biochemistry - How does a plant grow before photosynthesis is possible?

There are quite a few questions and thoughts in there, I'll try to cover them all:

First, to correct your initial word equation: During photosynthesis, a plant translates CO₂ and water into O₂ and carbon compounds using energy from light (photons).

You are correct to assume the C is further used for the growing process; it is used to make sugars which store energy in their bonds. That energy is then released when required to power other reactions, which is how a plant lives and grows. C is also incorporated into all the organic molecules in the plant.

Plants require several things to live: CO2, light, water and minerals. If any of those things is missing for a sustained period, growth will suffer. Most molecules in a plant require some carbon, which comes originally from CO₂, and also an assortment of other elements which come from the mineral nutrients in the soil. So the plant is completely reliant on minerals.

Most plants, before a leaf is established or roots develop, grow using energy and nutrients stored in the endosperm and cotyledons of the seed. I whipped up a rough diagram below. Cotyledons are primitive leaves inside the seed. The endosperm is a starchy tissue used only for storage of nutrients and energy. The radicle is the juvenile root. The embryo is the baby plant.

When the seed germinates the embryo elongates, the endosperm depletes, the testa ruptures, and the cotyledons emerge from the seed. The cotyledons are green, and like leaves can photosynthesise, so as soon as they are in the light the plant is able to make carbon compounds. The radicle elongates at the same time, and becomes the root, so the plant is very quickly able to obtain fresh nutrients from the soil (or whatever it's growing in).

At all stages of a plant's life it is using both energy stored in carbon compounds (from CO₂) and nutrients which it took up via its roots. At no point does the plant start to depend solely on the CO₂ in the air for its growth.

You are right that the way in which plants acquire energy and nutrients prior to leaves and roots being established varies between plants. Above I outlined the way most plants use. But there are lots of variations. For example, orchids have very tiny seeds, some barely visible to the naked eye, like specks of dust. They have no endosperm or storage tissue, so they have to rely on a symbiotic mycorrhizal fungus to get carbon and nutrients. The fungus grows through the coat of the orchid seed, then provides everything the growing plant needs until it has its own leaves. Then the orchid repays the fungus by providing sugars.

There are lots of other examples, but we could go on all day!

structural biology - Can protein structure be determined by X-Ray Diffraction in a single image?

Not by analysing a single protein. There is work with x-ray lasers.

You have to take a simultaneous image of millions of proteins and use that to get a structure. It's not quite prime time. People are also doing this with electron beams in electron microscopes.

These methods will reconstruct 3D models of the molecules, sometimes in states which cannot be obtained from crystallography. Examples being the structure of the many megadalton nuclear pore complex, and the f-actin fiber. The classic study is 3d model of bacteriorhodopsin, the first membrane protein structure which was at molecular resolution (this was a crystalline sample though).

While in principle, it sounds much simpler - get a pure sample of your protein, or complex and freeze it down and zap it with an Xray or Electron beam, its a lot more work to reconstruct the image and can take as long or longer than getting an x-ray structure. The resolution is also usually poor as the crystal will reinforce coherence, that is all the proteins are aligned in the same way and have close to the same 3d shape in a crystal.

gel electrophoresis - How do I deal with sticky and viscous samples from cell lysates?

To check a protein expression I pelleted a small amount of E. coli before and after induction and lysed them by redissolving them in SDS-PAGE loading buffer and heating them to 95 °C for 1 minute.

This lead to a solution with some very sticky and viscous parts in it, that make pipetting the sample into the gel wells extremely annoying. As far as I heard, this is probably genomic DNA, and my usual way to deal with this is to centrifuge the samples and only pipet a small part out from the top. This does seem to help sometimes, but not always.

How can I avoid the formation of that sticky and viscious stuff or how can I avoid pipetting that stuff into my wells?

pharmacology - In what ways, if any, does administration of rapamycin *not* mimic calorie restriction?

Rapamycin specifically inhibits the mTOR pathway (mTOR = mammalian target of rapamycin), which has numerous downstream functions including protein biogenesis, regulation of cell cycle, immune function and apoptosis. The upstream effectors of mTOR include growth factors and amino acid availability, so you can certainly see that the lifespan enhancing effects of caloric restriction will be (at least in part) mediated by the mTOR pathway.

But there are key differences. mTOR also receives signals relating to DNA damage and inflammatory changes (to name just 2) that are essential for healthy survival. So any direct inhibition of this pathway will affect all the functions - I can't find the reference now, but I have definitel read in one of the numerous rodent studies that rapamycin treated mice have reduced immune function (i.e. the lifespan increased effects can only be seen in a controlled lab environment - in the wild mTOR-inhibition to this degree would be a disadvantage).

I think therefore it is fair to say that the effects of caloric restriction on longevity are mediated by mTOR, but administration of rapamycin is not an equivalent treatment.

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Update

Really great review came out last month (http://www.ncbi.nlm.nih.gov/pubmed/22500797) - I recommend you give that a skim if you want detail!

human biology - How are proteins formed?

Essentially, yes, "proteins that we consume form new proteins that are different".

The processes are each of them topics for themselves. In short, consumed proteins are digested by peptidases (enzymes) in the stomach, breaking them down into their consituent amino acids. These are absorbed in the gut and transported in the blood to all cells. These take up amino acids and attach them to tRNA molecules which are used in translation to form new body proteins.

human biology - Why do people look different after a long sleep?

What happens during a long sleep that makes people look odd when they have just woken up? Why doesn't the same phenomenon occur in the case of a person who lies down for an extended period of time, but stays awake? I've noticed that some nights seem to make a bigger difference than others in the appearance of the sleeper, but haven't noticed a pattern.

biochemistry - ATP cost for gene expression

The cost of transcribing and translating a hypothetical average gene in yeast has been calculated as 551 activated phosphate bonds ~P per second (Wagner, 2005).

The median length of a yeast RNA molecule is 1,474 nucleotides, and
the median cost of precursor synthesis per nucleotide (derived from
the base composition of yeast-coding regions) is 49.3 ∼P. With a
median mRNA abundance of R = 1.2 mRNA molecules per cell and a median
mRNA decay constant of dR = 5.6 × 10−4 s−1, the mRNA synthesis costs
calculates as 49.3 × 1,474 × 1.2 × (5.6 × 10−4) = 48.8 ∼P per second
and cell. This is a fraction 48.8/1.34 × 107 = 3.6 × 10−6 of the total
RNA synthesis cost per second. The median length of a yeast protein is
385 amino acids, with a combined biosynthesis and polymerization cost
of 30.3 ∼P per amino acid. The median abundance is 2,460 protein
molecules per cell. No currently available data allows a meaningful
estimate of the median protein half-life, but a protein of an
intermediate half-life (see below) of 10 h (decay constant dP = 1.92 ×
10−5 s−1) yields an overall synthesis cost of 30.3 × 385 × 2,460 ×
(1.92 × 10−5) = 551 ∼P s−1.

For your question about a single gene, the cost would be 49.3 * 1474 ~P for the mRNA and 30.3 * 385 ~P for the translation, which would result in around 84 thousand ~P. This is probably a very misleading statistic as you can transcribe multiple proteins from a single mRNA.

How the cost of mRNA synthesis and translation are calculated is described in detail in the paper. A large part of the cost comes from the synthesis of the basic building blocks, the nucleotides and the amino acids.

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human biology - When to measure resting heart rate and blood pressure for following day-to-day trend?

I would be very surprised if the time of day made a difference. I've personally never heard mention of such a phenomenon in discussions with intensive care practitioners (where of course HR and BP are measured constantly). However this is only the case during rest, this paper (on horses) suggests that there is some difference in HR and BP after exercise depending on time of day.

You are right to attempt to control it however, I suppose. It doesn't matter when you take your measurements, as long as you take them at the same time each day.

I would say that one of your first suggestions of taking the measurements almost immediately after waking. Of course you would have to be relaxed as you assembled your equipment then rest for a few minutes in the posture you have chosen (this will matter) before taking the measurements but otherwise I can't see you having too great a problem.

human biology - How reversible is decerebrate posturing caused by brain stem damage?

This is a follow-up question to How likely would Abraham Lincoln be to survive his wounds today?

You don't have to see a CT scan or autopsy to know if the brainstem is
injured (directly or indirectly), if it doesn't work right.

The description of the first doc at the scene mentioned that Lincoln
was not breathing, and one pupil was dilated (the latter a clear and
unequivocal sign of dysfunction of the third cranial nerve or the
upper brainstem, from where it comes). Unfortunately, the second doc
described the enlarged pupil being the right one (it's extremely
unlikely to have been one and then the other - one of the doc's was
probably mistaken as to the side).

By 3 hours after injury, both pupils were fixed and dilated, and
Lincoln showed extensor (decerebrate) posturing - again, all signs of
profound brainstem dysfunction (but not yet brain death, though pretty
close to it).

Now, what is decerebrate posturing? See http://en.wikipedia.org/wiki/Abnormal_posturing#Decerebrate

Decerebrate posturing is also called decerebrate response, decerebrate
rigidity, or extensor posturing. It describes the involuntary
extension of the upper extremities in response to external stimuli. In
decerebrate posturing, the head is arched back, the arms are extended
by the sides, and the legs are extended.[6] A hallmark of decerebrate
posturing is extended elbows.[12] The arms and legs are extended and
rotated internally.[13] The patient is rigid, with the teeth
clenched.[13] The signs can be on just one or the other side of the
body or on both sides, and it may be just in the arms and may be
intermittent.[13]

A person displaying decerebrate posturing in
response to pain gets a score of two in the motor section of the
Glasgow Coma Scale (for adults) and the Pediatric Glasgow Coma Scale
(for infants). Decerebrate posturing indicates brain stem damage,
specifically damage below the level of the red nucleus (e.g.
mid-collicular lesion). It is exhibited by people with lesions or
compression in the midbrain and lesions in the cerebellum

biochemistry - Could hydrogen replace oxygen in cellular respiration?

No, hydrogen could not replace oxygen because it has entirely different characteristics. The most important one is probably its electronegativity - oxygen 'pulls' electrons much 'stronger' than hydrogen.

Basics: Reduction potential

Oxygen is the so-called terminal electron acceptor of the electron transport chain in eukaryotes. You can see "reduction potential" as a kind of stored "energy" which molecules have, similar to the power stored in batteries (very similar actually). To make this text a bit shorter I'll call it "RP" from now on.

One maybe confusing detail is that a substance with low RP has "more energy" than a substance with high RP, so it is the opposite way of thinking.

In very generalised terms, metabolism means that molecules with a low RP (glucose) are oxidised (burned) and turn into molecules with much higher RP (CO₂). Coupled with this, a different molecule with very high RP (oxygen) is reduced and becomes a molecule with slightly lower RP (H₂O). (You may have heard this before - it's called a redox reaction.) *

The important part is that the RP "released" by the oxidisation (burning) is larger than the RP "taken up" by the reduction. The surplus leaves as energy - heat and light if you just burn the glucose. This is a spontaneous process, meaning it will occur just by itself - even if it takes a long time if nobody drops a match on it.

The idea of metabolism is to let that process happen - but use as much of the energy it releases as possible. This works by not just letting it burn, but intercepting that burning process at different stages so that at each step a bit of the RP can be taken off and stored in something else. This "something else" is NAD which I'm sure you've encountered before. Each step that glucose is burned down, another bit of NADH is made, which then has a respectable reduction potential.

NADH (leaving out NADPH here which is a bit different) is channeled into a process called oxidative phosphorylation which retrieves the reduction potential in an actual form of energy.

Basics: Terminal electron acceptor

Finally, the reduction potential I've been talking about all the time is really just electrons, involved in bonds which are "happy" to react. Passing down the reduction potential as I've explained is really a passing down of electrons into more and more stable, less reactive bonds. That's why it's called "electron transport chain". At the end of oxidative phosphorylation, those electrons are dropped onto O₂ and make it into H₂O. That's why O₂ is called the "terminal electron acceptor".

Why Hydrogen can't replace Oxygen

Now to come back to why hydrogen cannot perform oxygen's function in our body. We use glucose as our source of reduction power and oxygen as our terminal electron acceptor. O has a high electronegativity (3.5) so it pulls electrons strongly towards it. H's electronegativity is only 2.1, so it's much weaker. O as a terminal electron acceptor works because it pulls them much stronger than H when they bond, so an O-H bond is almost like giving oxygen an electron. In order for hydrogen gas (H₂) to perform the same function, it would need to be possible to drop electrons onto hydrogen in a bond where it pulls them much stronger than the other partner. They do exist, and such compounds are called hydrides. But the catch is: unlike H₂O, these are normally strong reducing agents, meaning that hydrogen would rather not be in that bond. This not a feasible option for cell respiration, at least in humans, because it requires a lot of RP input. Making oxygen into H₂O does not require a lot, it's a very cheap electron acceptor.

I hope I was able to put this in understandable terms. Let me know if I need to clarify anything.

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*It works with other molecules than glucose->CO₂ / O₂->H₂O too, many prokaryotes do that and in fact that's how batteries work

t7 promoter - What determines a successful protein expression in E. coli?

I found a very nice paper: Designing Genes for Successful Protein Expression, which covers most factors that determine protein expression. I post parts of it, because I am sure it will be useful to some of you.

Translation can be controlled at the level of initiation and elongation. Initiation of translation is primarily dependent on the sequence of the ribosome binding site (RBS) and early mRNA secondary structure. Other determinants of protein expression are less well understood but equally potent.

1. Initiation of translation

A key component affecting initiation of translation in prokaryotes is the RBS that occurs between 5 and 15 bases upstream of the open reading frame (ORF) AUG start codon. Binding of the ribosome to the Shine–Dalgarno (SD) sequence within the RBS localizes the ribosome to the initiation codon... Affinity of the RBS for the ribosome is a critical factor controlling the efficiency with which new polypeptide chains are initiated. This interaction is in competition with possible base-pairing interactions involving the RBS region that may form within the mRNA itself. Thus, SD sequences with weaker base pairing to the ribosome are more susceptible to interference from mRNA structure. However, some experiments suggest that SD sequences with too strong affinity can be deleterious, particularly at lowering temperatures, by stalling initial elongation. Also critical is the distance between the RBS and the start codon with 5-7 bases from the consensus SD AGGAGG being optimal.

Numerous lines of evidence suggest that the initial 15–25 codons of the ORF deserve special consideration in gene optimization. Studies have shown that the impact of rare codons on translation rate is particularly strong in these first codons, for expression in both Escherichia coli and Saccharomyces cerevisiae. In E. coli, peptidyl-tRNA drop-off during translation of the initial codons appears to be accentuated by the presence of rare NGG codons. These effects appear to be independent of local mRNA secondary structure. It is also true that expression may be recovered by 5' sequence replacement even for sequences that do not show especially strong mRNA structure or contain rare codons or other obvious deleterious elements in this region.

2. Codon bias

The second way in which host codon frequencies can be used is to match the host codon frequencies in the designed gene. This can be done simply by choosing each codon with a probability that matches the host codon frequency...Using sets of genes broadly varied in gene design features, Welch et al. found that variation in synonymous codon usage frequencies had a profound effect on the amount of protein produced in E. coli, independent of local 5’ sequence effects. Variation of at least two orders of magnitude in expression was seen due to substitution beyond the initial 15 codons of the ORF. This variation was strongly correlated with the global codon usage frequencies of the genes, although the codon frequencies found in the highest expressed variants did not correspond to those found in the genome or in highly expressed endogenous genes of E. coli. Multivariate analysis showed that the frequencies of specific codons for about six amino acids could predict the observed differences in expression. It is not clear what the biochemical basis is for this correlation.

3. mRNA structure and translational elongation

While much evidence suggests that mRNA structure can interfere with translational initiation in both prokaryotes and eukaryotes, the effects of structure on elongation are less well understood. This in part may be due to intrinsic helicase activity of ribosomes, which allows translation through even very strong hairpins and may preclude many structures from limiting the translation rate in either prokaryotes or eukaryotes. Perhaps more importantly, mRNA structure is difficult to predict, particularly for actively translated messages which are in continuous flux between various folded and unfolded states.

4. Protein-specific factors providing additional complexity

The protein may be particularly unstable in the host, especially if it is poorly folded due to inherent instability, lack of sufficient prosthetic factors, or improper post-translational modification... Expression of secreted and membrane proteins may be limited by mechanisms for directing these proteins to the membrane. It is even possible that the protein amino acid sequence may limit translational efficiency. For example, proline is thought to be slowly translated in E. coli, regardless of which codon is used.

Expression of the protein may be toxic to the cell leading to instability of the expression vector or host suppression of protein synthesis...A common strategy to reduce toxicity is to lower expression to tolerable levels. Promoters varied in strength can be valuable tools for finding an optimal expression rate for maximal yield...One potential way to avoid toxicity of some proteins is to direct expression to the periplasm or media. This may be accomplished by N-terminal fusion of a secretion signal sequence.

For more information, please read the whole paper. I also recommend reading Design parameters to control synthetic gene expression in Escherichia coli.

genetics - What is the relative power of GWAS studies in different species?

question looks like it's been dormant for a while, but i think there's some discussion to be had here-

I would argue that in many (most?) of the model organisms, power would be much greater than humans. Frequently (worms, mice, plants, yeast) you can work with basically isogenic inbred lines. I would argue this is much more important than long haplotypes:
a) less importantly, no heterozygosity.
b) more importantly, you can re-phenotype the same line repeatedly to directly estimate experimental/environmental variation, and get a very precise estimate of actual E(phenotype | genotype). this is opposed to humans, where you have the one individual, so you just have to pray that your experimental/environmental variation is low and your heritability is high in the sample population.

For example, for just 100 inbred lines of a plant, you get massive, beautiful GWAS peaks for many phenotypes: Atwell et al. 2010. Human GWAS sample sizes generally have to be in the 1000s before they are sufficiently powered (ref).

This subject is discussed a little further here.

In direct reference to the long haplotypes, note that power and mapping precision are different things. That is, your power to detect an association can be extremely high, but you might have a very broad chromosome interval that you will then have to go hunting in to find the causal locus. Note that this problem might actually be worse with large effects, which are probably under selection if they are interesting, and thus there is likely to be substantial linkage disequilibrium between the causal locus and surrounding regions. Of course, if the effect size is small (generally the case in humans though not in other orgs), this is less of an issue.

pharmacology - Is it better to take a half dose of paracetamol and a half dose of ibuprofen together rather than a full dose of either?

Whilst Alexander Galkin gives some great information, I think there's a fundamental reason why that particular pain relief strategy is best, and it hasn't been mentioned yet.

The reason is simply that when you take Ibuprofen or Paracetamol (a full dose) you can only take it once every 4 hours. However, the pain relief doesn't last for 4 hours, so if you take either or both together every four hours you will experience pain after the medication wears off and before you are allowed your next dose.

So instead you can follow either of two strategies:

1. Take a full dose of Paracetamol at 0 hours, 4 hours, 8 hours, as well as taking a full dose of Ibuprofen at 2 hours, 6 hours, 10 hours. This will give you less time when no pain medication is in effect. But if your pain is inflammation related you might want to keep the anti-inflammatory effects of Ibuprofen topped up, so you can...


2. Take a half dose of Paracetamol + a half dose of Ibruprofen every 2hours. This gives you a mix of pure pain relief and anti-inflammation and ensures minimum time when each medication has no effect.

I don't think human biology has to come into it (apart from the fact that you can take the two substances together with no ill effects), it's just a good pain-avoidance strategy.

human biology - How exactly does long-term tension (over months) build up in the ligaments, tendons, muscles?

Tension is not a quantifiable thing, rather an abstract concept to describe feelings of stress in muscles. I think I see what you are getting at but I would separate it into two questions.

"Is massage provably beneficial to muscles under physically induced stress?"

Emphatically yes, however there are so many different types of massage, some are more medically viable than others. Most massage stimulates muscles to encourage a release of tightening, much like exercise does, though I would assume that there's also a psychosomatic that plays a significant role as well due to human contact.

The other question, more the point "Are massage therapist's claims about relief true?"

Some are and some aren't. Unless it's by a medical professional (prescribed physical therapy for instance) they can claim whatever they like. "Reiki contributes to your body balance and wellness by attuning your energy" This can not be proven, but not really disproven either, so they can claim it all they want though it's purely anecdotal (if not outright fabrication on the part of less ethical individuals.)

In summary: Massage has tangible benefits, but if the claims of the therapist sound too good to be true, they probably are.

biochemistry - When collecting cell lysates for a Western blot, how do I induce di-sulfide bonds?

I would like to conduct a simple dimerization experiment for some protein I'm collecting from a cultured cells. My thought is, that if I'm running a non-reducing, denaturing PAGE gel, then removing beta-mercaptoethanol/DTT from the sample buffer should be enough to allow di-sulfide bonds to form.

I have seen several authors incubate the cells first with a drug called BSS that diffuses across membranes and creates protein cross-links. I may be wrong, but the use of this drug seems more appropriate when trying to follow up with an immunoprecipitation or a pull-down assay.

If anyone has any experience with this, could you please enlighten me?

What is special about the N3 buffer for Qiagen minipreps?

N3 and P3 are different. N3 contain chaotropic agents. P3 does not.
Mainly there are two types of DNA purification kits on the market: silica membrane (glass fiber) based and DEAE anion exchange based. The silica membrane based method requires high concentration of chaotropic agents (salts that can change the water structure dramatically, GuHcl, GuSCN, NaI, are a few of them) present in the mixture before loading on to the spin column. DEAE based method, on the other hand, bind DNA based on the charge property on the DEAE resin which largely depends on the pH and ionic strength of the buffer. Chaotropic agents are not needed for DEAE based method.
In our lab we make our own kit using components and buffer recipe provided by Epoch Life Science for both mini prep and maxi prep. Their mini spin column and maxi DEAE column can be purchased without buffer. Their mini spin column also works perfectly with buffer from Qiagen and Lifetech. That's how we used up our leftover buffer from our old kits.

pharmacology - Looking for a cancer drug target database to guide sequencing of patient tumor DNA

I have a question I would like to pose to the community. I have recently received access to a bench-top ion torrent DNA sequencer. Our idea is to use this machine to sequence the DNA from patient’s tumors in order to guide treatment options. My job is to identify a list of all currently used anti-neoplastic drugs along with their known targets (i.e., specific genes and mutations) and accession numbers. I would like to put these data in a table in which each row corresponds to a different drug.

For example, a row in the table might read (column names are indicated in brackets): [disease] breast cancer, [drug] trastuzumab, [drug target] HER2/neu receptor, [gene] ERBB2, [location] chr17:37844393-37884915, [mutation type] amplification, [accession number] ENSG00000141736. The pathologists would then be able to use this database in order to select appropriate genes for sequencing whenever they receive a tumor specimen. If the patient’s tumor had an amplified ERBB2 gene, they could be given trastuzumab.

Currently our study is in pre-planning stages (i.e., we won’t actually be testing this on patients any time soon). I would appreciate it if anyone could give me on advice on how to go about creating such a database. I am aware of online databases including COSMIC, Sanger's Cancer Gene Census, and the Potential Drug Target Database (PDTD), but they don’t have everything that I’m looking for. I am familiar with R and could use it to combine data from multiple sources if necessary. If anyone else has comments or suggestions for further reading that would also be appreciated. Thanks!

Note: This question has also been posed on a Research Gate forum: http://www.researchgate.net/topic/Cancer_Biology/post/Looking_for_a_cancer_drug_target_database_to_guide_sequencing_of_patient_tumor_DNA

genetics - Relative Property of Alleles

Sure it is!

When we say that an allele is dominant on another what we really mean is that the phenotype obtained from that specific mutation of the gene somehow "masks" the phenotype of the other.

The easiest example would be that of pigment. A gene X could code for an enzyme which produces a red pigment. A mutation in X could result in a allelic variant x coding for a non-functional enzyme that does not produce pigment. In this case X will be dominant on x, as in the presence of both the pigment will still be produced (because you will have half functional and half non-functional enzyme molecules).

Things can, however be slightly complicated.

For instance, if the allele R codes gives a red pigment and r gives a white pigment Rr could give pink. This is called incomplete dominance.

On the other hand you could have co-dominance, where WW is white, ww is black and Ww is black and white.

The example you are asking for is called serial dominance and the "classical" example given is that of the rabbit coat color where you have for alleles with a dominance hierarchy like:

c+ > c^ch > c^h > c

c+ gives a fully functional enzyme, called tyrosinase
c gives a non-functional enzyme, resulting in an albino phenotype
c^ch (chincilla phenotype) and c^h (hymalayan phenotype) give partly functional enzyme (interestingly, the latter gives a temperature sensitive fur color).

In reality the rabbit coat genetics is even more complex, see this documents (PDF) for a more complete list of all the various loci and possible alleles involved.