Non-functional DNA: non-functional vs. inconsequential.

Each copy of the human genome consists of about 3,200,000,000 base pairs, and includes about 500,000 repeats of the LINE-1 transposable element (a LINE) and twice as many copies of Alu (a SINE), as compared to around 20,000 protein-coding genes. Whereas protein-coding regions represent about 1.5% of the genome, about half is made up LINE-1, Alu, and other transposable element sequences. These begin as parasites, and some continue to behave as detrimental mutagens implicated in disease. However, most of those in the human genome are no longer mobile, and it is possible that many of these persist as commensal freeloaders. Finally, it has long been expected that a significant subset of non-coding elements would be co-opted by the host and take on functional roles at the organism level, and there is increasing evidence to support this.

A notable fraction of the non-genic portion of human DNA is undoubtedly involved in regulation, chromosomal function, and other important processes, but based on what we know about non-coding DNA sequences, it remains a reasonable default assumption — though one that should continue to be tested empirically — that much or perhaps most of it is not functional at the organism level. This does not mean that a search for the functional segments is futile or irrelevant — far from it, as many non-genic regions are critical for normal genomic operation and some have played an important role in many evolutionary transitions. It simply means that one must not extrapolate without warrant from discoveries involving a small fraction of sequences to the genome as a whole.

More generally, it has been known for more than 50 years that the total quantity of DNA in the genome is linked to nucleus size, cell size, cell division rate, and a wide range of organism-level characteristics that derive from these cytological features. Thus, large amounts of DNA tend to be found in large, slowly dividing cells, which in turn typically make up the bodies of organisms with low metabolisms, slow development, or other such traits. On this basis alone, one would expect to see consequences for the organism if a large quantity of non-coding DNA were eliminated from or added to the genome, even if most of the particular elements in question were neutral or detrimental under normal circumstances. Non-functional is not equivalent to inconsequential. This is especially true when there are factors operating at different levels, for example when an abundant and diverse collective of entities includes components that are variously neutral, beneficial, and detrimental to a host.

Though they cannot prove an argument, analogies are often useful for understanding an issue. In this capacity, consider the following:

  • There are roughly 1013 to 1014 individual microorganisms living in your digestive tract (Gill et al. 2006), which is on par with, or perhaps even 10x larger than, the number of cells making up your own body. It is also two or three orders of magnitude larger than the number of humans who have ever lived, and of the number of stars in the Milky Way galaxy.
  • The assemblage of microorganisms in your intestines comprises some 500 species, most of which have never been cultured in the lab or studied in detail (Gilmore and Ferretti 2003). To put this diversity in perspective, there are only about 5,000 species of mammals on Earth today.
  • The combined “metagenome” of the microorganisms in your gut contains at least 100 times as many genes as your own genome (Gill et al. 2006).

We do not know the specific characteristics of many of the microorganisms in the gut. However, we do know that at least some of them are essential, or at least highly beneficial, for human health. Several of the species found in the gut are important mutualists, assisting with digestion and in return drawing nutrients from the food that we consume. In this sense, it is hard not to agree with Gill et al. (2006), who argue that “humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes”.

The question is, are all 10,000,000,000,000+ microbial cells that we carry with us functional for our well-being? Some certainly are. But many, maybe even most, are probably commensal freeloaders who neither harm nor benefit us, though of course their total abundance is limited to what can be carried by the host without deleterious consequences. By contrast, some gut bacteria are implicated in gastrointestinal disorders. A few are actively parasitic, but their numbers may be kept in check by our own immune system or through competition with non-pathogenic species, or because they kill the host or are killed by antibiotics. Some, such as the well known Escherichia coli, can be harmless or deadly depending on the presence of particular genes. Thus, the total number of microorganisms, and the relative diversity of species that this encompasses, is influenced by a complex interaction of factors internal to the gut (e.g., who invades, which microorganisms are already present, how efficiently they reproduce) and higher-level conditions (e.g., human immune response, dietary effects on which nutrients are present, positive or negative effects on the host).

What we know about bacteria and other microorganisms makes for a reasonable default assumption that much or even most of what is found in the gut is not there because it provides a direct benefit to humans. On the flipside, we have good reason to expect that some, perhaps even a large fraction, of these organisms are beneficial. Therefore, we require evidence to show that any particular species is functional from the human point of view, and that its abundance is determined on this basis. The search for such evidence is important, but it occurs against a backdrop of realizing that bacteria could be there for their own benefit only, whether or not that has any adverse effects on our well-being as hosts. Establishing that a specific strain of bacteria in the digestive tract is beneficial does not justify the conclusion that all bacteria in the gut are mutualistic. It does not even imply that all individuals of the helpful strain are essential, because the optimal abundance for the host and the pressures for reproduction of the microorganisms may not converge on the same quantity.

If one were to remove the microorganisms from the gut, or to significantly alter their species composition or abundance, one would expect to see consequences for host health. This would be true even if most of the particular organisms in question were neutral or detrimental in normal circumstances. As with non-genic elements in the genome, this means that even if many organisms in the gut are non-functional from the host’s perspective, their presence is not inconsequential for the biology of an animal carrying them.


The junk DNA collection.

In this post, I will maintain an up to date list of substantive posts dealing with the topic of “junk DNA” on this blog and various others.

Genomicron


Sandwalk

See also

Quintessence of Dust

Pharyngula



Non-functional DNA: quantity.

In my previous post, I noted that because of what we understand about the nature, origins, and cross-taxon quantitative diversity of the various sorts of non-genic DNA in large eukaryote genomes, the default assumption is that much or even most of it is not functional at the cell and organism levels. Thus, the burden of proof rests with authors who claim that a large fraction, or indeed most or all, of this DNA is functional for the organisms in which it occurs.

This should not be construed as claiming that all non-genic DNA is assumed to be non-functional. I have pointed out in various preceding posts that even those who postulated non-adaptive explanations for its existence did not rule out — and indeed, explicitly favoured — the notion that a significant portion would turn out to serve a function. You need not take my word for this, as it is not difficult to find unambiguous statements from the original authors themselves.

For example, here are Orgel and Crick (1980) who, along with Doolittle and Sapienza (1980), first proposed the concept of “selfish DNA” in detail:

It would be surprising if the host genome did not occasionally find some use for particular selfish DNA sequences, especially if there were many different sequences widely distributed over the chromosomes. One obvious use … would be for control purposes at one level or another.

Here, too, is Comings (1972), the first person to use the term “junk DNA” in print and the first to provide a substantive discussion of the topic. (The term was coined by Ohno in 1972, but Comings’s paper appeared in print first, citing Ohno as ‘in press’, and Ohno used the term only in the title).

These considerations suggest that up to 20% of the genome is actively used and the remaining 80+% is junk. But being junk doesn’t mean it is entirely useless. Common sense suggests that anything that is completely useless would be discarded. There are several possible functions for junk DNA.

The use of the terms “selfish DNA” or “junk DNA” has changed over time, and both are now often applied to all non-genic DNA, rather than to the sequences to which they originally referred (i.e., transposable elements and pseudogenes, respectively). Moreover, it seems that many authors — at least those whose studies focus primarily on protein-coding genes and DNA sequencing — believe that the assumption has been that all non-genic DNA is “junk” in the sense of totally non-functional. However, amidst any such assumptions there has always been a diversity of views on the subject, ranging from assuming that most non-genic DNA is non-functional (as in the quotes above) to expecting it all to be functional — the latter being a position held by strict adaptationists, and a large part of the motivation for proposing the alternative view of selfish DNA the first place.

As with many issues in evolution, this is a matter of relative quantity, not an exclusive dichotomy. We may reasonably expect a significant fraction of non-genic DNA to show evidence of function, and the pursuit of such evidence is a valid and important endeavour. It does not follow, however, that the pendulum must be perceived to swing from entirely functional to entirely non-functional and back again. We will undoubtedly refine our estimates of the amount of non-genic DNA that is mutualistic at the organism level, how much is commensal, and how much is best characterized as parasitic in nature.

As it stands, the evidence suggests that about 5% of the human genome is functional at the organism level. The total may be higher — as noted, Comings suggested 20% is actively utilized. It is conceivable that 50% or more of the genome is functional, perhaps in structural roles or some other higher-order capacity. It would require evidence to support this contention, however, and the question would remain as to why an onion requires 5x more of this structural or otherwise essential DNA, and why some of its close relatives can get by with half as much while others have twice the onion amount. There is nothing remarkable about onions in this sense, by the way — animal genome sizes alone cover a more than 7,000-fold range, and even among vertebrates there is a 350-fold difference. The range among single-celled protozoa is at least 30,000-fold, though even higher estimates have been presented.

The take home message is simply this. What we know about eukaryote genomes suggests that there are many mechanisms that can add non-coding DNA that do not require it to be functional. This does not in any way preclude the possibility of, or invalidate the search for, function in some, many, or possibly even most of those non-coding components. How much proves to be functional is an empirical question, and at present the indication seems to be that most non-genic DNA is non-functional. That said, non-functional is not the same as inconsequential.

________

Comings, D.E. 1972. The structure and function of chromatin. Advances in Human Genetics 3: 237-431.

Doolittle, W.F. and C. Sapienza. 1980. Selfish genes, the phenotype paradigm and genome evolution. Nature 284: 601-603.

Ohno, S. 1972. So much “junk” DNA in our genome. In Evolution of Genetic Systems (ed. H.H. Smith), pp. 366-370. Gordon and Breach, New York.

Orgel, L.E. and F.H.C. Crick. 1980. Selfish DNA: the ultimate parasite. Nature 284: 604-607.



Junk at Sandwalk.

Anyone who reads this blog but not Sandwalk (if any) should go right now and see Larry’s posts on junk DNA. Although I do not care so much for the term “junk DNA” because often it is employed ambiguously, Larry is careful to define it explicitly as sequences for which the evidence indicates nonfunction. The posts on the distinct components of the genome that are considered junk under this definition are:

Junk in your genome: LINEs

Junk in your genome: SINEs

Junk in your genome: pseudogenes

Junk in your genome: protein-encoding genes

A collection of related posts is compiled under Theme: genomes and junk DNA.

Enjoy!


Incidental DNA revisited.

Note – this post has been updated since originally posted.

In the recent exchange regarding my post about genome size and code bloat, one of the authors of the study in question made the following claim:

In its conclusion prof. Gregory suggests that we claim that “Non-coding DNA does
accumulate “so that” it will result in longer-term evolutionary advantage”.
We ABSOLUTELY NEVER stated such a non-sense. It is curious that the same accuse was moved by prof. Gregory in its article “Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma”, that we cite in our paper, to an article by Jain that we also cite in our paper. So, either prof. Gregory has a very poor opinion of our intelligence, or he thinks that we do not read the articles that we cite. Let us state, unambiguously, what we and Jain really say: “IF does exist a mechanism for genome size increase, THEN maybe the resulting long-term advantage can overcome the short-term disadvantage” (Jain was referring to the selfish dna as the genome increasing mechanism while we do not give any preference). Prof. Gregory reverts the implication: “IF there is a long-term advantage THEN the mechanism of genome increase is the product of selection”, and then explains us that it can’t be true. Incidentally, in the case of Jain, I think that what he was really intending can be clearly understood just by the title: “Incidental DNA”.

When someone suggests that one has misinterpreted the claims of an author, the appropriate thing to do is to consult the original article to be sure. So, I looked up the Jain (1980) letter, some quotes from which are given here (with emphasis):

Natural selection is concerned not only with the existing variability but even more so with mechanisms which ensure its continued availability. If there is intragenomic selection leading to rapid build-up of some of the DNA sequences (the selfish DNA of Doolittle and Sapienza and Orgel and Crick) we must treat this part of DNA as incidental to the fundamental process of mutability so vital for ensuring continued supply of raw material for the production of new genes. It does not follow that all of the DNA produced in this manner will, in fact, acquire a function. A large part of it (or even all of it) may not do so and may be eliminated only on an evolutionary time scale. Meanwhile, new DNA of the same and similar kind may continue to be produced so that at a given point of time there will always be large amounts of non-specific DNA. This fraction is best described as ‘incidental’ rather than ‘selfish’ DNA. We may call it incidental because it is a byproduct of the inherent property of mutability of the genome, a characteristic to which natural selection attaches great importance even if it leads to the production of repeated sequences and a wasteful deployment of energy. Viewed in this light, non-functional DNA is very much a product of natural selection — a selection operating for mutability per se. Its relative abundance is probably a function of its nonfunctional nature for any other DNA which carries information of one kind or another would create genetic imbalance and would be quickly rejected.



Nature places considerable premium on playing safe so that it will not run short of raw material even if this means indiscriminate production leading to sequences which are destined to remain functionless.

Now, Dr. Musso may interpret this very differently, but I take it to mean that Jain argued that non-coding DNA was preserved by natural selection specifically because it may become useful as a source of new genes. Moreover, this would have to be non-coding DNA that was preserved in this way because adding coding regions for future use would create complications in genic function. I have discussed in various posts (e.g. here, here) why this notion is untenable.

UPDATE: My interpretation of Jain (1980) was that he was arguing that non-coding DNA is preserved by selection because it contributes to mutability. Further discussion with Jonathan Badger, and another re-read of Jain (1980) in the context of alternative interpretation, has bolstered the conclusion that he was in fact suggesting something different from what I said. The much more reasonable interpretation, and what I now think he was actually arguing, is that the genome is inherently unstable for reasons unrelated to non-coding DNA and that this is maintained by selection (though, it must be said, not in the usual sense but interlineage level) and the accumulation of non-coding DNA is a byproduct of this. I will accept that the authors of the paper that began the discussions saw it this way — though their phrasing “IF does exist a mechanism for genome size increase, THEN maybe the resulting long-term advantage can overcome the short-term disadvantage” is easily confused with arguing that non-coding DNA generates some long-term advantage that overcomes its immediate disadvantage (rather than representing a side-effect of some other process with a long-term advantage). And then, there is still the issue of what the original article stated:

From this point of view, we can think of TMs in our simulations as organisms trying to increase their gene pools adding new genes assembled from junk DNA. If the organisms possess more junk DNA it is possible to test more “potential genes” until a good one is found.

Though I doubt he will read this post, I do apologize to Dr. Jain if indeed I misinterpreted his argument. That said, I do think his phrasing of selection is imprecise and that this probably contributed to the confusion. In my original citation written 8 years ago, I cited Jain as an example of a “noncoding DNA is there because it might be useful” line of thinking, and while he may have been an inappropriate example, this notion is still around and needs to be fixed. In any case, I have not changed my opinion that the article that started this discussion drew undue links between a model and biological genome evolution, and that their results have little bearing on the genome size question.

________

Update, part two

I hate to keep updating this post (though I have preserved the original form with strikeouts), but I just knew I was not the only person to have interpreted Jain (1980) as suggesting that noncoding DNA was preserved because of its potential long-term benefits. It seems W.F. Doolittle (an originator of the “selfish DNA” idea, and whose paper Jain was commenting on) got the same impression. I will quote at length from Doolittle (1982), in which he discussed the varying reactions to the notion of selfish DNA shortly after it was proposed (italics in original, most in-line references omitted).

(c) The long-term evolutionary advantage of genomic rearrangements. Transposable elements promote genetic rearrangements, and the kinds of rearrangements (transpositions, deletions and inversions) seem similar in both prokaryotes and eukaryotes. This (and the occasional turning on and off of genes adjacent to the site of insertion) appears to be all that many, perhaps most, transposable elements actually do for the organism which bears them and it does not seem to be a good thing. Selection operating on individuals should eliminate such elements. Thus many have claimed that transposable elements are maintained because they play important “evolutionary roles”. This is not a straw man which Carmen Sapienza and I set up in order to have a hypothesis against which to pit the notion of selfish DNA. I can only document this with quotations not, I hope, taken out of context:

“Whether they (insertion sequences) exert functions at these positions or are simply kept in reserve as prefabricated units for the evolution of new control circuits remains unclear.”

“It is possible that the sole function of these elements is to promote genetic variability…”

“A tenable hypothesis regarding the function of transposition is that it allows adaptation of a particular cell to a new environment.”

“All these alterations could lead to changes in structural gene function and in the control of gene expression and could provide organisms with a means of rapid adaptation to environmental change.”

Evolutionary roles have similarly been invoked for heterochromatic highly repetitive DNAs, whose presence does affect recombination in neighbouring and distant regions and whose characteristics may (although the experimental evidence is not strong) affect chromosome pairing.
Neither we [Doolittle and Sapienza] nor Drs Orgel and Crick denied that transposable elements or heterochromatic highly repetitive DNAs have such evolutionary effects, nor that these effects might not be important, perhaps even as the basis for macroevolutionary change. What we were arguing against was the assumption that these elements arose through and are maintained by natural selection because of these effects.
This assumption is often only implicit in the writings of many who suggest that the only roles of mobile dispersed and tandemly reiterated DNAs are evolutionary ones. Thus we have been accused by some of these of misrepresenting their positions and thus indeed of attacking straw men after all. I apologize to those who feel we have put words in their mouths. But I do not see how statements that the only “functions” of transposable elements or highly repetitive DNAs are to generate or modulate genetic variability can mean anything other than that natural selection maintains, and probably even gave rise to, such elements through selection for such “functions”. Shapiro (1980) has been brave enough to articulate this view outright:

“Why, then, are insertion elements not removed from the genome? I think the answer must be that there is a selective advantage in the ability to generate new chromosome primary structure.”

Those who speculate on the function of excess DNA have formulated this position in a more extreme way. For instance, Jain (1980) states

“at a given point of time there will always be large amounts of non-specific DNA. This fraction is best described as ‘incidental’ rather than ‘selfish’ DNA. We may call it incidental because it is a byproduct of the inherent property of mutability of the genome, a characteristic to which natural selection attaches great importance even if it leads to the production of repeated sequences and a wasteful deployment of energy. Viewed in this light, non-functional DNA is very much a product of natural selection — a selection operating for mutability per se.

The question of whether natural selection operates in this way, that is of whether the evolutionary process itself evolves under the direct influence of natural selection, lies at the root of the real controversy over whether self-maintaining, structured, genomic components without phenotypic function can properly be called “selfish”. This may seem like a small and metascientific quibble. In fact it is not; it is one of the most troublesome questions in evolutionary biology today. It manifests itself in debates over the origin and maintenance of mechanisms involved in the optimization of mutation rates, recombination, sexual reproduction, altruistic behaviours of all sorts and even speciation. Such mechanisms are not clearly advantageous to, and can be detrimental to, the fitness of the individual. Yet they may increase the long-term survival properties of the group to which the individual belongs, thus seeming to be the product of what has been called “group selection”.

________

Doolittle, W.F. (1982). Selfish DNA after fourteen months. In: Genome Evolution (G.A. Dover and R.B. Flavell, eds.), Academic Press, New York, pp.3-28.

Jain, H.K. (1980). Incidental DNA. Nature 288: 647-648.



Welcome Pharyngulanchers.

I see PZ has linked here in a discussion of yet more IDist nonsense regarding junk DNA. To help you find what you’re after, here is a list of relevant posts on the subject:


How much DNA could be deleted from the human genome?

Larry Moran asks an interesting set of questions about human DNA:

How much of it could be removed without affecting our species in any significant way in terms of viability and reproduction? Or even in terms of significant ability to evolve in the future? In other words, how much is junk?

The options are:

  • None
  • less than 10%
  • between 11% and 49%
  • between 50% and 74%
  • between 75% and 89%
  • 90% or more

I hope people take his poll, because I think it will be intriguing to see what most people think. However, I have to admit that I won’t really be able to vote, for the reason I outlined in the comments to his article:

I think this is where a distinction between nonfunctional and inconsequential is important [see Effect versus function for more details]. In terms of whether most DNA is functional, I would agree on the basis of what we currently know that much of it could be deleted in principle. However, this would also affect cell size, and therefore organs, and therefore organisms. And yet, I would not necessarily consider the influence of DNA amount on the cell as a function. It could very well be that there is upward pressure from transposable elements and other mechanisms that cause DNA to accumulate, and downward pressure against this accumulation through selection on organisms. The balance that has been reached is not necessarily adaptive for either side. However, deleting a lot of it could have impacts on development and morphology nonetheless. Maybe it would even be a beneficial change, maybe deleterious, but I can’t assume that there would be no effect.

In some ways, it’s a little like asking, how much of the bacteria in your gut could you kill without having an effect on your health? In principle, a lot of it is clearly not functional, and none of it is there just to function on your behalf. But if you cleared the gut of all bacteria, or even just the commensal and parasitic species, would there be no effect? And if there were adverse consequences, would you take this as evidence that all those bacteria had a function after all?



Signs of function in non-coding RNAs in mouse brain.

Over on his blog, Greg Laden points to some new work by John Mattick’s group on non-coding RNA expression in mouse brains. It’s interesting stuff, and worth a look. Please bear in mind as you do, however, that non-protein-coding but functional RNA is nothing new. Ribosomes are made of non-coding RNA, for one thing. Sadly, Greg seems to have bought into the distortions (several promoted by Mattick) about what people have said about non-coding DNA.

The “Junk DNA” story is largely a myth, as you probably already know. DNA does not have to code for one of the few tens of thousands of proteins or enzymes known for any given animal, for example, to have a function. We know that. But we actually don’t know a lot more than that, or more exactly, there is not a widely accepted dogma for the role of “non-coding DNA.” It does really seem that scientists assumed for too long that there was no function in the DNA.

As I have noted, people have been proposing functions for non-coding DNA since the beginning. As I noted in one of my first Genomicron posts,

Those who complain about a supposed unilateral neglect of potential functions for non-coding DNA simply have been reading the wrong literature. In fact, quite a lengthy list of proposed functions for non-coding DNA could be compiled (for an early version, see Bostock 1971). Examples include buffering against mutations (e.g., Comings 1972; Patrushev and Minkevich 2006) or retroviruses (e.g., Bremmerman 1987) or fluctuations in intracellular solute concentrations (Vinogradov 1998), serving as binding sites for regulatory molecules (Zuckerkandl 1981), facilitating recombination (e.g., Comings 1972; Gall 1981; Comeron 2001), inhibiting recombination (Zuckerkandl and Hennig 1995), influencing gene expression (Britten and Davidson 1969; Georgiev 1969; Nowak 1994; Zuckerkandl and Hennig 1995; Zuckerkandl 1997), increasing evolutionary flexibility (e.g., Britten and Davidson 1969, 1971; Jain 1980; reviewed critically in Doolittle 1982), maintaining chromosome structure and behaviour (e.g., Walker et al. 1969; Yunis and Yasmineh 1971; Bennett 1982; Zuckerkandl and Hennig 1995), coordingating genome function (Shapiro and von Sternberg 2005), and providing multiple copies of genes to be recruited when needed (Roels 1966).

I am not about to claim that the study hasn’t shown evidence of function for these non-coding regions. I think it’s quite interesting, and it wouldn’t surprise me if lots of non-coding RNA turned out to have a regulatory function. But let’s be realistic with this. The authors consider a “long” non-coding RNA transcript to be >200bp. So let’s just round up and say 1,000bp for convenience. They identified around 850 potentially functional sequences (and ~500 that do not show evidence of functional expression, at least in the brain) and estimate that there are 20,000 of them all told. 1,000bp x 20,000 = 20Mb. The mouse genome is about 3Gb. In other words, this study, even read generously, has identified possible function for 0.7% of the mouse genome.

In summary, cool research. Important question, neat result. But let’s not start the usual extrapolationfest that normally accompanies such publications.


Junk DNA and ID redux.

Just a reminder, these are the important points under discussion:

* Proponents of ID themselves clearly suggest that “junk DNA” will mostly or all be functional.

* No unambiguous explanation has been given for why ID must assume that non-coding DNA is functional, especially since they say nothing can be known about the designer or the mechanism.

* The existence of much non-functional DNA would not necessarily refute the idea of design, as many human-designed structures have redundant, non-functional, or even counterproductive characteristics. It would, however, challenge certain assumptions about the designer and the mechanism, which again is why these must be made explicit if the junk DNA argument is to be invoked. Therefore, this is only a useful prediction if one includes details about the mechanism of design.

* The demonstration that all or most non-coding DNA is functional would not support ID to the exclusion of evolution, because a strict interpretation of Darwinian processes has always been taken to propose function as well.

* The demonstration that all or most non-coding DNA in the human genome is functional would still leave the question unanswered as to why the designer put five times more in onion genomes.

* Many functions that have been proposed or demonstrated are dependent on the process of co-option, the same process that is involved in the evolution of complex features.

* Evidence for function in non-coding DNA comes from analyses using evolutionary methods. Other approaches, such as deleting some, have not supported the hypothesis that it is functional.

* The current evidence for function, and other details about how non-coding DNA forms, both suggest that most non-coding DNA is non-functional, or at least that this is the most plausible condition pending much more evidence.

Feel free to comment, but please address these points directly.