CharonY

Regarding GFP, it depends, how do you want to detect the GFP? Moreover, what is the strength of the promoter controlling the gfp gene? Regarding siRNA I have not enough personal experience on this issue. I can only imagine this happening if there is a massive production if the respective siRNA. I can more easily imagine a transfer of the whole vector, though.

I am still not quite clear what the objective of the experiment is supposed to be. Do you think that radiation might cause mutations that in turn increase resistance to UV radiations? Improbable, but not impossible. However you will only end up (if you are lucky) with UV resistant strains. To demonstrate which mutations were caused is not possible within a normal project budget though (less if it is some kind of course work or undergrad project). Let's go to the suggestions: - Sequence the genome: in theory it would work, but the price tag and effort to sequence even a small bacterial genome is enormous with traditional Sanger. Newer parallel sequencing methods based e.g. on pyrosequencing are cheaper, but still cost around 15-20k$ per single run. That's about the budget for a phd student for a year. -use BLAST: BLAST is basically a quick global alignment program used to identify homologous genes/proteins. It is unsuited to detect point mutations (the most likely mutations caused by UV radiation). But regardless, as you got no idea what is going to be mutated you either have to sequence the whole genome, or pick up potential genes involved in UV protection (the P. aeruginosa genome is published), make PCR and check the products for mutations. - demonstrate that UV causes mutation: that's easy to do (as pointed out by DrDNA), though I am not sure whether that's what you want to see. My final suggestion depends on what kind of project this is (e.g. practical course, undergrad studies, etc.). You can, for instance make random mutants on a global scale using marker carrying mutations (e.g. transposons) and then select for mutant strains that become UV resistant. Using the marker you can then identify the gene that has been mutated. However chances are rather low that mutations will generate resistant strains. And it is somewhat work intensive to create a transposon bank and screen it.

While that may be off-topic, I'd be interested to know how biology training is less rigorous than physics, for instance. What one can immediately agree on is the better mathematical foundation of most physics projects, but what else?

Of all the answers Ecoli is the only one really hitting the spot. BTW, the OP is a bit unclear which amino group is supposed to be protonated: the one in the backbone, involved in peptide bonds, or that in the residues of a few amino acids (e.g. lysine, asparagine). Specifically this bit is misleading: In a protein the amino group would form a peptide bound and thus will not be free for protonation anymore. Regardless on the details, under physiological conditions peptides are always in aqueous conditions. Thus they are surrounded by a given concentration of H+ or OH- ions, depending on the pH of the solution. Actually MrSandman already demonstrated that the charge is a protonation event (and not a loss of electron charge): See? NH2 -> NH3+ (addition of H+, or proton). Unfortunately here is another serious misconception what amino acids are. Maybe you should check on Wikipedia (did I really say that). The backbone of all amino acids is (+)H3N-CHR-COO(-). The "R" is just for residues as the various amino acids simply have different residues. The simplest amino acid is glycine in which R is simply a hydrogen. Now, if amino acids form proteins it is realized by above mentioned peptide bonds under loss of a water molecule: (+)H3N-CHR-CONH-CHR-COO(-) Note that the bonds are not depicted properly here in this text form, but you'll find information about that basically everywhere (how about textbooks )

First, please use less exclamation marks. Second, as you correctly noted, Prevotella and Bacteroides were initially regarded as one genus. As such the diagnostic tools used to identify them (usually specific 16s primers) were not able to distinguish between them.

Lactose is split into glucose and galactose which can be used normally as carbon sources. If it is not metabolized (as in those with lactose intolerance) the lactose stimulates bacterial growth in the bowels with rather negative effects.

What do you mean with "immediately get disappeared"? Are you referring to cell lysis?

Here they are: - Act with skill and care, keep skills up to date - Prevent corrupt practice and declare conflicts of interest - Respect and acknowledge the work of other scientists - Ensure that research is justified and lawful - Minimise impacts on people, animals and the environment -Discuss issues science raises for society -Do not mislead; present evidence honestly Most successful Profs are disqualified for the first point, though

Well, genes encoded within transposons and viral components (unless deactivated) are coding for functional proteins of course. But agreed, probably the promoters of ID are more referring to genes coding for more complex cellular features than that. That I understood, but as I meant that this is just an assumption. I have for instance not found any data as of how much of the library contained bacterial sequences. If it was truly inserted in the genome, one would expect a similar frequency as for human inserts. Undeniably in almost any eukaryotic library you will find bacterial DNA that are really contaminations and just claiming all of them to be inserts is imo a bit far fetched. The inheritence of Wolbachia sequences is somewhat convincing, the sequencing of a wild-capture is not. The latter by itself does not give information about stability, not about presence as you cannot track down the time of infection. Moreover, apparently not all Drosophila lines were cured of Wolbachia, first (S2). As Drosophila are commonly infected with WolbachiaTracking more (cured) generations would be a better indicator of stability. Regarding the expression of genes, apparently the authors normalized the Wolbachia genes against the act5 gene (unless I misunderstood it), but this is technically not correct as it does not account for sequence dependent differences in the PCR efficiency. I'd love to see the cut-off.

Different kinds of cells have different selective pressures in maintaining small genomes. Basically, unicellular organisms, especially prokaryotes usually keep a smaller, more densely packed genome than eukaryotes. Pioneers theory regarding charges does not really make sense, btw. DNA in eukaryotes is located in the nucleus, a compartment well away from the cell membrane. Prokaryotic DNA on the other hand is free in the cell, closely associated with the membrane, but they do not accumulate that amount of so-called junk DNA (the potential function of these have been mentioned by various posters already). Moreover the charge is evenly distributed across the DNA. Thus a larger DNA has the same charge per stretch as a small one. If that wasn't the case gel electrophoresis wouldn't work as it does.

I wonder why they would argue like that at all. Genetic mobile elements (like transposons, viruses or integrons) are known for a long time to integrate new genes into organisms. What is surprising here is the sheer amount of genes in one go. On the other hand if one just takes a look at how many sequences found in databases are of putative viral origin it ain't that much after all. Still, I see the problem of distinguishing sequences "real" chromosomal sequences from co-purified intracellular parasite sequences before any more conclusions can be drawn. Also it would be interesting to see how stable these insertions are. As for instance plants bioengineered by Agrobacterium often do not inherit the additional information (as it harshly reduces fitness). Also, sequences from mobile genetic elements appear to be stable mostly in an (apparent) inactive form.

Just to clarify, evolution requires that there are a means to have genetic variation. Mutation is one of these. However, mutation itself does not lead to evolution. In other words, wherever you heard that mutations equals evolution or is the only force accountable for evolution, it is plainly wrong. Mutation is but one of the mechanisms of evolution.

This question is either crap or cunningly formulated (I opt for the first). There are both unicelluar prokaryotes as well as eukaryotes (protista). However, there are also prokaryotes with multicellular stages (e.g. certain myxobacteria). Not knowing for which levels this questions are intended for I do not really want to comment on the answers in depth.

Bacteria do not that commonly insert pieces of DNA (either accidentally or with purpose, so to speak) in eukaroytes. At least, to date relatively few examples are known. The majority of these are parasites, of which Agrobacterium is probably the best known one. It injects genes controlling growth and opine production into its host plants. The plants is induced to produce tumour-like cells that overproduce opines which is then used by the bacterium. This process is also exploited for biotechnological purposes for the creation of transgenic plants. This is an example for natural directed mutagenesis. The large scale DNA exchange as described in the article is of course less common (hence the high-profile publication in Science). For example, mitochondria formation occurred only once until today. Viruses on the other hand are nothing more than DNA/RNA injectors, however their entire genome consist only of a couple of genes. Their contribution to evolution of e.g. the human species is well recognized, though. Most genomes, even those that have a selection on genome size, like for instance prokaryotes, are abundant with phage sequences (often inactive) or other mobile genetic elements (e.g. transposons, IS-elements and so forth). In evolutionary terms these additions of external DNA basically enhance the overall variety of the gene pool. They can be essentially regarded as a kind of mutagen.

Why would you want to finish off all the taxi-drivers and burger flippers? ;P

Viruses of course do not grow. Agents that deactivate viruses are aggressive substances as hydrogen peroxide, bleach etc. that would of course also harm cells. Also for the record, fungi are eukaryotes, most substances harming them is also potentially toxic for other eukaryotes, as humans. That being said, fungi cells do possess certain features distinguishing them from mammalian, as the presence of ergosterines in the cell membrane. These are then potential targets of antifungal substances. However, due to the large similarities between mammalian and fungal cells there are overall less potential targets. Finally, for bacteria there are a number specific agents like antibiotics. Their specificity for bacteria is of course, as mentioned, due to the large cellular differences between prokaryotic and eukaryotic cells. Examples are prokaryotic ribsomes (a common target for antiobiotics), which are structurally very different to eukaryoticy ones, or bacterial cell walls (eukaryotes either have none or e.g. in plants one with a very different chemical composition), bacterial LPS and so on. In essence these compounds simply target elements of the target cell that are not present or are structurally different in mammalian cells.

Precisely. To date there is no compelling evidence whether mobiles are harmful to health. It is only known that they do have physiological effects (as arguably any environmental stimuli), and this paper is to my knowledge the first that describes mechanisms involved. At the moment this and similar papers are only giving food for thought.

D'oh, I see now. One small problem is possibly that even at a higher band a low amount of small cDNA might be co-isolated (agarose gel are not that stringent in seperation). That kind of contamination usually doesn't hurt for direct cloning, though they might get co-amplified in PCR. But that protocol is short enough to be worth a try nonetheless

I do postgenomics stuff (proteomics, transcriptomics, metabolomics) for the most part. Also gearing towards biophysical methods a bit.

I just stumbled about this interesting paper. It has been reported earlier that EM fields of mobiles effect cells, including expression changes. Here a direct mechanism has been elucidated in which the EM field induces NADH oxidases to release reactive oxygen species, which in turn activate a regulatory cascade. Abstract: The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes. Friedman et al. Biochem. J. (2007) 405 (559–568)

Hmm i probably don't get it. Lemme see if I understood you: First you add a random primer with a 5`tag for RT. Result are random cDNA fragments from long and short RNAs with a tag. Due to the vast amount of small RNAs probably the majority of the cDNA will have a small RNA origin, unless they are lacking the random primer binding site. Then you add a primer complementary to the tag and make another one sided PCR. Am I already missing something here? Because I do not see how the additional PCR step helps to select for the larger fragments. Or was your plan just to get enough cDNA and simply separate it on gel? In that case I'd either use a polyA tailing reaction to the RNA to get polyA mRNA, or use a terminal to label the cDNA. That way you can use two primers to increase your cDNA yield and at the same time increase your chance to get your full mRNA sequence.

Ah, regarding the fishing idea, I omitted that I would have used biotinylated DNA, so that I can digest them away with DNAse. I was very unclear and imprecise. Sorry about that (lack of coffee and such). Your approach probably would als work, but wouldn't the small RNAs also be reamplified at a higher rate? Personally I prefer to recover DNA of specific lengths with nano-LC then to reisolate them from a gel, but that is down to personal preference (and equipment).

Too bad I am caffeine resistance, then

Biology was always the most attractive natural science for females (maybe except psychology, though it can be disputed whether psychology as a whole plays in the same league as biology) and their numbers are still increasing. In the last few years in many universities (depending on country) there are already more female starting to study biology than males. Many males interested in biology move over to the new established fields of biotechnology and bioinformatics instead. That was just to follow my usual habit of random quoting and then adding something vaguely related to it. Now to another part: interest in science is of course a good thing. One problem is however is that in order to truly understand something at least a minimum of effort has to be invested (regardless whether you are trained scientist or layman). Quite a lot of people shy away from that part. Also, coffee might help, but tea is also acceptable. Though I suspect that sleep deprivation is more indicative.

Sorry, I have to retract part of my statement. I remebered using a pH 8 buffer at one point, but it wasn't my storage buffer. Elastase has a higher solubility and activity at alkaline pH (that's when I used a pH 8 buffer), but for storage I also used a buffer around 5.5 in order to avoid activity loss. So ideally store it as powder. After resuspending it, you should aliquot it (for one assay each) and store at -20°C which should work for at least a month (if memory serves). At 4°C it loses activity after a week or so (in my hands).