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DNA replication: isolating temperature sensitive mutants

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Issues of the organisation of DNA replication in a bacterial cell
Sydney Brenner Scientist
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One of the important experiments which was very tricky to do was of course to show – in addition to all the things we had done with F factors – that it… that the DNA transferred from the male into the female bacterium was of course one-time replicated. This we proposed to do by a Meselson-Stahl experiments. Meselson and Stahl had labelled DNA with heavy atoms, nitrogen and carbon. And they had shown then that DNA replicated semi-conservatively – that is, the daughter molecules had one old strand, for example, light, and the new, and the new molecules had a heavy strand. So we proposed to use this. This was extremely difficult experiment to do at the time, because you had to have many labels radioactive to mark the parental DNA. It had to be done… we had to make sure that we destroyed all unconjugated cells, and that we only retrieved DNA from the cells. And eventually they were such a minority of the population. However, the experiment was done and in fact did show that the DNA that came across was indeed a hybrid DNA, had been replicated once. So what we were able to exclude quite conclusively that in fact the DNA had... that the DNA was transferred as one piece of DNA. However, we could not say for certain that it had been copied in the donor cell, because one… the equivalent theories, which is obvious when you think about it is that rather than have a replication driving the DNA across in the male, you could equally well have an enzyme in the female which pulled it across by exactly the same method – that is, replicated in the recipient cell. And we could not resolve those questions. In fact, very much later it was shown that the replication did take place in the… in the recipient, although this is still not completely decided, but as many questions in science are never decided they just vanish; this one is of no consequence. One of the other features of, of our experiment was that in somehow the DNA had to be attached to some part of the cell, and in bacteria that have no nucleus, no nuclear membrane, this DNA had to be attached to the cell membrane. And the idea was that if it was attached at a given point, which is another part, only one point would be required for the attachment, then when it copied, the new copy would be attached, remain attached by one strand. And then the idea was you would grow the membrane in between the two copies and that's the way they segregated to opposite cells. I think that even today that's not… I mean, many people believe that that's the way it happens, but it's still not completely proven. So those issues of the organisation of DNA replication in a bacterial cell are indeed still open questions. I have to say that in doing this work there, I had an echo of something I had done ten years previously, or more, in South Africa, where I had been using little pieces of liver in a Beams and King turbine ultracentrifuge, an air turbine which I'd built myself. And I noticed in centrifuging these cells and cutting sections of them, that the… that the DNA, or as we called it then, chromatin, had not sedimented completely to the bottom of the nucleus, but seemed to be connected to the top of it by strands. And in fact I was so impressed by this that I thought that that proved that, that the chromosomes were still attached to the nuclear membrane. And I wrote a paper on this which was published in Experimental Cell Research in 1953, called The Chromatic Nuclear Membrane, which was the result of that experiment and which showed DNA attached to this. And of course in the last few years this has now come back again and DNA seems to have in higher organisms some attachment to the cells. My idea was then that maybe this was involved either in chromosome segregation or, as I believed, possibly in expression. So that the… the good genes were stuck on the membrane, they could get their stuff over into the… into the cytoplasm. And they weren't in blocks of what were called heterochromatin in those days.

South African Sydney Brenner was awarded the Nobel Prize in Physiology or Medicine in 2002. His joint discovery of messenger RNA, and, in more recent years, his development of gene cloning, sequencing and manipulation techniques along with his work for the Human Genome Project have led to his standing as a pioneer in the field of genetics and molecular biology.

Listeners: Lewis Wolpert

Lewis Wolpert is Professor of Biology as Applied to Medicine in the Department of Anatomy and Developmental Biology of University College, London. His research interests are in the mechanisms involved in the development of the embryo. He was originally trained as a civil engineer in South Africa but changed to research in cell biology at King's College, London in 1955. He was made a Fellow of the Royal Society in 1980 and awarded the CBE in 1990. He was made a Fellow of the Royal Society of Literature in 1999. He has presented science on both radio and TV and for five years was Chairman of the Committee for the Public Understanding of Science.

 

 


Listen to Lewis Wolpert at Web of Stories

 

 

Tags: South Africa, Experimental Cell Research, 1953, The Chromatic Nuclear Membrane, Matthew Meselson, Franklin Stahl

Duration: 6 minutes, 33 seconds

Date story recorded: April-May 1994

Date story went live: 24 January 2008