 5′ (five prime) end to the 3′ (three prime) end. So, while one strand runs from 5′ to 3′ (the sense strand), the other runs 3′ to 5′ (the antisense strand). 

Jacinta: Right, so what we’re talking about here is DNA replication, which involves breaking those hydrogen bonds, among other things. 

Canto: Yes, so that backbone, or double backbone whatever, where the strands run anti-parallel, is a phosphate-sugar construction, and the sugar is deoxyribose, a five-carbon sugar. This sugar is oriented in one strand from 5′ to 3′, that’s to say the 5′ carbon connects to a phosphate group at one end, while the 3′ carbon connects to a phosphate group at the other end, while in the other strand the sugar is oriented in the opposite direction. 

Jacinta: Yes, and this is essential for replication. The protein called DNA polymerase should be introduced here, with thanks to Khan Academy. It adds nucleotides to the 3′ end to grow a DNA strand…

Canto: Yes, but I think that’s part of the zipping process rather than the unzipping… it’s all very complicated but we need to keep working on it…

Jacinta: Yes, according to Khan Academy, the first step in this replication is to unwind the tightly wound double helix, which occurs through the action of an enzyme called topoisomerase. We could probably do a heap of posts on each of these enzymes, and then some. Anyway, to over-simplify, topoisomerase acts on the DNA such that the hydrogen bonds between the nitrogenous bases can be broken by another enzyme called helicase.

Canto: And that’s when we get to add nucleotides. So we have the two split strands, one of which is a 3′ strand, now called the leading strand, the other a 5′ strand, called the lagging strand. Don’t ask.

Jacinta: The leading strand is the one you add nucleotides to, creating another strand going in the 5′ to 3′ direction. This apparently requires an RNA primer. Don’t ask. DNA primase provides this RNA primer, and once this has occurred, DNA polymerase can start adding nucleotides to the 3′ end, following the open zipper, so to speak.

Canto: The lagging strand is a bit more complex though, as you apparently can’t add nucleotides in that other direction, the 5′ direction, not with any polymerase no how. So, according to Khan, ‘biology’ adds primers (don’t ask) made up of several RNA nucleotides.

Jacinta: Again, according to Khan, the DNA primase, which works along the single strand, is responsible for adding these primers to the lagging strand so that the polymerase can work ‘backwards’ along that strand, adding nucleotides in the right, 3′, direction. So it’s called the lagging strand because it has to work through this more long, drawn-out process.

Canto: Yes, and apparently, this means that you have all these fragments of DNA, called Okazaki fragments. I’m not sure how that works…

Jacinta: Let’s devote our next post on this subject entirely to Okazaki fragments. That could clarify a lot. Or not.

Canto: Okay, let’s. Goody goody gumdrops. In any case, these fragments can be kind of sewn together using DNA ligase, presumably another miraculous enzyme. And the RNA becomes DNA. Don’t ask. I’m sure all will be revealed with further research and investigation.

References

Leading and lagging strands in DNA replication (Khan Academy video)

https://www.quora.com/What-is-DNA-unzipping

https://www.yourgenome.org/facts/what-is-dna-replication

Written by stewart henderson

February 26, 2020 at 10:59 pm

a DNA dialogue 3: two anti-parallel strands

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but why the twist? – we don’t know yet

Jacinta: Ok so these two strands of DNA are described as anti-parallel. Is this just intended to confuse us?

Canto: Apparently not, in fact it’s quite essential. The useful q&a site Quora has good info on this, and understanding it in all its complexity should help us to understand DNA general – it’s one of a thousand useful entry points.

Jacinta: Yes, and I’ll try to explain. It became clear to us last time that the strands or ribbons twisted round in a double helix, called the backbone of the molecule, are made from phosphate and deoxyribose sugar, covalently bonded together. That means tightly bonded. Between the two strands, connecting them like ladder rungs, are nitrogenous bases (this is new to us). That’s adenine, thymine, guanine and cytosine, bonded together – A always to T, and C to G – with weak hydrogen bonds. We’ll have to look at why they must be paired in this way later.

Canto: It’s called Chargaff’s base pairing rule, which doesn’t tell us much.

Jacinta: And, according to a respondent from Quora, ‘the two strands of DNA are anti-parallel to each other. One of them is called leading strand, the other is lagging strand’. But I don’t quite get this. How are there two strands of DNA? I thought there was one strand with two sugar-phosphate backbones, and a rung made up of two – nitrogenous nucleobases? – weakly connected by hydrogen bonds.

Canto: I think the idea is there are two strands, with the attached bases, one next to another on the strand, and weakly attached to another base, or set of bases each attached to another phosphate-sugar backbone. As to why the whole thing twists, rather than just being a straight up-and-down ladder thing, I’ve no idea. Clearly we’re a couple of dopey beginners.

Jacinta: Well, many of the Quora respondents have been teaching molecular biology for years or are working in the field, and just skimming through, there’s a lot be learned. For now, being anti-parallel is essential for DNA replication – which makes it essential to DNA’s whole purpose if I can call it that. I’ll also just say that the sugars in the backbone have directionality, so that the way everything is structured, one strand has to go in the opposite direction for the replication to work. If for example the strands were facing in the same direction, then the base on one side would connect to a hydrophobic sugar (a good thing) but the base on the other side would be facing a hydrophilic phosphate (a bad thing). Each base needs to bond with a sugar – that’s to say a carbon atom, sugar being carbon-based – so one strand needs to be an inversion of the other. That’s part of the explanation.

Canto: Yes, I find many of the explanations are more like descriptions – they assume a lot of knowledge. For example one respondent says that the base pairs follow Chargaff’s rule and that means purines always pair up with pyrmidines. Not very helpful, unless maybe you’re rote-learning for a test. It certainly doesn’t explain anti-parallelism.

Jacinta: Well, although we don’t fully understand it yet, it’s a bit clearer. Anti-parallelism is an awkward term because it might imply, to the unwary, something very different from being parallel. The strands are actually parallel but facing in the opposite direction, and when you think about the structure, the reason for that becomes clearer. And imagining those backbone strands facing in the same direction immediately shows you the problem, I think.

Canto: Yes and for more insight into all that, we’ll need to look more closely at pyrmidines and purines and the molecular structure of the backbone, and those bases, and maybe this fellow Chargaff.

References

https://www.quora.com/Why-are-DNA-strands-anti-parallel

https://slideplayer.com/slide/13304243/

Written by stewart henderson

January 21, 2020 at 2:38 pm

epigenetics and imprinting 5: mouse experiments and chromosome 11

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something new, since Carey’s book was published – a healthy mouse, from entirely maternal DNA, with healthy offspring – and in 2018 a healthy bi-paternal mouse was created

 

So we were looking at how we – mammals amongst others – are engaged in a kind of battle for the best way to ensure our genetic survival into the future, beyond our insignificant little selves. This battle begins in the very early phase of life, as zygotes multiply to form a blastocyst. 

Remember from my last post on this topic, the male mammal is interested in the offspring above all else. He’s even happy to sacrifice the mother for the sake of the child – after all there’s plenty more fish in the sea (or mammals in the – you know what I mean). The female, on the other hand, is more interested in self-preservation than in this pregnancy. She wants more than one chance to pass on her genes.

So, by the blastocyst stage, cells have differentiated into those that will form the placenta and those that will form the embryo itself. Experiments on mice have helped to elucidate this male-female genetic struggle. Mouse zygotes were created which contained only paternal DNA and only maternal DNA. These different zygotes were implanted into the uterus of mice. As expected, the zygotes didn’t develop into living mice – it takes DNA from both sexes for that. The zygotes did develop though, but with serious abnormalities, which differed depending on whether they were ‘male’ or ‘female’. In those in which the chromosomes came from the mother, the placental tissues were particularly underdeveloped. For those with the male chromosomes, the embryo was in a bad way, but the placental tissues not so much.

In short, these and other experiments suggested that the male chromosomes favoured placental development while the female chromosomes favoured the embryo. Thus, the male chromosomes are ‘aiming’ to build up the placenta to drain as many nutrients as possible from the mother and feed them into the foetus. The female chromosomes have the opposite aim, resulting in a ‘fine balance’ in the best scenarios.

Further work in this area has identified particular chromosomes responsible for these developments, and some of the epigenetic factors involved. For example, mouse chromosome 11 is important for offspring development. When the offspring inherits a copy of chromosome 11 from each parent, the offspring will be of normal size. If both copies come from the mother it will abnormally small, while if both come from the father it will be abnormally large. These experiments were carried out on inbred mice with identical DNA. Nessa Carey summarises:

If you sequenced both copies of chromosome 11 in any of the three types of offspring, they would be exactly the same. They would contain the same millions of A, C, G and T base-pairs, in the same order. But the two copies of chromosome 11 do clearly behave differently at a functional level, as shown by the different sizes of the different types of mice. Therefore there must be epigenetic differences between the maternal and paternal copies of chromosome 11.

So this means that chromosome 11 is an imprinted chromosome – or at least certain sections of it. This is the same for other chromosomes, some of which aren’t imprinted at all. But how is it done? That’s the complex biochemical stuff, which I’ll try to elucidate in the next post on this topic.

Footnote: the photo above shows a bi-maternal mouse with healthy offspring, and further work in deleting imprinted genetic regions has allowed researchers to create healthy bi-paternal mice too. There’s a fascinating account of it here.

References:

Nessa Carey, The epigenetics revolution, 2011

https://www.the-scientist.com/news-opinion/first-mouse-embryos-made-from-two-fathers-64921

Written by stewart henderson

January 19, 2020 at 12:26 pm

A DNA dialogue 1: the human genome

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what genomics tells us

Canto: I’m often confused when I try to get my head around all the stuff about genes and DNA, and genomes and alleles and chromosomes, and XX and XY, and mitosis and meiosis, and dominant and recessive and so on. I’d like to get clear, if only I could.

Jacinta: That’s a big ask, and of course we’re both in the same boat. So let’s use the magical powers of the internet to find answers. For example, here’s something that confuses me. The Human Genome project, which ended around the year 2000, involved a mapping of the whole human genome, and that includes coding and non-coding genes, and I think it was found to contain 26,000 or so – what? Letters? Genes? Coding genes? Anyway there’s a number of questions there, but they’re not the questions that confuse me. I don’t get that we now, apparently, have worked out the genetic code for all humans, but each of us has different DNA. How, exactly, does our own individual DNA relate to the genome that determines the whole species? Presumably it’s some kind of subset?

Canto: Hmmm. This article from the Smithsonian tells us that the genetic difference between human individuals is very tiny, at around 0.1%. We humans differ from bonobos and chimps, two lineages of apes that separated much more recently, by about 1.2%….

Jacinta: Yes, yes, but how, with this tiny difference between us, are we able to use DNA forensically to identify individuals from a DNA sample?

Canto: Well, perhaps this Smithsonian article provides a clue. It says that the 1.2% difference between us and chimps reflects a particular way of counting. I won’t go into the details here but apparently another way of counting shows a 4-5% difference.

Jacinta: We probably do need to go into the details in the end, but clearly this tiny .1% difference between humans is enough for us to determine the DNA as coming from one individual rather than 7 to 8 billion others. Strangely enough, I can well believe that, given that we can detect gravitational waves and such – obviously using very different technology.

Canto: Yeah the magic of science. So the Human Genome Project was officially completed in April 2003. And here’s an interesting quote from Wikipedia:

The “genome” of any given individual is unique; mapping the “human genome” involved sequencing a small number of individuals and then assembling these together to get a complete sequence for each chromosome. Therefore, the finished human genome is a mosaic, not representing any one individual.

Of course it would have to be a mosaic, but how can it represent the whole human genome when it’s only drawn from a small number? And who were these individuals, how many, and where from?

Jacinta: The Wikipedia article does give more info on this. It tells us that the project isn’t really finished, as we’ve developed techniques and processes for faster and deeper analyses. As to your questions, when the ‘finished’ sequencing was announced, the mosaic was drawn from a small number of anonymous donors, all of European origin.

Canto: But we all originated from Africa anyway, so…

Jacinta: So maybe recent ‘origin’ isn’t so important. Anyway, that first sequencing is now known as the ‘reference genome’, but after that they did sequence the genomes of ‘multiple distinct ethnic groups’, so they’ve been busy. But here are some key findings, to finish off this first post. They found some 22,300 protein-coding genes, as well as a lot of what they used to call junk DNA – now known as non-coding DNA. That number is within the mammalian range for DNA, which no doubt surprised many. Another blow for human specialness? And they also found that there were many more segmental duplications than expected. That’s to say, sections of DNA that are almost identically repeated.We’ll have to explore the significance of this as we go along.

Canto: Yes, that’s enough for starters. Apparently our genome has over 3 billion nucleobase pairs, about which more later no doubt.

References

http://humanorigins.si.edu/evidence/genetics

https://en.wikipedia.org/wiki/Human_Genome_Project

Written by stewart henderson

January 13, 2020 at 11:48 pm

epigenetics and imprinting 3: at the beginning

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stuff that can be done with iPS cells

A zygote is the union of two gametes (haploid cells), the sperm and the egg. It’s the first diploid cell, from which all the other diploid cells – scores of trillions of them – are formed via mitosis.

What’s interesting about this from an epigenetic perspective is that gametes are specialised cells, but zygotes are essentially totipotent – the least specialised cells imaginable – and all this has to do with epigenetics.

I’m not entirely clear about what happens to turn specialist gametes into totipotent zygotes, and that’s what I’m trying to find out. I’m not sure yet whether zygotes immediately start differentiating as they divide and multiply or whether the first divisions – in what is called the zygote phase, which eventually forms the blastocyst – form an identical set of zygotes. 

The two-week period of these first divisions is called the germinal phase. During this phase zygotes divide mitotically while at the same time moving, I’m not sure how, from the fallopian tube to the uterus. Apparently, after the first few divisions, differentiation starts to occur. The cells also divide into two layers, the inner embryo and the outer placenta. The growing group of cells is called a blastocyst. The outer layer burrows into the lining of the uterus and continues to create a web of membranes and blood vessels, a fully developed placenta.

But, as Nessa Carey would say, this is a description not an explanation. How does this initial cell differentiation – into the outer layer (trophectoderm), which becomes the placenta and other extra-embryonic tissues, and the inner cell mass (ICM) – come about? Understanding these mechanisms, and the difference between totipotent cells (zygotes) and pluripotent cells (embryonic stem cells) is clearly essential for comprehending, and so creating, particular forms of life. This PMC article, which examines how the trophectoderm is formed in mice, demonstrates the complexity of all this, and raises questions about when the ‘information’ that gives rise to differentiation becomes established in these initial cells. Note for example this passage from the article, which dates to 2003:

It is now generally accepted that trophectoderm is formed from the outer cell layer of the morula, while the inner cells give rise to the ICM, which subsequently forms the epiblast and primitive endoderm lineages. What remains controversial, however, is whether there is pre-existing information accounting for these cell fate decisions earlier than the 8-cell stage of development, perhaps even as early as the oocyte itself. 

The morula is the early-stage embryo, consisting of 16 totipotent cells. The epiblast is a slightly later differentiation within the ICM. An oocyte is a cytoplasm-rich, immature egg cell.

Molecular biologists have been trying to understand cell differentiation by working backwards, trying to turn specialised cells into pluripotent stem cells, mostly through manipulating their nuclei. You can imagine the benefits, considering the furore created a while back about the use of embryonic stem (ES) cells in medical treatments. To be able to somehow transform a liver or skin cell into this pluripotential multi-dimensional tool would surely be a tremendous breakthrough. Most in the field, however, considered such a transformation to be little more than a pipe-dream.

Carey describes how this breakthrough occurred. Based on previous research, Shinya Yamanaka and his junior associate Kazutoshi Takahashi started with a list of 24 genes already found to be ‘pluripotency genes’, essential to ES cells. If these genes are switched off experimentally, ES cells begin to differentiate. The 24 genes were tested in mouse embryonic fibroblasts, and, to massively over-simplify, they eventually found that only 4 genes, acting together, could transform the fibroblasts into ES-type cells. Further research confirmed this finding, and the method was later found to work with non-embryonic cells. The new cells thus created were given the name ‘induced pluripotent stem cells’, or iPS cells, and the breakthrough has inspired a lot of research since then.

So what exactly does this have to do with epigenetics? The story continues.

Written by stewart henderson

January 6, 2020 at 5:28 pm

epigenetics and imprinting 2: identical genes and non-identical phenotypes

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I’ve now listened to a talk given by Nessa Carey (author of The epigenetic revolution) at the Royal Institution, but I don’t think she even mentioned imprinting, so I may not mention it again in this post, but I’ll get back to it. 

The talk was of course easier to follow than the book, and it didn’t really teach me anything new, but it did hammer home some points that I should’ve mentioned at the outset, and that is that it’s obvious that genetics isn’t the whole story of our inheritance and development because it doesn’t begin to explain how, from one fertilised egg – the union of, or pairing of, two sets of chromosomes – we get, via divisions upon divisions upon divisions, a complex being with brain cells, blood cells, skin cells, liver cells and so forth, all with identical DNA. It also doesn’t explain how a maggot becomes a fly with the same set of genes (or a caterpillar becomes a butterfly, to be a little more uplifting). These transformations, which maintain genetic inheritance while involving massive change, must be instigated and shaped by something over and above genetics but intimately related to it – hence epigenetics. Other examples include whether a crocodile hatchling will turn out male or female – determined epigenetically via the temperature during development, rather than genetically via the Y chromosome in mammals.   

So, to add to the description I gave last time, the histone proteins that the DNA wraps itself round come in batches or clusters of eight. The DNA wraps around one cluster, then another, and so on with millions of these histone clusters (which have much-studied ‘tails’ sticking out of them). And I should also remind myself that our DNA comes in a four-letter code strung together, out of which is constructed 3 billion or so letters.

The detailed description here is important (I hope). One gene will be wrapped around multiple histone clusters. Carey, in her talk, gave the example of a gene that breaks down alcohol faster in response to consumption over time. As Carey says, ‘[the body] has switched on higher expression of the gene that breaks down alcohol’. The response to this higher alcohol consumption is that signals are generated in the liver which induce modifications in the histone tails, which drive up gene expression. If you then reduce your alcohol consumption over time, further modifications will inhibit gene expression. And it won’t necessarily be a matter of off or on, but more like less or more, and the modifications may relate to perhaps an endless variety of other stimuli, so that it can get very complicated. We’re talking about modifications to proteins but there can also be modifications to DNA itself. These modifications are more permanent, generally. This is what creates specialised cells – it’s what prevents brain cells from creating haemoglobin, etc. Those genes are ‘tightened up’ or compacted in neurons by the modifying agents, so that, for example, they’re permanently unable to express the haemoglobin-creating function.

All of this is extremely fascinating and complex, of course, but the most fascinating – the most controversial and headline-creating stuff – has to do with carrying epigenetic changes to the next generation. The inheritance of acquired characteristics, no less. Next time.

References

What is epigenetics? with Nessa Carey – The Royal Institution (video)

The Epigenetics Revolution, by Nessa Carey, 2011

Written by stewart henderson

January 3, 2020 at 3:58 pm

epigenetics and imprinting 1 – it’s complex

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A useful summary - not explained in the text

The last book I read was The Epigenetics Revolution by Nessa Carey, though I’m not sure if I’ve really read it. So much of it was about persisting with the next sentence though I hadn’t fully understood the previous one. Biochemistry does that to me – too many proteins, versions of RNA, transposons, transferases, suppressors, catalysers, adjuvants and acronyms. And in the end I’m not at all sure how much progress we’re making in this apparently tantalising field.

So I’m going to pick out imprinting for starters, as a way of familiarising myself a little more with the epigenetic process of leaving tabs or marks on specific genes.

I know nothing about imprinting. Isn’t it what female birds do with their offspring, even when they’re still in the shell? Here’s how Wikipedia introduces it :

Genomic imprinting is an epigenetic phenomenon that causes genes to be expressed in a parent-of-origin-specific manner. Forms of genomic imprinting have been demonstrated in fungi, plants and animals. As of 2014, there are about 150 imprinted genes known in the mouse and about half that in humans. Genomic imprinting is an inheritance process independent of the classical Mendelian inheritance. It is an epigenetic process that involves DNA methylation and histone methylation without altering the genetic sequence. These epigenetic marks are established (“imprinted”) in the germline (sperm or egg cells) of the parents and are maintained through mitotic cell divisions in the somatic cells of an organism.


This suggests that it’s not something life-forms do, it just happens. But there are a number of mysterious terms here that need exploring – ‘a parent-of-origin-specific manner’, ‘DNA methylation’ and ‘histone methylation’.

Briefly, to get all that DNA (between 2 and 3 metres to each nucleus) to fit inside that tiny space you need some expert packaging, and that’s where histones come in. They’re proteins that DNA gets wound around, like cotton reels, and together the histones and the DNA are called chromatin. They’re also divided into sections called nucleosomes.

DNA methylation is when a methyl group, derived from methane (CH3), is added to the DNA, affecting its activity, including repressing gene transcription. Histone methylation is when methyl groups are added to amino acids in histone proteins. Again these can repress or enhance gene transcription, depending on the amino acids and how they’re methylated.

The parent-of-origin thing is most interesting to me, and needs a bit more explaining. When a human sperm cell enters an egg cell, as the first step in fertilisation, it carries its load of 23 chromosomes in what is called a pro-nucleus. In a sense a sperm cell, much smaller than an egg, is nothing but a pro-nucleus surrounded by a membrane, with a tail for motility. Once inside the egg, the tail and the membrane are shed. The egg cell also has its load of 23 chromosomes in its pro-nucleus, but this is considerably larger than the male – and the human egg cell in its entirety has about 100,000 times the volume of a sperm cell. The point is that the differences in the male and female pro-nuclei have a lot to do with epigenetic effects including imprinting, which affect phenotypic traits, including disease prone-ness and structural effects in animals and plants. Tracing these effects in molecular terms to either parent therefore becomes a priority.

So, this is a little starter in what is an overwhelmingly complex topic. I shall return to it.

Written by stewart henderson

December 31, 2019 at 10:11 am