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Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells
~]ONITOR nities for investigation of gene function. Bearing in mind the the size of the target sequence compared with the genome as a whole, it might have been expected that homologous recombination would be the weak link in the technique, but as it turns out, getting the targeted embryonic stem (ES) cells into the germ line of chimeric mice has proved more problematical. Two recent reports confirm that this problem is being overcome. Zijlstra et al. disrupted the 132microglobulin gene, which encodes a component of class I MHC molecules. Schwartzberg et al. introduced
less dramatic alterations into the tyrosine kinase-encoding c-abl protooncogene (just mutating the terminal third of the protein). Both have obtained germ-line transmission of the targeted gene, but this is strongly dependent on the genotype of the host blastocyst into which manipulated ES cells are introduced. Interestingly, the heterozygous mice produced by both groups are phenotypically normal; crossing heterozygotes to give mice homozygous for the disrupted gene should, all being well, begin to give meaningful results about gene function in development, a~
A sperm supplied product essential for initiation of normal embryogenesisin Caenorhabditis elegans is encoded by the paternal-effectembryonic-lethal gene spe-II
Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation
Another role for melanocytes: their importance for normal stria vascularis development in the inner ear
M.P. KING AND G. ATrARDI
K.P. STEEL AND C. BARKWAY
Science 246, 500-503
Development 107, 453--463
D.P. HILL~ D.C. SHAKES, $. WARD AND S. STROME
Mammalian mitochondria are beginning to be opened up to bona fide genetic manipulation. King and Attardi have generated two cell lines devoid of mitochonddal DNA, by long term exposure to ethidium bromide. These cell lines rely totally on glycolysis for energy production, and are dependent upon uridine and pyruvate for growth, a feature that King and Attardi have now used to select for cells that have incorporated functional mitochondria from a number of donor cell lines, after fusion with cytoblasts. The respiratory characteristics of the viable 'cybrids' are distinct from those of the parent of the 'demitochondrialized' cell line and the cell line that donated the mitochondria, so mitochondrial anti nuclear genotypes, and their interactions, define the respiratory behaviour of a cell. Moreover, the powerful genetics possible in S. ceret,isiae should now be applicable to mammalian cells: nuclear suppressors of mtDNA mutations, and mtDNA suppressors of defective mitochondrial biogenesis, will be one obvious area to explore. The technique may also be relevant to the analysis of mitochondrial mutations giving rise to neuromuscular disease. Perhaps an answer will be found to the vexing problem that mammalian mitochondria are organizationally compact and neat, while those of fast-growing yeast are, in comparison, a mess. a~
What links white cats, Dalmation dogs and deaf mice? The answer, of course, is melanocytes. Many animals with coat colour abnormalities also have hearing abnormalities, and melanocytes appear to link these disorders, although no role for melanocytes in hearing has been defined. Steel and Barkway investigated melanocyte function in the stria vascularis of the cochlea in the mouse mutant viable dominant spotting, whose neural crest cells are defective and fail to give rise to melanocytes in the skin. Morphologically the stria vascularis consists of three layers - epitheliumderived marginal cells on the luminal surface, basal cells of unknown origin, and scattered intermediate cells, some of which are melanocytes. During devek)pment, the marginal cell layer forms an immense number of processes, which interdigitate with underlying cells. The stria vascularis also has a conveniently assayed function: it produces the endocochlear potential (EP), a resting potential in the endolymph of around +100 mV in mice. The authors followed stria structure and EP levels in mutant mice at differing times after birth: the EP is abolished at all stages, while the interdigitation of marginal cells is greatly diminished. Thus melanocytes are important for normal development and function of the stria vascularis, and the challenge now is to pinpoint how.
mitosis in all eukaryotic cells. However, in fission and budding yeasts, CDC28/cdc2 is also known to be required earlier in the cell cycle, at the G1-S transition (a point termed 'start'). This has led to the suggestion that it may play an analogous role in controlling the initiation of DNA synthesis. However, Shuster and Byers show here that during meiosis, budding
yeast cdc28 mutants (and two other 'start' mutants) block the meiotic cycle after completion of premeiotic DNA synthesis. This illustrates that initiation of DNA synthesis does not necessarily require CDC28/cdc2. Instead, the function of CDC28/cdc2 at 'start' may be to commit cells to nuclear division, an event that under some circumstances occurs after DNA synthesis, a~
Germ-line transmissiou of a disrupted I]2-microglobulingene produced by homologous recombiHation in embryonic stem cells M. Z~LSrRA ET AL
Nature 342, 435--438
Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells P.L SCHWARTZBERG, S.F. GOlF AND EJ. ROBERTSON
Science 246, 799-803 Gene targeting in mammalian cells, if perfected, offers a wealth of opportu-
Dev. Biol. 136, i54-166 While maternal effect genes have cropped up repeatedly in studies of embryogenesis (e.g. the battery of genes identified in Drosopbila), paternal effect genes are rather more scarce. Hill et al. have isolated and I~gun to characterize a C. elegans paternal effect gene, spe-ll: normal oocytes fertilized IW sperm from homozygous spe-ll mutants develop abnormally, while oocytes from such mutants give rise to normal embryos when fertilized by wild-type sperm. Spermatogenesis-defective (spe) mr,rants are generally identified as worms that are self-sterile (C. elegans is hermaphroditic and, in the absence of a mate, self-fertilizes) but produce offspring when crossed with normal m~:les, spe-ll is exceptional because most spe mutants affect sperm development/function rather than development of the fertilized embryo. The role of spe-ll + is unclear, but it appears to be expressed during spermatogenesis, and mutants affect early (one-cell stage) activation events in embryogenesis, and prevent correct mitotic spindle formation and cytokinesis. Interestingly, the two paternal effect genes identified in Drosophila, pal and ms(3)K81, appear unrelated to spe-ll, affecting mainly chromosome behaviour in the early embryo.a~,
Pachytene arrest and other meiotic effects of the Start mutations in Saccbaromyces cerevisiae E.O. SHUSTER AND n. BYERS
Genetics 123, 29--43 The CDC28/cdc2 gene encodes a protein kinase that is part of maturationpromoting factor, which plays a critical role in controlling the timing of
TIG JANUARY 1990 VOL.
m
6 NO. 1
less dramatic alterations into the tyrosine kinase-encoding c-abl protooncogene (just mutating the terminal third of the protein). Both have obtained germ-line transmission of the targeted gene, but this is strongly dependent on the genotype of the host blastocyst into which manipulated ES cells are introduced. Interestingly, the heterozygous mice produced by both groups are phenotypically normal; crossing heterozygotes to give mice homozygous for the disrupted gene should, all being well, begin to give meaningful results about gene function in development, a~
A sperm supplied product essential for initiation of normal embryogenesisin Caenorhabditis elegans is encoded by the paternal-effectembryonic-lethal gene spe-II
Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation
Another role for melanocytes: their importance for normal stria vascularis development in the inner ear
M.P. KING AND G. ATrARDI
K.P. STEEL AND C. BARKWAY
Science 246, 500-503
Development 107, 453--463
D.P. HILL~ D.C. SHAKES, $. WARD AND S. STROME
Mammalian mitochondria are beginning to be opened up to bona fide genetic manipulation. King and Attardi have generated two cell lines devoid of mitochonddal DNA, by long term exposure to ethidium bromide. These cell lines rely totally on glycolysis for energy production, and are dependent upon uridine and pyruvate for growth, a feature that King and Attardi have now used to select for cells that have incorporated functional mitochondria from a number of donor cell lines, after fusion with cytoblasts. The respiratory characteristics of the viable 'cybrids' are distinct from those of the parent of the 'demitochondrialized' cell line and the cell line that donated the mitochondria, so mitochondrial anti nuclear genotypes, and their interactions, define the respiratory behaviour of a cell. Moreover, the powerful genetics possible in S. ceret,isiae should now be applicable to mammalian cells: nuclear suppressors of mtDNA mutations, and mtDNA suppressors of defective mitochondrial biogenesis, will be one obvious area to explore. The technique may also be relevant to the analysis of mitochondrial mutations giving rise to neuromuscular disease. Perhaps an answer will be found to the vexing problem that mammalian mitochondria are organizationally compact and neat, while those of fast-growing yeast are, in comparison, a mess. a~
What links white cats, Dalmation dogs and deaf mice? The answer, of course, is melanocytes. Many animals with coat colour abnormalities also have hearing abnormalities, and melanocytes appear to link these disorders, although no role for melanocytes in hearing has been defined. Steel and Barkway investigated melanocyte function in the stria vascularis of the cochlea in the mouse mutant viable dominant spotting, whose neural crest cells are defective and fail to give rise to melanocytes in the skin. Morphologically the stria vascularis consists of three layers - epitheliumderived marginal cells on the luminal surface, basal cells of unknown origin, and scattered intermediate cells, some of which are melanocytes. During devek)pment, the marginal cell layer forms an immense number of processes, which interdigitate with underlying cells. The stria vascularis also has a conveniently assayed function: it produces the endocochlear potential (EP), a resting potential in the endolymph of around +100 mV in mice. The authors followed stria structure and EP levels in mutant mice at differing times after birth: the EP is abolished at all stages, while the interdigitation of marginal cells is greatly diminished. Thus melanocytes are important for normal development and function of the stria vascularis, and the challenge now is to pinpoint how.
mitosis in all eukaryotic cells. However, in fission and budding yeasts, CDC28/cdc2 is also known to be required earlier in the cell cycle, at the G1-S transition (a point termed 'start'). This has led to the suggestion that it may play an analogous role in controlling the initiation of DNA synthesis. However, Shuster and Byers show here that during meiosis, budding
yeast cdc28 mutants (and two other 'start' mutants) block the meiotic cycle after completion of premeiotic DNA synthesis. This illustrates that initiation of DNA synthesis does not necessarily require CDC28/cdc2. Instead, the function of CDC28/cdc2 at 'start' may be to commit cells to nuclear division, an event that under some circumstances occurs after DNA synthesis, a~
Germ-line transmissiou of a disrupted I]2-microglobulingene produced by homologous recombiHation in embryonic stem cells M. Z~LSrRA ET AL
Nature 342, 435--438
Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells P.L SCHWARTZBERG, S.F. GOlF AND EJ. ROBERTSON
Science 246, 799-803 Gene targeting in mammalian cells, if perfected, offers a wealth of opportu-
Dev. Biol. 136, i54-166 While maternal effect genes have cropped up repeatedly in studies of embryogenesis (e.g. the battery of genes identified in Drosopbila), paternal effect genes are rather more scarce. Hill et al. have isolated and I~gun to characterize a C. elegans paternal effect gene, spe-ll: normal oocytes fertilized IW sperm from homozygous spe-ll mutants develop abnormally, while oocytes from such mutants give rise to normal embryos when fertilized by wild-type sperm. Spermatogenesis-defective (spe) mr,rants are generally identified as worms that are self-sterile (C. elegans is hermaphroditic and, in the absence of a mate, self-fertilizes) but produce offspring when crossed with normal m~:les, spe-ll is exceptional because most spe mutants affect sperm development/function rather than development of the fertilized embryo. The role of spe-ll + is unclear, but it appears to be expressed during spermatogenesis, and mutants affect early (one-cell stage) activation events in embryogenesis, and prevent correct mitotic spindle formation and cytokinesis. Interestingly, the two paternal effect genes identified in Drosophila, pal and ms(3)K81, appear unrelated to spe-ll, affecting mainly chromosome behaviour in the early embryo.a~,
Pachytene arrest and other meiotic effects of the Start mutations in Saccbaromyces cerevisiae E.O. SHUSTER AND n. BYERS
Genetics 123, 29--43 The CDC28/cdc2 gene encodes a protein kinase that is part of maturationpromoting factor, which plays a critical role in controlling the timing of
TIG JANUARY 1990 VOL.
m
6 NO. 1