Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.     

PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes

PINCH1, an adaptor protein composed of five LIM domains, mediates protein-protein interactions and functions as a component of the integrin-integrin-linked kinase (ILK) complex. The integrin-ILK signaling complex plays a pivotal role in cell motility, proliferation, and survival during embryonic development of many animal species. To elucidate the physiological function of PINCH1 in mouse embryonic development, we have deleted the mouse PINCH1 gene by homologous recombination. Mice heterozygous for PINCH1 are viable and indistinguishable from wild-type littermates. However, no viable homozygous offspring were observed from PINCH1+/- intercrosses. Histological analysis of homozygous mutant embryos revealed that they had a disorganized egg cylinder by E5.5, which degenerated by E6.5. Furthermore, E5.5 PINCH1-/- embryos exhibited decreased cell proliferation and excessive cell death. We have also generated and analyzed mice in which PINCH1 has been specifically deleted in ventricular cardiomyocytes. These mice exhibit no basal phenotype, with respect to mouse survival, cardiac histology, or cardiac function as measured by echocardiography. Altogether, these data indicate that PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.     

Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development

The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 (gt/gt) embryos and Dhps (gt/gt) embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a (gt/gt) or Dhps (gt/gt) blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.     

Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but-in contrast to mice homozygous for a null K-ras allele-they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis.     

The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain

We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.     

Genetic evidence for interactions between yeast importin α (Srp1p) and its nuclear export receptor, Cse1p

The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature.     

Genetic evidence for interactions between yeast importin alpha (Srp1p) and its nuclear export receptor, Cse1p.

The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature. Received: 18 November 1998 / Accepted: 17 March 1999     

The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro.

Previous studies have suggested that the vav protooncogene plays an important role in hematopoiesis. To study this further, we have ablated the vav protooncogene by homologous recombination in embryonic stem (ES) cells. Homozygous vav (-/-) ES clones differentiate normally in culture and generate cells of erythroid, myeloid and mast cell lineages. Mice heterozygous for the targeted vav allele do not display any obvious abnormalities. However, homozygous embryos die very early during development. Crosses of vav (+/-) heterozygous mice yield apparently normal vav (-/-) E3.5 embryos but not post-implantation embryos (> or = E7.5). Furthermore, homozygous vav (-/-) blastocysts do not hatch in vitro. These results indicate that vav is essential for an early developmental step(s) that precedes the onset of hematopoiesis. Consistent with the phenotypic analysis of vav (-/-) embryos, we have identified Vav immunoreactivity in the extra-embryonic trophoblastic cell layer but not in the inner embryonic cell mass of E3.5 preimplantation embryos or in the egg cylinder of E6.5 and E7.5 post-implantation embryos. These results suggest that the vav gene is essential for normal trophoblast development and for implantation of the developing embryo.     

Disruption of the developmentally regulated Rev3l gene causes embryonic lethality

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.     

Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality

Mutations in the cytosolic enzyme phosphomannomutase 2 (PMM2), which catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, cause the most common form of congenital disorders of glycosylation, termed CDG-Ia. It is an inherited multisystemic disease with severe neurological impairment. To study the pathophysiology of CDG-Ia and to investigate possible therapeutic approaches, we generated a mouse model for CDG-Ia by targeted disruption of the Pmm2 gene. Heterozygous mutant mice appeared normal in development, gross anatomy, and fertility. In contrast, embryos homozygous for the Pmm2-null allele were recovered in embryonic development at days 2.5 to 3.5. These results indicate that Pmm2 is essential for early development of mice. Mating experiments of heterozygous mice with wild-type mice could further show that transmission of the female Pmm2-null allele is impaired.     

Targeted disruption of the mouse Asna1 gene results in embryonic lethality

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.     

Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.     

Molecular basis for the selective recognition and ubiquitination of centromeric histone H3 by yeast E3 ligase Psh1

Centromeres are chromosomal loci marked by histone variant Cen H3(centromeric histone H3) and essential for genomic stability and cell division. The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast Cen H3(Cse4) for ubiquitination and controls the cellular level of Cse4 for proteolysis,but the underlying mechanism remains largely unknown. Here, we show that Psh1 uses a Cse4-binding domain(CBD, residues 1-211) to interact with Cse4-H4 instead of H3-H4, yielding a dissociation constant(K_d) of 27 nM. Psh1 recognizes Cse4-specific residues in the L1 loop and a2 helix to ensure Cse4 binding and ubiquitination. We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination. Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding. Together, our study reveals a novel Cse4-binding mode distinct from those of known Cen H3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.