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TRENDS in Genetics Vol.17 No.8 August 2001

RNAi surges on: application to cultured mammalian cells The discovery that potent, sequence-specific inactivation of gene function can be induced by double-stranded RNA (dsRNA) has led to a revolution in reverse genetic analysis. The apparent ubiquity of the biochemical pathways involved means the approach, termed RNAi (for dsRNA-mediated interference has been demonstrated in various species of protozoa, hydra, flatworms, roundworms, arthropods and vertebrates, many not previously thought of as good genetic systems. For mammals, however, it was thought RNAi might be suitable only for studies on the oocyte and preimplantation embryo. In mammalian cells other than these, dsRNA triggers a nonspecific inhibition of protein synthesis that overwhelms any sequence-specific RNAi effect. Tuschl and colleagues1 have now demonstrated that the provision of an intermediate of the RNAi pathway can induce the sequence-specific effect without activating the general inhibition of translation in mammalian cells. Elbashir et al.1 showed that synthetic, 21-nucleotide RNAs, called small interfering RNAs (siRNAs), base paired such that they have

two-nucleotide 3’ overhangs, can specifically inhibit gene expression in cultured cells. Upon co-transfection with siRNAs, specific reductions in luciferase reporter gene expression occur in tissue cultured cells from human, monkey and mouse, as well as Drosophila melanogaster. In the mammalian cells, the effect was not as absolute as in the insect cells, with reductions in luciferase activity of up to 25-fold. Expression of one of the luciferase genes was not affected in one of the human cell lines examined. The study also examined endogenous genes. Again, the researchers used specific siRNAs, introduced into cultured human cells by liposome-mediated transfection, and measured the levels of the targeted endogenous proteins using antibodies. Although no reduction was observed in vimentin levels, a 90% reduction in lamin A/C protein was observed and lamin B1 and NuMA (nuclear mitotic apparatus protein) were also markedly reduced. Perhaps further developments, such as the use of multiple siRNAs aimed at a single gene, might improve the inhibition. The siRNAmediated RNAi effects might not be as

complete or as reliable, with regard to cell type or target gene, as other systems, but the applicability of RNAi in mammalian systems has been extended substantially by this work. The development of siRNAs is another step in the realization of the enormous potential of RNAi. The technique should be immediately applicable to reverse genetic analysis of the genes found in the human genome sequence. The pace of developments in the RNAi field gives the impression that any obstacle to a potential application of the technology will be overcome. If the spreading of the RNAi effect between cells throughout an organism could be obtained in mammals, in the same way as occurs in the nematode Caenorhabditis elegans, then RNAi could herald a new epoch in medicine. 1 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498

Ian A. Hope [email protected]

Cytoplasmic incompatibility and maternal-haploid Cytoplasmic incompatibility (CI) is an intriguing phenomenon, widespread in arthropods, that is induced by the maternally inherited endocellular bacteria Wolbachia. It can be described as an embryonic mortality occurring in crosses between infected males and uninfected females, whereas fertility is normal in the reverse cross, or if both partners are infected. The genes involved are unknown, but there is a general consensus that CI involves at least two bacterial functions: mod (for modification) and resc (for rescue). mod, the ‘poison’, is deposited on paternal chromosomes before Wolbachia are shed from maturing sperm, whereas resc, the ‘antidote’, is expressed by Wolbachia in infected eggs and saves the embryo from death. Embryonic mortality is due to the loss of paternal chromosomes, occurring at the first mitotic division. Chromosomes behave normally until metaphase, but paternal chromatids http://tig.trends.com

fail to separate in anaphase, resulting in the formation of a chromatin bridge in telophase. Loppin et al.1 recently provided an accurate cytological description of maternal-haploid (mh), a Drosophila melanogaster maternal-effect mutation. In fertilized eggs produced by mh homozygous mothers, paternal chromosomes are lost during the first mitosis or soon after, resulting in haploidy and embryo death. This observation demonstrates that paternal chromosomes are not functional after fertilization, unless maternal factors render them so. The authors note that the resemblance with CI is striking: in crosses involving mh homozygous mothers, paternal chromosomes behave exactly as in crosses where CI occurs (that is, in crosses between males infected by Wolbachia and uninfected females). What is the link between mh and CI? We see two hypotheses.

First, mh and mod might target the same sites on paternal chromosomes. Indeed, mod could occupy the target sites of mh, and thus disrupt the normal processing of anaphase. By interacting with mod, resc would allow the mh activity, and restore embryonic viability. Identifying the mh targets could then provide an indirect way to identify the mod function. Another, more speculative, hypothesis is that mh and resc are paralogous genes. In other words, the mod–resc system might be present in both the host and the Wolbachia genomes. Gene exchanges from the symbiont to the nucleus (or the other way round) could explain this pattern. Under this hypothesis, the mh phenotype would result from the same molecular process as CI: the inability of mh homozygous mothers to produce a ‘resc-like’ function and, thus, to save the embryo from the effect of a ‘mod-like’

0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.

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function expressed in the germline of the males. We have tested experimentally whether there is any functional redundancy between mh and the resc allele harboured by a Wolbachia variant present in some natural populations of D. melanogaster (namely, wMel): mh homozygous females, even when infected by wMel, are totally sterile.

TRENDS in Genetics Vol.17 No.8 August 2001

Thus, it seems that the resc allele harboured by wMel is not able to mimic the ‘resc-like’ function presumably missing in mh mutants. However, this is not surprising: Wolbachia biology teaches us that multiple and nonredundant resc alleles do exist. Wolbachia sequencing projects will soon reveal whether this proposition has any value.

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1 Loppin, B. et al. (2001) Paternal chromosomes incorporation into the zygote nucleus is controlled by maternal haploid in Drosophila. Dev. Biol. 231, 383–396

Sylvain Charlat [email protected] Hervé Merçot [email protected]

In Brief

Reversible regulation of mouse genes

Free bioinformatics education

All undergraduates studying genetics are taught about the lac operator–repressor system of E. coli. Now, this system can be hijacked to provide regulation of genes in the mouse. Previous attempts to use the lac operator system in mammals in vivo failed because the bacterial lacI coding sequences become heavily methylated and consequently silenced. A group led by Heidi Scrable made the lacI sequence more like eukaryotic sequences so that it could pass through the germline and embryogenesis without losing the ability to be transcribed. Mice expressing the altered lacI repressor system ubiquitously were crossed with mice that express a tyrosinase transgene that controls coat color. The promoter region of the transgene contained the lac operator sequence, which binds the repressor. Putting the lactose analog IPTG in the drinking water changes the coat color of the doubletransgenic mice. This system has the advantage over others, such as Cre/loxP, in that when IPTG treatment stops, the effects are reversible. Scrable and colleagues suggest that by targeting endogenous promoters, ‘endogenous loci can be switched on and off repeatedly to create reversible models of human disease and normal development in the mouse’. [Cronin, C.A. et al. (2001) Genes Dev. 15, 1506–1517] AI http://tig.trends.com

There is an immense need for bioinformaticists all over the world, but bioinformatics courses are only being offered in a few universities. Now, a worldwide consortium of six universities is offering free online bioinformatics courses to everyone. Experts from Stanford University, the Karolinska Institute, Uppsala University, the University of Western Cape, the National University of Singapore and the University of Sydney have come together to provide this unique Global Genomics and Bioinformatics Unified Learning Environment (GLOBULE). Audiovisual lectures in genomics, bioinformatics and medical informatics are offered, available in a modular format to allow students to choose their own topics of interest. The course is designed not only to help undergraduates with a basic knowledge of biology, mathematics and programming, but also to develop the skills of graduate students and professional bioinformaticists. (http://www.s-star.org) AP

Timing HIV infection Simultaneous monitoring of more than 7000 genes over 72 hours produced the first sequential record of the steps leading to immune cell death following HIV infection. The microarray results demonstrate how efficiently and rapidly HIV takes over the cell and inserts its own viral blueprints for destruction, suppressing vital survival and repair functions, and inducing the cell to kill itself. Within hours of entering the immune cell, HIV suppresses genes that regulate and maintain a constant and healthy internal environment, cripples enzymes essential for function of the mitochondria, and suppresses the genes involved in DNA repair, rendering the cell incapable of mending HIV-induced damage. Only 30 minutes after exposure to

HIV, more than 500 genes were shut down in the infected cells. Conversely, nearly 200 genes were activated in the infected cells compared with normal cells. These included genes associated with cellular defense against invasion, and ‘suicide’ genes that normally remain dormant until switched on as part of the normal cycle of cell death. ‘Better understanding of the steps involved in HIV destruction of immune system cells opens the door to new investigations of methods to potentially block or prevent HIV infection,’ said Jacques Corbeil, assistant professor of medicine at the University of California, San Diego. [Corbeil, J. et al. (2001) Genome Res. 11, 1198–1204] PL

When is a clone not a clone? Despite recent successes in cloning animals of several different species using nuclear transfer (NT), the process is far from efficient. The production rate of cloned offspring is low because of increased abortions, and many fetuses that survive show abnormal symptoms, such as high birth-weight. Two recent studies, one examining cloned mice and the other cloned cattle, show that these problems might arise because of incomplete epigenetic reprogramming of donor DNA. Early in normal development, genomewide demethylation occurs, followed by re-methylation of appropriate genomic regions. This process erases the gametic methylation patterns inherited from the parents and is a prerequisite for normal development. Both studies found that the methylation status of genes in animals generated by NT differed from controls (i.e. normally mated mice and IVF-generated cattle, respectively). Importantly, the mouse study also detected differences in methylation between cloned animals, indicating that clones might not actually be perfect copies of one another. [Kang, Y.K. et al. (2001) Nat. Genet. 28, 173–177; Ohgane, J. et al. (2001) Genesis 30, 45–50] AI

0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.