The Central Dogma of Molecular Biology

The Central Dogma of Molecular Biology

The 1950s witnessed an explosion of groundbreaking discoveries in the biological sciences, the most central of which was the discovery of the molecular structure of DNA, deoxyribonucleic acid. Commonly credited to James Watson and Francis Crick, the double helix discovery saw contribution from Rosalind Franklin and Maurice Wilkins, the latter of whom were included in the three-way split of the Nobel Prize rewarding their discovery. Franklin tragically passed away at the age of 37, and could not be awarded the Nobel Prize, which is not given posthumously.

The double helix itself consists of a sugar-phosphate backbone, each unit of which is attached to a nucleobase; adenine, cytosine, guanine or thymine. Each base attached to the ribose sugar and phosphate group is known as a nucleotide. The amount of adenine is equal to tyrosine, and likewise with cytosine and guanine, a property that was discovered by Erwin Chargaff. It follows that adenine always binds to tyrosine, and cytosine to guanine. Each nucleoside acts like a letter in the code with which life is written. The binding pattern and shape of the helix allows the two strands to separate, and for each strand to act as a template for the opposite strand to be synthesised. Thus, the discovery of the DNA structure allowed an understanding of the way in which DNA is replicated.

(From Wikipedia)

DNA serves as the blueprint of life, and is copied virtually unchanged as cells divide. It also serves as the template with which proteins are made, using another code of amino acids. However, it was long known that eukaryotic proteins are formed in the cytoplasm of a eukaryotic cell, while DNA resides in the nucleus. Therefore, an intermediate molecule, known as messenger RNA (mRNA), exists to bridge this gap. The central dogma of molecular biology, then, as it was dubbed by Crick, describes the process by which DNA is replicated, transcribed into RNA, and translated to produce proteins, the building blocks of life and the molecular machines which carry out the functions required by the body.

An idea often repeated by the general public is that out of 3 billion base pairs of DNA in each of our cells, only 3% are functional, while the remaining 97% are referred to as “junk”.  Formally the protein-coding regions are known as exons, while the bits in-between are introns. A single gene comprises of one or more exons interspersed by introns, which are spliced out post-transcription.

Why such a system exists is rather uncertain. The reasons include a variety of things such as the fact that exons appear to be able to be differentially spliced, producing different proteins. Thus a smaller amount of DNA is capable of producing the complexity of the human body. Another is that certain DNA elements known as transposons are able to relocate themselves into other regions of the chromosome. These so-called jumping genes find a tract of unused DNA and settle there. Natural selection ensures unfavourable transposon relocations, i.e. those that disrupt gene codes, do not persist. In fact, the accumulation of non-essential DNA could perhaps reduce the likelihood that functional DNA is damaged. What is becoming more and more certain is that many regions of DNA previously thought to be “junk” in fact do have a role.

These non-coding DNA are transcribed into RNA which do not ultimate serve as a protein template. Some of their functions have been known for decades, such as transfer RNA, ribosomal RNA and regulatory RNA. Increasingly, non-coding RNA transcripts are being found to somehow affect the levels of transcription of the protein-coding RNA. These act as fine-tuning mechanisms that can dictate when and where to produce more of a protein, and how much. Often they are affected by environmental cues, which are also important in ordering the direction of growth for organisms.

Epigenetic effects, which are modifications to DNA which does not alter its nucleotide sequence, are also important in affecting the way proteins are expressed. Each cell in our body contains the same DNA, yet different types of cells transcribe different RNA and thus produce different proteins. Modifications such as DNA methylation are responsible for highlighting which genes should be turned on or off in a cell, and these are also inherited as cells replicate. The structure with which DNA are packaged in chromosomes, by means of chromatin packaging, is also involved. DNA is wound tightly around protein complexes called histones, which inhibit the DNA from being transcribed, unless an unwinding process occurs.

So this is the means by which DNA serves as the genetic code upon which all life is written. DNA is transcribed into RNA, which is translated into amino acids, with the help of other RNA molecules and proteins which act on transcripts. These amino acids form the proteins which serve as the structural and functional backbone of all living creatures.

Another post about honours

I haven't posted here for a while. But my honours is year is drawing to a close and today I presented my research to a room of my colleagues and several academic staff. I've always had an issue with nerves and today was no different. Everyone seemed confident and knowledgeable, and there I was, sweating beads and unable to formulate entire sentences. A few people nodded along, though, so I must have made some semblance of coherence.

My talk itself discussed the background to my research, based on cisplatin and several of its analogues, and presented what results I have so far (which I certainly won't be posting here). Cancer is such a huge and massive field that affects so many lives, and I feel fortunate to be partaking (however minutely) in the fight against it. Although many treatments for cancer exist, as we currently understand it, cancer is so overwhelmingly prevalent due to the fact that it is a naturally attractive state for individual cells to attain. Just as many humans aspire to immortality, the cells inside them may have already discovered what it takes to become immortal: becoming a tumour.

It's a bleak outlook to consider that when fighting cancer, we are fighting against natural selection itself.It's depressing to think that selection occurs at multiple levels, and that we are in constant battle against our environment and also our genes. Humans, let alone humanity, cannot achieve perfection, though perhaps it'd be nice to be proven wrong here. I don't expect I'd live to see that day.

Anyway, so far I haven't really talked about honours at all, oops. I suppose I did as well as I could have in my seminar, and since my supervisors have generally been impressed with my writing, it should give me some hope that my thesis would be more well received than my verbal presentation. I essentially completely failed in accomplishing half of my initial project aims, so that's a bummer there too. Hopefully if I stay on to do a PhD I'll be able to make something more meaningful happen out of that. Stupid DNA, get into this other DNA! I should get around to submitting my PhD application though. Sheeeeit I've been busy, all right?

Also I miss my sister.

:3

Hi All

Not much of interest going on of late, so why am I updating my blog? Because I want to, I guess. Just going to be talking about a bunch of inane stuff. I went to the Blue Mountains last Sunday, which was pretty random, but fun. Saw my friend JAAAAAAAAAAASON, who lives in Adelaide now, so it was nice to meet up with him again after a few months. Took 3 hours to get there, milled around for around an hour, then went back home. Fun times. I took this stupid panorama shot:

Blurry as heck. Aw yeah. At least it's not quite Winter yet so it wasn't that cold when we got up there. Also the Three Sisters aren't as big as I remember them. Oh well.

Two days before that I was on this train:

I sat on it for about 15 minutes before it decided to just stop. Apparently it had hit someone, so it stuck around for about an hour while Police and stuff were milling about. Made me late for a meeting with my supervisor, but at least I didn't get hit by a train. RIP =(

And I'm in the mood for showing off soooo here are some personal bests that I have racked up, in two completely different things:

Yuuuup, on the left is my current record for jogging 9km (walk breaks between laps not included; lol I died on the last lap) aaaaand on the right is my ZOOZOO BUBBLE record. I'm planning on running the City2Surf, which is 14km, this year, so I need to up the number of laps I do (but it's so time-consuming (and tiring (brackets whoo))). I don't plan to be a ZooZoo pro-gamer, though, so I'm fine with this score (for now (Can't beat Doa's score anyway (brackets ahoyyy))).

ZOOOOOOZOOOOOOOOOO

As far as my honours project is concerned, I've got some sets of of preliminary data results already, but they're kinda iffy and funky. The cisplatin is damaging as intended, but some aspects of the process appear to be undesirable. So for the next couple of weeks I'm gonna just be working on locking down those issues. I probably shouldn't go into any more details about my project for IP-protection purposes. But thanks for your interest.

I'm going now.

Bye.

Identification of the DNA Adduct Sites of Cisplatin and its Analogues

Cancer claims the life of over 7 million people worldwide every year, a number that is set to rise over the next few decades. It is a disease that is diverse in its effects, targeting virtually every tissue in the body, yet are characterised by several common hallmarks (Figure 1). Hanahan and Weinberg in 2000 classified these as evasion of apoptosis, self-sufficiency in growth signals and insensitivity to anti-growth signals, sustained angiogenesis, limitless replication potential, and tissue invasion and metastasis. Metastasis refers to the secession of a malignant tumour from its central mass, entering the blood stream and re-establishing itself elsewhere.

Figure 1: Hanahan and Weinberg’s 6 Hallmarks of cancer

The current treatment regimes to combat cancer primarily consist of surgery, radiation therapy and chemotherapy, which will be the focus of this research. Cisplatin (Figure 2) is one of the most commonly used drugs in clinical cancer treatment, as it is highly effective against testicular, ovarian (the so-called “silent killer”), cervical, non small-cell lung, and head and neck cancers. Its mechanism of action involves the formation of adducts on DNA which hinder polymerase activity. However it possesses various toxic side effects, some of which are irreparable, and often meets with resistance by cells.

Figure 2: The chemical structure of cisplatin

Cisplatin is normally inserted into the body intravenously, where it will begin to diffuse into the cells. There is mounting evidence that certain proteins are involved in transporting it across the cell membrane. Once it enters the cell, an aquation reaction replaces one of the chlorine atoms, allowing this to form a bond with the N7 of a purine nucleotide, usually guanine. This is repeated to form a second bond, shown to occur most commonly on an adjacent guanine, but less commonly forms an ApG or GpNpG adduct, or form an interstrand adduct. The cisplatin adduct produces a kink (which various NMR studies show to vary between 40° to 60°) that hinders the progress of DNA polymerase along the template. Figure 3 represents this process. Unless repaired, this leads to apoptosis.

Figure 3: The binding mechanism of cisplatin on adjacent guanine nucleotides

Despite its widespread use, cisplatin has numerous toxic side-effects including neurotoxicity, gastrointestinal toxicity, nephrotoxicity (kidney damage), ototoxicity (hearing loss) and hematologic (blood) toxicity. Patients universally experience nausea and vomiting within hours of treatment, which can often be severe. Damage to renal function in particular is often irreparable, due to accumulation of cisplatin in the kidney.

Another cause for concern in the use of cisplatin is the development of resistance to the drug. This may be caused by a change in expression levels of genes which are thought to be responsible for cisplatin transport into the cells. There is some contention regarding whether or not cisplatin diffuses via active transport, and to what degree it occurs. It's possible that cisplatin does diffuse passively until a certain concentration, at which point further transport requires active protein assistance. Other mechanisms of resistance involve an increase in the proficiency of the repair mechanisms (primarily nucleotide excision repair) which are able to remove the DNA adducts. Increased expression of glutathione and metallothionein also contribute to cisplatin resistance due to competition for binding.

Other similarly related platinum compounds have also been developed in response to the toxicity and resistance problems. Carboplatin and oxaliplatin (Figure 4a) are currently used clinically worldwide, while nedaplatin sees more limited use in Japan. Carboplatin, while significantly reducing the toxic side-effects compared to cisplatin, has the same active form and is thus similarly unviable against cisplatin-resistant cells. Although cisplatin is able to bind onto DNA, it does so in a time-consuming manner since it has no natural affinity for nucleic acids. Thus analogues such as 9AmAcPtCl₂ (Figure 4b) are being studied, where the 9-aminoacridine moiety is able to intercalate with DNA.

a)

b)

Figure 4: The chemical structures for (a) carboplatin and oxaliplatin, clinically successful cisplatin analogues, as well as (b) 9AmAcPtCl₂, currently studied by the lab

My research will focus on comparing the adduct formation patterns of cisplatin, carboplatin and other analogues including 9AmAcPtCl₂ and related analogues, namely 7-methoxy-9AmAcPtCl₂, 7-fluoro-9AmA-cPtCl₂ and 9-ethanolamine-AcPtCl₂. Apparently my lab doesn’t currently have stocks of oxaliplatin but hopefully this will change soon. Due to the frequency of binding with GG dinucleotides, telomeric sequences (with their TTAGGG motif), CpG islands and other sequences of repeated guanines are expected to be preferentially targeted by cisplatin. Telomeres are of particular interest due to their expansion in tumour cells, while CpG islands are of interest due to their association with gene promoters. Specific promoters of genes of interest are proposed to be cloned into plasmids along with telomeric repeats and CpG islands. Several sequences have been previously analysed, including the transferrin receptor protein 1 (TFRC) and retinoblastoma protein (Rb1) promoter regions. These will be treated with cisplatin and analogues, then processed through a linear amplification assay using the fluorescent Rev2 primer (Figure 5) and sequenced to identify the location and frequency of DNA adducts formed by the platinum compounds. Fragment analysis will be performed at the Ramaciotti Centre.

Figure 5: The linear amplification method used to identify sites of cisplatin adduct formation

The results from the fragment analysis are presented as an electropherogram with peaks corresponding to the location of DNA adduct sites. The intensity of the peaks correspond to the abundance of adducts at that site. In this example set of data of T7.CpG.G10 DNA from Julie’s thesis (Figure 6), we can see that treatment with 3.0μM cisplatin produces mild peaks at the T7 region and higher peaks at the G10 and G5 regions, as expected with cisplatin’s preference for binding to tandem guanine repeats. The 3.0μM 9-aminoacridine analogue shows greater binding with the CpG region than with cisplatin. The right-most peak shows that a large amount of DNA remains unbound by the drugs at this concentration, as the linear amplification was able to continue to the end of the target sequence.

Figure 6: Example of an electropherogram obtained through fragment analysis of cisplatin-damaged DNA, in this case the T7.CpG.G10 plasmid construct.

A secondary aim will be to construct more plasmids to study, which will be started once I collect sufficient data from the cisplatin and analogue damage experiments on the presently available plasmids. Several lab techniques will be relevant to this aim which I would not have performed for the first set of experiments. DH5α E. coli cells will be grown on LB media and CaCl₂-treated to become competent for plasmid transfection. Following transfection, a midi-prep is performed to extract the plasmids from the cloned bacterial cells. The plasmid DNA is digested with PvuII to obtain the desired target sequence. This process is outlined in Figure 7.

Figure 7: Outline of the process involving construction of plasmids leading to cloning in DH5α cells. Plasmid extraction using a midiprep kit is carried out followed by digestion of the plasmid with PvuII to isolate the target sequence.

The genes of interest to be cloned are those which have been previously shown by microarray analysis to be differentially regulated following cisplatin treatment. These are BRCA2, MT1L and IL-1β, and have interesting properties besides being significantly regulated by cisplatin. Mutation in BRCA2 has been shown to be strongly associated with the development of breast and ovarian cancers, the latter being a clinically important target of cisplatin. As shown in Figure 8, women with BRCA2 mutations have a 23% overall lifetime risk of developing ovarian cancer.

Figure 8: Overall lifetime risk of ovarian cancer in BRCA1 and BRCA2-mutated women

The metallothionein gene product acts as a metal ion scavenger which can interact with cisplatin in the cell, such that increase in MT1L expression is associated with higher cisplatin resistance. There is some contention with this, however, and several studies agree that metallothionein expression levels do not affect cisplatin resistance to the same degree as glutathione. The interleukin-1β gene product is involved in the pathway to apoptosis, for example following cisplatin treatment. The interleukin-1β converting enzyme (caspase 1) has been previously shown to be induced by cisplatin.

So far I have produced an electropherogram of a linear amplification based on cisplatin-damaged T7.G10 DNA (Figure 9). Though similar to Julie’s results, it uses a slightly different DNA strand, essentially missing the CpG region between the telomeric repeats and the 10 tandem guanines. 30μM of cisplatin evidently produces so many adducts that virtually no strand of DNA was extended wholly.

Figure 9: Electropherogram of fragment analysis of T7.G10 plasmid treated with cisplatin, amplified with FAM-Rev2 primer

Zelda Symphony and other stuff

So on Saturday I went to the Sydney Opera House to watch the Legend of Zelda: Symphony of the Goddesses (Fig 1). I was a bit apprehensive about it because 1) the ticket was pretty expensive and I thought I shouldn't have forked out so much when a cheaper ticket would let me hear the same things (and I don't really need to watch the orchestra play) and 2) because I had already listened to most of the songs through the CD that came with Skyward Sword.

Figure 1. Program leaflet for The Legend of Zelda: Symphony of the Goddesses presented by the Sydney Symphony at the Sydney Opera House Concert Hall

Plus, I went by myself because I didn't really bother going around asking, "Hey, you want to listen to a bunch of people play music from this video game series I like? Oh, you don't like that video game series? Oh you like it, but not the music (haha, nobody exists in this category)?" So I sat awkwardly by myself (after waiting awkwardly by myself outside) while everyone around me was either dressed up as a Zelda character or wearing a Zelda t-shirt or playing Zelda on their 3DS. I wore a Pokemon t-shirt.

But then the music dimmed, and the Sydney Symphony started playing the Prelude, and the tears started flowing.

The concert hall was packed (Fig 2) and it was full of people who love Zelda and love its music. They were people who, like me, were drawn in by the magic of the ocarina and the windwaker and ... wolf howling. Strangely they played very little of the music from Skyward Sword, but you can probably see there are two harps there. They played much better than Link on his little dinky Goddess Harp.

Figure 2. The packed Concert Hall and the Sydney Symphony accompanied by Cantillation, conducted by Eimear Noone.

Anyway, after the prelude I stopped crying, since the initial shock of hearing my favourite music being played live wore out, and so I simply sat in admiration of show that Sydney Symphony, Cantillation and Eimear Noone had set out for us. Eeimar Noone, by the way, is also the composer for the music to StarCraft 2, Diablo 3 and World of Warcraft, so as far as video game music goes, she's a pretty big deal. And her passion for it really showed as she conducted the Symphony. And the cheers that erupted when she drew forth the Windwaker itself to conduct the Windwaker Symphony movement was spectacular, only beaten out by the cheers for the second and third encores, which played Gerudo Valley (personally one of my favourite pieces) and the Majora's Mask medley (which I am ashamed to say I have not played!)

Speaking of Gerudo Valley, the concert inspired me to finally return to Ocarina of Time to try to beat the Gerudo Fortress, which I had previously ragequit at due to sheer amounts of bullshit. It wasn't as difficult this time around, so I quickly beat the mini-dungeon and I'll try to finish the game sometime in the next couple of weeks. Since uni is starting soon and all, so I should probably get that done and dusted.

Oh, and I did say other stuff.

But I think that's enough for now.

I guess I'll just say I watched Django Unchained and I liked it? It was reaaaaaaaaaaaaally long and it felt like it was never going to end. But then it did, and it was an all right ending. I guess. Lots of blood as per Tarantino standards.

All right I'm off.

Honours just around the corner

So my supervisor returned from her holiday yesterday, and I suppose this means I'll be starting my actual honours soon. Meaning that I'm not going to have so much time for gymming and dungeon-crawling (yes I've been playing Dungeons and Dragons, say hi to my level 5 Goliath Paladin). And now is the time to reflect on what I've done in the past two-and-a-half months of holidays. Hmmmm. Not too much.

I decided to pick up learning guitar a week ago-ish (found out I have the wrong type of guitar but I'm too cheap/lazy to go out and buy another one). So I'm terrible and I slightly regret not starting to learn 2 months (years) ago. Learning Chinese also hasn't progressed very much. I should be practising writing and reading full sentences (and not the wishy washy sentences in songs that don't make sense half the time) but I'm too stuck on picking up single words that I probably won't use and trying to learn those.

I improved with my swimming somewhat; I managed to swim 600 metres last week, though my friend swam double that amount in roughly the same time. Still, being able to run 9000 metres makes me feel good. My goal is 12km in one hour, but I didn't go running last week because of rain/laziness so I might not be able to reach that in the time I set myself (before honours starts, heh). At least the city-to-surf isn't for another few months, and I should be able to get there by then.

Anyway, here's half a short-story I wrote a while ago that I still don't know how to end (hence only putting up half):

Sandy emerged from the crevice, ragged and dirty, gasping for clean air. She had not realised how deep it had been, and how quickly light ceased to penetrate the musty atmosphere trapped between the ancient granite. The skin on her arms had been cut both on the way down, and again on her way up.

“Well maybe you can grab some long-sleeves and a helmet-light, and have another go?” said Alex.

Alex Winter watched as her friend silently complied, donning a bright red jacket, and grabbed onto the rope that fed into the chasm. It would be very hot in there with the jacket, compounded by what she believed to be sulfur fumes swirling between the narrow rock walls.

Sandy ignored them, descending deeper than she had before. She thought about possibly acquiring a face mask, in case she ever needed to do this again. She hoped she wouldn’t. Her nose squirmed and contracted in protest, and her eyes began to sting and water. The pupils would dilate to accommodate for the reduction of light; she wondered if it would cause the fumes to affect her more.

Suddenly she reached the bottom. Or rather, the point at which the crevice no longer plummeted vertically down. The light from her hard-hat illuminated more dust than anything else in the cave, but it was still welcome. Now she won’t trip over her feet and embarrassingly small rocks.

Tentatively, Sandy took a step forward. She didn’t notice her breathing becoming more laborious, despite the stench that reeked heavier than before. She also didn’t notice something was drawing her in deeper, and when she finally became aware of her surroundings, she could no longer see any light besides the one radiating from her forehead. She started to panic.

Sound wouldn’t travel here from the surface, would it? she thought, as she peered at her surroundings. She was in a completely non-descript cavern, perhaps eight metres wide, but the ceiling was much higher than she thought possible. It seemed to reach further than she remembered climbing down. Just a trick of the light, she thought.

Alex wouldn’t be able to hear her down here. Sandy recalled agreeing that 3 tugs on the rope meant she found something noteworthy, while 4 meant she was in trouble. Either way, Alex would have come rushing down to her, leaving the third member of their party at the surface.

Was the fact that this cavern appeared taller than was possible noteworthy? Sandy didn’t think so. Yet she could see nothing else. No markings on the walls and no other exit save for the one she had just entered by. She took careful steps around the cavern floor, feeling for loose stones and unnatural edges. Nothing.

Well, only one thing for it now, she thought, as she leapt at the wall and found easy handholds. Expertly she scampered up the rocky wall, but the higher she climbed, the further away the ceiling appeared to be. All right, that’s noteworthy, she thought. But before she could tug on the rope, she felt three sharp tugs at her waist. She’s out of rope. 

Destroying my muscles

So that they may rise again from the ashes and engulf me in a criss-cross of myosin and actin filaments bulging at every junction. Then I shall open jars between my gargantuan biceps and straddle mountains with my iron thighs. Or something. Is that how it works?

I'm new to this gym thing.

And while I'm not new to swimming, it's an activity that leaves me more breathless (ha!) than I remember. Also I think I'm not moving my arms right, and it's probably terrible. Also my sister says I'm afflicted with "vertical swimming", whereby my feet drag across the bottom of the pool as they kick. This is equally if not more terrible.

Is there some way of measuring the density of my bones or my bone/muscle mass ratio? Because I think it's considerably higher than most other people. And despite what a doctor told me some years back, I think I have terrible lung capacity. And this tendency to panic when my face isn't in contact with air. I suppose these things will improve with time, but seriously I can't make my legs kick up properly =( Back to the kiddie pool practising kicks against the wall, I suppose. Can't be blaming my body for the shitty way my body moves. That's probably a fault of a different part of my body which is not familiar with how leg muscles attach to hips and such.

Gym feels easier initially but by the next day I'm horribly afflicted by can'tstraightenmyarms syndrome, which put me at a severe disadvantage in Jungle Speed. That game destroys friendships almost as fast as Mario Party!

Anyway I'll just leave this mess here:

今天, 我到游泳池去游了， 然后我去了健身房。我只能游泳三百米。 我的朋友能游泳一公里多。我希望我也能游一公里多。