CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

Mammalian and yeast ras gene products: biological function in their heterologous systems.

Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.

Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

The Yck2 yeast casein kinase 1 isoform shows cell cycle-specific localization to sites of polarized growth and is required for proper septin organization.

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP-Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP-Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.

Confirmation of locus heterogeneity in the pure form of familial spastic paraplegia.

Familial spastic paraplegia (FSP), characterized by progressive spasticity of the lower extremities, is in its "pure" form generally of autosomal dominant inheritance pattern. Hazan et al. [Nat Genet 5:163-167, 1993] reported tight linkage of a large FSP family to the highly polymorphic microsatellite marker D14S269 with z (theta) = 8.49 at theta = 0.00 They further demonstrated evidence for locus heterogeneity when they showed that 2 FSP families were unlinked to this region. We have subsequently studied 4 FSP families (3 American, one British) and excluded the disease locus in these families for approximately 30 cM on either side of D14S269, thereby confirming evidence for locus heterogeneity within the spastic paraplegia diagnostic classification.

Functional characterization and visualization of a GABAA receptor-GFP chimera expressed in Xenopus oocytes.

The GABAA receptor is a ligand-gated chloride channel belonging to the superfamily of ligand-gated ion channels of which the nicotinic acetylcholine (nACh) receptor is prototypic. In the central nervous system the GABAA receptor mediates fast neuronal inhibition. To facilitate the study of this receptor, a GABAA receptor-green fluorescent protein (GABAAR-GFP) chimera was constructed by fusing green fluorescent protein (GFP) to the C-terminus region of the GABAA receptor alpha1 subunit. When expressed in Xenopus oocytes, this chimera responded in a manner indistinguishable from the wild-type GABAA receptor with respect to agonist potency, receptor desensitization, allosteric modulation, rectification, and ion selectivity of the channel. The addition of GFP to the GABAA receptor alpha1 subunit did not appear to alter the assembly or efficiency of expression of the GABAA receptor complex. The GABAAR-GFP chimera generated a strong fluorescent signal that was restricted to the animal pole of the oocyte plasma membrane. This signal was readily detectable using either epifluorescence or laser confocal microscopy. To confirm the extracellular location of the GFP portion of the chimera, non-permeabilized oocytes were immunolabeled with an anti-GFP antibody. Fluorescence microscopy showed that GFP was located extracellularly since it was accessible to the GFP antibody. These results confirm the predicted extracellular location of the C-terminus of the GABAA receptor alpha1 subunit and also demonstrate that GFP retains its fluorescent property when expressed extracellularly. The usefulness of the GABAAR-GFP chimera in receptor trafficking was investigated using non-hydrolyzable GTP analogues since GTP binding proteins participate in protein transport in oocytes. Microinjections of GTP-gamma-S but not GDP-beta-S reduced both GABA-gated chloride currents and cell surface GFP fluorescence in oocytes expressing the GABAAR-GFP chimera indicating that the chimera undergoes internalization upon stimulation of oocyte GTP-binding proteins. The results of the present study show that the GABAAR-GFP chimera is functionally similar to the wild-type GABAA receptor and can be used to study receptor trafficking in living cells. This is the first demonstration of a ligand-gated ion channel-GFP chimera for an ion channel belonging to this superfamily and also is the first example of the fusion of GFP to an extracellular domain of an integral membrane protein.

Interpersonal relationship quality in young adulthood: a gender analysis.

This study explored gender differences in the degree to which parent-child dyads and family system variables are associated with relationship quality in late adolescence and early adulthood. It was hypothesized that the quality of mother-child and father-child relationships, as well as family adaptability and family cohesion, would be positively correlated with the quality of intimate relationships in late adolescence and young adulthood. Further, it was hypothesized that correlations would vary according to gender. The sample was composed of 50 males and 48 females between the ages of 18 and 24. The results indicated that the relationship of family factors to the intimate relationships of young adults was similar for males and females. Specifically, a positive relationship with mother and greater adaptability in the family system during adolescence were related to more positive intimate relationships in young adulthood.

Family characteristics and adolescent substance use.

Using self-report questionnaire data from high school students (N = 253), the relation between adolescents' perceptions of family characteristics and adolescent substance use patterns were examined. Results indicated that adolescents' perception of maternal substance use, family hardiness, and age of the adolescent were significant predictors of adolescent substance use. Implications are presented.

Atherosclerotic occlusive disease of the aorta.

Aortoiliac occlusive disease is a common manifestation of atherosclerosis. Signs and symptoms include intermittent claudication, diminished femoral pulses, and impotence in males. During the assessment process, the coronary, renal, cerebrovascular, and distal extremity vessels must also be evaluated. Treatment options include conservative measures including angioplasty, as well as surgical intervention including aortic reconstruction or extra-anatomic bypass surgery.

A gas chromatographic method of measuring residual water in freeze-dried smallpox vaccine.

A new method for the accurate determination of residual water in freeze-dried vaccines is described. Tests may be carried out on very small samples in sealed ampoules. The method is based on the adsorption of water from the vaccine with benzene and its estimation by gas chromatography. It appears that the residual water and total nitrogen content are important factors in the stability of the vaccine.

TFS1: a suppressor of cdc25 mutations in Saccharomyces cerevisiae.

The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.

RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation.

Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family. These two genes, RAS1 and RAS2, constitute an essential gene family since spores with disruptions of both genes fail to grow. We report here that strains containing RAS2 disruptions have three distinct phenotypes. First, they fail to grow efficiently on nonfermentable carbon sources. Second, they hyperaccumulate the storage carbohydrates glycogen and trehalose. Third, diploid cells homozygous for the RAS2 disruptions sporulate on rich media. Extragenic suppressors have been isolated that suppress the gluconeogenic defect. These suppressors fall into at least three complementation groups, mutations in two of which bypass the normal requirement of RAS for cell viability, allowing cells containing neither RAS gene to grow. The phenotype of the RAS2 mutant and extragenic suppressors implicate RAS with some function in the normal response to nutrient limitation.

Restriction endonuclease analysis and ribotyping differentiate Pasteurella haemolytica serotype A1 isolates from cattle within a feedlot.

Pasteurella haemolytica serotype A1 isolates were collected from cattle within a feedlot during an outbreak of bovine respiratory disease. Genetic heterogeneity among the isolates was examined by restriction endonuclease analysis (REA), ribotyping, and analysis of plasmid content. The susceptibilities of isolates to several antibiotics were also examined. Five different REA patterns and three different ribotypes were observed among the isolates. Fifty percent of the isolates had an identical REA type, ribotype, and plasmid profile. Examination of the plasmid content of the isolates revealed that most (73%) carry a single plasmid which encodes beta-lactamase, 13.5% carry two plasmids, and 13.5% carry no plasmid. The data reveal the presence of genetic differences among isolates of P. haemolytica A1, associated with shipping fever pneumonia within a closed feedlot, and suggest that a combination of REA, ribotyping, plasmid analysis, and antibiotic susceptibility determination will be useful in analyzing the molecular epidemiology of this disease.

Molecular genetic analysis of distal mouse chromosome 6 defines gene order and positions of the deafwaddler and opisthotonos mutations.

Two neurological mutants deafwaddler (dfw) and opisthotonos (opt) and a cluster of three Shaker-like potassium (K) channel genes Kcna1, Kcna5, and Kcna6 were all independently mapped to distal mouse chromosome six (Chr 6). In this study, genetic and molecular techniques were employed to assess directly the linkage of the two mutants and to investigate the likelihood that a mutation in one of the three K channel genes may underlie dfw and/or opt. Genetic crosses testing for allelism showed that the dfw and opt mutations complement each other. Additional crosses demonstrated that the mutants are separated by a recombination distance of 3.1 +/- 1.8 cM. Microsatellite marker analysis of the crossover chromosomes recovered from the opt, dfw recombination study indicated that opt maps centromeric to dfw. The location of the K channel genes relative to the dfw mutation was determined by mapping these genes and 15 microsatellite markers in an intersubspecific backcross (IB) segregating for dfw [(CAST/Ei-+/+ x C3HeB/FeJ-dfw/dfw) x C3HeB/FeJ-dfw/dfw]. Analysis of the backcross progeny positioned the dfw locus in the interval between the microsatellite markers D6Mit11 and D6Mit55, D6Mit63. The K channel cluster maps telomeric to dfw. This study establishes the gene order cen-opt-dfw-Rho (D6Mit44)-Kcna1, Kcna5, Kcna6 on distal mouse Chr 6 and suggests that the neurological mutants opt and dfw affect two different genes, neither of which is caused by a mutation in any one of the three clustered K channels.

Activation of protein kinase C induces gamma-aminobutyric acid type A receptor internalization in Xenopus oocytes.

The inhibition of gamma-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABAA receptors expressed in Xenopus oocytes. PMA (5 nM) reduced the GABA response in oocytes expressing the alpha1 beta2 gamma2L receptor construct, as measured by the two-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes expressing a receptor construct from which the known PKC phosphorylation sites were absent, alpha1 beta2(S410A), were comparably inhibited. Phorbol 12-monomyristate (PMM; 5 nM), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0.5 microM) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABAA receptor alpha1 subunit/green fluorescent protein (GFP) chimera (alpha1GFP) was used to visualize GABAA receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing alpha1GFP beta2 gamma2L and alpha1GFP beta2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABAA receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM. Experiments with bd24, a monoclonal antibody which recognizes an extracellular epitope of the alpha1 subunit, were used to demonstrate that PMA, but not PMM, decreases alpha1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the alpha1 beta2 gamma2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABAA receptor through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation.

Identification of Rgp1p, a novel Golgi recycling factor, as a protein required for efficient localization of yeast casein kinase 1 to the plasma membrane.

The Yck1p and Yck2p casein kinase 1 isoforms in yeast are essential peripheral plasma membrane-associated protein kinases with roles in endocytosis, cellular morphogenesis and cytokinesis. The membrane targeting of these cytoplasmically oriented protein kinases requires normal secretory pathway function, but specific targeting factors have not been identified. To learn more about Yckp targeting, we characterized mutations that cause synthetic lethality with impairment of Yck function. We report here that these include mutations in two gene products that function in protein trafficking. One of these is the previously described t-SNARE Tlg2p, which participates in recycling of proteins to the Golgi. The other is a previously uncharacterized protein, Rgp1p, which appears to have a similar function. Loss of either Tlg2p or Rgp1p causes inefficient localization of Yck2p, suggesting that its transport may be directed, in part, by a targeting factor that must be recycled back to the Golgi.

Clinical features of infections due to Escherichia coli producing heat-stable toxin during an outbreak in Wisconsin: a rarely suspected cause of diarrhea in the United States.

In September 1994, a foodborne outbreak of enterotoxigenic Escherichia coli (ETEC) infection occurred in attendees of a banquet in Milwaukee. E. coli was isolated from stool specimens from 13 patients that were comprehensively tested; isolates from five patients were positive for E. coli producing heat-stable toxin, were biochemically identified and serotyped as E. coli O153:H45, and were all resistant to tetracycline, ampicillin, sulfisoxazole, and streptomycin. Diarrhea (100%) and abdominal cramps (83%) were the most prevalent symptoms in 205 cases; vomiting (13%) and fever (19%) were less common. The median duration of diarrhea and abdominal cramps was 6 days and 5 days, respectively. In the United States, health care providers rarely consider ETEC as a possible cause of diarrhea in their patients, and few laboratories offer testing to identify ETEC. Hence, outbreaks of ETEC infection may be underdiagnosed and underreported. As in this outbreak, the relatively high prevalence of diarrhea and cramps lasting > or = 4 days and the low prevalence of vomiting and fever can help distinguish ETEC infection from Norwalk-like virus infection and gastroenteritis due to other causes with incubation times of > or = 15 hours and can provide direction for confirmatory laboratory testing.

Physical and genetic maps of the deafwaddler region on distal mouse Chr 6.

The deafwaddler (dfw) mutation, displaying motor ataxia and profound deafness, arose spontaneously in a C3H/HeJ colony and was mapped previously to distal mouse Chr 6. In this study, a high-resolution genetic map was generated by positioning 10 microsatellite markers and 5 known genes on a 968-meioses intersubspecific backcross segregating for dfw [(CAST/Ei(-)+/+ x C3HeB/ FeJ-dfw/dfw) x C3HeB/FeJ-dfw/dfw], giving the following marker order and sex-averaged distances: D6Mit64-(0.10 + 0.10 cM)-Pang-(1.24 + 0.36 cM)-Itpr1-(0.62 + 0.25 cM)-D6Mit108-(0.52 + 0.23 cM)-D6Mit54-(0.21 + 0.15 cM)-D6Mit23, D6Mit107, D6Mit328-(0.72 + 0.27 cM)-D6Mit11-(0.21 + 0.15 cM)-dfw-(0.93 + 0.31 cM)-Gat4, D6Mit55-(0.10 + 0.10 cM)-D6Mit63-(0.31 + 0.18 cM)-Syn2-(0.62 + 0.25 cM)-D6Mit44 (Rho). Female and male genetic maps are similar immediately surrounding the dfw locus, but show marked differences in other areas. A yeast artificial chromosome-based physical map suggests that the closest markers flanking the dfw locus, D6Mit11 (proximal) and Gat4, D6Mit55 (distal), are contained within 650-950 kb. The human homologues of the flanking loci Itpr1 (proximal) and Syn2 (distal) map to chromosome 3p25-p26, suggesting that the human homologue of the dfw gene is located within this same region.

Rapid scan phase modulator for interferometric applications.

A new rapid scan phase modulator has been studied in a range of interferometric applications from the visible to the millimeter wave region. The modulator consists of two parallel mirrors mounted on a rotating platform. It causes little attenuation and is capable of large changes in optical path length at high speeds. The system is relatively insensitive to vibration and can yield a nearly flat transmission envelope.