Analysis of Biological Development (K. Kalthoff)

Updates to Topic 19: Genetic and Paragenetic Information

Answers to Questions in Text

Prion Protein (p. 486)

  1. The experiment described above proves that prion precursor protein is necessary for contracting and transmitting scrapie. What kind of experiment would prove that the transition from prion precursor protein to prion protein (PrPC to PrPSc) by itself – without a chance for viral contamination – is sufficient to cause scrapie? Answer: Purify PrPC, find a way of chemically transforming it into PrPSc, and inoculate test animals with either preparation. The animals inoculated with PrPC should stay symptom-free, whereas the animals inoculated with PrPSc should develop scrapie.
  2. Suppose you would graft brain tissue from mice carrying wild-type prion genes into inoculated knock-out mice. Which tissue(s) would you expect to develop the signs of spongiform encephalopathy, the grafted tissue, the host brain, or both? Remember that the prion precursor protein is tethered to the plasma membrane. Answer: The graft, but not the host tissue, should develop spongiform encephalopathy. This experiment was actually done, and the results were as expected (Brandner et al., 1997, Nature 379: 339-343).
  3. Assuming that the biological functions of prion precursor protein are equivalent in mice, sheep, and cattle, would it be possible to generate sheep and cattle whose meat could be eaten by humans without any risk of contracting spongiform encephalopathy? Answer: Yes, by developing a gene knock-out technology for sheep and cattle and then using nuclear transfer (see Section 7.7) to generate sheep and cattle without genes for prion precursor protein.


Clarifications and Corrections

New Review Articles

Collinge J. (2001) Prion diseases of humans and animals: their causes and molecular basis. Annu. Rev. Neurosci. 24: 519-550

Dobson C.M. (2002) Protein-misfolding diseases: Getting out of shape. Nature 418: 729-730

Hinchcliffe E.H. and Sluder G. (2001) "It takes two to tango": understanding how centrosome duplication is regulated throughout the cell cycle. Genes & Devel. 15: 1167-1181

Prusiner S.B. (2004) Detecting mad cow disease. Scientific American July 2004: 86-93

Roth M.G. (1999) Inheriting the Golgi. Cell: 559-562

Stearns T. (2001) Centrosome duplication: A centriolar pas de deux. Cell 105: 417-420

New Research Articles

Castilla J, Saa P, Hetz C, Soto C. (2005) In vitro generation of infectious scrapie prions. Cell 121: 195-206
The authors show that the conversion of normal cellular prion protein (PrPC) into protein-resistent prion protein (PrPres) can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.

Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB. (2004) Synthetic mammalian prions. Science 305: 673-676.
Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

Mead S. et al. (2003) Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics. Science 300: 640-643

O'Connell K.F., Caron C., Kopish K.R., Hurd D.D., Kemphues K.J., Li Y. and White J.G. (2001) The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with with distinct maternal and paternal roles in the embryo. Cell 105: 547-558
The authors investigate the control of centrosome duplication during the cell cycle. The zyg-1+ gene encodes a protein kinase that is specifically required for daughter centriole formation. Loss-of-function mutants form monopolar mitotic spindles organized by a single centrosome. Zyg-1 protein localizes transiently to centrosomes and acts at least one cell cycle prior to each spindle assembly event. Paternal zyg-1 protein is required during the first cell cycle and maternal zyg-1 thereafter.

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