Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast.

The MKC7 gene was isolated as a multicopy suppressor of the cold-sensitive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-alpha-factor and other secretory proproteins at pairs of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene, a previously isolated multicopy suppressor of the pro-alpha-factor processing defect of a kex2 null. Multicopy MKC7 suppressed the alpha-specific mating defect of a kex2 null as well as multicopy YAP3 did, but multicopy YAP3 was a relatively weak suppressor of kex2 cold sensitivity. Overexpression of MKC7 resulted in production of a membrane-associated proteolytic activity that cleaved an internally quenched fluorogenic peptide substrate on the carboxyl side of a Lys-Arg site. Treatment with phosphatidylinositol-specific phospholipase C shifted Mkc7 activity from the detergent to the aqueous phase in a Triton X-114 phase separation, indicating that membrane attachment of Mkc7 is mediated by a glycosyl-phosphatidylinositol anchor. Although disruption of MKC7 or YAP3 alone resulted in no observable phenotype, mkc7 yap3 double disruptants exhibited impaired growth at 37 degrees C. Disruption of MKC7 and YAP3 in a kex2 null mutant resulted in profound temperature sensitivity and more generalized cold sensitivity. The synergism of mkc7, yap3, and kex2 null mutations argues that Mkc7 and Yap3 are authentic processing enzymes whose functions overlap those of Kex2 in vivo.

Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms.

Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by mutations in the steroid 21-hydroxylase gene (CYP21). Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed "gene conversions," although the mechanism by which they are generated is not well understood. To assess the frequency at which these different recombination events occur, we have used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal individuals. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were never detected in the matching leukocyte DNA. Gene conversions in the same region occur in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggest that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, gene conversions must occur by a mechanism distinct from unequal crossing-over.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

Mutations in the gene encoding the alpha subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa.

Mutations in the genes encoding two proteins of the retinal rod phototransduction cascade, opsin and the beta subunit of rod cGMP phosphodiesterase, cause retinitis pigmentosa (RP) in some families. Here we report defects in a third member of this biochemical pathway in still other patients with this disease. We screened 94 unrelated patients with autosomal dominant RP and 173 unrelated patients with autosomal recessive RP for mutations in the gene encoding the alpha subunit of the rod cGMP-gated cation channel. Five mutant sequences cosegregated with disease among four unrelated families with autosomal recessive RP. Two of these were nonsense mutations early in the reading frame (Glu76End and Lys139End) and one was a deletion encompassing most if not all of the transcriptional unit; these three alleles would not be expected to encode a functional channel. The remaining two mutations were a missense mutation (Ser316Phe) and a frameshift [Arg654(1-bp del)] mutation truncating the last 32 aa in the C terminus. The latter two mutations were expressed in vitro and found to encode proteins that were predominantly retained inside the cell instead of being targeted to the plasma membrane. We conclude that the absence or paucity of functional cGMP-gated cation channels in the plasma membrane is deleterious to rod photoreceptors and is an uncommon cause of RP.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.

The silver gene of Drosophila melanogaster encodes multiple carboxypeptidases similar to mammalian prohormone-processing enzymes.

The silver (svr) gene of Drosophila melanogaster is required for viability, and severe mutant alleles result in death prior to eclosion. Adult flies homozygous or hemizygous for weaker alleles display several visible phenotypes, including cuticular structures that are pale and silvery in color due to reduced melanization. We have identified and cloned the DNA encoding the svr gene and determined the sequence of several partially overlapping cDNAs derived from svr mRNAs. The predicted amino acid sequence of the polypeptides encoded by these cDNAs indicates that the silver proteins are members of the family of preprotein-processing carboxypeptidases that includes the human carboxypeptidases E, M, and N. One class of svr mRNAs is alternatively spliced to encode at least two polyproteins, each of which is composed of two carboxypeptidase domains.

Regulation of yeast phospholipid biosynthetic gene expression in response to inositol involves two superimposed mechanisms.

Transcription of phospholipid biosynthetic genes in the yeast Saccharomyces cerevisiae is maximally derepressed when cells are grown in the absence of inositol and repressed when the cells are grown in its presence. We have previously suggested that this response to inositol may be dictated by regulating transcription of the cognate activator gene, INO2. However, it was also known that cells which harbor a mutant opi1 allele express constitutively derepressed levels of target genes (INO1 and CHO1), implicating the OPI1 negative regulatory gene in the response to inositol. These observations suggested that the response to inositol may involve both regulation of INO2 transcription as well as OPI1-mediated repression. We investigated these possibilities by examining the effect of inositol on target gene expression in a strain containing the INO2 gene under control of the GAL1 promoter. In this strain, transcription of the INO2 gene was regulated in response to galactose but was insensitive to inositol. The expression of the INO1 and CHO1 target genes was still responsive to inositol even though expression of the INO2 gene was unresponsive. However, the level of expression of the INO1 and CHO1 target genes correlated with the level of INO2 transcription. Furthermore, the effect of inositol on target gene expression was eliminated by deleting the OPI1 gene in the GAL1-INO2-containing strain. These data suggest that the OPI1 gene product is the primary target (sensor) of the inositol response and that derepression of INO2 transcription determines the degree of expression of the target genes.

Selective vulnerability of late-generated dopaminergic neurons of the substantia nigra in weaver mutant mice.

In homozygous weaver (wv/wv) mutant mice, nearly 50% of the dopaminergic substantia nigra neurons degenerate by postnatal day 20. We have now determined that the total number of dopaminergic neurons in the ventral midbrains of a litter of obligatory homozygous weaver pups and a litter of normal wild-type control pups indicates that no significant differences are present between groups at birth. To test the hypothesis that the subsequent degeneration of these neurons is linked to their time of origin, [3H]thymidine autoradiography was combined with tyrosine hydroxylase immunocytochemistry to construct neurogenetic timetables on postnatal day 20 in wild-type mice and weaver homozygotes. Both groups have the same span of neurogenesis but have statistically different proportions of neurons generated on specific days. In wild-type mice, more than half of the dopaminergic neurons originate on or after embryonic day 12. In contrast, over two-thirds of the surviving dopaminergic neurons in homozygous weaver mice originate on or before embryonic day 11. Our data suggest that the weaver gene does not interfere with the generation of dopaminergic neurons, but it preferentially kills late-generated dopaminergic neurons between birth and postnatal day 20.

Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.

Previous studies in transgenic mice and cultured cells have indicated that the major enhancer function for erythroid cell expression of the globin genes is provided by the heterodimeric basic-leucine zipper transcription factor NF-E2. Globin gene expression within cultured mouse erythroleukemia cells is highly dependent on NF-E2. To examine the requirement for this factor in vivo, we used homologous recombination in embryonic stem cells to generate mice lacking the hematopoietic-specific subunit, p45 NF-E2. The most dramatic aspect of the homozygous mutant mice was an absence of circulating platelets, which led to the death of most animals due to hemorrhage. In contrast, the effect of loss of NF-E2 on the erythroid lineage was surprisingly mild. Although neonates exhibited severe anemia and dysmorphic red-cell changes, probably compounded by concomitant bleeding, surviving adults exhibited only mild changes consistent with a small decrease in the hemoglobin content per cell. p45 NF-E2-null mice responded to anemia with compensatory reticulocytosis and splenomegaly. Globin chain synthesis was balanced, and switching from fetal to adult globins progressed normally. Although these findings are consistent with the substitution of NF-E2 function in vivo by one or more compensating proteins, gel shift assays using nuclear extracts from p45 NF-E2-null mice failed to reveal novel complexes formed on an NF-E2 binding site. Thus, regulation of globin gene transcription through NF-E2 binding sites in vivo is more complex than has been previously appreciated.

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Cryptorchidism and homeotic transformations of spinal nerves and vertebrae in Hoxa-10 mutant mice.

Homozygous mice mutated by homologous recombination for the AbdB-related Hoxa-10 gene are viable but display homeotic transformations of vertebrae and lumbar spinal nerves. Mutant males exhibit unilateral or bilateral criptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and sterility. These results reveal an important role of Hoxa-10 in patterning posterior body regions and suggest that Hox genes are involved in specifying regional identity of both segmented and nonovertly segmented structures of the developing body.

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.

Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.

A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.

A yeast gene has been identified by screening for DNA replication mutants using a permeabilized cell replication assay. The mutant is temperature sensitive for growth and shows a cell cycle phenotype typical of DNA replication mutants. RNA synthesis is normal in the mutant but DNA synthesis ceases upon shift to the nonpermissive temperature. The DNA2 gene was cloned by complementation of the dna2ts gene phenotype. The gene is essential for viability. The gene encodes a 172-kDa protein with characteristic DNA helicase motifs. A hemagglutinin epitope-Dna2 fusion protein was prepared and purified by conventional and immunoaffinity chromatography. The purified protein is a DNA-dependent ATPase and has 3' to 5' DNA helicase activity specific for forked substrates. A nuclease activity that endonucleolytically cleaves DNA molecules having a single-stranded 5' tail adjacent to a duplex region copurifies through all steps with the fusion protein.