Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes

We have analyzed the Drosophila immune response in domino mutant larvae, which are devoid of blood cells. The domino mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of domino larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that domino mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (immune deficiency larvae) and in mutants that are deficient for humoral melanization (Black cells larvae). However, if domino was combined with either the immune deficiency or the Black cell mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide Drosophila a highly effective defense against injury and/or infection.

Identification of morc (microrchidia), a mutation that results in arrest of spermatogenesis at an early meiotic stage in the mouse

The microrchidia, or morc, autosomal recessive mutation results in the arrest of spermatogenesis early in prophase I of meiosis. The morc mutation arose spontaneously during the development of a mouse strain transgenic for a tyrosinase cDNA construct. Morc −/− males are infertile and have grossly reduced testicular mass, whereas −/− females are normal, indicating that the Morc gene acts specifically during male gametogenesis. Immunofluorescence to synaptonemal complex antigens demonstrated that −/− male germ cells enter meiosis but fail to progress beyond zygotene or leptotene stage. An apoptosis assay revealed massive numbers of cells undergoing apoptosis in testes of −/− mice. No other abnormal phenotype was observed in mutant animals, with the exception of eye pigmentation caused by transgene expression in the retina. Spermatogenesis is normal in +/− males, despite significant transgene expression in germ cells. Genomic analysis of −/− animals indicates the presence of a deletion adjacent to the transgene. Identification of the gene inactivated by the transgene insertion may define a novel biochemical pathway involved in mammalian germ cell development and meiosis.

Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene Lhx8

Formation of the mammalian secondary palate is a highly regulated and complex process whose impairment often results in cleft palate, a common birth defect in both humans and animals. Loss-of-function analysis has linked a growing number of genes to this process. Here we report that Lhx8, a recently identified LIM homeobox gene, is expressed in the mesenchyme of the mouse palatal structures throughout their development. To test the function of Lhx8 in vivo, we generated a mutant mouse with a targeted deletion of the Lhx8 gene. Our analysis of the mutant animals revealed a crucial role for Lhx8 in palatogenesis. In Lhx8 homozygous mutant embryos, the bilateral primordial palatal shelves formed and elevated normally, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. Because development of other craniofacial structures appeared normal, the impaired palatal formation in Lhx8-mutant mice was most likely caused by an intrinsic primary defect in the mesenchyme of the palatal shelves. The cleft palate phenotype observed in Lhx8-mutant mice suggests that Lhx8 is a candidate gene for the isolated nonsyndromic form of cleft palate in humans.

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Phosphorylation Controls Timing of Cdc6p Destruction: A Biochemical Analysis

The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell cycle. Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed. First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase. However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p. SCF-Cdc4p– and N-terminal phosphorylation site–dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle. The stable cdc6 mutants show no apparent replication defects in wild-type strains. However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex.

High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Δ mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of ∼100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of ∼480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.

Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation

We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum. Our analysis provides important insights into the molecular pathogenesis of inherited prion disorders in humans.

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin β6 subunit (β6−/−), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8m) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

Mutation R120G in αB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function

αB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in αB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92–95]. By using α-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G αB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G αB-crystallin to unfolding α-lactalbumin enhanced the kinetics and extent of its aggregation. R120G αB-crystallin became entangled with unfolding α-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G αB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G αB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G αB-crystallin has decreased β-sheet secondary structure and an altered aromatic residue environment compared with wild-type αB-crystallin. The apparent molecular mass of R120G αB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type αB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G αB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of αB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

Human sulfite oxidase R160Q: Identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation

Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.

A premature-termination mutation in the Mus musculus cyclin-dependent kinase 3 gene

Our understanding of the mammalian cell cycle is due in large part to the analysis of cyclin-dependent kinase (CDK) 2 and CDK4/6. These kinases are regulated by E and D type cyclins, respectively, and coordinate the G1/S-phase transition. In contrast, little is known about CDK3, a homolog of CDK2 and cell division cycle kinase 2 (CDC2). Previous studies using ectopic expression of human CDK3 suggest a role for this kinase in the G1/S-phase transition, but analysis of the endogenous kinase has been stymied by the low levels of protein present in cells and by the absence of an identifiable cyclin partner. Herein we report the presence of a single point mutation in the CDK3 gene from several Mus musculus strains commonly used in the laboratory. This mutation results in the replacement of a conserved tryptophan (Trp-187) within kinase consensus domain IX with a stop codon. The protein predicted to be encoded by this allele is truncated near the T loop, which is involved in activation by CDK-activating kinase. This mutation also deletes motif XI known to be required for kinase function and is, therefore, expected to generate a null allele. In stark contrast, CDK3 from two wild-mice species (Mus spretus and Mus mus castaneus) lack this mutation. These data indicate that CDK3 is not required for M. musculus development and suggest that any functional role played by CDK3 in the G1/S-phase transition is likely to be redundant with another CDK.

Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa

Length change mutation at the Ms6hm hypervariable mouse minisatellite locus was analyzed in C57BL/6N × C3H/HeN F1 mice and the F1 of the reciprocal cross born to irradiated male parents. Spontaneous mutant frequencies were 8.4% and 9.8% for the paternally derived and maternally derived C3H/HeN alleles, respectively. The mutant frequencies for the paternally derived allele increased to 22% and 19% when the male parents were irradiated with 6 Gy at the postmeiotic spermatozoa stage and the spermatogonia stage, respectively. These increases in the mutant frequency were at least 10 to 100 times higher than those expected from the frequency of hits to the 3- to 4-kb allele, suggesting that the length change mutation at this minisatellite locus was not a targeted event due directly to DNA damage in the region. Further analysis demonstrated that the mutant frequency increased also at the maternally derived C3H/HeN allele to 20% when the male parents were irradiated at the spermatozoa stage. This increase in the maternal allele mutation was not observed in F1 born to irradiated spermatogonia. The present study suggests that introduction of DNA damage by irradiated sperm triggers genomic instability in zygotes and in embryos of subsequent developmental stages, and this genomic instability induces untargeted mutation in cis at the paternally derived minisatellite allele and in trans at the maternally derived unirradiated allele. Untargeted mutation revealed in the present study defines a previously unnoticed genetic hazard to the maternally derived genome by the paternally introduced DNA damage.