Dr. Andy

Reflections on medicine and biology among other things

Thursday, March 24, 2005

Too Weird

While all anyone can talk about is Terry Schiavo, who is, after all, just a single person without much cognitive function left, a letter in this week's Nature (confusingly, short articles in this most prestigous of scientific journals are called letters) challenges much of what we know of genetics.

As I briefly summarized yesterday, plant genetecists at Purdue have observed unusual reversions of a mutant gene to the wild-type sequence in the plant Arabidopsis. They study a gene called HOTHEAD, that causes unusual fusion of flower petals when mutated. They observed that a substantial fraction of offspring of parents with normal flowers and were puzzled. Sequencing showed that in each case the gene was identical to the "normal" sequence. They did a pretty good job of ruling out contamination of there stocks or pollenation by wild-type plants by doing experiments like looking at embryos or cross-breeding different strains.

Then they tried to exclude the possibility that another DNA sequence in the genome was acting as a "template" to restore the correct information. This kind of process would be unusual and interesting, but probably not revolutionary. In this they did a less good job, in my opinion.

They noted the sequence alwasy went back to the parental, and cite, but don't show, more data showing sequences can be recovered several generations later. They note that other related genes have other sequence differences that aren't found in the reverted gene, but don't do a search of the Arabidopsis genome to exclude a stretch of matching DNA outside a known gene, despite the fact that the Arabidopsis genome is sequenced.

They hand-wave a bit about RNA storage of genetic data, but there are several problems with this.

First, they don't attempt to document wild-type RNA sequences. This would be pretty straightforward using RT-PCR (first RNA is reverse transcribed into single-stranded DNA and then amplified by PCR). Interpretation of data MIGHT be hard because only a few percentage of RNAs would carry it and PCR enzymes are error prone, but I think you have to try.

Second, this would imply a complex genetic apparatus of which we have remained blissfully unaware despite all our knowledge of cell biology, having sequenced the genome of multiple organisms, etc. Just think of all the genes that would be needed to store copies of RNA (which would have to be passed on in pollen), activate some sort of DNA repair enzymes that use RNA as a template only under certain conditions of stress, make the repairs, etc. How many generations is RNA retained and when is it replaced. Recall that RNA is far less stable than DNA and therefore more prone to damage.

Additionally, I'd be a lot more confident in their data with a few more controls. One good one they didn't do was to make plants with genes that were slightly altered via silent polymorphisms. Would those then be altered back in HOTHEAD deficient plants? What if they were introduced into a strain with slightly different sequences. Would those be reverted too. This would be a lot of work (I know n0thing of the details of plant work, I purposely ignored those sections druing grad school), but before you publish a paper challenging the basis of genetics you have got to have your I's dotted and T's crossed

Overally, I'm a skeptic. I suspect they have a gene conversion going on and just haven't recognized the template yet.


At 4:43 PM, Anonymous Scott Kern said...

I'm a cancer geneticist who spends a lot of time trying to expunge mutation artifacts from our genetic studies. I'm appalled at the sloppy presentation of the arabidopsis reversions in Nature. No supplemental data, a methods section shorter than the Abstract, elimination of methodology in the text, etc.

In the first paragraph of the second page, did they really use allele-specific PCR to "clearly" show a conclusion? In other words, they purchased the chosen sequence by mail, then found the same sequence when the mail-order oligos were used in an artifact-prone method. All without showing any controls for the specificity of the allele-specific method?

In figure 1a, what was the restriction site used? Did it occur naturally in the sequence (I don't see it in the figure showing the sequences), or was it artifactually introduced during PCR? Why does the top allele in Figure 1A appear so much brighter than the restriction-cut allele just below it - are the alleles not in molar equivalence?

In the Southern blot, what was the probe?

What were the primer sequences for all the assays used?

If the altered sequences were found by one method, could they be found also by another method having non-overlapping artifactual tendencies? I note that they didn't use phage lifts or allele-specific ligation to quantitate the allelic ratio of the revertant alleles, as had been done 15 years earlier to provide the necessary controls for the study that showed infrequent mutant genes in stool samples of colorectal cancer patients.

Why no negative controls - such as non-embryoid parts of the hth/hth and HTH/HTH plants whose DNA had been isolated in minute amounts similar to the technique used for the seeds.

Why no primary data from gene sequencing in any of the figures? Did Nature refuse to publish the primary data even in supplemental form?

Do we really think that 38% of samples had gene reversion (figure 2)? That would be a higher rate of gene conversion than any other recombination system, including yeast.

Why no coded samples? When the infrequent gene mutations in pancreatic ductal lesions and in stool samples of pancreatic cancer patients were found 10 years ago, the samples were tested blindly. Everyone would do this, or some similar form of investigator blinding. And why not do the studies in a lab that had never previously seen studies of the HTH gene? This has been done when other authors wanted to quantitate gene mutation prevalence rates in cancer patients.

I suppose Nature will once again call in the Amazing Randi to investigate the authors, just like they did with their water memory paper in 1988.

I don't claim to know whether the data in this paper are valid, but certainly the methods are not presented in adequate detail nor in adequate confirmatory depth to judge. Until then, discussing this paper is a waste of time.

At 2:30 PM, Blogger jon said...

I was looking at your posts about bone cancer and found a good article about the same bone cancer info too...

God luck with it : )


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