Various functions of BACH1 and the other clone, BACH2, which also is regulated by heme, have emerged in not only erythropoiesis but also iron and heme metabolism, immune response and cancer progression.Those Simple Moments That Make It All Worth It For us, it took another two decades to come to a reasonably correct answer on the function of the heme–BACH1 axis in erythropoiesis. It did not take a second before he saw the tubes and suggested carefully, "This may be a heme protein."Ī beauty emerged: Heme, which is massively synthesized during erythropoiesis, would regulate the activity of this transcription factor, BACH1, coordinating heme synthesis and globin gene expression.Īs so many scientists know, the simplicity of a model does not guarantee a simple way forward. Then our lab head, Norio Hayashi, who has a long track record in enzymes of heme synthesis, happened to walk by. We were troubleshooting in front of the cold room, with the tubes in his ice box. When he came out of the cold room, he was disappointed, thinking that his purification was a total failure. The other student, Tatsuya Oyake, expressed one clone in E. One student, Ken Itoh, got two promising candidate cDNA clones in two hybrid screenings using MafK, one of the subunits of nuclear factor erythroid 2, as a bait. The laboratory where I started as a new faculty member, led by Norio Hayashi and Masayuki Yamamoto, focused on erythroid cells: Colleagues were working on the regulation of heme synthesis during erythropoiesis, and I was trying to identify transcription factors that would regulate globin genes by binding to their superenhancers, the locus control region, together with two graduate students. That morning, I went to lab, added the correct amount of protein, and this time, the Western blot worked. I bolted awake, ran to my laptop, checked the Excel sheet and voila - the Excel sheet math equation was incorrect! I dutifully added my dream concentrations and realized … the math was wrong. But to no avail - the Western blot had won.īut that night, in my dreams, I went over the Excel sheet provided by my PI that provided a handy plug-in for calculating the amount of protein to add to the Western blots. I had tried optimizing the denaturing steps, removed the denaturing step, changed the blocking buffer, modified the run times … I had tried everything I could think of and everything my helpful colleagues had recommended. Day after day, I had been troubleshooting these Western blots. I needed to measure the protein levels of the sodium–hydrogen exchanger isoform 3 and the colonic HK-ATPase in mouse colon by Western blot. I gloried over ion transporter mRNA, enjoyed functional assays in tissue cultures, reveled in measuring fecal ion concentrations by flame photometry - but then I encountered the dreaded Western blot. Horrible Western blots … the bane of my existence.Ī nitrocellulose membrane stained for protein detection during Western blotting.Īs a graduate student, I was fascinated by the beautifully orchestrated events of ion transport of the intestine. I was deep in REM sleep, dreaming of Western blots. I was in bed huddled under a big down comforter, which enveloped my whole body like an amoeba. It was a cold night the wind was blowing, and the branches of a nearby tree were scratching against the roof of my apartment. I think a child lives inside every scientist, a child whose curiosity is challenged by mysteries, who wants to make visible the invisible and to magnify the microscopic and who wants to look back into time as far as the origins of everything - of cells, life, the earth and the universe. This was one of the most exciting moments of my career as a biochemist and the closest I ever came to realizing my childish fantasy of time travel. I also observed that α- and β-tubulin were themselves about 40% to 45% identical, so I now perhaps was seeing more than 2 billion years into the past to the time of the first eukaryotes, one-celled organisms that lived on a planet that would be unrecognizable to us. I had no idea what that animal looked like, but I knew something about its tubulin. I felt like I was looking perhaps 700 million years into the past. This meant that I had just learned something very intimate about the common ancestor of vertebrates and echinoderms, an organism whose appearance and morphology were unknown and only could be guessed at. I realized that chicken and sea urchin α-tubulin were about 95% identical and that the same was true of the β-tubulins. The results came off the sequencer around Christmas Day. At that time, very little was known about tubulin sequences. My aha moment came in 1972 when I was a graduate student in Dow Woodward's laboratory at Stanford determining the first 25 amino acids in the sequences of alpha- and beta-tubulin from chickens and sea urchins.
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