Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

p53 deficiency does not affect the accumulation of point mutations in a transgene target.

DNA repair is required by organisms to prevent the accumulation of mutations and to maintain the integrity of genetic information. Mammalian cells that have been treated with agents that damage DNA have an increase in p53 levels, a p53-dependent arrest at G1 in the cell cycle, and a p53-dependent apoptotic response. It has been hypothesized that this block in cell cycle progression is necessary to allow time for DNA repair or to direct the damaged cell to an apoptotic pathway. This hypothesis predicts that p53-deficient cells would have an abnormal apoptotic response and exhibit a "mutator" phenotype. Using a sensitive assay for the accumulation of point mutations, small deletions, and insertions, we have directly tested whether p53-deficient cells exhibit an increased frequency of mutation before and after exposure to DNA-damaging agents. We report that wild-type and p53-deficient fibroblasts, thymocytes, and tumor tissue have indistinguishable rates of point mutation accumulation in a transgenic lacI target gene. These results suggest that the role of p53 in G1 checkpoint control and tumor suppression does not affect the accumulation of point mutations.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by approximately 35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than approximately 35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial, mutations. The most prominent feature of the ENU-induced base-pair mutations in testicular germ cells sampled 55 days after treatment is that 70% are induced in A.T base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.

Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress.

We have discovered that three longevity mutants of the nematode Caenorhabditis elegans also exhibit increased intrinsic thermotolerance (Itt) as young adults. Mutation of the age-1 gene causes not only 65% longer life expectancy but also Itt. The Itt phenotype cosegregates with age-1. Long-lived spe-26 and daf-2 mutants also exhibit Itt. We investigated the relationship between increased thermotolerance and increased life-span by developing conditions for environmental induction of thermotolerance. Such pretreatments at sublethal temperatures induce significant increases in thermotolerance and small but statistically highly significant increases in life expectancy, consistent with a causal connection between these two traits. Thus, when an animal's resistance to stress is increased, by either genetic or environmental manipulation, we also observe an increase in life expectancy. These results suggest that ability to respond to stress limits the life expectancy of C. elegans and might do so in other metazoa as well.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

Increased expression in adipocytes of ob RNA in mice with lesions of the hypothalamus and with mutations at the db locus.

The gene product of the recently cloned mouse obese gene (ob) is important in regulating adipose tissue mass. ob RNA is expressed specifically by mouse adipocytes in vivo in each of several different fat cell depots, including brown fat. ob RNA is also expressed in cultured 3T3-442A preadipocyte cells that have been induced to differentiate. Mice with lesions of the hypothalamus, as well as mice mutant at the db locus, express a 20-fold higher level of ob RNA in adipose tissue. These data suggest that both the db gene and the hypothalamus are downstream of the ob gene in the pathway that regulates adipose tissue mass and are consistent with previous experiments suggesting that the db locus encodes the ob receptor. In db/db and lesioned mice, quantitative differences in expression level of ob RNA correlated with adipocyte lipid content. The molecules that regulate expression level of the ob gene in adipocytes probably are important in determining body weight, as are the molecules that mediate the effects of ob at its site of action.

Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations.

The human VHL tumor suppressor gene has been implicated in the inherited disorder von Hippel-Lindau disease and in sporadic renal carcinoma. The homologous rat gene encodes a 185-amino acid protein that is 88% sequence identical to the aligned 213-amino acid human VHL gene product. When expressed in COS-7 cells, both the human and the rat VHL proteins showed predominant nuclear, nuclear and cytosolic, or predominant cytosolic VHL staining by immunofluorescence. A complicated pattern of cellular proteins was seen that could be specifically coimmunoprecipitated with the introduced VHL protein. A complex containing VHL and proteins of apparent molecular masses 16 and 9 kDa was the most consistently observed. Certain naturally occurring VHL missense mutations demonstrated either complete or partial loss of the p16-p9 complex. Thus, the VHL tumor suppressor gene product is a nuclear protein, perhaps capable of specifically translocating between the nucleus and the cytosol. It is likely that VHL executes its functions via formation of specific multiprotein complexes. Identification of these VHL-associated proteins will likely clarify the physiology of this tumor suppressor gene.

A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica.

Mutations causing mitochondrial defects were induced in a virulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. Virulence on apples and chestnut trees was reduced in four of six extensively characterized mutants. Relative to the virulent progenitor, the attenuated mutants had reduced growth rates, abnormal colony morphologies, and few asexual spores, and they resembled virus-infected strains. The respiratory defects and attenuated virulence phenotypes (hypovirulence) were transmitted from two mutants to a virulent strain by hyphal contact. The infectious transmission of hypovirulence occurred independently of the transfer of nuclei, did not involve a virus, and dynamically reflects fungal diseases caused by mitochondrial mutations. In these mutants, mitochondrial mutations are further implicated in generation of the attenuated state by (i) uniparental (maternal) inheritance of the trait, (ii) presence of high levels of cyanide-insensitive mitochondrial alternative oxidase activity, (iii) cytochrome deficiencies, and (iv) structural abnormalities in the mtDNA. Hence, cytoplasmically transmissible hypovirulence phenotypes found in virus-free strains of C. parasitica from recovering trees may be caused by mutant forms of mtDNA.

Efficient induction of point mutations allowing recovery of specific locus mutations in zebrafish.

A technique is described that greatly increases the efficiency of recovering specific locus point mutations in zebrafish (Danio rerio). Founder individuals that were mosaic for point mutations were produced by mutagenizing postmeiotic gametes with the alkylating agent N-ethyl-N-nitrosourea. Under optimal conditions, each founder carried an average of 10 mutations affecting genes required for embryogenesis. Moreover, approximately 2% of these founders transmitted new mutations at any prespecified pigmentation locus. Analyses of new pigmentation mutations confirmed that most were likely to be point mutations. Thus, mutagenesis of postmeiotic gametes with N-ethyl-N-nitrosourea yielded frequencies of point mutations at specific loci that were 10- to 15-fold higher than previously achieved in zebrafish. Our procedure should, therefore, greatly facilitate recovery of multiple mutant alleles at any locus of interest.

Adaptive mutations in Escherichia coli as a model for the multiple mutational origins of tumors.

The cells in most tumors are found to carry multiple mutations; however, based upon mutation rates determined by fluctuation tests, the frequency of such multiple mutations should be so low that tumors are never detected within human populations. Fluctuation tests, which determine the cell-division-dependent mutation rate per cell generation in growing cells, may not be appropriate for estimating mutation rates in nondividing or very slowly dividing cells. Recent studies of time-dependent, "adaptive" mutations in nondividing populations of microorganisms suggest that similar measurements may be more appropriate to understanding the mutation origins of tumors. Here I use the ebgR and ebgA genes of Escherichia coli to measure adaptive mutation rates where multiple mutations are required for rapid growth. Mutations in either ebgA or ebgR allow very slow growth on lactulose (4-O-beta-D-galactosyl-D-fructose), with doubling times of 3.2 and 17.3 days, respectively. However, when both mutations are present, cells can grow rapidly with doubling times of 2.7 hr. I show that during prolonged (28-day) selection for growth on lactulose, the number of lactulose-utilizing mutants that accumulate is 40,000 times greater than can be accounted for on the basis of mutation rates measured by fluctuation tests, but is entirely consistent with the time-dependent adaptive mutation rates measured under the same conditions of prolonged selection.

Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists.

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivity of some breast cancers to tamoxifen treatment.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

Gain of function mutations for paralogous Hox genes: implications for the evolution of Hox gene function.

To investigate the functions of paralogous Hox genes, we compared the phenotypic consequences of altering the embryonic patterns of expression of Hoxb-8 and Hoxc-8 in transgenic mice. A comparison of the phenotypic consequences of altered expression of the two paralogs in the axial skeletons of newborns revealed an array of common transformations as well as morphological changes unique to each gene. Divergence of function of the two paralogs was clearly evident in costal derivatives, where increased expression of the two genes affected opposite ends of the ribs. Many of the morphological consequences of expanding the mesodermal domain and magnitude of expression of either gene were atavistic, inducing the transformation of axial skeletal structures from a modern to an earlier evolutionary form. We propose that regional specialization of the vertebral column has been driven by regionalization of Hox gene function and that a major aspect of this evolutionary progression may have been restriction of Hox gene expression.

Identification of mutations in the Wiskott-Aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at DXS6940, adjacent to the disease gene.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The disease gene has been localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The function of the encoded protein remains undetermined. In this study we have characterized mutations in 12 unrelated patients to confirm the identity of the disease gene. We have also revised the coding sequence and genomic structure for the WAS gene. To analyze further the transmittance of the disease gene, we have characterized a polymorphic microsatellite at the DXS6940 locus within 30 kb of the gene and demonstrate the inheritance of the affected alleles in families with a history of WAS.

The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.

The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation.