Analysis of Biological Development (K. Kalthoff)

Updates to Topic 15: The Use of Mutants, DNA Cloning, and Transgenic Organisms in the Analysis of Development

Answers to Questions in Text

Polyspermic Sea Urchin Eggs (p. 376/377)

  1. Could Boveri (1902) have explained his results by the hypothesis that each blastomere simply needed a minimal number of chromosomes? Answer: No, the surviving blastomeres were not necessarily the ones with the greatest numbers of chromosomes.
  2. How are the disasterous consequences of multiple fertilizations avoided in normal development? Answer: By blocks to polyspermy. See Section 4.5.

Mutagenesis Screen for Genes Controlling Early Development in Drosophila (p. 381/382)

  1. Why did Nüsslein-Volhard et al. (1984) mate single males in the F1 generation whereas groups of males were mated in the P and F2 generations? Answer: Single males were used in the F1 generation because each male was the founder of a mutant line, and because all individuals from a given line should have the same mutagenized 2nd chromosome.
  2. Less than 10% of the lines showing embryonic lethality showed deviations from the normal body pattern that could be discerned in cuticle preparations. Why may the larvae of the other 90% of the lines have been unable to hatch? Answer: One large group of mutants formed an apparently normal body pattern but did not develop far enough to form a cuticle. Another large group of mutants did form an apparently normal cuticle. Their inability to hatch may have been due to internal defects that left them unable to mount the pressure necessary to rupture the egg shell, or chorion.

Use of P-Elements for Genetic Transformation of Drosophila (pp. 396-398)

  1. One of the reasons for Rubin and Spradling (1982) to choose the rosy+ gene for their pioneering experiment was the fact that only a few percent of the normal XDH level are necessary to produce the red eye color of wild-type flies. Why was this an important consideration? Answer: Under these circumstances, the presence of the transgene would cause a phenotypic effect even if the transgene for unforeseen reasons would not be fully expressed. In the absence of a phenotypic effect, the investigators would not have been able to improve their experimental technique.
  2. The transposase activity required to insert the rosy+ transgene i209-214nto the host genome was provided by a separate “helper” plasmid coinjected with the pry1 plasmid. If the investigators had used instead a single plasmid containing both the rosy+ gene and the transposase gene, what would have happened with their transformant lines? Answer: The lines would have been unstable since the transgene would behave like a P element. (For the same reason, it was important to use embryos from a strain without P elements as recipients. ) The helper plasmids were injected in smaller numbers than the pry1 plasmids so that at least some of the pole cells that would form at the injection site would inherit enough transposase enzyme and pry1 plasmid to insert one of the latter, but no helper plasmid, into their genomic DNA.
  3. How do you think the investigators established the chromosomal site of transgene integration for each of their transformant lines? Answer: By in situ hybridization to polytene chromosomes (see Fig. 15.12).

Genetic Transformation of Mice by Transgene Injection into an Egg Pronucleus (p. 399/400)

  1. How do you think Palmiter et al. (1982) established which of the mice derived from injected eggs were transgenic for the MT-GH fusion gene? Answer: Genomic DNA samples from tail tips (missing from mice seen in Fig. 15.1) were prepared for Southern blotting (see Method 15.5). Upon probing with a fragment of the rat GH gene, the blots from seven mice showed the hybridization pattern expected after integration of the transgene.
  2. How could the investigators have tested their expectation that the MT-GH fusion gene would be expressed primarily in the livers and kidneys of the transgenic mice? Answer: By Northern blotting.



RNA interference (RNAi) seems to be built on a natural molecular pathway.

The method of generating the equivalent of loss-of-function alleles by RNA interference, or RNAi, mentioned on p. 395 has turned out to be widely applicable and very specific in its effects, presumably because it utilizes a molecular pathway that has evolved naturally. Variations of this pathway operate in the defense of animals and plants against viruses, in the destruction of erroneously produced RNAs, and in translational control. RNAi has become a powerful tool for researchers to study the biological function of any gene by destroying its mRNA transcripts. Several biotechnology companies are racing to turn RNAi into therapies that will stifle the expression of genes involved in cancer, viral infection, and other diseases.

The basic RNAi mechanism is shown in Fig. 15.1* (below), which is adapted from the review article of Lau and Bartel (2003) quoted below. The key elements of RNAi are small double-stranded RNA molecules that are about 22 base pairs long and called short interfering RNAs (siRNAs). They are produced by an enzyme called dicer from larger double-stranded RNAs, which may be replicating viral RNAs or erroneous transcription products. Cells unwind siRNA into single strands, which associate with multiple proteins to form an RNA-induced silencing complex (RISC). The RISC then captures any mRNA that hybridizes to the siRNA. If the match is perfect then the captured mRNA is cleaved into non-functional fragments, which are released and degraded while the RISC is re-used. Under these circumstances, a few siRNA molecules can cause the inactivation of many mRNA molecules. If the match between siRNA and its target mRNA is less than perfect, the RISC remains stuck to the mRNA and prevents its translation.

Using RNAi is much more convenient than working with transgenes, provided that a way can be found to ferry the siRNA or its larger precursor into the cells of interest. In plants and in nematodes, the RNAi response is somehow spreading far beyond the site where siRNA is injected otherwise introduced. In C. elegans, the functions of 1,722 genes have been studied individually by feeding the worms with bacteria engineered to produce a double-stranded RNA directed against a specific target mRNA (Kamath et al., 2003).

In a study using adult mice, McCaffrey et al. (2002) injected the liver with a reporter gene linking the hepatitis C virus to a gene encoding the firefly enzyme luciferase, which emits light when it reacts with its substrate, luciferin. The reporter gene was expressed in mouse liver as detected by whole body imaging (Fig. 15.2*, below)). The glow was reduced significantly when the transfected mice were co-injected with siRNA directed against luciferase mRNA while co-injection with unrelated siRNA had no such effect.

In some organisms, the RNAi pathway extends beyond the silencing of mRNA and acts on the genome (Matzke and Matzke, 2003). In yeast and plants, siRNA silences not only mRNA but also the encoding gene itself by DNA methylation and histone modification.

Related to siRNA are small regulatory RNAs referred to as microRNAs (miRNAs). Both siRNA and miRNA are cut by dicer from larger double-styranded RNA precursors. And both act either by causing the destruction of their target RNAs or by preventing their transl;ation. However, while siRNA come from the same genes that they ultimately silence, miRNAs come from different genes whose sole function is to produce the small regulatory RNAs. One of the first miRNAs to be discovered is the one encoded by the lin-4 gene of C. elegans (see p. 680). Since then, dozens if not hundreds of miRNAs have been identified in C. elegans, Drosophila, plants, and the human (Carrington and Ambros, 2003).


Fig. 15.1* Current model of RNA interference by short interfering RNA (siRNA)(from Lau and Bartel, 2003)

Top panel: Double-stranded RNA from various sources (a-c) is cleaved by dicer enzyme to produce fragments of about 22 nucleotide pairs called siRNA. Such fragments may also be prepared in vitro and introduced into the cell with liposomes (d). Bottom panel: The siRNA fragments separate into single strands, which combine with proteins to form RNA-induced silencing complexes (RISCs). Each RISC then binds mRNAs complementary to its RNA segment. If the match is perfect then the captured mRNA is cleaved into useless pieces. If the match is imperfect then the RISC remains stuck to the mRNA, thus inhibiting its translation.


Fig. 15.2* Demonstration of RNA interference in adult mice (from McCaffrey et al., 2002; see text above)

Review Articles on RNAi

Carrington J.C. and Ambros V. (2003) Role of microRNAs in plant and animal development. Science 301: 336-338.
Lau N.C. and Bartel D.P. (2003) Censors of the genome. Scient. Amer. August 2003: 34-41
Kamath R.S. et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231-237
Matzke M. and Matzke A.J.M. (2003) RNAi extends its reach. Science 301: 1060-1061
Novina C.D. and Sharp P.A. (2004) The RNAi revolution. Nature 430: 161-164

Research Articles on RNAi

Boutros M. et al. (2004) Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303: 832-835
Giraldez A.J. et al. (2005) MicroRNAs regulate brein morphogenesis in zebrafish. Science 308: 833-838
Kim J.K. et al. (2005) Functional genomic analysis of RNA interference in C. elegans. Science 308: 1164-1167
McCaffrey A.P., Meuse L., Pham T.-T.T., Conklin D.S., Hannon G.J. and Kay M.A. (2002) RNA interference in adult mice. Nature 418: 38-39
Rossi J.R. (2004) A cholesterol connection in RNAi. Nature 432: 155-156
Soennichsen B. et al. (2005) Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434: 462-469
Soutschek J. et al. (2004) Therapeutic silencing of an endogenous gene by systematic administration of modified siRNAs. Nature 432: 173-178


Review Articles on Topics Other than RNAi

Fraser S.E. and Harland R.M. (2000) The molecular metamorphosis of experimental embryology. Cell 100: 41-55
Hirsch N., Zimmerman L.B., and Grainger R.M. (2002) Xenopus, the next generation: X. tropicalis genetics and genomics. Devel. Dynamics 225: 422-433
Heasman J. (2002) Morpholino oligos: Making sense of antisense? Devel. Biol. 243: 209-214


Research Articles on Topics Other than RNAi

International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409: 860-921
Venter C.G. (2001) The sequence of the human genome. Science 291: 1304-1351

Two draft sequences of the human genome were published in print on 15 and 16 February 2001 by two competing teams, one publicly financed consortium writing in Nature and one privately funded group led by Craig Venter of Celera Genomics writing in Science. Both drafts cover about 84% of the entire genome and about 90% of the gene-rich euchromatin. The error rate for the euchromatic sequences is quoted as less than 1 base in 10,000.

One of the major surprises is the unexpectedly low total number of human genes, now estimated near 30,000 by the Consortium and near 40,000 by the Celera group. These estimates represent only about one half of earlier estimates, and only about twice the predicted numbers of genes for Drosophila (14,000 genes) and Caenorhabditis elegans (19,000 genes). These comparisons show that there is no simple relationship between an organism's number of genes and its anatomical and behavioral complexity. The human genes are also rely more on alternative splicing for generating a larger number of protein products.

The number of single nucleotide polymorphisms (SNPs) in the human genome is estimated at 1.6 - 3.2 million, or one SNP every 1,000 - 2,000 nucleotides. Analysis of these SNPs should be useful for many applications, ranging from customized drugs to detailed pedigrees of human populations.

Kamath R.S. et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231-237

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