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Genetic Imprinting and Cell differentiation
It does not seem possible that these two processes can coexist:
1) Genetic imprinting is the phenomenon where genes are expressed differently depending on the parent of origin:
1a. Methylated stretches of DNA are not transcribed.
1b. If the gene copy originating with mom is methylated but dad's copy is not, then only dad's copy will be expressed (e.g. Prader Willi syndrome).
1c. Methylation is preserved during cell divisions.
1d. Methylation is wiped at gametogenesis, when females will erase dad's imprints and re-imprint according to maternal imprint before meiosis.
But now I'm reading about the role of epigenetics in cell differentiation, and I discover:
2) Cell differentiation occurs with lineage-specific patterns of methylation.
2a. Immediately after fertilization (prior to the first cell division), the paternal genome undergoes demethylation.
2b. The maternal genome undergoes demethylation during the first few cell divisions.
2c. Cell differentiation is accompanied and perhaps even accomplished by progressive re-methylation following these "wipes."
Source for 2): http://labs.genetics.ucla.edu/fan/papers/HuangK_RM2010.pdf
"DNA methylation in cell differentiation and reprogramming: an emerging systematic view" Huang & Fan (2010). Regen Med. 5(4):531-44.
I suspect that there is no conflict and I merely misunderstand one, the other, or both. Otherwise, how can a pattern of methylation be wiped both during gametogenesis and early embryonic stages and still be inherited?

What if imprinted regions are immune to being wiped? Also, you may be confusing DNA methylation, and histone methylation (?). Classical biochemistry posits that DNA methylation states can be transmitted from a dividing mother cell to both daughter cells because after DNA replication the two daughter chromosomes will each be hemi-methylated, and a DNA methylase that finds a hemi-methylated site will methylate the other strand (like a corrective editing mechanism).
The third thing to consider in your question is that parental imprinting is established in the germ line. In terms of wiping other epigenetic marks during early embryogenesis, the only ones present would be the ones involved in gametogenesis.  In other words, as far as we know, genes activated in muscle development are never expressed after fertilization in the zygotic cells that will give rise to the primordial germ cells, so those muscle-specific epigenetic marks don't need to be wiped off in the embryo. Neither the sperm nor the egg ever expressed muscle myosin, etc.

Probably, there are narrow and wide definitions for imprinting. I am guessing the history of researches about imprinting would be like the followings.
Initially, maternal or paternal phenotype transmissions were recognized and some of such phenotype seemed because the maternal or paternal responsible gene does not work. As you know this is clearly imprinting.
Then, DNA methylation was discovered and turned out to be responsible for inactivation of imprinting genes defined above. However DNA methylation and repression of the expression by methylation occur to other genes, too. ES cells have distinct a DNA methylation pattern, which can probably explain the distinct gene expression pattern at least partially, I think. In addition, suppression of some tumor suppressor genes by methylation occurs during the course of carcinogenesis. It looks like DNA methylation regulates gene expressions as well as imprinting defined above. I know people call all gene regulations by DNA methylation imprinting. This is the wide definition.


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Why do urine have a chemical smell after drinking a lot of alcohol (red wine)?
When I've been drinking a lot of red wine my urine starts to have a chemical smell. How do that urine differ from normal urine?
Is that chemical smell a sign of liver issues? When I was younger I don't remember my urine having that smell after or during drinking lots of alcohol.

Welcome to Bio.SE! What attempts to answer this question have you already taken? We ask that all question posters here attempt to search for an answer to their own question and explicitly indicate what research they've already done, what they learned, and what is still confusing or unknown to them. Our goal is not to simply be an answer site, but rather a site that promotes self-learning with some expert help along the way :). Please take a moment to edit your post with this additional detail, and it will likely be received more positively by our community. Thanks!

Also, personal medical questions are considered off-topic on this site. You should edit your question to be more broadly applicable and less about a personal medical experience. Otherwise, our community will likely vote to close the question. Please [edit] accordingly. Thanks!

Its a very specific question and those are simply not on internet. You should know this.
You misunderstand. If it were a medical issue I would go to a real doctor and not ask sketchy strangers about it. Im asking because of curiosity.

What is your issue about how I formulated the question? Its short and to the point. I see no issue with it. Do you mean I should formulate it as a hypothetical question? That would be ridiculous nonsense and unnecessary.

Some things you can do to conform to our expectations here and to improve the response your question receives: 1) to avoid looking like a personal med question, indicate explicitly (i.e., by linking/citing specific examples) how others have experienced the same thing; 2) be more specific about the smell -- compare it to something; 3) to demonstrate research effort: do an extensive online search for your question (e.g., Google, Google Scholar, PubMed, NIH, etc.,) and explicitly report back where you looked (with which search terms), what you found out, and what you'd still like to know.

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Are there hobbyist microscopes which can take digital pictures of cells?
I'm reading a biology textbook and it's suggesting things like taking onion skin etc. and looking at it under a microscope to see its cells.  I'm tempted to do this for fun, and maybe share photos of what I find on Facebook.  (I remember using a microscope in school, but all I ended up seeing was air bubbles.)  I'm wondering if there is some way of doing this as a hobbyist (on a budget of, say, $100).
I saw this on Amazon:

but I'm not sure if it can see individual cells.  It doesn't look like it can do that.
Question: Are there hobbyist microscopes which can take digital pictures of cells?

Yes, but I'm not sure about the price. Look for something with 400x magnification or more, and to see cells get a slide microscope with lower illumination. Some come with pre-prepared slides that are stained to give contrast. Many without built-in cameras are compatible with separately-sold cameras.

“Hobbyist” seems poorly defined here. Is that just the cost of under 100$, or are there other requirements?

It implies I'm not particularly knowledgeable about what features I should search for.  I'm probably only going to use it a handful of times (for fun), so it shouldn't be expensive.  I don't have additional equipment.  And I'll probably ask random people on the Internet for suggestions of cool things to take photos of.

Search for "usb digital microscope". These are quite cheap (generally under 100$) with up to 1000x magnification, they are eay to use, and they connect to your computer for taking images. Obviously, the image quality is way worse than using a professional tabletop microscope, but I consider them useful enough for a hobbyist.

@Domen this is a cool suggestion that could readily be an answer, IMO.

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How to keep Paraffin Sections from falling apart?
I've been trying to make 5 uM sections of injured mouse spinal cords and stain the tissue with Luxol Fast Blue and H&E. At first I was removing the bones from around the spinal cord, but the tissue just fell apart after I got it on the slide. I think because it had been severely damaged.

I thought I might be able to hold the tissue together better if I kept the bone, so I took a spine and decalcified it with 500mM EDTA for 1 week and tried again. But the bone tissue just pulled away from the spinal cord.

These were some of my better images. I have many others that are even less intact. I see the tissue fall apart on the slide during the drying step, after I put the section on the surface of a water bath and catch it on the slide. I have seen some sections spread all the way across the slide. I suspect it has something to do with water getting trapped under the paraffin section and not drying correctly.
How can I improve the procedure to keep the tissue in 1 piece?

What is your dehydration protocol? What are you using for a fixative? How experienced are you with histology generally? Brain and spinal cord can be tricky tissues to work with. My initial bet is on the dehydration being incomplete. Also a nice attempt on the decalcification and using the bone as a scaffold - I don't have a good sense on whether this is likely to cause other problems, but it seems most typical for people to dissect the cord out rather than keep it in situ. And as you noticed, it's going to be hard to keep the tissue anchored within the bone after the shrinkage of fixation.

@BryanKrause Fixation is done by transcardial perfusion of 10 mL PBS followed by 10 mL of 4% PFA. The spines are removed and stored in 4% PFA for 1-3 days, then 500 mM EDTA for 1 week, then back into 4% PFA until I have time for paraffin embedding, usually just overnight. I need to double check the dehydration protocol, it's done by a machine with multiple buckets and I was just told to put the samples in the basket and hit start. I'm pretty sure it's 70%, 90%, then 100% Dryzol followed by paraffin, but not sure about the time for each step. Total time is 24 hours.

@BryanKrause I have no experience with histology. My advisor just sort of dropped me into a project a year ago and expected me to learn animal surgery, gait analysis, histology etc with almost no training or supervision. Needless to say, this has been a slow project.

That's why I asked about your experience with histology - I think it's fairly typical to get thrown into a project like that, whereas professional histologists have a ~2-year professional degree program. Not to mention you are working with some of the most difficult tissues. I would try to find a histology expert that works in your department in another lab to help you out a bit (I myself am a novice at best). Your PFA is made fresh, right? No methanol?

I'm not familiar with "dryzol" but if you are dehydrating in ethanol some people use several sequential baths in absolute ethanol at the end (again, this should really be absolute ethanol, 100% ethanol, not "reagent ethanol" or some similar alcohols mix that has methanol or isopropyl in it).

The PFA is purchased premade. Dryzol is a mixture of ethanol, methanol, and isopropanol, not sure of the ratios.

Let us continue this discussion in chat.

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When doing minipreps, do "good" plasmids produce more DNA than "bad" plasmids?
I've been doing minipreps for close to 10 years using the standard qiagen kits and DH5a e. coli. I typically do 24 minipreps at a time and then identify the good plasmids by enzyme digestion, PCR, sequencing, etc. Something I've noticed is that the good plasmids often seemed to have higher DNA concentrations than the bad plasmids. I thought it was just superstition, but I looked back at a couple of recent miniprep batches and did statistics, and found that the good plasmids had a statistically significantly higher amount of DNA. I don't see this pattern all the time, but I have seen it when using InFusion based cloning and traditional restriction enzyme based cloning. I don't have access to all of my miniprep data, so I can't check everything for statistical significance.

So, why would "good" plasmids have better yields of DNA than "bad" plasmids? Has anyone else noticed this pattern?
As far as I knew, as long as the plasmid carried a working antibiotic resistance gene and the bacteria could replicate it efficiently, then the plasmid should be ok. Could the bad plasmids have been cases where the bacteria manages to ligate the vector backbone together but creates a sequence that doesn't replicate well? Could it be a timing issue, where the bad plasmid doesn't start replicating until later, so that the bacterial growth is delayed? I have not weighed the bacterial pellets, so I can't say anything about bacterial growth vs plasmid yield.

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How can two biological sequences be anything but homologous?
If we consider homology between two nucleotide sequences as a yes/no answer to whether they have shared a common ancestral sequence, then given that all life share common ancestry and sequences are carried by living things, then how can two biological sequences be anything but homologous?
One possibility I can think of is perhaps natural transformation in bacterial---but even so, the DNA sequences picked up must have come from a biological source.
(I assume that it must be possible that there are sequences that are non-homologous, because otherwise there wouldn't be a need to point out which things are homologous, i.e. I assume that the concept of sequence homology would not be as widely used as it is right now)

There has been some discussion on my claim that homology should take a Boolean value. As I have stated in the comments, for this I refer to Inkpen, S.A. & Doolittle, W.F. J's "Molecular Phylogenetics and the Perennial Problem of Homology" in Mol Evol (2016), pp. 187.

Nevertheless, there is a general understanding that conflating the extent of similarity and the inference of common descent is always a misuse of the concept—that there can be no such thing as ‘‘percent homology.’

"If we consider homology between two nucleotide sequences as a yes/no answer to whether they have shared a common ancestral sequence..." But we don't. If you think we do, please cite a source that supports this idea. In fact, you are not asking a real biological question, but playing with words to expressing a philosophical opinion that is not the concern of this list.

I'm voting to close this question as off-topic because it is not about Biology in the terms of this list.

@David I refer to Inkpen, S.A. & Doolittle, W.F. J's "Molecular Phylogenetics and the Perennial Problem of Homology" in Mol Evol (2016), pp. 187. Quoting the article: "Nevertheless, there is a general understanding that
conflating the extent of similarity and the inference of
common descent is always a misuse of the concept—that
there can be no such thing as ‘‘percent homology.’"

@David I want to stress that I am not asserting my opinion. Rather, I am stating my premises which have lead to my conclusion. By doing so, I hope to expose these premises to the Q&A in addition to the actual question, to avoid an XY problem. I do not agree that I am 'playing with words'. I truly believe that there can be situations where non-homologous sequences can be created---just that I have not yet thought of them.

@ning You should edit your question and insert new information there. The paper/quote for example might get buried in the comments, or later even deleted...

@ning — There may be no such thing as "percent homology", so the terminology used (as I remember) is "percent identity". You might argue that identity is like pregnancy — all or nothing — but a metric of the percentage of amino acids that are identical (or 'similar') is both possible, useful and used. It is the basis of statistical comparisons (which I don't personally understand) that allow one to place a confidence value on two sequences being 'related'. If you don't like the term homologous sequences, you can use 'related' or 'structurally related' (to avoid any evolutionary implications).

Homology in this context is always a matter of degree exactly because of shared ancestry. Measures of homology are often in terms of "percent homology" (or more often "percent similarity" as evidence of homology) or some other measure that allows representation of that degree.
To emphasize why this makes sense, think about a receptor family. In an extant organism, you might have 3 receptors that all are homologous. However, if receptors 2 and 3 are more similar to each other than to receptor 1, then you can make some better assumptions about the history of the evolution of receptors 1 to 3, that there is likely a "common ancestor receptor" to both 2 and 3. There is more "information" if you consider the degree of homology rather than just its existence.
At some extremes, sequences could be so different that even if they share a lineage they may have now diverged to the point that they are not distinguishable from independent sequences. However, selective pressures prevent such complete reorganization in key sequences and so at least some homology can be maintained across very distantly related organisms.
As far as deciding what is "homologous" versus "not homologous", that is somewhat of an arbitrary threshold that depends on levels of evidence and the purpose for recognizing the homology.

It is interesting that you argue according to degrees of homology. I was taught that homology is strictly answered in binary, or at best likelihoods, and never as a degree. A degree of similarity can estimate a likelihood (but not degree) of homology. Frankly, I do not agree with looking at homology in terms of degrees, but then your answer would not make sense unless I do. Perhaps you could elaborate on why homology should be thought of in degrees?

@ning It all depends on what your goals are. I added a paragraph, does that help at all?

@BryanKrause I feel it is important to stress that degrees of homology as represented by percentage similarity is a very flawed metric, given that degrees of homology is discrete in a certain sense, determined by degrees of common ancestry. See for example Smith & Pease, 2017


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Heritability in population genetics
What is the difference between narrow sense heritability and broad sense heritability?
Does heritability in the broad sense refer to the degree of genetic determination?
Heritability in the narrow sense refers to additive effect of alleles , what does it actually mean?

The answer to this question should give you everything you need.

Heritability in the narrow sense
Heritability in the narrow sense $h_N$ is the genetic variance $V_G$ over the overall phenotypic variance $V_P$
$$h_N = \frac{V_G}{V_P}$$
More information at Why is a heritability coefficient not an index of how “genetic” something is?.
Heritability in the broad sense
The heritability in the broad sense $h_B$ is the additive genetic variance $V_A$ over the overall phenotypic variance $V_P$
$$h_B = \frac{V_A}{V_P}$$
Decomposing of the genetic variance
Adding together the additive genetic variance $V_A$, the dominance genetic variance $V_D$ (and the epistatic genetic variance $V_I$) sums up to the overall genetic variance $V_G$. Here I will ignore the epistatic genetic variance
$$V_G = V_A + V_D$$
, where
$$V_D = x^2(1-x)^2(2\cdot M_{12} - M_{11} - M_{22})^2$$
$$V_A = 2x(1-x)(xM_{11}+(1-2x)M_{12} - (1-x)M_{22})^2$$
, where $M_{00}$, $M_{01}$ and $M_{11}$ are the measurements of the phenotypic trait in genotypes 00, 01 and 11 respectively.
Highly related

Why is a heritability coefficient not an index of how “genetic” something is?
Additive genetic variance with nn alleles
Additive genetic variance with nn loci


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Need help identifying this wasp/hornet
I am currently on holiday in southern France and have noticed that the house we are staying in has somewhat of a hornet/wasp problem.
Is there anybody that can help in identifying it? My best guess is a European Hornet but I'm no expert so may be wrong.
Below are some pictures. Sorry they couldn't have been of any clearer quality, they were taken through a window, and I didn't particularly feel like going out to get any closer because I've heard that some hornets can be quite aggressive even if unprovoked.
From what I saw, it has visible fur, and is around 2cm in length. The feature that led me to it being a Hornet is that the waist gets smaller before the abdomen.

This looks like a European hornet Vespa crabro to me. Wasp-like appearance, red on head and thorax and base of the abdomen. And the legs are red/dark, not yellow.

Picture from wikipedia
Hornets can be aggressive, especially if you come near the nest. Usually, European hornets are actually not really interested in people (unlike other wasps). But if there are many around your house, it is likely the nest is near, so be careful and perhaps contact the owner of the house.

Ahh thank you! We just found their nest in the roof of the house we're staying in for the week. I've just been reading about the social behaviour of wasps/hornets. Fascinating! We'll leave them be, no need to antagonise. Thanks again

It is quite possible that what you are dealing with are Asian Hornets.
This is a link to an article that helped me answer your question.
http://www.telegraph.co.uk/news/worldnews/europe/france/6049302/Tourists-warned-as-Asian-hornets-terrorise-French.html

Welcome to Bio. Link-only answers are considered to be comments rather than answers. Look here to a good answer on a similar question. It includes a description of the species, a photograph taken from your source and perhaps additional information.

The link you shared explicitely says it is recognizable by its yellow legs and the picture shows a wasp with red on the abdomen. So Asian hornet? I don't think so.


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Can you identify this mosquitoe-like bug?
I'm wondering what is this bug, I have never seen it before and its behaviour is just weird for me, they just try to climb to the top of the window, then fall and start over again, I see no hole they are going through, they seem capable of flying but they don't.

(click to enlarge images)
Please help me know:
A) Which bug is it?
B) Is it harmful?
C) What business do they have with my window (What is attracting them?)
D) How do I get rid of them?
I'm in Mexico City and it was rainy up until this last days when they arrived, now it is warm, also I have some plants in the room but they don't seem to be interested to go near them.

Interestingly, one turned up on our kitchen table this afternoon. (Southern UK). Never knowingly seen one before. So, watching.

Unsure, but looks to be part of a mating swarm of ants. Not a problem if so.

Also unsure, but I think it's some kind of two-winged fly (Diptera); an in-focus photo showing wing venation would be a big help, but I suspect they're no longer around (it's been three weeks since this inquiry; the flight season for this critter might already be over).


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Why have parasites not evolved to be harmless?
I have yet to understand why so many viruses or bacteria haven't evolved to be harmless (specifically, I don't know of any harmless viruses). I think it would be greatly beneficial for a virus to control its population and use natural transfer channels that don't cause discomfort (like feces), since it would be allowed to stay in an organism indefinitely and would have a lifetime channel for transmission, hopefully getting to be the most common strain.
So, why have viruses not evolved to be harmless to their hosts?

Many are harmless. Some are not. They occupy different niches. If there are 6 billion humans, why would a virus care to take down a few? Plus, many don't kill, only harm. If the host lives, why would a parasite care if its host gets sick?

This is a good question. I know at least some models of pathogen spread have a parameter for lethality of the virus, and high lethality decreases pathogen spread. Part of the answer could be that high infecitivty (pathogen spread, would be selected for) is associated with high lethality/morbidity (not necessarily beneficial for the pathogen per se)

@Christiaan The parasite should care about the host getting sick because if so the host would evolve antibodies. Both could continue the biological arms race, but the side-channel of harmlessness seems pretty effective to me.

@CactusWoman Seems likely, please share them!

@chubakueno It's been a while since I've used those equations so I don't have them on hand, (hence me making a comment and not an answer), but I'll see what I can do

Although I still don't have a full answer, the wikipedia page on Optimal Virulence seems to be relevant: https://en.wikipedia.org/wiki/Optimal_virulence

The post "why-do-parasites-sometimes-kill-their-hosts?" is very much related.

You consist of more bacterial cells than human cells, and most of these are harmless or benificial.

Just a reminder, : "harmless" to humans does not indicate it's harmless for all other organisms.

Does this answer your question? Why do some bad traits evolve, and good ones don't?

There are actually many examples of viruses that have integrated into the human genome and were rendered harmless, perhaps even helpful.
This New Scientist article referencing this Nature article describes endogenous retroviruses, which are ancient viruses which have integrated into the human genome.

About 9 per cent of our genome is thought to have come about this way.
  Until recently, these viral relics were largely dismissed as inactive
  “junk” that ceased to have any impact on their host many thousands of
  years ago. The discovery that HERVK, the most recent ERV to make
  itself at home in our DNA – probably around 200,000 years ago – is
  active in human embryos challenges that notion.
Joanna Wysocka and her colleagues at Stanford University in California
  made the unexpected find while they were analysing gene activity in
  3-day-old human embryos, which are bundles of eight cells. Besides DNA
  from the parents, they found genetic material from HERVK. “The cells
  were full of viral protein products, some of which had assembled to
  form viral-like particles,” says Wysocka.
Further experiments revealed that the virus appears to produce a
  protein that prevents other viruses penetrating the embryo, suggesting
  it protects the embryo from dangerous circulating viruses, such as
  influenza. It also seems to play a crucial role in the genetic
  activity of the embryonic cells, helping to genetic instructions to
  the cellular protein factories.

Additionally, the endosymbiotic theory is also another possible example. It is thought that mitochondria and chloroplasts are previously free-living, possibly parasitic organisms that ended up integrating into the host cell and living as an organelle.
In short, parasites absolutely can and do evolve into harmless versions of themselves. There are already discovered examples, and as the human genome is better understood, doubtlessly more would be discovered.

Chloroplasts and mitochondria are unlikely to have been parasitic. They are rather tamed preys captured by unicellular predators.

Why do you think so, @WYSIWYG?

Pathogens don't evolve to be harmless to their hosts, because that doesn't benefit the pathogen.
For example, think about a pathogen, like cholera, that spreads by fecal/oral contamination.  How could it enhance its transmission? One obvious way is by increasing the amount of fecal contamination, which means causing diarrhea, which means harming the host.
Or think about a pathogen, like myxomavirus, that spreads via sand fleas.  The fleas bite one rabbit, jump to a new one, and infect it.  How could the virus enhance its transmission?  It turns out that sand fleas don't bite dead rabbits, so viruses that kill their hosts immediately lose out.  But on the other hand, perfectly healthy rabbits groom away the fleas, and those viruses lose out.  The viruses that transmitted best made their hosts very, very sick, too sick to groom, for a long time, to maximize transmission.
Or think about a pathogen that's transmitted through saliva, like rabies.  Maximizing the number of new hosts that get bitten, by damaging the brain, would help there.
There are generally balances for maximizing transmission.  Sexually-transmitted pathogens might not make the host sick for a longer time.  Respiratory agents might do better with hosts that can mingle more with potential new hosts, while fecal contamination doesn't need such a healthy host.  But in general, pathogens evolve toward maximizing transmission, not host health.
(You may wonder why pathogens don't change their mode of transmission - why don't fecal transmitters evolve the ability to spread by respiratory transmission? The answer is generally the same as any other "Why doesn't X evolve Y?" questions -- because the path toward a new complex ability means going through a phase where the pathogen is not as good at either ability, and gets out-competed by its more traditional relatives.)

It doesn't follow that if pathogens (like all evolved organisms) maximise fitness, they would necessarily harm their hosts. Symbiosis is not a zero-sum game. If a pathogen evolved into a mutualistic relationship, they could maximise fitness of themselves along with their host.

@MarchHo symbiosis actually is much more complicated than parasitism and involves an intricate genetic interaction. Pathogens usually have a reduced genome and to evolve into a harmless species they have to gain a function (which is more difficult than losing one).

@WYSIWYG Did you mean mutualism? Parasitism is a kind of symbiosis.

@MarchHo yes mutualism. AFAIK parasitism is not included in symbiosis (only mutualism and commensalism are considered symbiosis).

@WYSIWYG This paper suggests that parasitism is also a kind of symbiosis, and that this definition is widely accepted in biology textbooks.

Pathogens don't evolve to be harmless? That's a big assumption! Thinking of a bacterian flora in one's guts, I can imagine all sorts of evolutionary changes...

Please don't twist what I said.  I didn't say no pathogen has ever evolved to be harmless.  That would be silly, as would interpreting what I said that way.  (Spumaviruses are better examples than gut flora.)  But that evolution would be a byproduct of enhanced transmission, not an evolutionary pressure on its own.  I even pointed out conditions in which that could happen.  As for "imagining all sorts of evolutionary changes" that's lovely, but there's an extensive literature on the subject; you'd be better off using some research, logic, and reason than pure imagination.

I think the key to your answer is in the word "parasite": parasitic relationships always involve the parasite taking something from its host, causing it some (possibly slight) amount of harm. Once the parasite has evolved to cause an insignificant amount of harm to its host, we no longer call their relationship "parasitism" but "commensalism".
There are lots of commensal bacteria -- you have more in and on your body right now than you have cells in your body -- and many of them may have evolved from parasites.
All viruses need to hijack cellular machinery, particularly the protein generation machinary, in order to reproduce themselves. So it's hard to imagine a commensal virus that doesn't harm the cell it uses to reproduce itself. As @March Ho pointed out, however, they might eventually become incorporated into their hosts' genome, potentially giving that host additional capabilities that it might not have otherwise possessed.

Great question! Christiaan's comment is the best way to start thinking about this; why should the virus care? In other words, what determines the selection pressure to increase or reduce virulence?
The most common definition of virulence in ecology is the reduction in host fitness resulting from infection by a pathogen. If reductions in host fitness also reduce the probability of successful transmission then there is selective pressure on the virus to become less virulent. Conversely, if reductions in host fitness make no difference (or actively increase the likelihood of transmission) then there is no selective pressure to reduce virulence.
Consequences of pathogen infection which increase the likelihood of transmission via certain routes often reduce host fitness (the trade-off hypothesis). For example, diarrhoea increases the potential for faeco-oral transmission of the pathogen but increases the host's risk of death from dehydration. Differences in optimal virulence reflect differences in the degree to which host and pathogen fitness are correlated. If transmission is typically rapid and/or unrelated to host fitness then there is no selective pressure to reduce virulence.
As another simple illustration, a mosquito-transmitted pathogen that makes the vertebrate host sick (and thus less likely to react to mosquito feeding) will probably be transmitted more effectively, while one which makes the mosquito sick (and reduces its host-seeking frequency) will be transmitted less often. Vertically-transmitted viruses often become less virulent because host reproductive success and virus transmission are linked. This paper explores this theory with reference to a natural system (Plasmodium infection) and considers some implications that might not be immediately obvious, such as the consequences of better control via drug treatment etc.

I'm not convinced it strengthens my answer much (if anyone thinks it does I can edit it in) but I happened to come across this reference today which mentions that in WNV-infected bird species viraemia mostly correlates with mortality (although with some exceptions): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939481/

There is generally a pressure for parasites to be harmless towards their hosts (apart from the drain in resources), especially if those two species have co evolved for a long time.
This sets them apart from parasitoids. Though those terms are sometimes used interchangeably, a parasite requires its host to live on while a parasitoid aims to ultimately kill the host.
By far the most harmful parasites are those that did not co-evolve with their current host or ended up there by accident or in the wrong stages of their life cycle (for instance, the fish tapeworm in humans).
The same principle applies not only to parasitic animals but diseases in general, where most of the infections with the highest mortality rates are, at least initially, zoonotic in nature.

Welcome to BiologySE... we always appreciate references accompanying answers as well as questions. This is so others can read about and better understand what you are asking/saying.


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Why are two forward and reverse priming sites depicted here? what do they do?
This question is in regard to the baculovirus expression system.

(source: amsbio.com)
Why do we need to generate primers for the polyhedrin promoter and the baculovirus?
Primers are needed to amplify DNA regions of interest.Here, why exactly do we need to amplify these regions? and at which steps?
PS : I am not asking about the need for forward and reverse primers.

what is this slide and why does it have so many typos?

It's just extra information that you don't need to understand the part above. You could use these primers for colony PCR or sequencing when you'd use the plasmid (after transforming to E.coli for example), but they're not absolutely required.
Compare it with this map for example. There are also some primer sites listed, but you don't actually need those primers for anything.


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User friendly phylogenetic tree editing software
Background: I have Hackett 2009 (from birdtree.org) phylogenetic tree and I need to reorder it according to Prum 2015. I also work only with some species relevant to my project, not the entire tree.

I currently use Mesquite, and it is absolute purgatory. Doing any reordering in Mesquite takes insane amount of time. I'd also like to add higher taxon information to each species (like order) to help my my orientation in the data.
I understand some of Mesquite's shortcomings might be shortcomings of Nexus format. As I don't need to analyse the data myself, I can work in some better format like PhyloXML as long as it's possible to export back to Nexus format in the end.
Is there any way to do this? Some software with better visual editor (and overall better UX) or other method to do this in effective manner? I find it hard to believe that much bigger data is handled in such clumsy way.

Can you just start with the Prum tree and cut it down to your taxa?

If one of the answers is helpful for you, it would be a nice gesture towards the author to accept it. Additionally the system will not bump the question anymore.

Yes, I think that you should consider using MultiSeq which is a package of Visual Molecular Dynamics (VMD). You can use visual molecular dynamics to make phylogenetic trees based on sequence/structural/other statistical means.
If you want to check it out and see if it works for you, then install visual molecular dynamics: http://www.ks.uiuc.edu/Development/Download/download.cgi?PackageName=VMD
and try out these tutorials under Bioinformatics from the distributors:
http://www.ks.uiuc.edu/Training/Tutorials/
Note: VMD doesn't work very well in 64-bit Windows operating systems if that's what you use. To get it to work well, I suggest you install virtualbox from Oracle, and install your favorite free open-source Linux operating system into virtualbox or into your guest linux machine on your host machine.
I suggest installing your guest Linux OS on virtualbox with 1/2 the ram of your host physical computer allocated, and like 100 GB of space for when you discover that you love LINUX so much and have lots of information on there.
To full-screen your guest Linux OS, you need to install guest additions.
My favorite Linux OS is Kali Linux.
To install guest additions on Kali Linux and most linux guests on VirtualBox,
you have to do this:
But as I've discovered you have to upgrade Kali Linux to Kali Linux Rolling to get Kali Linux to fullscreen. How you do that is via this:
And you probably shouldn't do your work as user "root" the default user. You have too much power so to increase security create a new user on Kali-Linux:
If you are already using linux, then you won't have any problem with getting VMD to work. But by the way, you have to install VMD from the command terminal in linux and this is how you do it.
The instructions as for how this guy does it (and sorry he doesn't speak English) are in the README file when you download VMD.
Sorry, apparently, I don't have a high enough reputation to cite you all the links. I'm sorry that I had to remove them.

To install Guest Additions: https://www.youtube.com/watch?v=kmnyJ9x0szM

As I said, you have to upgrade Kali Linux to Kali Linux Rolling:
https://www.kali.org/news/kali-linux-rolling-edition-2016-1/
And you also have to install VMD:
https://www.youtube.com/watch?v=5TN_DV3F198
Also, you shouldn't run Kali Linux as "root" user so to create a new user:
https://www.linkedin.com/pulse/20140502074357-79939846-adding-a-new-user-in-kali-linux

If virtualbox still doesn't work for installing a linux distribution, you will have to "enable virtualization technology" in your system BIOS.

I had good experiences with Archaeopteryx. This is a java package so it should be easy to run but I recommend the BioLinux if you don't want to spend too much time installing and get straight to the biology. You can run it as a virtual machine or install it side by side. It is getting a bit older but everything works just out of the box and there is a wide array of tree editing tools installed. Just look through the package list.


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Can chitosan affect the absorption of unsaturated fatty acid?
I know chitosan will decrease the absorption of fat. However unsaturated fatty acid, such as DHA, is beneficial. Does chitosan effect the absorption of unsaturated fatty acid?

It seems that there is no difference in the effects of chitosan on the uptake of saturated and unsaturated fatty acids. The study cited below (which has been done on guinea pigs) fed the animals a diet which contained different digestion-resistant fibers (maltodextrin, cellulose and chitosan) and also fats. Then the fatty acid content of the feces was measured afterwards.
Only for the group which got additional feeding of chitosan showed a significant increase in the excreted fatty acids. This was true for the saturated (lauric, myristic and palmitic acid) but also for the unsaturated fatty acids (Oleic, linoleic and α-linolenic acid). This effect is specific for fatty acids, The uptake of neutral sterols and bile acids was not affected in any of the experimental groups.
The second paper suggests that chitosan forms an emulsion with fatty acids under the acidic conditions of the stomach. This emulsion is stable and later excreted with the feces.
References:

Selective in vivo effect of chitosan on fatty acid, neutral sterol
and bile acid excretion: a longitudinal study.
Interaction between chitosan and oil under stomach and duodenal
digestive chemical conditions.


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Localization of Protein Kinase-A
Is protein kinase-A located in the cytosol/cytoplasm of cells or in the plasma membrane?  Also, is it considered a receptor molecule since it is dependent on cAMP?
Any and all help is appreciated. Thanks!

The localization of the protein kinase A (PKA) cannot be explicitly answered, since the localization depends on the activation state of the protein. Lets have a look at the protein itself first, as this is important for the activation.
PKA consists of four subunits, two catalytic subunits and two regulatory subunits. See the image from the Wikipedia article on PKA:

Each regulatory subunit R is binding two cyclic AMP (cAMP) molecules and dissociates then from the protein complex. This sets the catalytic subunits free, which can then transfer phosphate groups from ATP to serine or threonine residues of target proteins. Among the target proteins is also the Phosphodiesterase (PDE) which gets activated by phosphorylation from PKA. Activated PDE hydrolyzes cAMP into AMP which reduces the amount of free cAMP which is necessary for PKA activation. PKA is also inactivated by phosphorylation.
In the cell you will find PKA almost everywhere. The traditional belief is that the protein is activated in the cytosol and enters the nucleus where it phosphorylates targets like CREB which then subsequently binds the the CRE sequence in gene promoters and activates this genes. Reference 1 shows that PKA is also activated in the nucleus, meaning that also the inactive form is present there. Reference 2 gives a nice summary of the article.
The regulatory subunit of inactive form of PKA binds to a special class of proteins: The A-kinase anchor proteins (AKAP). These are membrane bound and allow the association of PKA to membranes, although the protein itself is not directly binding to the membrane. The AKAP family is rather big (at least 50 family members are known today), which probably allows the localization of PKA at different membranes. Have a look at references 3-5 for details on the localization and the anchoring.

References:

Regulation of nuclear PKA revealed by spatiotemporal manipulation of
cyclic AMP.
Protein kinase A activated in the nucleus as well as the cytoplasm
The where's and when's of kinase anchoring
Localization of A-kinase through anchoring proteins.
The biological functions of A-kinase anchor proteins

It is present throughout the cytosol but can also be anchored to plasma membrane and mitochondria via indirect interactions. Can also be translocated to the nucleus. (See here).
It is not called a receptor for cAMP (though the mechanism is homologous).


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Why are there no artificial wombs yet?
If the conditions within the womb are mimicked, and proper amniotic fluid with constantly recycling nutrients is maintained, is it not possible to obtain an artificial womb? Is there anything missing? I think, artificial womb, if possible, could open new ways to study human anatomy and development and further the research studies. Starting with test tube fertilization, one could observe the entire process of growth of a zygote to a fetus.

It is impossible at present to reproduce all the conditions of a womb.
A womb is more than a warm fluid-filled home for nine months. A few days after fertilization, the trophoblast portion of the conceptus attaches itself to the uterine wall where it develops into a placenta and umbilical cord while the rest (the epiblast) becomes the baby.
Throughout the pregnancy, the mother delivers oxygen, nutrients and hormones through the her blood flow to the placenta, while carrying away waste. The womb also prevents infection of the placenta and amniotic fluid.
In addition, the placenta contributes to amniotic fluid production. Production and reabsorption of amniotic fluid is a very dynamic process; it isn’t just there. It is estimated that more than 95% of amniotic fluid turns over on daily basis, and the fluid composition changes throughout pregnancy.
While cell cultures of the womb's endometrium have been grown, there is nothing artificial which is close to this kind of environment yet, nor is there likely to be in the near future.

I think the trophoblast entirely forms extraembryonic tissue.

Thanks for the insight, I hope it becomes a reality sometime in the future.

@a.aniq I'm afraid your not seeing most of the (generally bad) implications of such invention, I hope it won't be created any time soon. Sometimes it's better to don't create sth.

@Mithoron I understand your concern, but artificial sperm and eggs have already been created, has it changed our culture? It is just for research purposes, nothing else. International Regulatory committees will absolutely put restrictions on it's use. And only a handful of authorised individuals will be allowed access to this technology, if it becomes viable sometime in the future. Who knows? Maybe something good will be discovered which will accelerate our endeavour further. We can use nuclear energy to destroy our world, and also to create a viable energy source. It's up to us to decide.

Artificial amniotic fluid has been used on goats, and for premature human babies.

In the 1996 experiment, Thomas Shaffer, a scientist at Temple University, used an oxygenated liquid in a clinical trial with thirteen infants born at twenty-three to twenty-four weeks who were not expected to survive, and seven babies were discharged healthy. Jonathan Knight, An Out of Body Experience, 419 NATURE 106, 106-07 (2002); Partial Liquid Ventilation with Perflubron in Premature Infants with Severe Respiratory Distress Syndrome, 335 N. ENG. J. MED. 761, 761 (1996)
(documenting scientific results from Shaffer's experiment).

Also in 1996, Japanese professor Dr. Yoshinori Kuwabara of Juntendo University, successfully gestated goat embryos in a machine that holds amniotic fluid in tanks.
Dr. Helen Hung-Ching Liu, Director of the Reproductive Endocrine Laboratory at the Center for Reproductive Medicine and Infertility at Cornell University.  In 2003, her team was able to grow a mouse embryo, almost to full term, by adding engineered endometrium tissue to a bio-engineered, extra-uterine "scaffold." Recently she grew a human embryo, for 10 days in an artificial womb. However, legislation imposes a 14-day limit on any research project of this nature.
Even if we do get a working artificial womb - we don't know how much we might be missing.  Something that appears to work, may not be as good as the natural system.  And we will have some difficulty in testing it in non-animal applications.
I'm trying to find the laws, but I believe we're currently precluded from pursuing artificial wombs mostly because of legal measures, not particularly by engineering problems.  Also, allegedly, the US is one of the most un-regulated first world nations for this type of research (while saying you can only research this for 14 days, smh).

Your first sentence is misleading. You have over-estimated the benefits of the technology you advocate. Note Partial-Liquid ventilation of perflubron a nebulized solution (gas/droplet mixture). Full liquid resulted in improvements on parameters but death of all neonates on removal of fluid, imparting no benefit whatsoever. Of embryos - goat or human - several days survival (not what you infer) was the max. Please link sources to your citations so that your interpretation can be verified. In the case of Hung-Ching Liu, you are totally misrepresenting her work and her findings.

Umm, 7 babies were discharged healthy seems like a benefit.

Partial-Liquid ventilation is not artificial amniotic fluid. It seems like you still don't get the point. The fact is that your answer is essentially incorrect.


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Why are haploid cancer cells not killed by immune system?
I have seen haploid cancer cells (I think it was leukemia cells) in a lab.
Sperms and eggs are haploid but are not destroyed by the body because they are protected by other cells surrounding them.
My questions :

How are haploid and diploid cells differentiated by our immune system ?
Why are the haploid cancer cells not killed ? I know I could as well ask why any cancer cells are not killed but I want to know if there is anything specific about these cells ?
How are these cells generated ? (By meiosis ?!)

Normally , tumor cells are destroyed by NK cells. Is this a case of autoimmunity ?

No, autoimmunity causes the body to attack its own cells. The other thing: Haploid cancer cells don't really make sense, since they cannot divide due to the half set of chromosomes. I only did a short search in the Pubmed but besides artificially generated haploid stem cells and "near haploid" cancer cells, nothing pops up. Do you have any references?

@Chris I do not have references but I have seen the cells myself.I had gone for a camp at Stanford University and our instructor had shown them to us. I am sure they were haploid.

@chris But aren't tumor cells our own cells too ?

What makes you sure they were haploid? Yes, tumor cells are our own cells - but out of control. So it makes sense that the immune system removes them. In an autoimmune disease all cells of a certain type are attacked.

@chris The person working on them told us that they were haploid.

Thats interesting. Did they tell you the name of the cell line?

I don't have an answer but - "Near-haploid karyotype in a squamous cell lung carcinoma"  - http://onlinelibrary.wiley.com/doi/10.1002/gcc.2870070405/abstract

@chris No they didn't tell us the exact name but they told us that they were from a patient with leukemia.

@Chris correctly references the KMB-7 cell line.  Being haploid, it is straightforward to do experiments that involve knocking out a gene (and cited refs).

'Chris' mentioned cancer cells are our own but' out of control'. Why doesn't this being 'out of control' restrict their immuno- suppressant abilities?

Now that I did some research on this, it seems to be a very rare condition of acute lymphoblastic leukemia (ALL), see this article:"Chromosomes and causation of human cancer and leukemia. XLVII. severe hypodiploidy and chromosome conglomerations in ALL.".
This article ("Origin of near-haploidy in malignant hematopoietic cells.") hypothesizes that this might happen through gradual chromosome loss during cancer formation and that in the end one clone with a lower than normal number of chromosomes.
This seems connected to a much worse outcome of the cancer than for hyperdiploidies (more chromosomes than normal). See the "Atlas of Genetics and Cytogenetics in Oncology and Haematology".
They also define why haploid (or near haploid means: Less than thirty chromosomes, down to 23 have been observed). This is not the normal haploidy found in germ cells. Se also "Isolation and Characterization of a Near-Haploid Human Cell Line".
Since these chromosomal changes occur inside the nucleus I think this is mostly invisible to the immune system - so there is probably no difference how these cells escape the immune system compared to other cancer cells. I think its possible that due to the chromosomal loss some proteins which control apoptosis are lost, so the cells escape this fate as well.
Regarding the immune question, you can either start with the following articles or simply go over to Pubmed and choose some articles. In this field there is a lot of research going on.

Does the immune system see tumor as foreign or self?
The tumor microenvironment and its role in promoting tumor growth
Tumor Evasion from T Cell Surveillance

If there are access problems, let me know.

The recent publication in pancreatic neuroendocrine cancer https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054670/ revealed about one third of patients have a half of the genome haploid. They sequenced 57 samples (WES) and defined a group of samples MEN1 mutant and +-12 haploid chromosomes. As expected the group had the worst prognosis.


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Why do body parts sometimes get a itching and tingling feeling when falling asleep?
Just before I fall asleep I sometimes experience an itchy and stinging feeling in my body parts. It can be that the nose or neck suddenly start to itch or a tingling stinging feeling in the legs. Sometimes my fingers also start to do small uncontrolled movements.
What is this about?

This is not a duplicate.

Not a dupe of http://biology.stackexchange.com/questions/31107/involuntary-twitches-and-apnoea-during-early-phases-of-sleep

@FloriOn Why is this marked as a duplicate? My question is not about PLMD nor apnoea.

Sorry everyone, removed.

Maybe you're being allergic at something from your bed (maybe dusts). Try sleeping with clean bed clothing and pillows (and yourself too) and see if the itching persists.


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Methylation Data for Erythrocytes (Red Blood Cells)?
Why is it that white blood cells (leukocytes) are highly characterized in DNA methylation pure cell line profiling, but I can't find any profiles of red blood cells (erythrocytes)? Is blood considered a pure tissue type?
I'm following the deconvolution of DNA methylation data by cell type (white and red), but I can't do this without pure cell line methylation data. Can anyone help me understand either:

Why red blood cell DNA methylation data has not been purified and collected yet.

OR if it has...

Where can I access the methylation data for red blood cells (erythrocytes)?

Thanks so much for any help!!!

The answer is relatively simple: Mature erythrocytes do not have a nucleus and thus you cannot study methylation patterns there. You can do this in young, developing erythrocytes which still have their nucleus.


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What is the heredity model of the following heredity tree?
I have the following heredity tree:

and I need to decide which heredity model it fits the most, with the least number of assumptions, from the following models:

autosome dominant
autosome recessive
X-linked dominant
X-linked recessive
Y-linked
mitochondrial

I think the answer is autosome recessive because of the following analysis:

and the only assumption which is significant (the other assumptions are about the non-family members which are that they are carriers of the hilliness, it isn't said that the illness is rare, so in examining any model different assumptions about the out-of-family members are made) is the assumption on the last generation where there is two ill persons instead of one if we cross their parents alleles: AA, Aa, aA, aa, but we got 2 aa and AA, Aa, but that can be explained by little statistics in the family, so it is hardly even an assumption at all.
Then I've been told that it is a rare illness and that it autosome illness and not a linked one, its also said as a clue that there may be some Mendelic exceptions (known exceptions from the Mendel heredity models).
So In my understanding its more probable that the model with this data is autosome dominant with father genomic imprinting the following way":

As you can notice for the classic autosome dominant model the circled one should be black (ill) but they are not, so my assumption according to the clue that the ill alleles are imprinted in fathers (males) and expressed only when they inherited from mothers (females).
Till this part I would like you to check me did I any wrong in this analysis, but my major question that made me doubt my analysis is the following:
In addition to previous data, is is now given the the ill girl in the 4 generation (which is black circle on the image) got pregnant from one of two males:
male A: healthy male with both parent healthy and and Homozygous both.
male B: ill male with one parent Homozygous and the second parent Heterozygous
It is also given that no matter who is the father, the chances of getting an ill child are 50%.
From the previous analysis I decided that the heredity model is dominant autosome, so the first contradiction to that, that one of the parents of male B is Heterozygous, means he carries the ill allel and the healthy allel, but then in dominant model he has to be ill, but it said both parent are healthy.
The second thing that should correlate to the model is the chances computation. If the girl is Aa as was established by previous analysis then if the father is male A, i.e. his alleles are Aa (because he is ill with parents which are aa and Aa), then the chances for a ill child are 3/4 because the options are {(A_father, A_mother),(A_father, a), (a, A_mother), (a,a)} and if the father is male B then his alleles are aa because his parent are both aa, then the chances of an ill child are 1/2, so the total chances are (3/4 + 1/2) / 2 which is 5/8 which isn't 50%.
Or maybe this is wrong for male 1, because if the ill phenotype is delivered only by women (mothers) because of male genome imprinting, then for the option {(A_father, A_mother),(A_father, a), (a, A_mother), (a,a)} only two will make the child ill and not 3 : (A_father, A_mother) and (a, A_mother), then the calculation will be (1/2 + 1/2) / 2 which is 50% indeed, and for the parent of male B being Heterozygous but not ill can be explained by the assumption that he/she got the allel from his father.
Is it correct that the exception of genome imprinting is for the whole illness and not for some ancestry ?
I'm not sure if genome imprinting is a phenomena for the ill allele (the whole illness) or per concrete ancestry? (in the second option it will be that actually I do another assumption on the male B ancestry, that the imprinting is also as in the given ancestry tree).
But this make some sense' isn't it ?
It made me doubt because if the model was autosome recessive, then the computation would give me exactly 50% without thinking and explaining about the male's B Heterozygous parent (which then would be healthy just a carrier), but then the previous tree seem to be much more complicated because if you only look at the first generation in this model:
if the father is AA (A - healthy dominant allele, a - ill recessive allele) and the mother is aa, then they shouldn't have any ill children (because their cross is {Aa, Aa, Aa, Aa}), but they do, that's why I started to doubt is it dominant autosome as I concluded.


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What kind of bug is this? Small, slower than an ant, black head, red body
It is very small but it is slower than an ant. It has a black head and six legs with a red body. I’m in North Carolina.

Welcome to Biology.SE. Please [edit] your post to include as much of the following information as possible to improve your chances of getting a good answer: 0) size estimate; 1) photos from multiple angles if possible; 2) the location (e.g. country and region — the more specific the better) where you observed this organism; 3) habitat information — e.g.s: (the building is) in a forest (conifers vs. deciduous), next to water (lake vs. river vs. ocean), etc.; and 4) any other behavior you observed. See detailed tag info page.

It actually looks like a bug to me - a true bug (Hemiptera) nymph.  Unfortunately, it doesn't resemble anything I am familiar with, but luckily there are lots of bug specialists around - someone should be able to point you in the right direction!


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What is the name of this spikes insect?
Location is South India.
Behavior: It is not moving.
Month: May,
Place: No people come. (Actually species chooses the place on my next door, where neighbors left 2 months ago)

Can you add some more details? Location, type of habitat, size, how long it has been there, do you see them often etc.

@arboviral I added new HD image, is that quite enough to ID the species?

The Creature is "Debris-carrying Lacewing.".

(Source)

Attention: I get a high-risk warning when clicking the link!

Well then, I'm not going to click on it...


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Is this plant "Hedera canariensis"?
Location: South India
I found this plant beside bus stop.

Side view of the Plant.

Actually, Wikipedia says Hedera canariensis is "native to the Atlantic coast in Canary islands and northern Africa", but it is growing in India, so is it really Hedera canariensis?

What makes you think that could be Hedera canariensis? It does not look anything like it ...

Looking at your profile, you are using this community to provide the species identification for everything you find, until you find something nobody knows and you can finally declare you found an unknown species. For one, it is highly unlikely you will find an unknown species near a bus stop. Or anywhere near where humans are. Otherwise, please start to read up on how to identify plants and do some preliminary work. We are not some kind of "free expert service".

@AlwaysConfused Well! your plants exactly match with my plant, but I added side view of my plant. does it ring any bells?


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Why does my gel have such poor resolution?
Brand new user to bio stack exchange here. I ran this gel (0.9% agarose, run starting at 60V then to 105V over ~30-45 min) with DNA samples containing 20uL of DNA and 1X tracking dye in each of the wells. I do not know why the resolution of this gel is so poor. I'm assuming it's from DNAse activity, as this is what my lab tech said it probably was. I'm pretty sure I did everything right regarding the procedure. I'm not sure what other info I could provide, other than the samples were prepared and stored at -20C a week prior to the gel being run. Here's a picture of the gel below.

Are you running the products of a restriction digest? How clean is the water that your DNA is dissolved in (e.g. nuclease free)? What size fragments are you hoping to see?

Yes, we used nuclease free water in the samples. I was expecting ~5.8kbp and 0.7kbp fragments in the second lane, ~6.8kbp fragment in the the third lane... I could go on to explain all of them if its necessary, but fragments were expected in each lane.

That your marker is also poorly resolved suggests that your gels system is the problem. Can you assign sizes to the marker bands?

@canadianer By marker do you mean my ladder in the leftmost lane? If you're asking for a picture of what the ladder sizes should look like heres a picture.

How did you set up your gel?

My diagnosis: no buffer added when the gel was cast. I see this every year when a class of students pours and runs gels.

@AlanBoyd My suspicion, too.

@AlanBoyd, oh, so using water instead of buffer? I always thought that made the gel just melt when voltage was applied. (Or so we've been warned)

Although your tech is referring to your DNA samples, your ladder also indicates there is some room for improvement of your running technique. -20C for a week should not have impacted the quality of the DNA assuming that it was good quality going into the freezer. You can mitigate DNAase activity by adding EDTA to your storage buffer, which will pull metal ions out of solution. Just make sure to take that into account if you're doing a reaction later that requires metal ions (like PCR).
DNA
Focusing first on the DNA samples. There are a few things to check.
How much did you load? I see you stated 20uL, but what was the concentration? It it more important to know the amount of ng being used as the sample. Various stains have different concentrations they can detect (EtBr, SYBR, etc.) I would check the specifics of the stain you're using to make sure you're loading an appropriate amount of DNA.
Are these PCR products? If so, how many cycles was your PCR run? I have seen similar results from PCR if the samples are run for too many cycles. Non-specific products begin to be produced in later cycles, and will cause degradation of your specific products. I would also re-check your primers, the extension time, and the polymerase you're using. It is more difficult to do extensions of the 5.8kb and 6.8kb sizes you mentioned, but not impossible.
Are these restriction digests? Do you have a control well showing the undigested plasmid, or other source? Your source should have a clean band first to expect a clean band in the results. If your source is clean and bright, and the digest looks like the samples above, check your digest buffer and enzymes.
Electrophoresis
There are several things which can improve the quality of an agarose gel. Thermofisher provides a guide for their tips and tricks. Below are things that I have found useful in my own experience.

The time between casting the gel and running it. A common practice is to make gels and store them in TBE/TAE buffer until they're ready to be used. In my experience, the quality of the gel will degrade over time, yielding diffuse bands. The best results are achieved with a freshly poured and cast gel.
Loading technique. Carefully loading the samples into the wells so that they settle as closely to the bottom without mixing with the surrounding buffer is key. The best results are achieved by having your pipet tip touching the bottom of the well, and slowly pulling it out as you release your sample so the sample does not have to settle down into the well on its own.
Run immediately after loading, and image immediately after running. Diffusion will cause nice tight bands to become poor the longer the samples sit in the well.
Voltage, timing, and agarose concentration. Getting these right takes practice. You'll want to try to run your gel as quickly as possible. The more time it sits in the gel, the more diffusion will occur. However, have the voltage too high, and you risk melting your gel. Vary the concentration of agarose to target the size of DNA you're after. I have previously used anywhere between 0.5% and 1.5%.
Prestaining the gel or poststaining? I prefer prestaining for the stains that work well, as it reduces the time the samples will sit in the gel allowing diffusion to occur.
I am looking at your ladder, and the link to the provided standard, and it is difficult to tell which bands are which. The 3.0kb fragment should stand out as the brightest, however the bottom band in your gel is the brightest. Is this the 3.0kb band? Maybe. It's difficult to count the number of bands above it to confirm. If it's not, which might be indicated by the fact that none of your samples seem to have run to the end of the gel, then this would be the 0.5kb band. If it is the 0.5kb band, the ladder intensity doesn't match up with your expected intensity. Double check your ladder so you can know what sizes you're looking at.


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Do the recent findings of an ape-like creature in Europe with human-like teeth change the Out of Africa narrative?
http://www.telegraph.co.uk/science/2017/05/22/europe-birthplace-mankind-not-africa-scientists-find/
Two fossils of an ape with human-like teeth were discovered in Greece, and Bulgaria, and they are about 7.2 million years old.
Does this change the narrative of the 'Out of Africa' theory?
Have scientists in the relevant fields commented on this yet?

Of course every researcher wants to find something exciting and new. As the linked Telegraph article identifies their fossiles as the oldest hominins ever found, arguing that the lineages between pre-humans and chimpanzees therefore split in Europe, with pre-humans then migrating back to Africa to be consistent with other evidence.

Does this change the narrative of the Out of Africa theory?

Not at all since the Out-Of-Africa hypothesis is about the African origin of all modern humans (Homo sapiens), i.e. that the species originated in Africa and then spread over the rest of the world. It is not about where possible, early ancestors may have originated. The ape from the article, Graecopithecus freybergi is claimed to be one of the oldest Homininesa, but that does not change the claim of the Out-Of-Africa hypothesis.
Reference
a Fuss, Jochen, et al. "Potential hominin affinities of Graecopithecus from the Late Miocene of Europe." PloS one 12.5 (2017): e0177127. https://dx.doi.org/10.1371/journal.pone.0177127


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PCR a plasmid with self-complimentary regions?
I am trying to do a PCR using a plasmid as the template, pretty basic stuff. But the problem is that my forward primer just happens to bind to another location on the plasmid in the reverse orientation, in between the proper forward binding site and the reverse binding site. This isn't an example of non-specific binding, the sequences are perfect because the plasmid contains self-complimentary regions and my forward primer just happens to land right on it.
Here is a diagram:

Simply optimizing the melting point or magnesium concentrations probably won't help, because the sequences are the same. I'm doing this cloning to insert a reporter gene into the vector, and I need to place it very precisely, so I don't know if I can order a new set of primers, the insert needs to fit there.
The bad PCR product is about 500 bases shorter than the good product, but I only see the bad band on gel when I run a normal PCR. If I reduce the amount of forward primer and increase the reverse oligo, I can see the good band, but the bad band is still there and the total yield is much lower.
So my idea right now is to do an asymmetric PCR using the reverse primer only, then add forward primer to create the double-stranded DNA, then separate the good and bad bands using gel electrophoresis. But I don't have a clue how many cycles I should run each step, or how much forward primer to add, etc. Additionally, asymmetric PCR has low yield due to arithmetic amplification, but once I add the forward primers it can begin symmetric PCR and geometrically amplify both bands, but I think the shorter bad band wins out.
Does anyone here have experience with this sort of complex PCR, or suggestions to improve my yield?

What's the problem wtih using a lower percentage gel and running it for a long time on low voltage to separate the two bands and cut out only the longer one you want?

Can you amplify two separate regions that meet at a restriction site and reconnect them by ligation? Without knowing the exact sequences and purpose, I'm afraid it's hard to give clear recommendations, because the best way to approach this kind of thing tends to be highly specific.

@Armatus When doing the PCR using equal amounts of both primers, I did not see the desired 3000 base band, only the bad 2500 base band on gel. The ladder had bands at both 3000 and 2500, so I know the bands should have resolved if they were both present in the PCR sample. The shorter band must be winning out. I was able to see both bands when I used much more reverse primer than forward primer, but the bands were very faint.

@JackAidley I designed the cloning procedure around InFusion cloning, but that system can work with multiple fragments as long as they have a 15 base overlap. I can probably design a couple more oligos that bind on top of a restriction site. That will let me cut the plasmid and PCR the shorter pieces with overlapping ends in separate reactions, then mix all the parts together for the InFusion reaction. I'll let you know how it works, but it will take time to get the new oligos.

@user137: if you combine the fragments by PCR won't you encounter the exact same problem with your fusion (aka stitch) PCR?

@JackAidley I don't think so. If I find a good restriction site I can cut the plasmid at that site and create a new set of forward and reverse oligos that bind at the cut end. I can run 1 reaction using the blue primer from above and the new reverse primer, and the other reaction using the green primer and the new forward primer. The blue forward primer can still bind to the bad red site, but if the plasmid is cut between the red and blue sites it shouldn't amplify. I've done a similar thing using this same plasmid at a different site, where I had direct repeats around an EcoRV site.

@user137: That's not what I meant. You suggested combining the fragments at the end using an overlap; wouldn't your fwd/rev primers for the final product be your current ones?

How much longer would the forward primer have to anneal uniquely to the one locus?

@JackAidley The InFusion reaction doesn't use PCR to assemble the fragments. Each fragment is created by separate PCRs, but the assembly is carried out using proprietary enzymes that can attach fragments with a 15 base overlap. This manual might explain more than I can

@swbarnes2 At least 9 bases, but it's already 30 bases long. Of course a 39 or 40 base oligo isn't impossible.

@user137 great that should work then.

From experience, your present problem will not completely go away no matter what you do.  The idea for asymmetric PCR is not a bad idea. However, you will only get linear amplification of the desired product from the initial single primer, so you will not "see" any product even after 30-40 cycles.  This first single primer PCR might also cause downstream problems with amplification of undesired products made during the second PCR (junk products).
I agree with comment #1, but then neatly cut the "visible" "good" band out of the gel.  Suspend (crush) the agarose band in sterile TE buffer, and then use an aliquot of that as template for a secondary PCR (use equal primers - maybe 30 cycles).  This should provide more than enough "good" starting template.
Alternately, you could design a new forward primer that does not have a bad secondary location on your plasmid.


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What are some of the general characteristics of the DH5 alpha strain?
I can not find some useful sources unfortunately.
Please tell me about some important characteristics of DH5 alpha.
What makes DH5 alpha suitable for the gene cloning?

There are two possible sources: One is the article from the Bethesda Research Laboratories (reference 1), which names a few, the other is the book "DNA cloning: A practical approach" (see reference 2)., which sits here in my bookshelf.
Taking both together, the DH5 alpha strain is derived from the DH5 strain with the introduction of a few additional features. The naming DH are the initials of Douglas Hanahan which developed the strain. The strain is easy to transform with high efficency.
The new features of DH5 alpha are the recA and the endA1 mutations:

The endA1 mutation inactivates an intracellular endonuclease that
degrades plasmid DNA.
The recA mutation eliminates homologous recombination. This reduces the chance for deletions and plasmid multimerization.

The DH5 strain also has the following features:

The hsdR17 mutation eliminates the restriction endonuclease of the
EcoKI restriction-modification system, so DNA lacking the EcoKI
methylation will not be degraded. DNA prepared from hsdR strains that
are wt for hsdM will be methylated and can be used to transform wt E.
coli K-12 strains.
Δ(lacZ)M15 is the alpha acceptor allele needed for blue-white
screening with many lacZ based vectors.

Of special interest are the references 3 and 4, which gives you all the details you want to know.

References:

Bethesda Research Laboratories. 1986. BRL pUC host: E. coli DH5α
competent cells. Focus 8(2):9. The article is on page 13 in this paper.
DNA Cloning: A Practical Approach. Glover, D. M. (ed.), 1985, Vol. 1, p. 109, IRL Press, McLean, Virginia
Choosing a Competent E.coli Strain
E. coli genotypes

Oh, I've forgotten about it. Sorry!


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human anti-mouse antibody
I have heard about human anti-mouse antibodies (HAMAs) and read that HAMAs neutralize murine antibodies, therefore decreasing the effectiveness of those murine antibodies. Is this true that HAMAs neutralize the murine antibodies? How?

@user137 please don't answer questions in comments. Write a full answer instead.

@MattDMo It's fixed, just took me a while to get the protein sequences aligned. I don't like to make answers without some evidence.

@MattDMo - I'm curious, this is second comment regarding answering in comments I've seen today. I wasn't aware this is normal policy. Personally I often leave a brief answer in a comment if I don't have time (or patients) to write a full good quality answer - I just put the comment in, if someone wants to take it for an answer thats fine by me. Perhaps there needs to be a meta post to discuss policy? (I don't know the extent of the answer that was left as a comment here, but certainly on the other I saw a user was told to put a quick one line comment in to a proper answer)

@GriffinEvo see this meta post for a previous discussion on the phenomenon, and of course this recent related post as well. Regarding the (now-deleted) answer-as-comment here, it was probably 6 or 7 lines long, and covered all the points in the answer below, with the exception of the alignment.

As mentioned in the comment, all antibodies have constant regions and variable regions. The variable regions are the binding sites on the ends of the two arms, and the constant regions are the rest of the molecule. When humans are injected with murine antibodies, the immune system recognises and sets up an immune response to them, through generation of HAMA. This affects the efficacy of the antibody because it is attacked the same way a pathogenic substance would be. Antibodies have different effects, but the most important one in this instance is through opsonisation (http://en.wikipedia.org/wiki/Opsonin#Examples). Because antibodies have two arms, they can bind multiple targets and cause a grouping and clumping. This also causes activation and phagocytosis through phagocytes (generally macrophages) which will ingest and destroy the therapeutic antibodies.
Antibodies that are mostly human with just mouse variable regions have been created to try and counter this problem, known as chimeric antibodies. Humanised antibodies are also available, which are entirely human with only the very tips of the variable regions (known as the hypervariable regions) are from mice. This reduces the problem as far as possible (http://en.wikipedia.org/wiki/Fusion_protein#Chimeric_protein_drugs).

It's common for the human immune system to create antibodies against many proteins, even some human proteins. Hemophiliacs who receive regular doses of clotting factor proteins often develop neutralizing antibodies against the clotting factor proteins, even though they are a human protein1.
Therefore it's not surprising that antibodies would be developed against mouse antibodies. There are differences between mouse and human antibodies, and human antibodies may bind to these different regions. I found the amino acid sequences for human2 and mouse3 immunoglobulin gamma heavy chain constant region and aligned them by hand and marked the matching amino acids:
Human: MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARECDNWF DPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPC      PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
       |   |  |||| || |||||||||||||||| ||||  ||||||||||| | | |||||| ||||||| |       || | |||||||||||||| |  || ||| |||| ||||||      | |||||  ||||| |  ||| ||||    | |    |||||| |||| ||| |||| | | |||||| ||| |||  || ||||||    ||  | | |  | | |||| ||     |   ||||      ||| | |||||| |||  || |||| || |||||||||  || |   | |  |||| | |    | |||| |||| |   ||||  ||| |||| || || ||| |||| ||  | |||| |||    |    ||||||| || | || ||| ||   | ||| | |||||||| | ||||    | |     ||| | || | | |  |  | ||||
Mouse: MDSRLNLVFLVLILKGVQCEVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYINSGSTTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARELWLRRIDYWGQGTTITVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQS GLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

As you can see, there are a lot of matching residues, but several differences. Keep in mind that antibodies also contain light chains, and that there are several other types of antibodies, I just used the IgG heavy chain as an example.

is there a way to prevent this? Also, how does this neutralize the antibodies?

This could be prevented by using human antibody genes in cell culture instead of growing antibodies in mice and extracting them from the blood. As far as neutralization goes, the human antibody may just mark it for uptake by white blood cells.

user137's comment about opsonized particles being marked for phagocytosis by macrophages is right on. There are a number of ways that antibodies can be neutralized (e.g. steric hinderance), but being eaten up definitely does the trick.  However, growing antibodies in culture won't solve the problem. They are recognized, in large part, just by their Amino Acid sequence, rather than any culture conditions (you might make an argument for glycosylation). The variability of antibodies will always make them vulnerable to immune responses. There are even HAHA (human anti-human antibody responses.


text:
Question about cutting gene in Plasmid
I am designing a researching proposal for the class. Because it uses microinjection to up-regulate the gene in C. elegans, the plasmid pCFJ104 - Pmyo-3::mCherry::unc-54 sequences has been chosen. But because the plasmid already has a unc-54, I wonder if there a way that to cut the unc-54 out of the plasmid?

Can you give more information on what the unc-54 gene does? Try and be a bit more clear maybe even a picture or link of the plasmid with the unc-54 location, thanks.

unc-54 encodes the major bodywall muscle myosin. In these plasmids the only portion from unc-54 is usually the 3'-UTR and the polyA addition site (AATAAA). myo-3 is either another BWM myosin, or a pharyngeal-specific myosin. This is providing the tissue-specific promoter. The expression plasmid drives expression of the mCherry fluorescent reporter in muscle cells.

pCFJ104 - Pmyo-3::mCherry::unc-54  is the plasmid I choose, here is the link: https://www.addgene.org/19328/; because the experiment need to up regulate a gene that is also in the muscle, so before doing the microinjection, the unc-54 need to be cut out before insert the gene I use. So I am wondering how to cut the gene out. Thanks!

Following the link you provided confirms that the C. elegans expression vector will express your insert of choice in the transgenic animals' pharyngeal muscle cells (driven by the myo-3 promoter).
From examining the two images of the plasmid's restriction map on the page you linked to, I infer that a SacI x XbaI double digest should excise the mCherry insert, and be suitable for sub-cloning your fragment of choice.
However you will need to confirm this for yourself. In general, I think it extremely unlikely that you would find an individual on Biology SE who could provide such precise information as "what enzyme sites should I use for my sub cloning experiment."
A general approach would be to

Research to find out if you can obtain a fasta file containing the complete DNA sequence of the starting plasmid.
Search GenBank to see if there is a record containing annotations for the starting plasmid sequence--that will tell you the start points and end points of each functional region.
If the plasmid is not in a sequence database try using the sequence in a BLAST search of a Non-redundant sequence database. In this example you would have retrieved high scoring hits on the unc-54 gene, the myo-3 gene, the mCherry gene, the beta-lactamase gene from pMB9/pBR322/pUC, and the E. coli lacZ gene (among others).
You should also consult the original publication (if there is one) because it may contain additional useful information, including how to contact the authors--who may have sequence files, useful maps, etc.
With the sequence in hand you will be able to create your own restriction map using a suitable software program (there are many commercial ones), e.g. the EMBOSS suite of open source bioinformatics software.

In your case most of this work was done for you because the AddGene site had two annotated restriction maps that showed almost all of the information you needed.  N.B. One map had the locations of the useful restriction enzyme sites, and the other map had the annotations showing the extent and locations of the worm fragments and the reporter gene. It was only by integrating the information from both maps that I was able to suggest one possible solution to your query.


text:
Why some parts of the human body have immune privilege?
Why have the eye and CNS have immune privilege? Why does the body not develop tolerance against their tissue and instead risk their damage in case an accidental immune cell infiltration?
Wiki:

...in immune privileged sites, tissue grafts can survive for extended periods of time without rejection occurring

i added quote from wiki and a link, please feel free to remove it if you feel so. but I would encourage you to read that wiki page on immune tolerance

The immune privilege is happening for brain, CNS, eye, testis and the uterus. All these tissues (or structures) share that they cannot be regenerated once they get damaged by an overshooting immune reaction. Infections in the eye followed by a strong immune response often lead to the loss of the eye, in rodents loss if immune tolerance against the fetus leads to abortion.
So basically this is a protective mode for these tissues to protect them from a stong immune reaction. See the references for some more details (especially on how this works).
References:

See no evil, hear no evil, do no evil: the  lessons of immune
privilege
What is immune privilege (not)?
Immune privilege or privileged immunity?
Immune Privilege and the Philosophy of Immunology

Each immune privileged tissue has a unique function. The eye must protect the light path and signals that stimulate the retina, and photoreceptors to preserve vision. The testis has to protect the sperm as they proceed to the epididymis where they mature. The maternal reproductive tract has to protect its eggs both before and after fertilization and thus has developed specialized mechanisms to modify the body’s response to foreign antigens. These unique challenges require different solutions and lead to unique immune privilege mechanisms. However, although microenvironment and the stromal cells that carry out the particular function may vary between tissues, and consequently the mechanisms that promote regulation may be unique to that cell or tissue, the goal is the same: limit collateral damage to preserve tissue integrity and maintain homeostasis to the extent possible, without compromising host defense.


text:
Why is second pKa value of phosphoric acid different across different sources?
Am I missing something very fundamental here for phosphoric acid's second pKa value?

I check the Wikipedia page and these are the values: (1) 2.148, (2) 7.198, (3) 12.377
Then I check Sigma-Aldrich website and it says: (1) 2.15, (2) 6.82, (3) 12.38

Why is the second pKa value different? Any ideas?
I need to prepare potassium phosphate buffer for my proteins and got stuck with this simple problem.

for the reasons that alan outlines below you usually prepare buffers and then adjust them with a pH meter rather than preparing them from a recipe.

Yes, I did exactly that; I was just curious why there are two different values :). Also, I didn't want to introduce a lot of ions by using excessive acid/base when adjusting pH. It is best to start with the correct recipe to end up with the correct molarities of ions in the solution.

I used to mix 1M stocks of each H2KPO4 and HK2PO4 to avoid unknown NaCl from NaOH and HCl, but this was also wasteful...

Someone will probably come along and move this to "chemistry", but here goes anyway.
The answer is: it depends upon how you measure it.
See:

Jungas, RL (2006) Best literature values for the pK of carbonic and phosphoric acid under physiological conditions. Analytical Biochemistry 349 1-15

The author states that   "..the best value for the true thermodynamic second dissociation constant of phosphoric acid in pure water at 38°C is 7.180, rounded to the nearest 0.005."
He then goes on to re-analyse the data reported in:

Cohn, EJ (1927) The activity coefficients of the ions in certain phosphate solutions: a contribution to the theory of buffer action. J Am Chem. Soc. 49 173-193

and concludes that under physiological conditions of ionic strength and pH the best estimate is 6.795.


text:
What is the translation termination efficiency in mammalian cells?
When I express proteins in bacteria I put at least two stop codons at the end of the gene to increase the termination efficiency. Is this the case in eukaryotic cells too? If I put a single stop codon is there a risk for the ribosomal complex to readthrough and continue translation?

There is no such practice as far as I know. Personally I have never done that and things work fine. You can add a polyadenylation signal, though.
Is this a synthetic gene or something ?

From what I understand the eRFs are not perfect but they still work better than RF1 and RF2 which allow for a non trivial amount of readthrough.

This is a synthetic gene. I already designed a primer with 3 stop codons at the end of the gene. I will post here again if I see any improvement in gene expression (with compared to other similar constructs in our lab).

There is a paper for transfection of mammalian cells here which has a bit of comparison of stop codon and protein yield.
Otherwise this article suggests having a 'rare' codon after the stop to prevent readthrough. But yes, I'm not sure why one wouldn't simply put two stops after each other.


text:
Where to put the gene after eukaryotic promoter for best expression levels?
As far as I know there is an optimum distance between a promoter and the gene for the best expression levels. What is that distance for common promoters like CMV, SV40? If you have a first hand experience about this (e.g. if you have cloned the gene way downstream of the promoter and didn't/did see expression) and share it I will be grateful.
If you have comprehensive knowledge for other promoters please feel free to add them in the list too; they don't need to be common. I just want to hear people's experience on the subject.

a 10bp linker is sufficient. From all your other posts I gather that you are trying to build a synthetic system. My personal strategy for things like these is to pick up sequences from the vectors in your lab that you know work quite well.

Eukaryote is rather general - different eukaryotes have vastly different regulatory systems. Are you talking about something like human or something like yeast?

Take a look at the sequences for some common/popular vectors for your target organism. As WYSIWYG pointed out, it doesn't need to be very big, and it can vary somewhat depending on where in the multiple cloning site you insert your gene of interest.

Thank you all for your comments. I did look to other common constructs and decided to leave around 40 bp between promoter and the initiation codon (this was the minimum distance I could leave because of the restriction site limitation). @Bitwise, I am using mammalian cells (specifically; fibroblast and MDCK cells).

I used pEYFP-N1 (stock vector) where the spacing between the CMV promoter and the EYFP start codon is ~88 nt, and the expression is high.
I also cloned a protein 5' of the EYFP, only 4 nt after the CMV promoter. The expression of this fusion was also high. Because I was only using the chimeric protein in experiments, I did not try to assess differences in EYFP expression.
If you look here: https://synbiota.com/projects/19/sequences you'll see the basic pEYFP-N1 vector and the separation between the CMV promoter and the EYFP coding sequence.
On wiki: "The TATA element and BRE typically are located close to the transcriptional start site (typically within 30 to 40 base pairs)."


text:
What keeps mitochondria from multiplying out of control?
What keeps mitochondria from multiplying out of control and killing the cell?
According to endosymbiotic theory, mitochondria were once free-living bacteria that got ingested but not metabolized by the host cell. What prevents mitochondria from replicating out of control and overpowering the host cell? The question was inspired by this brainstorming session about engineered endosymbiosis.

Could you please add some more details or show some sign of research on your own? E.g. any citations on mitochondrial replication?

It seems like a pretty straightforward and self-sufficient question. What would be the purpose of adding such a paragraph? I'm not against it, I'm just trying to understand the rationale

well, as your accepted answer begins, "I'm guessing at the motivation of your question..." I think jamesqf does a good job of rephrasing the question into something that's a lot easier to answer.

err. i mean acvill. those are more answerable questions. Additionally, you should have provided your brainstorming link in the first place if that was where you were coming from.

I hope this fixes it

I'm guessing at the motivation for your question -- In the evolutionary history of eukaryotic cells, mitochondria were once free-living bacteria.  What prevents them from acting as intracellular pathogens and replicating out-of-control?
Yes, by endosymbiotic theory 1, mitochondria were once free-living prokaryotic organisms.  This is evident because they have their own DNA, undergo replication by fission, and have characteristics similar to proteobacteria.
However, some of the genes required for mitochondrial replication have been moved to the nuclear genome.  This means that, while mitochondrial replication is not necessarily tied to cell replication, factors encoded in the nucleus are necessary for mitochondrial replication.2

https://en.wikipedia.org/wiki/Symbiogenesis
Ali, et al. Nuclear genetic regulation of the human mitochondrial transcriptome. eLife 2019;8:e41927.

That's all I needed to hear:) The question was actually motivated by this brainstorming session about engineered endosymbiosis https://brainstorming.com/r/s164

I think we might have to go back further than the present situation.  A bacterium couldn't become a successful endosymbiont if it multiplied out of control and killed its host, and it would seem that this non-lethal behavior must have existed long before evolution moved the genes around.

Good point. I will set the question as not answered to see if we can come up with something that accounts for this. Dirigible did solve the question I had in mind, but now that you mention this plot twist, I would love to know:)

jamesqf's point is a good one.  However, the original question was "What keeps mitochondria from multiplying out of control and killing the cell?", not "What factors contributed to the intracellular symbiosis that led to mitochondria?" Post a new question (with preliminary research / thoughts) if you want a new answer. It's poor etiquette to post a question, accept an answer, add more questions, and remove your accept.

understood. will fix it

@RetardiGrade Thank you. Of course, if someone provides a better answer to your original question, feel free to accept that answer. https://meta.stackexchange.com/a/93970/722486.  I encourage you to ask you "sub-question" as a new question, as it will likely generate interest.

@RetardiGrade I think you should look up retrograde signalling between the Nucleus and mitochondria if you would like to know the molecular mechanism regulating mitochondrial numbers.