Discovery of Okazaki fragments - Does anyone know? (Dec/04/2004 )

Hi,

we all know replication... lagging strand... OKAZAKI fragments. (short version)

Has anyone got a clue about how Mr. R. Okazaki discovered those framents.

Any hints (even just as to where to look) are greatly appreciated.


Thanks says:

bruno

Reiji Okazaki (1930-1975), a Japanese biochemist, born in 1931 in a town called Shiratori town of Hiroshima City, graduated in 1953 from Nagoya university, died age 44.

In 1968, Reiji Okazaki and his colleagues exposed the replicating DNA to short pulses (about five seconds) of tritiated radioactive nucleotides, followed by the addition of an excess of normal cold (nonradioactive) nucleotides. This sort of pulse-chase experiment resulted in label being present only in the DNA that was synthesized during the short period of the pulse. Soon after the pulse, they isolated the DNA and separated the individual strands from one another in alkaline solution. The various pieces of DNA could then be sorted out by size: the alkaline solution of DNA was placed on a “sucrose gradient” and spun in an ultracentrifuge. The bigger pieces of DNA settled more rapidly in such a sedimentation velocity experiment as this (the sucrose served to stabilize the resulting separations until the investigator could look at them). The scientists then looked for the presence of label on the spun pieces of DNA. Label occurred on two sizes, one very long, and the other only on small fragments of 1000 to 2000 nucleotides in length.

To read more, check this link http://www.mhhe.com/biosci/genbio/raven6b/...hink/10-lab.pdf

Thank you very much paulina. You're fantastic.

/bruno

I still have probelms with the results, at least as they are explained by most texts. The pulse should end up in both the leading and lagging strands equally, since both are adding bases at the same rate. Yet the data seem to show all of the pulse going into small pieces (the fragments) initially and only showing up in larger pieces later (after ligation). But shouldn't the large leading strand show up at the same time as the fragments?