text:
Requirement of energy for metabolic processes
Sorry if this is too elementary, but can someone explain why the following reaction can occur without a net influx of energy from some other process?

$C_6H_{12}O_6 + 6O_2 \to 6 CO_2+6H_2O$

And why does this one need an influx of energy?

$6CO_2+6H_2O\to C_6H_{12}O_6+6O_2$

Wait is the second one hydrolysis?

Changed "influx" to "requirement" in your title, as we don't really use that word in metabolism  in the sense you describe. The energy is generally provided by the use of ATP in reactions, which is hardly coming from the outside, as influx implies. Left it in the question body, but change makes a better title for indexing etc. Hope that's OK with you.

I’m not quite sure which of two questions you are asking here, but as this is basic to understanding metabolism, I think an answer to both questions will be of most general utility, even if you personally already understand the answer to the first.
[1] The Gibbs Free Energy Change in a chemical reaction determines whether it will proceed spontaneously

If a reaction involves a negative change in Gibbs Free Energy (ΔG), it
  will proceed spontaneously; if not, an input of energy is needed to drive it.

The breakdown of glucose to carbon dioxide and water has a large –ve ΔG and therefore does not require input of energy — the reverse reaction has an equivalent +ve ΔG and therefore requires an input of energy to drive it.
This basic aspect of chemical reactions emerges from the laws of thermodynamics, and is explained in detail in section 1.3.3 of Berg et al., available on-line.
[2] Reactions that break bonds tend to involve negative changes in free energy
Why does the oxidation of glucose have a –ve ΔG, and its synthesis a positive –ve ΔG? This is because glucose oxidation involves breaking covalent bonds, and its synthesis making covalent bonds.
To understand the thermodynamic contribution of bond energies, the account in section 2.4 of Lodish et al. (available on-line) is recommended. In brief, ΔG = ΔH – TΔS, where H is enthalpy and S is entropy. The bond energies contribute to ΔH, and there is also generally in increase in entropy when two moieties become one, as mentioned in the answer by @MangoPrincess.
Footnotes

Not all chemical or biochemical reactions involve net formation or breakage of bonds — for example molecular rearrangements. In these cases more sophisticated chemical considerations are needed to understand the experimentally determined changes in free energy.
The importance in discussing biochemical reactions in terms of Gibbs Free Energy changes is that an energetically favourable reaction can be coupled to an energetically unfavourable one to drive the latter, and it is the quantitation of the ΔG that allow one to predict or understand this. It is also important as ΔG can be quantitatively linked to oxidation–reduction potentials.

Is my second equation an example of hydrolysis? and the first one a dehydration reaction? Studying them more closely, that is what it seems like.

@Anonymous 1. Each equation is just a representation of the overall balance of constituents of glucose. You cannot consider them as a reaction as they are the balance of many reactions. 2. The overall reactions are oxidation of glucose and reduction of carbon dioxide — not hydrolysis and dehydration. 3. The equations omit the oxidising and reducing agents NAD+/NADH and FAD/FADH2 — not to mention ATP and ADP — so are incomplete. 4. I‘ll look at the individual reactions in another comment as I’m running out of characters.

@Anonymous 4 cont. You can see tables which classify the inidividual reactions of glycolysis and the TCA cycle (for the oxidation of glucose) in Berg et al. Table 16.3 and Table 17.2. The reactions that break C–C bonds are not hydrolyses but an aldol cleavage and decarboxylation/oxidations. Hydrolysis breaks a bond by adding water, for example the peptide bond ~CO–NH~ becomes ~COOH + ~NH2. Other examples are hydrolysis of phosphodiester and glycosidic bonds.

The first reaction is catabolism. It does require some energy input in the beginning ("energy investment" of glycolysis) but ultimately leads to net energy release through "energy payoff" of glycolysis, citric acid cycle, and finally electron transport chain and oxidative phosphorylation.
The final result is oxidation of glucose, and reduction of oxygen.. At various points, electrons transfer to NAD+ and FAD. Then, the reduced forms of these 2 electron carriers (NADH & FADH2) go to the electron transport chain, where electrons transfer to oxygen at the end (reduction of oxygen). Meanwhile, protons were pumped into the intermembrane space to create the proton gradient that drives ATP synthesis.
The second reaction is anabolism. The Calvin Cycle requires energy from ATP and NADPH to create the necessary bonds to build up sugars.
In terms of bioenergetics, degradation of a molecule is exergonic. This is favorable if you think about the second law of thermodynamics: there is more entropy when there are multiple, separate sub-units, while there is more order when one molecule made up of those sub-units. Synthesis of a molecule is endergonic (requiring energy). It IS favorable in the sense that we get sugar to eat and burn in the future.
In summary:

Breaking down a molecule = catabolism = release energy from chemical bonds
Building up a molecule = anabolism = absorb energy to FORM chemical bonds (think of the resources needed to construct a new building)

Note my answer leaves out specific details about the processes, but I hope this is basic enough to answer your question!
Source: Lehninger Principles of Biology, David Nelson & Michael Cox

Welcome to Biology.SE. Please add some references that give both evidence for your claims and input for further reading. With these basic processes, Wikipedia articles should suffice.

But if I didn't recognize those processes as glycolysis and the citric acid cycle, how would I know? I'm searching for a more generalized way of figuring out my question.

Oxygen has a high electronegativity, in other terms it really likes electrons.

In this scale neon has the highest electronegativity of all elements,
  followed by fluorine, helium, and oxygen.

https://en.wikipedia.org/wiki/Electronegativity#Allen_electronegativity

These are redox reactions. In the first reaction carbon was oxidized 0 -> +4 and oxygen was reduced 0 -> -2. In the second reaction the reverse happens. Nomen est omen oxygen is an oxidizing agent, so it does not like the reactions where it will be oxidized, since by oxidation it loses electrons. And it likes electrons. Really.
Forcing somebody to do something they don't want, always requires energy, and oxygen is not an exception. ;-)
Just to mention there is always an activation energy regardless of the exothermicity of the reaction. Remember; sugar doesn't burn spontaneously at room temperature... These are multi step reactions and enzymes lower the activation energy of each step, that's why you don't need to invest much by the first reaction. Ofc. a similar reaction can happen by burning sugar, but in that reaction the activation energy is much higher, and it is coming from the heat of the gas flame.

It's a bit misleading to say "these are redox reactions". The equations representing the overall process are, but they represent a whole series of reactions, only some of which are redox reactions. And the principle of whether a reaction requires energy is applicable regardless of whether the reaction is redox. The point about activation energy is important, but one needs to stress that in metabolism the reactions proceed at room temperature etc. because of the enzyme lowering the activation energy (or raising the ground state).

@David I did not want to be perfectly accurate with this answer. Ofc. I could have described it in more detail, but I don't think that would be necessary. If you feel the need you can always edit it, but please try to keep the same length and simplicity.


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Is a Linker sequence mandatory in primers for PCR Cloning?
I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no linker sequence before/after the restriction site in the primer. What measures should I take to be successful in the cloning?

You can add the restriction recognition site directly next to your sequence, this is not a problem. However, a lot of restriction enzymes have a lower or no activity, if the restriction site is directly at the end of a DNA strand and require some additional bases here.

i was using bamhI and hindIII for digestion ... do i need to order a new primer set ... and is there any specific linker sequence for respective restriction enzymes?

This is the website you need, and yes, most of the time it's better to have several extra bases at the start of your primer.
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

source: Stack Exchange

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sort: votes


text:
Evolution of complex organs
If it evolved, how is a feature selected through a long time span with useless function before the feature becomes complete?
For example, an eye is a very complex organ consisting of countless small features. According to evolutionism, every such small feature should be formed by evolution over long time. But most intermediate features would be useless until the small incremental features are accumulated to provide any useful function good enough to be selected by nature. How do evolutionists explain this intermediate useless features and natural selection? I would appreciate if someone doesn't direct me to another long article but provide an essential succinct explanation. (think of all the organs' intermediate state.. would they survive?)

You really should try to write a short comprehensible title. And this is a question and answer forum, not a debating society or a text book. If I suggest you read The Blind Watchmaker this is because Dwarkins went out of his way to argue this very point in that book. Why should we provide you with a summary? In any case, as I believe you already stated that your attitude to evolution was based on religious belief, you're not likely to be convinced by argument.

This question has been asked many times on this site. See for example http://biology.stackexchange.com/questions/49209/how-did-the-cardiovascular-system-evolve

"an essential succinct explanation" of a complex process is never helpful. Learn about it if you really want to know what it's all about. As a Christian, learning molecular biology and evolution actually made me appreciate the complexity of creation and the compassion shown for the adaptability of creation to change. Read something by Francis Collins, a believer and the head of the Human Genome Project and current head of the National Institutes of Health. It's enlightening.

@anongoodnurse I disagree that "an essential succinct explanation" is never helpful. Having a very basic framework on which to build a deeper understanding can be, and is, vital. While 'Simple systems develop, interact, grow interdependent.' is utterly simplistic, it captures the thrust of the process and gives something to build on. Simple explanations should not be dismissed so readily.

You're working under the false and foolish assumption that intermediate traits are useless and should be selected against.

A little bit of terminology first
"evolutionism" refers to a set of beliefs from the 19th century. Evolutionary biology is much more than "evolutionism" and I suppose you are more interested is the current day understanding rather than a historic perspective on the question, is that right?
An "evolutionist" is a person that believes in evolution without necessarily understanding it (as by opposition to "creationist"). Someone who study evolutionary processes is called an evolutionary biologist (or often evolutionary geneticist although it does not exactly refer to the same field).
Issue with the question
It is a very standard question as it is a common fake argument from religious extremists that refuse to accept the theory of evolution. A good answer is necessarily long as (1) there are a number of processes of interest and (2) within the question it is clear that the person asking it has very little understanding in evolutionary biology. I will try to keep as introductory and short as possible. Asking for a short answer for this question is like asking for the name of the most populated city in the USA and authorized to use only one word in the answer (while "New York" is in two words).
Answering to a specific case
For the reasons explained above, it is often easier (faster) to explain for a specific case than in theory for a general case. Here are two examples from Biology.SE

How did the cardiovascular system evolve?
When did vision evolve for the first time? and What is the minimum eye?

Source of information
There are good books designed for people that know nothing to evolutionary biology to get an introductory answer to that question. The Blind Watchmaker is one such book for example. More generally speaking, one might want to have a look at  Understanding Evolution by UC Berkeley, a very introductory source of information on evolutionary biology.
Key Concepts to answer your question
Quickly speaking here are a series of quick concepts that are necessary to understand in order to answer this question.

Genetic drift

There are a number of different forces that affect evolutionary processes. Layman often think of natural selection as being the only force causing evolution but this is wrong. Genetic drift refers to the random sampling of individual at one generation to build up the successive generation. The relative importance of drift to natural selection in driving evolution of a population depends on the effective population size. Have a look at Why is the strength of genetic drift inversely proportional to the population size?.
Typically a phenotypic feature that explain little in the variance of fitness in the population might well increase in frequency in the population just through drift.

Fitness effect of intermediate states

It is common for layman to assume that intermediate states ought to be deleterious. This is a typical logical fallacy called argument to ignorance. In reality the intermediate states might well be neutral or only slightly deleterious (and therefore be mainly affect by genetic drift) or might even be beneficial.

Existence of intermediate states

Darwin framed his theory into the idea that evolution is a very gradual process. As we know today, this is far from being true. Evolution can actually occur through a big steps. There are single mutations that double the amount of genetic material, there are single mutations that completely affect the physiology or anatomy of an individual. There are cases of horizontal gene transfer (transfer of genetic material between species) as well that can cause sudden important change without having to get through intermediate stages. It is sometimes wrong to assume that there must be intermediate steps.

Phenotypic flexibility

While you haven't raised this question, a common creationist argument is to say that if a mutation occurs to make a bone a big longer, then one organism would simultaneously need mutations that would make muscles, nerves and other tissues longer accordingly. In reality our developmental process is quite flexible to an environmental change or to a mutational change. This flexibility is very much used in medicine and agriculture.

Why do we talk about the "Theory" of evolution
This is just a sidenote but you might want to have a look at this post

Thank you for explaining the down vote so that I can improve my post accordingly. Note that the downvote appeared about 10 seconds after I posted my answer. It seems therefore impossible that the one who down voted had time to read my answer.

Not the down voter, but it bears mentioning that it's 'beliefs', not 'believes', also 'believes', not 'believe'.

Sure, I explained why the answer has to be of a certain length.

While I agree that your answer is illuminating, thorough and good, the core concept I think can be expressed in a shorter phrasing: 'Simple stuff developed, interacted, got more complicated.' It's not a complete answer, nor even ultimately a satisfactory one, but it does get a core point across, and leaves something to build on. Biology is necessarily full of these kinds of simplifications, because life is just… It's just all over the place, man. It's crazy. Complete lunacy. A good and complete answer can't be shorter, but a very rough and ultimately helpful outline can be, I feel.

I'm not the down voter. I read your answer but again another long not-convincing words evading my core question. I'm on something else but will try to add more comments later. I already read about eye evolution but not convincing at all. Still seems like a far-fetched theory which fails to explain things.

You should probably follow an introductory course to evolutionary biology then and you will be able to read more advanced explanations. I also think that going through example specific would help a lot (but I recognize the interest of a question that seek for general explanations). Typically, you said that something still remained unclear to you about the evolution of the eye so you might want to investigate this particular subject in a post.

my question is not about the eye, can any one suggest any 'intermediate organ?' don't you all think we should be seeing many such intermediate organs having meaningless features?

To put it succinctly: None of the parts of any complex organ present in humans today appeared as they are now.
The parts of the organs we have now are useless in isolation because they have over the last ~500 Ma developed to rely more and more on each other, becoming more and more interdependent.
To use the eyes as an example, the very earliest ones (some of which are still found today, in mollusks, for example) were just light-sensitive skin spots or light-sensitive clusters of core cells in the nervous system. Additional features developed around these, and over time became more interdependent.

The question was not specific to the eye and discussion on the evolution of the eye already exist on Biology.SE

@Remi.b: The eye makes a good case study. And a discussion on the issues with the underlying question already exists on Biology.SE (I assume).

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Are plants actual oxygen factories?
Is the oxygen exhaled by plants during the day compensated by inhalation of carbon dioxide at night?

All green plants do Photosynthesis as well as Respiration.
Plants respire during the day and night, but they only photosynthesis during the day when it is light:
there is equations for aerobic respiration and photosynthesis:
C6H12O6 + 6O2 → 6CO2 + 6H2O (aerobic respiration)
6CO2 + 6H2O → C6H12O6 + 6O2 (photosynthesis)
In terms of reactants and products, they are the reverse of each other. In terms of gas exchange:
Process        -------Carbon dioxide    -----------------Oxygen
Respiration     -----------Out  ----------------------------    In
Photosynthesis  -------In         ----------------------------    Out

Photosynthesis is only in the presence of Sunlight . So it occurs only during the day .
For photosynthesis they require Carbon Dioxide gas ( CO2 gas )
This gas enters the leaves through tiny pores on them called stomata .
The byproduct of photosynthesis is Oxygen gas ( O2 gas )
It is either passes out through the same stomata OR used for respiration

For respiration they require O2 gas .
It enters the leaves through the same pores or stomata .
The by product of respiration is CO2 gas .
During day time it is immediately used for photosynthesis .
During night time the CO2 gas is released in the atmosphere

Net gas exchange
The net (overall) effect depends on the time of day and the light intensity. Photosynthesis doesn’t occur at night. When there is no photosynthesis, there is a net release of carbon dioxide and a net uptake of oxygen.
If there is enough light during the day, then:
the rate of photosynthesis is higher than the rate of respiration
there is a net release of oxygen and a net uptake of carbon dioxide

(consider CAM plants)
in a plant using full CAM, the stomata in the leaves remain shut during the day to reduce evapotranspiration, but open at night to collect carbon dioxide (CO2)
https://en.wikipedia.org/wiki/Crassulacean_acid_metabolism#During_the_night

this link help you understand the process better by simple language :
http://www.bbc.co.uk/education/guides/zxtcwmn/revision/1

It's impossible for a plant to use more oxygen in the long term. As @BlueFoxy explained, whenever a plant is using oxygen it is to oxidise sugar ($C_6H_{12}O_6$). These sugars were made by the plant itself using $CO_2$, $H_2O$ and sunlight, so it can't oxidise more sugar than it produced in the first place.
If the plant is growing then some of the carbon removed from $CO_2$ will be used to form the plant's tissues, causing a net increase in oxygen in the atmosphere. If the plant isn't growing it'll probably be oxygen neutral.
There are probably some weird parasitic plants that break this pattern by stealing other plants' carbon, but in general the rule holds.

"There are probably some weird parasitic plants that break this pattern by stealing other plants' carbon" - Monotropha uniflora is parasitic on mycorrhizal fungi, so does this indirectly.

@AlwaysConfused good catch - I'd forgotten about dodder. However, Cuscuta reflexa can actually photosynthesize a bit; a species which is thought to be entirely dependent on the host plants for nutrition such as C. europaea would be an even better example.


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Is there any reason an arbitrary DNA sequence such as 5'-ACACACACAC-3' would not exist in nature?
I am making a DNA model for my class, and I looked up some examples and they all have different sequences of bases. If I choose an arbitrary sequence like:
5'-ACACACACAC-3'
3'-TGTGTGTGTG-5'

or
5'-ATATATATAT-3'
3'-TATATATATA-5'

Is there any reason it would not exist in nature?

What do you mean by left and right? Or the order of bonds changing? Have you consulted a basic book of biochemistry or molecular biology or looked at the DNA entry for Wikipedia. I have voted close this question as unclear. You need to put more effort into understanding DNA before making models.

How do you know I've put no effort into understanding DNA? I've researched this question for a clear answer but have found nothing, which is why I asked here. Stop acting as if an unclear question is an "inconvenience" to the forum as two others kindly answered it. You can't just close a question because it is unclear - a simple edit will fix it. P.S: I don't own a biochemistry or molecular biology book.

This is not a forum, but a question and answer site that has certain rules about questions to maintain its usefulness and standards. You can read them here: http://biology.stackexchange.com/help/how-to-ask. Questions should demonstrate effort — yours does not. The fact that you do not own a biochemistry book is no justification and suggests you are in the wrong place — there are many online resources on DNA. One of the use of the comment facility is to ask for clarification and improvements in the question. This I did and you have not responded.

You appear to be building a model of a double-stranded DNA 10-mer but are afraid that the sequence of bases you intend to use might be disallowed by some biological or chemical principle of which you are unaware, as your knowledge in these areas is minimal. I have edited your question to this effect. It is such a broad and basic question that I believe the it should be closed: there is no reason any random DNA sequence would not be found in some genome. However, the repetitive sequence proposed may mislead your audience into thinking this is a necessary feature of DNA. I would chose another.

There is no reason. Look up PDB search for your sequence and its likely its already been identified somewhere. What cant be done yet is, take and arbitrary sequence, and say with any certainty what its is for.. that we cant do. Best is comparison against what is already known from any homology

The proposed sequences are so short, it's almost certain they exist in nature.

You must first understand a few things:

What are proteins made of
What is DNA made of
What is the relationship between DNA and proteins

From DNA to proteins
DNA is transcribed into RNA which is then translated into protein. The protein, then has some effect (e.g. could be an enzymatic effect) such as affecting the level of sugar in the cell, digesting some lipids or affecting the expression of other genes.
More information on Khan Academy > Central Dogma
What is DNA made of?
DNA (or at least the genetic code) is made 4 different molecules which are called nucleotides. We abbreviate their names by the letters A,T,C and G.
What are proteins made of?
Proteins are made of amino-acids. There are ~20 different amino acids.
Genetic code
For the interest of your question it is now essential to understand how does the succession of nucleotides A,T,C,G in the DNA is reflected in the protein. This is called the genetic code.
We call a series of three nucleotides, a codon. AAT is a codon for example. Each codon (except special codons) correspond to a specific amino acid. This correspondance is called the genetic code. Here is the universal genetic code (where the T has been replaced by U because RNA contains U instead of T)

Consider for example the codon UAU and AUU (where only the order changes) The first one codes for the amino acid Tyr while the other codes for Ile. So yes, the order matters.
Information theory
Globally speaking, for any information the order of a sequence is of fundamental importance! More information on Khan Academy > Information Theory.

So, because the order of the bonds differs every time, I could theoretically make a DNA model like this, and it would be correct? AT CG AT CG AT CG AT CG AT CG

Please delete the reference to the central dogma. You do not need to give a name to the principal you state and it is not Crick's Central Dogma, which is basically that the information in DNA can go to protein but not in the other direction. I document this in detail in answer to http://biology.stackexchange.com/questions/49201/crick-s-central-dogma-counter-cases/49210#49210

@David I agree it is not worth using this term after all. I was tempted to use it as a consequence of the link to a Khan Academy video that is called central dogma. Is it better now?

You're still using the term "Centra Dogma"...

@Deadfult what does that sequence actually mean? Is it 5'-ATCGATCGATCGATCGATCG-3'? That's fine, as long as you include the antiparallel 5'-CGATCGATCGATCGATCGAT-3' as the paired strand.

DNA has a polarity that is based on the numbering of the carbon atoms in the deoxyribose molecules in the sugar phosphate backbones of the two complementary strands.
So, for example, the 3'-OH of one base's sugar is attached, via a phosphate, to the 5'-OH of the sugar on the adjacent base in the strand.
Therefore, we say, that one end of a single-stranded piece of DNA has a 5'-end, and at the other end a 3'-end.
Go no further until you understand this concept and can draw a picture of this.
In Watson and Crick's model for double-stranded DNA the two complementary strands are anti-parallel. In other words, if you imagine the double helix like a ladder, then one of the legs of the ladder is oriented in the 5' --> 3' direction, while the other, complementary strand is oriented in the 3' --> 5' direction.
When we describe a DNA sequence, by convention we read the sequence strands in the 5'--> 3' direction.
So now you should be able to state your query more succinctly.

Yes they do matter. Imagine reading just one side of the DNA strand. This is how the code is actually read, so the order would change that side's reading.

ACAAGATGCCATTG

is different than

ACAAGATGCCATTC

Notice the different last base making the message different. This can affect transcoding to proteins.
Just like the change of one letter can change the meaning of the english word "petty" to be "patty". The same is true for any encoded message.

Alright, so let's say I made my model like this then:
AT
CG
AT
CG
AT
CG
AT
CG
AT
CG
Theoretically, this would be correct right?

Correct for what? I have no idea what you are asking


text:
How do DNA-binding proteins recognize the correct DNA base pairs?
My professor posed this question to the class today - "How do DNA binding proteins specifically bind to base pairs?"
He alluded to the different arrangements of hydrogen-bond donor and acceptors in A-T, T-A, C-G, and G-C base pairs. However, they are not always significantly different in some pairs (i.e. the minor groove of the C-G and G-C base pairs have the same acceptor - donor - acceptor pattern).
So how would a binding protein know the difference between C-G and G-C on the minor groove of a DNA base pair? How does a DNA binding protein know it's reached the right base pair, more generally?
Thank you!

The major groove contains more information and can distinguish between C-G and G-C

There are different kinds of DNA binding domains; the ones involved in base identification in the major groove can differentiate between different base pairs of the same nucleotides i.e. AT vs TA because they bind to functional groups on a nucleotide and not the base-pair per se. The stereochemistry i.e. which nucleotide is in the major groove, is important. The location of the nucleotides in the major groove also is a factor (Figure 1).

Figure 1: DNA sequence identification by Helix-Turn-Helix transcription factor. This is the example case of the Engrailed protein in Drosophila. Reproduced from Paro and Sauer (1992) [1].

You can see that Adenine is recognised differently than Thymine. In many cases such as that of zinc finger proteins (ZF), sequence identification usually happens for dinucleotide pairs such that each finger (a modular unit) can identify a di/tri-nucleotide (Figure 2,3). An array of fingers in case of ZF and helices in case of Helix-Turn-Helix motifs can recognize a unique stretch of DNA sequence (which is certainly more unique than individual base pairs). Usually the binding of the fingers are co-operative which leads to increased affinity and specificity of the protein-DNA binding [2,3]. Analogous mechanism may exist for other kinds of DBD too (I haven't specifically looked for that). Moreover, many DNA motif binding proteins oligomerize, which is again co-operative. This increases the specificity and the overall binding kinetics becomes sigmoidal and thresholded (see Hill kinetics).
Binding mechanisms are best understood for ZF proteins that led to the development of zinc finger nucleases which was one of the first modern methods of targeted genome editing [4] (though they have now been obsoleted by CRISPR-Cas system).

Figure 2: DNA sequence identification by zinc finger proteins. This example considers the Zif268 protein. Reproduced from Paro and Sauer (1992) [1].

Figure 3: A more detailed depiction of Zif268-DNA interaction. Reproduced from Durai et al. (2005) [4].

References:

Pabo, Carl O., and Robert T. Sauer. "Transcription factors: structural families and principles of DNA recognition." Annual review of biochemistry 61.1 (1992): 1053-1095.
Krishna, S. Sri, Indraneel Majumdar, and Nick V. Grishin. "Structural classification of zinc fingers SURVEY AND SUMMARY." Nucleic acids research 31.2 (2003): 532-550.
Lee, Juyong, Jin-Soo Kim, and Chaok Seok. "Cooperativity and Specificity of Cys2His2 Zinc Finger Protein− DNA Interactions: A Molecular Dynamics Simulation Study." The Journal of Physical Chemistry B 114.22 (2010): 7662-7671.
Durai, Sundar, et al. "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells." Nucleic acids research 33.18 (2005): 5978-5990.


text:
Does rainwater contain many fewer micro-organisms than river water?
From watching many documentaries on micro-organisms, I can tell water typically contains quite a lot of them.
But what about rainwater? (before it hits the ground). I know nothing about any micro-organisms that live in the sky or clouds. Does rainwater contain fewer, and less dangerous, micro-organisms?

If my response answers your question, please accept it. Otherwise, please leave a comment about how I could further answer your question. Thanks.

@theforestecologist It's almost a complete answer. Your last link says 80,000 bacteria per milliliter of riverwater. But I could not find a number per mL for rainwater (rain before it hits the ground).

Found a study for you and have updated my answer. Please accept if all is ok.

Who voted to close this as "homework??" 1. This question was not easy to answer, so it is not trivial (i.e., even after an initial search, very little reputable sources can be found). 2. it's already well-received with a well-received answer. I vote to keep open.

There seems to be a basic problem with the question, which is that not all rivers are alike.  Indeed, I would think that the number of microorganisms varies greatly within a single river, e.g. the Mississippi/Missouri near its headwaters, and just downstream of New Orleans.

typically something like a million times less. only think that filters water on earth is sand percolation, and the UV status of the atmosphere makes it pretty hostile to animals. it's a big UV and mechanical filter up there.

According to a number of citations listed on Kenyon College's MicrobeWiki, rain can contain microorganisms via a process called "bioprecipitation."

Essentially, microorganisms, dust and other small particles get swept up into the atmosphere, and cold temperatures cause atmospheric water vapor to freeze around the organism/particle. Once the ice-covered particle picks up enough mass, it falls from the sky and reaches earth as precipitation (likely rain if temperature is warm enough).

Here is a schematic from Wright et al. (2104) of how an increase in relative humidity, due to a cold-frontal passage, can trigger biological ice nuclei release followed by seeding of the frontal cloud band with the lofted ice nuclei:

 So How Many (and Which) Bacteria Are in Rain?
Which Bacteria?
According to Wikipedia:

The ice-nucleating bacteria currently known are mostly plant pathogens

According to MicrobeWiki, these include:

Exserohilum turcicum, Pseudomonas viridiflava, Pseudomonas fluorescens, Pantoea agglomerans and Xanthomonas campestris.
The most well described organism that demonstrates ice nucleation is Pseudomonas syringae, which was determined to specifically supply a source of ice nucleators by Leroy Maki in the 1970’s.

According to work by Natasha DeLeon-Rodriguez, Athanasios Nenes, et al. (full paper here and NPR story here), 100s of species of bacteria exist in the clouds and it's not uncommon to find almost 20 species in any given sample (60% of which on average were still living).

Here's a graph showing the relative amounts of various taxa found in their samples:

How Many Bacteria?
DeLeon-Rodriguez et al's (2013) results revealed an average bacterial concentration of somewhere between $5.1 × 10^3 cells /  m^3$ to $1.5 × 10^5 cells /  m^3$ of cloud (depending on method of counting).

Quickly doing some math (1 m$^3$ = 1000000 mL), their results suggest that, on average across a cubic meter of cloud, there is <1 bacteria / mL of moisture.

According to research by Joly et al (2014) that specifically examined ice nuclei (or "IN"):

there were 0 to ~220 biological IN/mL of cloud water (i.e. 0-22 per m$^3$ of cloudy air based on cloud liquid water content estimates) and these represented 65% to 100% of the total IN.

Comparatively, river water (and drinking water) can contain a lot more bacteria (i.e., 80,000 bacteria / mL) and other microorganisms.
However, this disparity is not always so great, especially if we focus at comparing counts of only one or a few species (and not total bacterial counts).

Based on research of a tropical reservoir by Kaushik et al (2014):

Levels of E. coli were found to be in the range of 0 CFU/100 mL – 75 CFU/100 mL for the rainwater, and were 10–94 CFU/100 mL for the reservoir water.

Note: CFU = colony forming units. (estimation of microbial numbers by CFU will, in most cases, undercount the number of living cells. [wikipedia]).

Citations

 DeLeon-Rodriguez, N., Lathem, T. L., Rodriguez-R, L. M., Barazesh, J. M., Anderson, B. E., Beyersdorf, A. J., ... & Konstantinidis, K. T. (2013). Microbiome of the upper troposphere: species composition and prevalence, effects of tropical storms, and atmospheric implications. Proceedings of the National Academy of Sciences 110(7), 2575-2580.
 Joly, M., Amato, P., Deguillaume, L., Monier, M., Hoose, C., and Delort, A. (2014). Direct Quantification of Total and Biological Ice Nuclei in Cloud Water. Atmos. Chem. Phys. Discuss 14(3):3707–3731.
 Kaushik R, Balasubramanian R, Dunstan H (2014) Microbial Quality and Phylogenetic Diversity of Fresh Rainwater and Tropical Freshwater Reservoir. PLoS ONE 9(6): e100737. doi:10.1371/journal.pone.0100737
 Wright, T. P., Hader, J. D., McMeeking, G. R., & Petters, M. D. (2014). High relative humidity as a trigger for widespread release of ice nuclei. Aerosol Science and Technology 48(11), i-v.
For further reading, also see the associated citations from MicrobeWiki.
I think you would also benefit by perusing the works of Pierre Amato.

A fairly recent study examined this as well: Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications

You cite two wildly disparate figures for bacteria in river/drinking water, the latter of which seems to be relatively close to the number in rain water. Is one more trustworthy than the other?

Two things to note: 1. One source presents "bacteria / mL" while the other is CFU (colony forming units) -- estimation of microbial numbers by CFU will, in most cases, undercount the number of living cells [wikipedia]. 2. Regardless, the Kaushik study looks at ONLY E coli in a reservoir (where enterobacteria such as E coli are typically managed to stay very low). I guess I could edit the answer to better contextualize all of this...

One source is talking about only E. Coli, while another source says 80k bacteria (any bacteria). They are not really comparing apples to apples. BTW in looking at the new link, I saw this: "Both rainwater and reservoir water samples were collected from the sampling locations at regular weekly intervals and within 24 h of rainfall events." This brings up a good question, namely how far apart rainfall events should be to avoid contaminating riverwater with rainwater.

@DrZ214 I've already addressed that in the above comment and in my answer edits. Your "This brings up a good question..." statement should be asked as a new question and is not relevant here.

Ofc it's relevant here. We do not want cross-contamination of riverwater with rainwater because we do not want to add the number of MOs in rainwater to the number in riverwater. Nevertheless, this latest edit answers the OP. Looks like riverwater has over 300x more MOs than rainwater.


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Effect of a doubling of the start codon in a gene
I am learning about frameshift mutations. Frameshifts can occur due to a nucleotide deletion. Suppose that due to a frameshift, because of a deletion somewhere upstream from the original start codon, two additional start codons are generated, just before the stop codon in the new reading frame. What would happen in terms of translation?
AUG-GCC-AUA-AUG--------UAA
Start       Start then stop

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This is an old question, but it should say, or the answers should say, whether they are referring to eukaryotic or prokaryotic systems. All the answers appear to assume eukaryotic systems.

There is a basic misconception in the question you have asked, which @biogirl has explained. There is only one start Codon in any mRNA and it defines the open reading frame.
All other AUGs in the open reading frame are simply codons that encode for the Amino Acid Methionine and have no function in the start of translation. There are factors other than AUG that determine the start of translation.
So a frame shift that gives you an additional AUG only means that you will have a different Amino Acid encoded for in the resulting polypeptide. A frame shift will generally completely alter the protein product of the gene. If however the frameshift does disrupt the start codon, then it is unlikely that you will have any translation what-so-ever, as the other elements necessary for determining the start of translation will likely not be present in other areas of the coding sequence. In prokaryotes, you need a Shine-Delgarno sequence to initiate translation, and in Eukaryotes, though all of the factors for translation start are not well understood many genes carry a Kozak sequence that indicates to the ribosome the start of the open reading frame.
The more important codons to look for are introductions of stop codons. These three codons, UAA, UAG, and UGA do not have tRNAs with complementary anticodons (for the most part, as tRNA genes can also sustain mutations that change their anticodon) and therefore all result in the termination of translation if the shifted frame results in the ribosome reading one of the three stop codons in frame.

Very good answer and +1. Just a nitpick: strictly speaking, there are actually tRNAs that recognize STOP codons. Such nonsense tRNAs can be the product of a specific mutation but also, as is the case for tRNASec, wild type sequences that insert a specific amino acid.

@terdon, Learn something new (or old as this was discovered in the 80's) every day. I had a hard time finding references, but Molecular Biology of the Cell 6th Edition mentions selenocysteine and it does indeed have an ACU anti-codon. It looked like there needs to be a secondary stem-loop structure in the mRNA for it to be incorporated instead of having a release factor bind and terminate transcription, but it definitely shows the remarkable flexibility and diversity of the genetic code. Thanks!

mRNAs can include multiple start codons (and thus multiple ORFs). Upstream AUGs and their effect on translational regulation are an active area of research. They are also sensitive to neighboring mutations. http://science.sciencemag.org/content/352/6292/1413

AUG functions as a start codon only when it is at the 1st position of the open reading frame.  Whenever AUG is present in between, it codes for methionine amino acid. Go through the basics of translation from a good book.

The start codon is not sufficient to start translation. A ribosomal binding site is also required. It's likely that translation would initiate at its normal location and then simply proceed through any additional start codons in the new ORF until it reaches the first stop codon.

That is half the story as it is only in Prokaryotes that the ribosome binds to the Shine-Delgarno sequence and the starting AUG. In Eukaryote small ribosomal subunits bind near the 5' cap and scan through the 5' UTR until the Start codon. In many mRNAs that is near the Kozak sequence, but not all Eukaryotic mRNAs have a Kozak sequence.

@AMR Nowhere in my answer did I make the claims you say I did. The ribosomal binding site is self explanatory, be it the SD sequence or 5' cap.

It is because you are answering a questionabout two AUGs, and you are saying that there needs to be a ribosomal binding site, which is correct, but when taken with the question implies that the ribosome is choosing between one or the other AUG based on where it binds, and that would only be relevant in the case of prokaryotes as it would bind to the AUG in the SD sequence. Eukaryotes would scan to the start site. AUG is simply the codon for methionine so even saying additional start codons as you have in your edit obfuscates the situation.

@AMR I really don't understand your point. The start codon is chosen based on where the ribosome binds. What is the controversy?

In Eukaryotes you can have an AUG or multiple AUGs in the 5'UTR, so your last comment doesn't hold, and your answer is not complete and is confusing. Eukaryotic translation does not necessarily begin with the first AUG encountered. And as I pointed out before, there is one start codon, which defines the open reading frame; any other AUGs are methionine codons or are in the UTRs and are untranslated, so no additional AUG is called a start codon.


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Do all restriction enzymes have palindromic recognition sites?
I am trying to find out the requirements for a segment of DNA to be the recognition site of a restriction enzyme.
Acording to the article on Restriction Enzymes in Wikipedia, "many of them" (referring to recognition sites) are ‘palindromic’ (in the sense that the sequence of nucleotides on one strand should read the same as the that on the other strand, from the opposite direction).
Is this true for all recognition sites?

Please read our homework policy which requires that you demonstrate that you have done some research to answer the question yourself before posting here. Did you read the Wikipedia article on this topic?

Yes i did, and it tells that "many of them" (referring to recognition sites) are palindromic and that left me in confusion. Does that mean there are recognition sites that aren't palindromic? Unless i didn't see some certain part in that article.

Instead of writing "I suppose" you should have stated that you had read the Wikipedia article, giving a link, and quoted the phrase, as in your comment. You can find the answer by reading further in the article, but so as not to discourage you, I will rephrase your question, and briefly answer it.

thanks and i apologize for the late reply.i get it now

The article cited in the question goes on to discuss the different classes of restriction enzyme, for which the recognition ‘style’ differs. Not all recognize palindromic sequences.
Type II restriction enzymes
These have been divided into a number of subclasses (reviewed by Pingoud and Jeltsch). Those regarded as orthodox — which are the ones generally used in DNA cloning — appear always to require palindromic sequences in their recognition sites, although those in some other classes do not (see list here).
This statement is subject to the proviso that you regard as palindromic 5-base recognition sites, such as AlwXI, GCAGC, and others with a ‘non-conforming’ central region, such as ApaBI, GCAN5TGC. The reasons for including the latter (which provides an alternative way of defining the requirements of the recognition sequence) can be seen by examining the symmetrical manner enzymes of this class bind the site, which can tolerate a non-symmetrical central region. This is described in more detail in an answer I gave to another SE question on restriction enzymes.
Type I restriction enzymes:

…cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site. Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors. The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides.

Type III restriction enzymes:

…recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20–30 base pairs after the recognition site.

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What is meant by translational repression by an upstream open reading frame?
The NCBI report on the human huntingtin gene contains the following statement:

“This gene contains an upstream open reading frame in the 5' UTR that
  inhibits expression of the huntingtin gene product through
  translational repression.”

But no specific reference it given. Can anyone explain what this means.

What I understood is that the promoter of the gene can only bind to repressors and inhibits translation. Am I right?

No. Please cite the source of your quotation. You say there are many mechanisms to achieve this, but do you know what “this” is? Do you know what translation is and what in a general sense repression would mean? (And how translation differs from transcription? Your talk of promotors suggests not.  Or is it this particular example? In which case, as stated, give us your source.

@David  https://www.ncbi.nlm.nih.gov/gene/3064  The last line of the Huntington gene summary is the source of the quotation. The reason why I say there are many mechanism for "this" is http://www.jbc.org/content/285/37/28506.full    https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007059   etc. I know the basic difference between transcription and translation. And repression, as I know, is the process of inhibitors binding to the promoter hence repressing transcription and translation.

No. Repression is a general English word meaning to subdue or put down, and a general molecular bioscience word to diminish or inhibit, which can be applied to many systems, and implies no mechanism and certainly does not require promoters, which are in no way involved in translation (at least in eukaryotes). I’ll look at the paper later.

@David oh..okay. Thanks!

Summary
The statement refers to the small but significant translation of short open reading frames (ORFs) of huntingtin mRNA, 5′ to the main open reading frame that encodes the large (3144 amino acid) functional product of the mRNA (and gene). It has been reported that the translation of these short ORFs decreases (i.e. inhibits) the translation from the main ORF. It is implied that this is a mechanism for regulating the expression of the gene, and, as it occurs on the mRNA in the cytoplasm, it is an example of ‘translational regulation’ — specifically inhibition. The precise mechanism of this regulation — although not unique to this mRNA — is apparently unknown.
Translation of ORFs in eukaryotic mRNA
Most eukaryotic mRNAs are functionally monocistronic — i.e. they encode only a single protein — unlike bacterial mRNAs. Initiation of protein synthesis involves selection of an initiation codon (usually AUG) by a complex of initiator-tRNA and the small ribosome subunit, but, in contrast to prokaryotes, the mechanism in eukaryotes involves scanning along the mRNA from the 5′ end. In the majority of cases the initiation codon of the protein product is the first AUG, but the significant number of exceptions suggest an effect of sequence context or mRNA secondary structure.
In recent years evidence has accumulated that in cases where the first AUG is not that of the main protein product, a small amount of translation from such 5′ upstream ORFs does often occur.

Diagrammatical representation of mRNA with upstream ORF (blue with initiation codon represented as an open triangle) and main ORF (black with initiation codon as closed triangle.)
Regulatory role of upstream ORFs
Johnstone et al. (2016) have reviewed a large body of evidence that indicates that upstream ORFs are translational repressors in eukaryotes. This has generally been established by measuring the extent of translation of the main ORF after manipulation or removal of the upstream ORFs. Much less is known about the actually mechanism of such regulation, which may vary from case to case, and certainly the paper on huntingtin only offered speculative possibilities. These include:

Binding of small peptide products of the upstream ORFs to the AUG of the main ORF
Pausing of ribosomal initiation complex at upstream ORFs impeding the progress of other to the AUG of the main ORF
Change of mRNA secondary structure by the binding of the ribosomal initiation complex at upstream ORFs increasing the difficulty of the initiation step in which the 5′ end is ’melted‘.

It also appears that the extent of the translation of upstream ORFs — and hence the extent of the repression — can itself be regulated by mechanisms such as the phosphorylation of initiation factor eIF2.

Seems odd that translation occurs in the so called untranslated region.

@canadianer — History. I didn't want to go into it in any more depth, but the initial assumption back in the 70s and 80s was that because mRNA was monocistronic (e.g. beta-globin) there was only one translated reading frame. It is obviously necessary to be able to name features of something one is discussing, and the regions of the mRNA 5' and 3' of the region coding for the protein were of interest — e.g. did they have any function regions, describing where the initiation complex started etc. At the time "untranslated region" (UTR) seemed appropriate. Knowledge changes, names stick.

Of course; just seemed amusing.

@David Thank you very much for your insight. I am new to the subject and have no background so this was really helpful.

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Why do antibiotics have a special importance when taking lactulose?
“Before taking lactulose, tell your doctor and pharmacist what prescription and nonprescription medications you are taking, especially antacids, antibiotics including neomycin (Mycifradin), and other laxatives.” (Quote from here)
Why do antibiotics have a special importance when taking lactulose?
Why is neomycin (Mycifradin) mentioned separately?

@winerd-> Sorry, you're correct. Still think you should have merged the questions though, since you are quoting the same passage and asking the same general question on two substances (feels more like a 1a, 1b question than two separate). The same answerer can most likely answer both at once.

@fileunderwater: No problem, thanks. Let's see if the answers will be similar or not. (I thought they would talk about different mechanisms, but indeed I could be wrong.)

From the Wikipedia article (I'm sure there's a scholarly reference, but I think this is well-known):

Lactulose works by relieving constipation through a number of different methods. Lactulose is made up of sugar molecules and is partly broken down by the bacteria that live in the lower part of the gut.

So, certain antibiotics affect (and reduce) the intestinal flora (and neomycin especially so, which is I believe the reason why it is mainly used in ointments), leaving the lactulose undigested and not as effective in forcing the body to retain water in the colon, which is the mechanism through which the drug acts.

Lactulose is an osmotic laxative. This means it works by drawing water into the colon (i.e. it retains a lot of water with it) and hence softens your stool in cases of constipation. As you can imagine, this interferes with absorption of most drugs including antibiotics and even food. Neomycin is especially toxic to gut flora (as mentioned above) and if taken with lactulose, it will linger in your GI tract for even longer and kill more gut flora. Your gut flora is important in protecting you against other harmful bacteria through competition for food, nutrients, etc. If you kill your flora, you are allowing other harmful organisms a field day in your intestine and they will grow and cause a condition known as pseudomembranous colitis (commonly caused by clostridium difficile infection). Also neomycin is mentioned separately because it has been associated with this condition which can sometimes be fatal (toxic megacolon, GI perforation, severe diarrhoea, etc)
In summary, you shouldn't be taking other medication whilst you're constipated anyway (especially when taking lactulose) because they won't get absorbed and may make the constipation worse.

Is there any evidence that lactulose decreases absorption thereby leading to an increased intestinal concentration?

"Neomycin ... if taken with lactulose, it will linger in your GI tract for even longer" - Are you sure about this? I thought that any laxative would REDUCE the time any substance spends in the colon.

@winerd but within the context of constipation, anything you take will linger in your GI tract for much longer. In theory, a laxative should increase your transit time but bearing in mind that lactulose interferes with absorption, this might be the case.

@AndroidPenguin, this just makes sense to me-just like having a diarrhoea which also interferes with absorption. Most laxatives will eventually cause diarrhoea which will interfere with absorption of other nutrients.

@John: I find it counter-intuitive that a laxative would increase transit time. Furthermore here it's stated for lactulose specifically and for many other laxatives that they "can cause SHORTER gastro intestinal transit time". Can you support your statement?

@winerd I meant it reduces transit time-you're right.


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What is the disadvantage of having no anticoagulation after pulmonary embolism?
Is chronic anticoagulation always given after pulmonary embolism?
What are the risks or disadvantages

if anticoagulation is not given?
if terminated early?

Not always, however typically it is given for at least a few months. Lack of anticoagulation therapy would greatly heighten the risk of having another PE or related cardiovascular events. At the worst side this could cause death or permanent disability, and PE related deaths are quite common. However this depends on the nature of the PE and risk factors. For example, obesity, cancer and the pill are all risk factors and if these were reduced then it is likely the person wouldn't have another PE despite not being on long term anticoagulation therapy. However, if there is an underlying cause even despite anticoagulation therapy a person may suffer a PE.
The problem with anticoagulation therapy is of course it stops your blood clotting. Thus haemorrhages are more common and also more severe. However in an individual receiving anticoagulation therapy, they typically have increased clotting so this may be balanced out by therapy.
Finally anticoagulation therapy is typically warfarin in the long term. Warfarin interacts with so many drugs including even antibiotics, it is teratogenic and quite sensitive to diet (any changes in Vitamin K will directly affect the effectivity of warfarin) thus frequent monitoring is required. This is annoying for the patient as they have to report for frequent blood tests as well as watch their diet etc.

It should be noted that the teratogenicity should not be too great of an issue as those trying to conceive can be switched to low molecular weight heparin

I summarize that specifically addresses my questions: Anticoagulation is typically given. Not giving anticoagulation risks another PE or related cardiovascular events, in the worst (not rare) case leading to death or permanent disability. -- Would the second PE be somehow triggered by the first one? Or by the same factors triggering the first one?

The latter, same triggers


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Help with designing the analysis of radio telemetry study
I was wondering if there are any spatial ecologists out there who can provide some insight on the trouble I'm having with my Masters research.
I am looking at the association a bat species to high density aggregations of a flowering plant. The bats were tracked throughout the night with a point taken at approximately 10 minute intervals. The bats were tracked over consecutive nights until an asymptote in their space use was reached. So now I have point patterns for each bat that pretty much covers the entirety of their space use at that period.
When the data is displayed visually there is a clear pattern of the bats that were tracked during the flowering period moving into the aggregations of the plant however they also spend a lot of time foraging around their roost sites.
I have simulated the point pattern of each bat 999 times by randomly shifting and rotating the points within the study window (the internal structure of the observed pattern was maintained in the simulations to account for autocorrelation). I then did randomisation tests to see if the average distance to the nearest plant aggregation for the observed data is within the lower 5% of the distribution of the averages for the simulated point patterns. However because the majority of the points collected are highly associated with the roosts the average distance is primarily effected by the position of the roost in relation to any of the plant aggregations.
I've been thinking of ways to disentangle the association with roosts and the association with the plant.
First I was thought about putting a buffer around the roosts and removing the points falling within these buffers from the analysis. The problem is deciding on a buffer. In the other areas I have been using a buffer equal to the circular error probable of the radio tracking error but placing this around the roost will only incorporate the points where the bat was actually inside the roost not foraging around it. All the bats have different foraging patterns around their roosts and I can't see how to assign a buffer distance that is not completely arbitrary.
The second method we have been looking into is taking out the points that are more closely associated (closer in distance) with the roost than the nearest plant aggregation and then running the randomisation to look at the association of the remaining points to the plant. I want to get an idea of whether or not removing the association with the roosts in this way will potentially bias the analysis.
Any thoughts or further suggestions?!
Thanks in advance!

Very interesting question. However, I would suggest to migrate this to CrossValidated, as probably the folks there will give more appropriate answers.

Yes, but I think the idea of comparing the data without plants with data that include plants is the right idea. Have you only measurements where plants are in the vicinity? Then only a realistic simulation of movement in the absence of plants can be a good model for comparison. But maybe someone else has already created a good simulation?

@ rwst Unfortunately there has been very little spatial work done on these bats. We tracked four bats before the plant came into flower an four during. That's probably not a large enough sample size to make any serious conclusions with so that's why I went down the road of simulating the bat locations but I'll look into it thanks!

@Georgia: I think the moderators can do that directly. I will flag for their attention.

@Georgia has already posted this herself to CrossValidated so I'll close it here


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(how) is it possible to choose the biological sex of a child?
I recently read of a study that investigated the ethics of child's biological sex choice in humans, and now I want to find out how this can/could be done. Does anyone know of scientific papers or journals that describe the methods in some detail? The answers I was finding in my own search were not as specific as I would like.

Do you mean sex selection? Keep in mind that gender and sex are not the same thing. If you mean gender then I think that this question is more suited to the cognitive sciences SE.

Yes you are right, I mean sex selection.

I mean that there is research involving giving couples the ability to choose whether their baby will be male or female.

The article you read had to do with ethics. So are you interested in ethics or in science? Are you interested in the methods that offer couples the opportunity to choose the sex of their baby or are you interest in how some species manage to chose the sex of their baby (which is particularly the case when there is environmental sex determination (ESD)). This wikipedia article might be a good start.

I read an article about ethics, but I wanted to learn more about the science behind giving humans the option to choose. I don't really care about the ethics at this moment, I'm more concerned with how it is done. I did some searches on Google, but I was getting generalized, nonspecific answers. I would prefer to find something from a journal or research paper that actually talks about the methods, success rate, and why it works (or why not).

Ok, so you are looking for a review (in a peer reviewed journal or not I suppose) of the methodologies that can be used for parents to chose the sex of their offspring, is that right?. You maybe want to make clear in your question that you are looking for a list of methodologies. The question is interesting (I am totally ignorant in the subject). I just wonder whether wikipedia is not already a very good source of information given how broad is your interest.

This question is too broad. Can you add some details

I looked at the wikipedia article you suggested; it does provide some detail, but I do still have more questions. I'll check the references there later in case those contain further detail. I did not realize wikipedia would contain that much information. Thank you for your help.

The main technique is sperm separation or sorting, where you separate sperm by gender, and only use the gender that you want. Existing techniques aren't perfect. AFAIK, they are typically less than 90% accurate, but it is still better than 50%. Typically, this involves some type of cell sorting device often with some kind of fluorescent tag marker.
The second technique is embryo screening. Often, with IVF, you screen and can confirm the gender that you want before implanting the embryo in the mother.
Links:
http://www.fertility-docs.com/programs-and-services/gender-selection/select-the-gender-of-your-baby-using-pgd.php
http://www.in-gender.com/Gender-Selection/MicroSort/


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Expression of eukaryotic ribosomal DNA
The Wikipedia page for rDNA says "Ribosomal DNA (rDNA) is a DNA sequence that codes for ribosomal RNA" Also, the figure next to it says "The gene segment of eukaryotic rDNA contains 18S, 5.8S, and 28S tracts and forms a tandem repetitive cluster". So, it gave me the impression that rDNA codes for both rRNA and the structural proteins of ribosome. Is that right?

Wrong. Read about ribosomal protein genes.

Thanks David! Could you tell me, what exactly is meant by "The gene segment of eukaryotic rDNA contains 18S, 5.8S, and 28S tracts and forms a tandem repetitive cluster; the 5S rDNA is coded separately. NTS, nontranscribed spacer, ETS, external transcribed spacer, ITS, internal transcribed spacers 1 and 2, numbered from 5' end."  in the picture (https://en.wikipedia.org/wiki/Ribosomal_DNA#/media/File:Eucaryot_rdna.png)

As well as messenger RNA (mRNA) that encodes protein, there are a variety of RNAs that have structural or regulatory roles and do not code for protein. Ribosomal RNAs (rRNA) are one such structural RNA. Like mRNA, rRNA (and other stable RNAs) are  specified by genes in the chromosomal DNA, although the details of their transcription (the RNA polymerase employed and the nature of the promotors) may vary.
The mature eukaryotic ribosome contains three species of rRNA that are analogous to those in prokaryotes — 18S (cf. 16S), 28S (cf. 23S) and 5S (sizes for mammals). However there is an additional 5.8S species in eukaryotes. The questions addressed in the article are: 1. What genes and what transcripts give rise to these mature RNA species? and 2. Is there a single gene for each species or multiple repeated genes? A third, implicit, question is whether the situation in eukaryotes is similar to that in eukaryotes.
The answer to question 2 is given in the phrase “forms a tandem repetitive cluster” — there are multiple copies of the genes for 18S, 28S and 5.8S rRNA, clustered together one after another. These genes are separated (‘spaced apart’) by regions of the genome that are not transcribed, and are termed non-transcribed spacers (NTS).
(There are also multiple copies of the 5S rRNA gene, although these are only clustered in some species.)
The answer to the first question is that the 18S, 28S and 5.8S rRNA are transcribed from a single gene and are initially present in a single transcript. This is processed by ribonucleases to generate the three mature species. If one compares the sequences of the mature species with that of the gene/transcript one can identify portions of the initial rRNA transcript that are not present in any of the three mature species. These are termed transcribed spacers. The one at the 5′ end of the transcript is termed the external transcribed spacer (ETS), and those that are between the three mature species are termed  internal transcribed spacer (ITS).

The situation in prokaryotes differs from eukaryotes in so far as the 5S rRNA gene is part of the gene/transcript containing the 16S and 23S rRNAs, lying at the 3′ end of this. This is mentioned in the Wikipedia entry for Ribosomal RNA, rather than that for ribosomal DNA, quoted in the question, and illustrated in the figure below taken from the teaching web pages of Ross Hardison at Penn State University. Notice the transfer RNA gene in the transcript between the portions resulting in the mature 16S and 23S rRNAs.

Thank Dav! You have been very helpful to me. Thank you so much!

"rRNA are synthesized in a different pathway. In the nucleolus, the rRNAs combine with ribosomal proteins to form the large and small subunits of the ribosome. Ribosomal proteins are synthesized in the cytoplasm and transported to the nucleus for subassembly in the nucleolus. They autoassemble in a long complicated maturation process which ends in the cytoplasm (ER)". https://www.nature.com/articles/35030006 says there's more rRNA than protein in the ribosome, so there are a lot of copies of those few transcripts. Did they succeed in producing ribosomes in-vitro from the separated ingredients?

@reuns — As I understand it you are asking either one or two supplementary questions. In answer to the question in your final sentence. Nomura reconstituted bacterial ribosomes in 1968 (if I remember rightly) and showed that resistance (or sensitivity — I forget) to streptomycin was a property of one specific protein (S12). People I knew were trying unsuccessfully to do the same thing for eukaryotes in the early 70s, but I imagine (and seem to remember) that was subsequently achieved. As assembly in vivo occurs on the rRNA precursors, the reassembly in vitro is somewhat artificial.

Tks, also I realize what I said was wrong (for prokaryote) the 30s subunit contains only one single stranded rRNA (secondary structures make it locally double stranded) of 1500 nucleotides each containing about 20 non-hydrogen atoms gives the 30000 rRNA atoms mentioned in the nature article. Thus (for the small subunit) it is about assembling 1 rRNA and 21 proteins which is doable and has been achieved since 40 years. This explains why the map of the 30s is described in term of 5' central and 3' parts


text:
Relative abundance of transcription factors and protein kinases
Are transcription factors and protein kinases only expressed at low levels in eukaryotes?
As regulatory proteins, I would expect their abundance to be lower than most other proteins, but I cannot find any published research to support that.

@l0110 Replacing makes the question "Are transcription factors..."

You are grouping genes by overall function "important cellular regulators" but asking a question about a putative shared characteristic "expressed at low levels" for genes which act via very different mechanisms. I would think looking into their mechanisms would help answer your question.

Summary

The abundance of protein kinases and transcription factors vary among and between these broad classes of protein.
General Considerations
Let us first think what might be expected for the abundance of transcription factors and protein kinases.
Protein kinases: These would be expected to be less abundant than their substrate proteins as they are enzymes catalysing a reaction. However some protein kinases are part of amplification cascades (e.g. MAP kinases) so the abundance of individual member would be expected to differ.
Transcription factors: Transcription factor is a portmanteau term that includes both proteins that act generally on promoters (e.g. the TATA-binding proteins TFIIA, TFIIB etc.) and ones that are specific for the promoters of particular genes or classes of genes (e.g. heat shock factor). The abundance of transcription factors might be expected to be inversely proportional to their specificity.

Correlation between Protein Kinases and Transcription Factors: I can see no reason to group these two classes together as the regulation of metabolic activity and gene expression are very different processes. All I would expect is that they would to less abundant than structural proteins such as actin or ribosomal proteins (although differing in abundance among themselves).
One Possible Approach

To the man with a hammer, everything looks like a nail. My particular hammer may not be the best tool for this particular nail, and there are certainly other tools available which other answers may mention, but it’s something you can do at home (without even adult supervision). The hammer is called FlyAtlas 2 and can be accessed at http://flyatlas.gla.ac.uk/FlyAtlas2/index.html. It allows one to examine the extent of expression of different genes in the tissues of the fruit fly, Drosophila melanogaster.
Some Words of Caution

Before looking at experimental data, one should be aware that the abundance of enzymes will vary depending on the particular tissue examined and the physiological conditions. One should also be aware that the data are for abundance of mRNA transcripts, which may not always be proportional to the amount of the encoded protein. Further, although the resource has been published in a peer-reviewed journal, as is normal in science, the referees have not repeated the experiments.
How to Proceed

Connect to http://flyatlas.gla.ac.uk/FlyAtlas2/index.html
There are two ways of proceeding here. One can go to the Category page, type in a term in either as a Category or Free Search, and select from the menu, as shown.
Alternatively go to the Gene page, click (or tap) on Gene Name and choose from the autocomplete. This gives fewer results than 2, but they tend to be grouped in a way that makes it easier to make repeated searches.
Click Search and tick Whole Body in the results (which may be downloaded in a spreadsheet-friendly format). An example is shown.

Results

A quick application of this approach shows that:

Protein kinases are expressed in whole flies in a ten-fold range, from just detectable (e.g. Cyclin-dependent kinase 2) to 20–40 FPKMs (protein kinase A, calmodulin-dependent protein kinase).

Some protein kinases are highly expressed, but only in specific tissues (e.g. Erk7 in testis).

Transcription factors are expressed in whole flies in a range from just detectable (e.g. Transcription factor AP-2) to about 10 FPKM (TF IIB, heat shock factor)

Some Transcription factors are tissue-specific (e.g. TF B4 in ovary and testis).

Comparative values of cellular enzymes are glycogen phosphorylase (c. 100), phosphofructokinase (c. 50), enolase (c. 200).

Thanks David. I usually use R or Python for these analysis but it is nice to know there is a website to do it. My own analyses showed these two groups of genes are expressed at low levels for mRNA and protein when comparing to other groups of genes in plants. Since I want to know if there is a reference there (that was the original question), I up-voted your answer but not accept it. But thanks again for your time and effort!

@l0110 The website is, like others of its type, a front end to a relational database, providing the general user with the ability to make a restricted range of queries. It also provides APIs for the programmer to make queries in whatever language he prefers (see online Docs). Although perhaps not of interest to you as a plant scientist, queries using the recent categorization of fly genes into classes on FlyBase might be a better way to approach the question. I imagine there are similar resources for Arabidopsis. Btw. I have deleted my comments on your question now.


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How valid are Koestler’s criticism of evolutionary theory?
I recently read Arthur Koestler's 1967 book The Ghost in the Machine. In it, Koestler criticises the neo-Darwinian theory of evolution—beneficial random mutations preserved by natural seleciton—as insufficient to explain the formation of complex forms like eyes and eggs. The issues Koestler has with the theory are ones that I've been trying to wrap my head around since before I read the book, but I'm aware that:
a) the book is half a century old;
b) Koestler was not a biologist or scientist; and
c) neo-Darwinian theory/the modern synthesis seems to have stood the test of time
so I'm wondering how accurate Koestler's account of the theory is, and if he is wrong what the retorts to his claims are. Here are two examples he gives of complex forms:

[The giant panda] has on its forelimbs an added sixth finger, which comes in very ‘handy’ for manipulating the bamboo-shoots which are its principal food [but] that added finger would be a useless appendage without the proper muscles and nerves [and t]he chances that among all possible mutations those which produced the additional bones, muscles and nerves should have occurred independently are of course infinitesimally small.

and

The decisive novelty of the reptiles was that, unlike amphibians, they laid their eggs on dry land...[b]ut the unborn reptile inside the egg still needed an aquatic environment...[i]t also needed a lot of food...[s]o the reptilian egg had to be provided with a large mass of yolk for food, and also with albumen—the white of egg—to provide the water. Neither the yolk by itself, nor the egg-white itself, would have had any selective value...[e]ach change, taken in isolation, would be harmful, and work against survival.

Instead of random mutations and external selection, he suggests that ‘internal selection’ works at all levels, from chemical upwards, to correct ‘misprints’ long before the developed organism is exposed to any sort of external selection. The implication that there must therefore be some plan towards which embryonic development works is supported by two examples:

the growing eye-bud of the embryo is an autonomous holon, which, if part of its tissue is taken away, will nevertheless develop into a normal eye

and

[the fruit fly has a recessive gene that when paired with another in a fertilised egg will produce an eyeless fly.] If now a pure stock of eyeless flies is made to inbreed, then the whole stock will have only the ‘eyeless’ mutant gene, because no normal gene can enter the stock to bring light into their darkness...within a few generations, flies appear in the inbred ‘eyeless’ stock with eyes that are perfectly normal.

His other main point is that evolution takes a zig-zag path, evolving down until reaching an evolutionary dead-ends before retracting to ‘an earlier or more primitive, but also more plastic and less committed stage—followed by a sudden advance in a new direction’. For example:

[A]mphibian...ancestry...goes back to the most primitive type of lung-breathing fish; whereas the apparently more successful later lines of highly specialised gill-breathing fishes all came to a dead end.

and

...the human adult resembles more the embryo of an ape than an adult ape

Is Koestler's science just faulty, or are these valid criticisms that've been resolved since?

I am entirely unfamiliar with Koestler, but based on the excerpts you posted it sounds like he just making assertions to justify his own hypothesis. Does he make reference to any actual experiments?

Regarding the panda example: polydactyly occurs in many mammals often as the result of a single mutation. This example is commonly used in teaching developmental biology as it shows the effects that single mutations can have on tissue patterning during embryogenesis. You can read the Wikipedia article for a primer on this. If such a mutation happens to have a selective benefit for pandas, it is entirely plausible that it could take hold in a population.

Regarding the reptile example: I don't have any specific information on this, but evolutionary theory does not say that deleterious mutations cannot become established in a population (assuming for the sake of argument the mutations leading to reptilian eggs were in fact deleterious). It depends on the strength of selection against them, among other factors such as genetic hitchhiking or heterozygote advantage.

@canadianer For the zig-zag theory of evolutionary progress he cites Garstang, for the embryonic tissue reformation Weiss.

Regarding the fruit fly example: It is difficult to comment without knowing the details of the actual experiment. A well studied gene in fruit flies is called eyeless (actually the Pax6 transcription factor with homologs in all sighted animals studied) because mutations to it can prevent eye development. In such mutants, it is hypothetically possible for back mutations to restore the original phenotype (I'm not aware if this has actually been observed, but I'm not aware of a lot of literature on fruit flies).

Maybe both. Certainly his understanding of limb developement doesn't match modern understanding. The quote you provide seems to indicate that he thought the appearance of an additional digit would require a multitude of coordinated mutations. Our current understanding seems to indicate that it's all about changes in the regulation of genes. For example in fruit flies mutations in the antennapedia gene can cause antenna to grow where legs should be or visa versa. Another example is polydactyly(extra toes) in cats which be be caused by mutations to a single regulatory region.

within a few generations, flies appear in the inbred ‘eyeless’ stock
  with eyes that are perfectly normal.

I'm not a fruit fly expert, but this seems highly dubious to me. There is a well studied eyeless mutation, but it's dominant, not recessive. Furthermore getting two copies of this mutant eyeless gene is lethal at the embryonic stage. This means that a colony of adult eyeless flys are necessarily all heterozygous. That is, they have one copy of the mutated eyeless gene, and one copy of the normal eyeless gene. By Mendel's laws, a quarter of the offspring from the completely eyeless generation are going to have two copies of the normal eyeless gene, and so will have normal eyes. I wouldn't be surprised if the flies with normal eyes outcompete the flies without eyes, so eventually the eyeless flies will disappear from the colony. I can't tell you whether Koestler has mis-characterized this gene, or is talking about some other gene all together. Citation needed.

Koestler's entire argument can be summed up is an argument of irreducible complexity, which always stems from a lack of understanding of the biological systems. All his arguments stem from a lack of knowledge about the things he is describing. Lets handle them in order.

Panda thumb. lack of understanding. The panda thumbs does not move, it is just a spur of bone. but becasue it is a wrist bone it still has muscles on it so the argument is wrong in two ways.

Reptile eggs. Amphibian eggs have yolks and albumen. so this again is just a lack of knowledge.

Internal selection is again based on a lack of knowledge, the "plan" embryonic development works from are called HOX genes, which regulate development. And there are  organisms without them and indeed behave just like you would expect. Eyeless fruit flies do not lack the genes to make eyes they have a mutation in the HOX genes that trigger eye development, (lake a "make eye here" on a blueprint) and just as single mutation can prevent the gene from functioning, another can make it functional again, we can can even trigger eye development in the wrong place, say on the legs.

Is just confusing, It sounds like he thinks there needs to be some overarching plan to evolution instead of the chaotic whatever works mess that actually happens. In which case he does not even understand what evolution is, so his arguments against it make even less sense. I certainly can't imagine why he would claim teleost fish are dead ends when they are the most numerous vertebrate on the planet.

Time — i.e. the advances in our knowledge of molecular genetics — has clearly disposed of Koestler’s arguments, as it has of many of those of people who cannot see the wood for the apparently diseased tree.
Other answers provide chapter and verse, but what I wish to contribute in this answer is something about the intellectual bias that influenced Koestler’s views. Most of us will be aware that many anti-evolutionary arguments are made from religious conviction, rather than from disinterested scientific rationalism, but the poster and the younger members of this list may not be aware of a different kind of intellectual bias.
Although Koestler is most famous for his book “Darkness at Noon” in which he attacked the evils of Stalinism, after which he rejected the Communist party, this did not change the (in many respects admirable¶) socialist principles that had lead to his joining the party in the first place.
So, what does this imply? It does not imply a rejection by Koestler of Darwin’s theory of evolution, but a rejection of the idea of a genetic mechanism in which man is subject to the dice of random mutation. He still clings to a doctrinal belief in Lamarckism in which man evolves by the incorporation of characteristics acquired by his own efforts†. It is ironic that despite repudiating Stalin, he hung on to Stalin’s dogmatism that led to Lysenko and the consequent murder of Soviet geneticists as well as the death by starvation of untold thousands of Russians.
And the moral for today? There are several. But it is politic to restrict myself to suggesting one should question the motives of those who cry “epigenetics” when they haven’t the first idea of what they are talking about.

¶ In my opinion. (Not particularly relevant, but to show I am not criticizing him from a right-wing standpoint.)
† The actual context at the time he wrote it was different — a rejection of social Darwinism. I personally regard the latter as having nothing to do with genetics or biology, but in order to repudiate the one, he seems to have returned to his repudiation of neo-Darwinism.


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Would it be possible to create a gel, holding viruses, that could alter the human genome when rubbed in?
I was wondering if it could be possible to engineer a virus, that when it comes into contact with human hair follicles, it could alter someones natural hair color.
Naturally blue hair anyone?
Doubtless there are loads of problems, but i was hoping they could be identified and potentially worked around.

You might take a look at Topical Gene Delivery on pubmed, but this also has papers that deliver to the eye, and any in vivo gene delivery is really hard right now. Also, blue pigment is not something you could easily make biologically. Blue birds use light interference to get their color.

So, i would need to somehow engineer biological microstructures, in order to reflect only a blue wavelength?                                                                          And when you speak of birds, you refer to plumage coloration, while i'm asking about hair specifically. There are some mammalian species that have blue or bluish hair. I'm wondering how to adapt that to a human.

Mamalian species with dye colored blue hair? I doubt that. This is probably some light effect.

Cats and rats can have a color that is classified as "blue", but it really looks more grey. Behold: http://messybeast.com/colour-charts.htm


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Why is a gene for a fluorescent protein used rather than one for an enzyme that produces fluorescent molecule?
Green fluorescent protein (GFP) is a small protein that emits bright green fluorescence in blue light. It was first isolated from the jellyfish.
The gene coding for GFP can be expressed in bacteria, and so it is often used as a marker to show successful uptake of a gene by the bacterium.
What is the advantage of using the gene for GFP to produce easily detectable fluorescence, rather than using a gene for an enzyme that produces a fluorescent substance?
Ps. The original question I got was to explain about the disadvantage, which is that GFP may not fluoresce very brightly and so may be difficult to detect.
Thank you for your help.

Lemme ask you same thing, in reverse: what is the advantage of using a gene for an enzyme that produces a fluorescent substrate, rather than using the gene for GFP to produce easily detectable fluorescence? If you're getting something done directly, why do the same thing indirectly?

Possibly because some people discovered how to induce GFP before anyone discovered an enzyme route?  If indeed anyone has done that.

If you used a substrate you would have to get it into the sample. This would add variability in terms of penetration, reaction time and diffusion of the reaction product away from the site of interest. Why bother? Just image the GFP directly with none of this hassle.

GFP has a lot of advantages:

The detection of the fluorescence works directly and doesn't need a lysis step or the uptake of a reagent.
Fluorescence can be detected directly using a fluorescence or confokal microscope with the cells. This can be done while growing them on plates (works for bacteria and eukaryotic cell culture).
You can sort labelled cells using a flow cytometer.
direct tagging of proteins and their subsequent localisation on the cell is possible.
GFP is rather stable in the cells and not toxic.

To add to this: GFP will not diffuse out of the cells (or even out of a vesicle) and you can engineer cells to encode your protein of interest with GFP attached. You can even add all sorts of localization tags to GFP and observe where the tag leads your GFP.

There are no genes that I know of that encode proteins, more specifically enzymes, that will catalysed the production of a fluorescent compound from intermediates present in the average cell. Presumably one would need a whole pathway.
So I suspect that the question has never really arisen.

IIRC, pseudomonas fluorescens produces a carbon-scaffold molecule around iron ions which produces a fluorescent small molecule. But that would probably require expressing multiple enzymes


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Emergence of novel protein interactions by mutation of amino acids
Are there any examples of proteins that, without coming from a recent duplication event, underwent a mutation(s) that caused it to have a novel interaction with a new ligand, substrate, other protein or molecule? By novel I imply that it doesn’t revert to an ancestral interaction or develop an interaction that is already performed by a close relative. I imagine that such mutations would be extremely rare, and that duplication is almost always necessary to have them fixed in a lineage.

Just to make sure I understand what you're saying... You're asking for an example of a protein which changed functions throughout generations without leaving a trace of the original protein or a paralog?

I edited your question a little to remove some typos, but also edited your title. "Genuine" isn't required as you explain this in your quesion, and best to keep it short, but avoid abbreviations like aa — need to spell in full for indexing.

Thank you. I meant a protein that acquired new interacting partners (ligands, proteins, etc) without being duplicated. Because I’m already aware of evidence of proteins acquiring new interacting partners by being first duplicated and while one of the copies maintained the original interactions the other acquired the new ones.

@Marlo, I have restructured my answer to address your question more directly and focus specifically on the AB polymorphism which does not involve gene duplication. Does this fit the bill? I don't quite understand the import of "an interaction that is already performed by a close relative". If the protein is distinct and original mutations occur in it independently, what relevence do the interactions performed by other proteins have? Is there some philosophical or other concern underlying your question? (Please include the @ handle in any reply — that way I'll be alerted.)

I must say that I am disappointed that you have not responded to my last comment. You appeared to be keen to have an answer as I understand you also posted to SE Bioinformatics, and you can hardly deny that I took your question very seriously and have supplied an extensive, detailed and fully documented answer. I see that you have been a member of the SE community for over two years, so I would expect you to realize that it would be courteous to let me know whether or not the answer is what you require.

Sorry, the reason is that just after reading your answer i started reading your other answer and from there a chain of articles regarding the topic, some theoretical and some experimental, and i still don't reach a conclusion if it's a satisfying answer or if i can formulate specific counterpoints to your post, i am still very much in the process of this. What i do recognize is that i find your answer more detailed than i expected and for this you have all my gratitude. Sorry that i wasn't courteous enough to provide at least one comment saying this.

The polymorphism in the gene product of the human ABO locus seems
  to provide an example of what the question demands. It does not
  involve a gene duplication or recovery from loss of function; rather it involves changes that cause
  interactions with a different ligand, and the mutations that produce
  these changes have been identified and examined at a molecular level.

I have described the biochemistry of the human blood group system in an answer I gave to a previous question, so the cartoons of the reactions are reproduced from that answer. The diagrams of the molecular interactions and the gene cluster are new.
Human AB polymorphism
The products of the A and B alleles of the human ABO locus are glycosyltransferases:

One of the enzymes recognizes the sugar, galactose (activated by UTP), whereas the other recognizes N-acetylgalactosamine. There is no doubt that they are the products of different alleles of the same gene, even though this is a member of a gene cluster (see below).
The structures of both these enzymes have been determined and the differences of the interaction with their substrates at the active site modelled as shown below:

There are four residues that differ critically between the two enzymes, three of which are shown. Two of these (Met/Leu 266 and Ala/Gly 268) make contact with the UDP-glucosyl substrates. The third (Ser/Gly 235) appears to affect the conformation of the H-antigen co-substrate.
How is a change in function possible without gene duplication?
The poster expresses the opinion (my rewording):

“I imagine that such mutations would be extremely rare, and that
  duplication is almost always necessary to have them fixed in a
  lineage.”

Why, then, is gene duplication not necessary in this case? The reason is that the enzymes involved are not indispensable: the product is an elaborated branched cell-surface polysaccharide, the function of which can be served by a range of variants. Indeed, the polysaccharide is functional without elaboration, as in the case of blood group ‘O’, where the enzyme is inactive. However, as discussed in my answer to the previous question, there may be a ‘hidden’ function that has driven the evolution of the polymorphism in humans. It has been suggested that the blood cell surface antigens were used by parasites such as the malarial parasite to enter the cells, and that variation provide resistance (albeit temporary, i.e. until the parasite evolved in response).
Footnote: Gene Clusters of Glucosylating enzymes
The question specifically asks for examples that do not come from a recent gene duplication. However I feel it important to mention the gene duplications that have produced such changes in the gene cluster to which the ABO locus belongs. This is to make it clear that the polymorphism is distinct from such examples.
Animals have the ability to vary the sugars on their cells by selecting from a large repertoire of genes with the potential to specify different glycosylating enzymes. An example is the GT6 gene family (glycosyltransferase family 6):

There are about seven members of this family, not all of which are present in every species. A paper by Turcot-Dubois et al. gives a detailed comparison of the genes in human, rat, toad and zebrafish. I just reproduce the gene map for the human cluster in which the four members of the family are indicated by solid triangles:

The enzymes specified by some of these gene have been characterized by X-ray crystallography, e.g. in this paper by Gastinel et al..


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What is the name of these beautiful white trees?
I found these beautiful white trees in an unknown video.

If you do a Google Image search with the picture in the question, you'll see the screenshot is from the 2000 movie The Patriot starring Mel Gibson. The movie's filming locations were mainly scattered around South Carolina, on the southern East Coast of the United States. I've spent a lot of time in the region, and the gray-ish fluffy stuff hanging off the branches is almost certainly Spanish moss (Tillandsia usneoides), which is very common in the area.

While it is more difficult to identify the tree, as the bark and leaves are not clearly visible up close, a good educated guess based on the size and shape of the tree would be live oak (Quercus virginiana), which also happens to be one of the primary hosts of Spanish moss.

Plot twist. I was on set. The Spanish moss on the large tree in front of Ben Martin’s (Mel Gibson) house was hung by hand using a cherry picker. Spanish moss is found closer to the coast. This scene was shot in the Rock Hill area of South Carolina