Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2Ek/Ek) was lethal in mid-gestation (the equivalent of mouse E9.5–E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long–Evans strain Tsc2Ek/Ek embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Suppression of an absolute defect in Type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.

Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that ‘hot-spot’ p53 mutants (which comprise ∼30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes

Intercellular signaling by fibroblast growth factors plays vital roles during embryogenesis. Mice deficient for fibroblast growth factor receptors (FgfRs) show abnormalities in early gastrulation and implantation, disruptions in epithelial–mesenchymal interactions, as well as profound defects in membranous and endochondrial bone formation. Activating FGFR mutations are the underlying cause of several craniosynostoses and dwarfism syndromes in humans. Here we show that a heterozygotic abrogation of FgfR2-exon 9 (IIIc) in mice causes a splicing switch, resulting in a gain-of-function mutation. The consequences are neonatal growth retardation and death, coronal synostosis, ocular proptosis, precocious sternal fusion, and abnormalities in secondary branching in several organs that undergo branching morphogenesis. This phenotype has strong parallels to some Apert's and Pfeiffer's syndrome patients.

Gain- and loss-of-function of Rhp51, a Rad51 homolog in fission yeast, reveals dissimilarities in chromosome integrity

Rad51 is crucial not only in homologous recombination and recombinational repair but also in normal cellular growth. To address the role of Rad51 in normal cell growth we investigated morphological changes of cells after overexpression of wild-type and a dominant negative form of Rad51 in fission yeast. Rhp51, a Rad51 homolog in Schizosaccharomyces pombe, has a highly conserved ATP-binding motif. Rhp51 K155A, which has a single substitution in this motif, failed to rescue hypersensitivity of a rhp51Δ mutant to methyl methanesulfonate (MMS) and UV, whereas it binds normally to Rhp51 and Rad22, a Rad52 homolog. Two distinct cellular phenotypes were observed when Rhp51 or Rhp51 K155A was overexpressed in normal cells. Overexpression of Rhp51 caused lethality in the absence of DNA-damaging agents, with acquisition of a cell cycle mutant phenotype and accumulation of a 1C DNA population. On the other hand, overexpression of Rhp51 K155A led to a delay in G2 with decondensed nuclei, which resembled the phenotype of rhp51Δ. The latter also exhibited MMS and UV sensitivity, indicating that Rhp51 K155A has a dominant negative effect. These results suggest an association between DNA replication and Rad51 function.

The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.

The weaver mutation in mice results in a severe ataxia that is attributable to the degeneration of cerebellar granule cells and dopaminergic neurons in the substantia nigra. Recent genetic studies indicate that the GIRK2 gene is altered in weaver. This gene codes for a G-protein-activated, inwardly rectifying K+ channel protein (8). The mutation results in a single amino acid substitution (glycine-->serine) in the pore-forming H5 region of the channel. The functional consequences of this mutation appear to depend upon the co-expression of other GIRK subunits--leading to either a gain or loss of function. Here, we show that G-protein-activated inwardly rectifying K+ currents are significantly reduced in cerebellar granule cells from animals carrying the mutant allele. The reduction is most pronounced in homozygous neurons. These findings suggest that the death of neurons in weaver is attributable to the loss of GIRK2-mediated currents, not to the expression of a nonspecific cation current.

A DAF-1-binding protein BRA-1 is a negative regulator of DAF-7 TGF-β signaling

We have identified homologs of a human BMP receptor-associated molecule BRAM1 in Caenorhabditis elegans. One of them, BRA-1, has been found to bind DAF-1, the type I receptor in the DAF-7 transforming growth factor-β pathway through the conserved C-terminal region. As analyzed using a BRA-1∷GFP (green fluorescent protein) fusion gene product, the bra-1 gene is expressed in amphid neurons such as ASK, ASI, and ASG, where daf-1 is also expressed. A loss-of-function mutation in bra-1 exhibits robust suppression of the Daf-c phenotype caused by the DAF-7 pathway mutations. We propose that BRA-1 represents a novel class of receptor-associated molecules that negatively regulate transforming growth factor-β pathways.

Abrogation of disease development in plants expressing animal antiapoptotic genes

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant–pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host–cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant–pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.

Galanin regulates prolactin release and lactotroph proliferation

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17β induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30–40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.

The STAR protein QKI-6 is a translational repressor

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3′ untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.

Gene replacement by homologous recombination in the multicellular green alga Volvox carteri

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.

The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.