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FIRST ISOLATION OF A GENE--lacZ

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GENE SEQUENCES IN OPPOSITE ORIENTATIONS

Soon after I joined Charlie Thomas' lab at Harvard Medical School, Dean Rupp, on a postdocfrom Yale, had the idea that we could determine whether one unique strand of DNA was transferred to recipient cells from Hfr or F' bacteria by transferring lambda and using the poly U,G technique (developed by Szybalski to separate the strands of lambda) after zygotic induction. (Poly U,G binds to a greater extent to one of the two strands, usually, and because it is heavily aggregated, the density in CsCl of the two DNA strands is significantly different,) As Dean suggested this idea, I immediately realized that if we transferred in the opposite direction, the opposite strand of lambda DNA ought to be transferred. This provided a perfect control for the experiment

I tried the poly U,G procedure to see whether strand separation was straightforward. As happens on rare occasions, the procedure worked easily, without any complications. We obtained the donor strains from Jon Beckwith's lab and I did the experiment. When transferred in one direction, most of the radioactive label was in one strand of the progeny phage, and when transferred in the opposite directions, most of the label was in the other strand. I presented this paper (Rupp and Ihler) at the Cold Spring Harbor Symposium. It was very well received, but I was petrified to discover Ohki_and_Tomizawa were going to present a talk on the same topic. Fortunately for us, their experiments lacked the elegant control of transferring in the opposite direction. We published this paper later in PNAS (Ihler and Rupp)

On his web site, Dean_Rupp looks as young as he did in 1969, lucky dog. Maybe it is an old picture. Rupp_and_Dan_Vapnek later directly showed that only one of the two strands of F is transferred. Also later, Ihler and Kawai used the asymmetrically labeled lambda phage to separately follow the fate of the two DNA strands after infection, and found, unlike T7, that one remains circular and the other ends in a concatemer, as would be predicted by the rolling_circle model of DNA replication.

ISOLATION OF lacZ FROM PHAGE WITH INVERTED lac SEQUENCES

Later I met Karin Ippen (Memorial) who was a postdoc in Jon Beckwith's lab, and we became friends. One day she asked me to go for a walk to discuss an experiment. I think she (and I) both had the walk together more in mind than this experiment. She said the Beckwith lab wanted to isolate a gene, but could not think of any way to do it. We unsuccessfully discussed various crackpot ideas and then walked back to Charlie Thomas' lab. I was making a phone call when it came to me how to do the isolation. Because transducing phages can pick up bacterial genes in either orientation, using various tricks that had mostly been developed in Beckwith's lab, it would be possible to separate the strands of lambda transducing phages that carried lac in opposite orientations. Then hybridization of the same strand of lambda DNA from both transducing phage would permit hybridization of lac DNA, but of course the lambda DNA strand would not hybridize to its identical strand. We had recently been using a single-strand-specific nuclease, to degrade single-stranded DNA in Charlie Thomas' lab, and so I realized that we could degrade the unhybridized DNA, leaving pure lac operon DNA.

I explained this idea to Karin, while I was on the phone complaining about a billing error to the phone company. She needed to return to her lab and so we didn't get a chance to discuss doing the experiment together. She explained the procedure to the other people in her lab, and I didn't hear anything more about it for a while.

Later, Karin told me that she was being pushed out the experiment by more aggressive people in her lab. So I decided that I had better do the isolation myself, especially since I had available all the techniques and reagents in connection with the Hfr transfer experiment and other experiments. As I recall, the phage strains used had already been developed previously in the Beckwith lab for other reasons and were available off the shelf (despite the impression of diligent design and construction that you might get from the paper). I separated the strands (fig 4 of the manuscript), digested the DNA (fig 7) and determined its molecular size (fig 8) by velocity sedimentation relative to beta-galactosidase (a nice touch, I thought, to size the DNA relative to its protein product). The first time I ran the nuclease-treated DNA, I did not find a peak of radioactivity. I decided that might be because of the background of degraded single-stranded DNA, so I acid-precipitated the DNA the next time, and there was a nice peak of radioactivity at the correct position. Subsequently, Lorne MacHattie, also in Charlie Thomas' laboratory, did the elegant EM visualizations of the S1-treated and untreated DNA. Larry Eron in Beckwith's lab did an RNA hybridization experiment, I guess to show that the DNA really was lac. This was the only experiment actually done in the Beckwith lab, and seems to have been included so that that some experiment in this paper was done in that lab. .

Since I had devised the procedure by which the DNA was isolated and since I had in fact been the only one to isolate lac DNA, I felt my name should be first on the paper, and told Jon Beckwith that. But he gave me a fish eye.

PRESS CONFERENCE

Subsequently, when the paper came out, Jon Beckwith and Jim Shapiro held a press conference, always a bad idea in my view. There was no consultation about this; I got a weird call from Jon Beckwith a day or two before. Bill Reznikoff told me later that the people in the lab had worked very hard and mostly fruitlessly to get Jon to call me prior to the press conference. But all he said on the phone was there was going to be a press conference. I said, "Um", and that was the end of the conversation.

The press conference proved to be highly controversial. My view of the press conference was that Jon Beckwith and Jim Shapiro had used a nice experiment, as a forum for other ideas and to promote a complex political agenda, and in the process trashed the experiment, while inappropriately taking credit for it, since neither had contributed to the experiments or to the invention of the procedure used. On the other hand, of course, Beckwith's lab was from the beginning the most exciting bacterial genetics lab in Boston, and both Jon Beckwith and Jim Shapiro have had very distinguished careers since. The tenuous connection between the experiment and the political message was that the isolation of a gene for the first time meant that a new era of molecular control of genetics was dawning and that considerable thought needed to go into the ethics and uses of the technology. Indeed a new world did dawn, but perhaps those who developed cloning deserve the credit. Many people now consider the press conference as prescient, and certainly Jon Beckwith, already widely respected and well into his distinguished career, was in a knowledgeable and advantageous position to see this future. Beckwith has written extensively on genetics and society (* **). Most of the scientific community reacted very badly to a characterization of genetics and science in general as potentially irresponsible in the new era of gene technology that was emerging. I know scientists who even now, 30 years later, are infuriated and contemptuous of this press conference. For myself, I do think the press conference was a direct precursor of later widespread attempts to regulate cloning and gene transfer, which possibly deterred some unwise experiments (although I don't know of any) but also caused a lot of provocative administrative hassle for people trying to do basic science research, before it was mostly done away with. The press conference probably did galvanize people in general to think about whether they want foreign genes in their tomatoes, but whether the people are coming to the right conclusions, or even what the right conclusions are, is not clear to me. Part of the problem is that Molecular Luddites seem to concentrate on preventing research rather than controlling the applications of that research, on the theory that if you don't know it, you can't use it.

An Inaccurate Rendering of History

In his book "Making Genes, Making Waves" (Harvard University Press, 2002) Jon Beckwith presents his recollections of this experiment (pp32-37). Most notably, he takes credit for the idea of using different DNA molecules whose only common segment was the gene for beta-galactosidase, hybridiing the DNA and using a single-stranded specific DNase to degrade the non-hybridizing DNA. However this was, in fact, my idea. Beckwith certainly had a lot of great ideas in his career, but I don't think this was one of them. Karin told me that they could not figure out any way to isolate the gene, and the procedure that I ultimately used was entirely my idea.

"Everyone in the lab was excited. Jim Shapiro offered to test out the idea for gene purification and within a short time believe that he had the process working." (p33). Since Jim at no time did any experiments on the gene isolation, this statement can only refer to the fact that I did the experiment and got it to work. FORTUNATELY I STILL HAVE THE LAB NOTEBOOKS.

Describing the press conference, Beckwith says ""We have no right to pat ourselves on the back", we announced" (p36). That was certainly true since they did not contribute to the procedure used nor the technology that made it possible (that all came from Charlie Thomas' lab). Genetic cloning has been plagued for decades by publicity seekers and savants whose message is half-truths at best. I think it is most unfortunate that Beckwith and Shapiro stated the gene controversy that way by taking credit from something they didn't really do and using that as a platform for their political ideas.

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MOLECULAR BIOLOGY PAPERS

Here is a heterogeneous collection of Molecular Biology papers on topics including bacteriophage lambda, RNA polymerase, long range base pairing in lambda and phiX.

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RED CELLS & LIPOSOMES AS CARRIERS FOR PROTEINS & DNA

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The idea for this project came to me in a dream, as it did for the discoverer of the structure of benzene. I had just read TMS Chang's excellent book, Artificial Cells, and was thinking about red cells while I was a first year medical student taking Physiology, I was trying to think of some way to put macromolecules inside red cells, and, in the dream, holes opened in the red cell membrane under hypotonic conditions and the proteins entered. When I awoke, I remembered the dream (unusual for me). After thinking about it with the rational mind, I decided it could not work because all the hemoglobin rushing out would prevent the proteins from entering and I set the idea aside as impractical. Later I decided it might work and did a few experiments to try it out.

Proteins and DNA up to a considerable size can enter through holes in the red cell membrane opened osmotically. Under hypo-osmotic conditions, water enters the cell, which swells to its maximum and then holes open, allowing the escape of hemoglobin and the cell contents. The holes subsequently reseal when the osmolarity is restored. If precautions are taken to avoid excessive loss of the cellular contents (small extracellular volumes, reseal with red cell juice, etc), the red cells can survive in the circulation with nearly the normal life span. If they are deliberately moderately damaged, they are taken up by splenic macrophage; greater damage causes them to be taken up mostly by liver macrophage (targeted delivery).

Our initial idea was to entrap glucocerebrosidase for delivery in Gaucher patients to macrophages loaded with undegradable glucocerebroside. We never actually tried this because we had no practical source of glucocerebroside (pre-cloning era), and subsequently these patients are treated by direct uptake of the enzyme. However, it seems very likely that the treament would have worked, and in fact, even now it might have specialized uses in Gaucher disease, since a red cell can deliver a huge amount of enzyme to individual macrophages.

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PROTEINS ENGINEERED TO INSERT IN MEMBRANES OF CELLS & LIPOSOMES

We devised this as a procedure to put proteins, antibodies, or enzymes on the OUTSIDE of red cells, or indeed any cells, or into liposomes. One potential use of this procedure is targeting of liposomes and delivery of the contents to specific locations. Another is to take advantage of the long circulatory life time of red cells (relative to free proteins) to attach antibodies to virus particles, bacteria or other pathogenic organisms. A third is to utilize enymes which might degrade excessive levels extracellular substrates without the need to transport the substrate into the red cell. We took advantage of the ability of the hydrophobic sequence of cytochrome b5 to spontaneously insert into membranes and attached this sequence to other proteins. Later others used the same procedure, but failed to cite this prior work.

DNA INJECTION ACROSS MEMBRANES

The process by which phage DNA enters cells is complicated by the complexity of the membranes, the viability of the bacterial cell etc. We used liposomes to simplify the membrane/cell problem and showed that DNA injection across the membrane needed only the phage receptor, lamB, and resulted in a long-lasting hole in the membrane.

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BARTONELLA BACILLIFORMIS

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MISCELLANEOUS

Ihler G, Chami-Stemmann H. 7-oxo-DHEA and Raynaud's phenomenon.
Med Hypotheses. 2003 Mar;60(3):391-7

Patients with Raynaud's phenomenon have abnormal digital vasoconstriction in response to cold. The pathogenesis remains unknown but may involve a local neurovascular defect leading to vasoconstriction. Diagnosis of primary Raynaud's phenomenon is based on typical symptomatology coupled with normal physical examination, normal laboratory studies and lack of observable pathology by nail fold capillaroscopy. Secondary Raynaud's phenomenon is known to occur associated with several connective tissue diseases, vascular injury due to repeated vibrational trauma, and other causes which produce demonstrable vascular and microcirculatory damage. Treatment of Raynaud's symptoms is conservative and aimed at prevention of attacks. Patients are advised to remain warm and, if possible, to live in warm climates. We suggest that an ergogenic (thermogenic) steroid, 7-oxo-DHEA (3-acetoxyandrost-5-ene-7,17-dione), which is available without prescription as the trademarked 7-keto DHEA, may be very helpful in prevention of primary Raynaud's attacks by increasing the basal metabolic rate and inhibiting vasospasm.

 

A paper in Lancet proposed that the Earl of Derby had been poisoned, based on an historical account give by John Stowe. However, as it turns out, this theory was already widely accepted in the 17th and 18th centuries. Here is the original letter to the Lancet editor, which was slightly trimmed by Lancet for space reasons.

 

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