Analysis of Biological Development (K. Kalthoff)

Updates to Topic 07: Genomic Equivalence and the Cytoplasmic Environment


Answers to Questions in Text

Cloning Dolly (pp. 162-164)

  1. The high failure rate of nuclear transfer experiment in frogs has been ascribed, for the most part, to the inability of nuclei from differentiated cells to re-adjust to the fast pace of DNA synthesis during cleavage divisions. Could this adjustment be easier in mammals? Answer: Yes, cell cycles during cleavage are 10-20 times longer in mammals than in frogs, making it much easier for mammalian nuclei to completely replicate their DNA between successive mitoses.
  2. The cell culture from which the donor cell for the cloning of Dolly was derived has been established from tissue of a pregnant ewe. Under these circumstances, could Dolly have been cloned inadvertently from a fetal cell rather than an adult cell? Answer: Yes, cells may detach from fetal villi in the placenta, enter the maternal blood stream, and be carried into other maternal tissues. However, the genetic test described on p. 164 indicates that Dolly's genome was derived from the ewe herself and not from her fetus.

Control of Gene Expression by Cytoplasmic Factors (p. 167/168)

  1. Why did DeRobertis and Gurdon (1977) inject Xenopus kidney nuclei into Pleurodeles oocytes rather than into Xenopus oocytes? Answer: Presumably the authors were heading off a potential criticism that the oocyte-specific proteins might have been encoded by long-lived maternal mRNAs and not by the transplanted nuclei. If this were so, then the oocyte-specific proteins should have beenPleurodeles oocyte-specific proteins. Since the proteins of the two species run differently on two-dimensional gels, and because the oocyte-specific proteins were indeed of the Xenopus variety, the investigators could safely conclude that the injected kidney nuclei had been reprogrammed in their gene expression pattern.
  2. What do the results show about the evolution of the signals that control gene expression? Answer: The signals and their receptor molecules must have been well conserved in evolution, since newts and frogs have been separated for more than 300 million years.

Comments

Clarifications and Corrections

p. 153, Fig. 7.5a: The arrow supposed to mark the chromocenter has been omitted in print. It is shown on this page to the right in a black-and-white version of the same picture.

p. 153, Fig. 7.5b: Labels from another photograph have been used inadvertently. The bands of highly coiled chromatin are the dark horizontal layers. The interbrands are the areas between the bands, with straight chromatin strands running more or less vertically.

p. 165, left column, the first three lines should read: "gestation period of mice, further basic research on mammalian cloning will proceed much faster than it would with large mammals."

New Review Articles

Lanza R. and Rosenthal N. (2004) The stem cell challenge.   Scientific American June 2004: 93-99
Rideout W.M. III, Eggan K. and Jaenisch R. (2001) Nuclear cloning and epigenetic reprogramming of the genome. Science 293: 1093-1098

 

New Research Articles

Rideout WM 3rd, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R. (2002) Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109(1): 17-27.
By genetic transformation of embryonic stem cells obtained after nuclear transfer from immonodeficient mice, the authors have developed a model for human cell replacement therapy.

Kikyto N., Wade P.A., Guschin D., Ge H. and Wolffe A.P. (2000) Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI. Science 289: 2360-2362
Nuclear transfer experiments were designed to test whether differentiated cells retain a full complement of genetic information. This is a very demanding test, as it also requires that all kinds of genes be accessible to the transcriptional machinery at the appropriate time. Quite possibly, this additional requirement is the reason why the success rate of nuclear transfer experiments is so low. Researchers are therefore focussing now on the "remodeling" process that nuclei undergo upon transfer to egg cytoplasm. The remodeling involves the loss of nuclears protein as well as the uptake of new cytoplasmic proteins. The authors of this study have developed a system for studying this process in vitro. They found that TATA-binding protein, a major general transcription factor (see p. 412/413 of text), is released as part of the remodeling process. The release requires a nucleosomal adenosine triphosphatase known as ISWI.

Wakayama T., Tabar V., Rodriguez I., Perry A.C., Studer L. and Mombaerts P. (2001) Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292: 740-743
One of the expected benefits of therapeutic human cloning is to provide any given patient suffering from a degenerative disease such as diabetes or Parkinson's with isogenic replacement cells, which are not rejected by the patient's immune system. This study describes essential parts of this procedure using the mouse model. The investigators obtained 35 cell lines by transfering nuclei from various somatic cells into enucleated eggs, which were then grown into blastocysts to harvest the inner cells mass. In vitro, these nuclear transfer-derived embryonic stem (ntES) cells gave rise to a wide range of somatic cell types including dopaminergic and serotonergic neurons. When added to fertilization-derived blastocysts, the ntES cells gave rise to both male and female germ cells, which contributed to viable offspring. The results demonstrate that mouse ntES cells are totipotent.


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