Mutant

About: Mutant is a research topic. Over the lifetime, 74520 publications have been published within this topic receiving 3477079 citations.

A M(r) 34,000 proinflammatory outer membrane protein (oipA) of Helicobacter pylori.

Abstract: The complete genome sequence revealed a family of 32 outer membrane proteins (OMPs) in Helicobacter pylori. We examined the effect of four OMPs (HP0638, HP0796, HP1501, and babA2) on the production of the proinflammatory cytokine, IL-8. Mutants of the four OMPs, as well as cagE and galE from H. pylori from the U.S. and Japan, were constructed by inserting a chloramphenicol-resistant cassette into the gene. Twenty-two pairs of parental and mutant H. pylori strains, as well as 160 clinical isolates (80 from Japanese and 80 from U.S.), were cocultured with gastric cancer cell lines. IL-8 production in the supernatant and adhesion was assayed by ELISA. HP0796, HP1501, babA2, and galE gene knockouts had no significant effect on IL-8 production. Knockout of the HP0638 gene in 81% of cag-positive strains reduced IL-8 production approximately 50%. The three cag-positive strains in which IL-8 levels were unchanged by HP0638 knockout had five or seven CT dinucleotide repeats in the 5′ region, resulting in a frame shift and truncation. Strains with naturally inactive HP0638 gene were all from the U.S.; Japanese strains were always “on” and thus, on average, may be more virulent. Although cag-negative isolates produced a limited IL-8 response, cag-negative strains that contained a functional HP0638 gene produced more than 3-fold greater IL-8 than cag-negative nonfunctional HP0638 strains. We hypothesize that functional HP0638 gene may be an important virulence factor in relation to the risk of clinically significant outcomes of H. pylori infection. We denote HP0638 gene as outer inflammatory protein (oipA).

Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses

Abstract: A two-fold difference in X chromosome dosage distinguishes males (XO) and hermaphrodites (XX) of the nematode Caenorhabditis elegans at the beginning of embryogenesis. As adults, individuals of the two sexes differ in size, anatomy, and behavior. About one-third of their somatic cells express sex-specific characteristics. How the X chromosome to autosome (X : A) ratio determines sex has been the subject of detailed genetic investigation (for review, see Meyer 1997). These studies have defined a regulatory pathway that transduces information about the X : A ratio to a gene known as tra-1. In all somatic tissues, tra-1 activity is necessary and sufficient to promote female differentiation (Hodgkin 1987). Inhibition of tra-1 activity results in male development. The tra-1 gene encodes a zinc finger transcription factor, TRA-1A, that is related to Drosophila Ci and the Gli proteins of vertebrates (Zarkower and Hodgkin 1992). Few targets of TRA-1A have been identified, but in at least two cell types, TRA-1A represses genes that would otherwise cause adoption of male-specific fates. The survival of the HSN neurons, which are required for egg laying in hermaphrodites, depends on the repression of the egl-1 gene by TRA-1A (Conradt and Horvitz 1999). Similarly, vitellogenin synthesis by intestinal cells in the hermaphrodite requires that TRA-1A repress the mab-3 gene (Yi et al. 2000). The role of tra-1 in the germ line is less well understood. Mutants lacking tra-1 activity, whether XX or XO, often exhibit limited spermatogenesis followed by oogenesis, suggesting that tra-1 is needed to sustain spermatogenesis (Schedl et al. 1989). On the other hand, strong gain-of-function alleles of tra-1 completely feminize the germ line, indicating that unregulated tra-1 activity can suppress spermatogenesis (Hodgkin 1980; 1987). Recent observations suggesting that TRA-1A may serve as both an activator and a repressor of the fog-3 gene, which is required for spermatogenesis, reinforce the idea that the function of tra-1 in the germ line is complex (Chen and Ellis 2000). Negative regulation plays a prominent role in C. elegans sex determination (Fig. ​(Fig.1A).1A). Thus, male development in XO animals requires the inhibition of tra-1 activity by three fem genes (Doniach and Hodgkin 1984; Kimble et al. 1984; Hodgkin 1986). In XX animals, the tra-2 and tra-3 genes indirectly activate tra-1 by inhibiting fem activity. Support for this model comes from the observation that mutational inactivation of any of the fem genes renders tra-2 and tra-3 dispensable for female development. For example, both fem-1 mutants and tra-2; fem-1 double mutants develop as true females as a result of unregulated tra-1 activity (Doniach and Hodgkin 1984). Figure 1 Model of somatic sex determination in C. elegans. (A) Genetic pathway regulating somatic sex determination. Barred lines indicate negative interactions, and arrows indicate positive interactions. The X/A ratio controls X chromosome dosage compensation ... The major product of tra-2 is a large membrane protein known as TRA-2A (Fig. ​(Fig.1B)1B) (Kuwabara et al. 1992). A direct interaction between the intracellular domain of TRA-2A and FEM-3 inhibits the masculinizing activity of the FEM proteins, and although the mechanism of inhibition remains to be determined, this interaction would seem sufficient to explain the feminizing role of TRA-2A (Mehra et al. 1999). In support of this idea, overproduction of the intracellular domain of TRA-2A as a soluble protein in the somatic tissues of XO animals is strongly feminizing (Kuwabara and Kimble 1995; Mehra et al. 1999). A 1.8 kb mRNA specific to the hermaphrodite germ line can encode a second TRA-2 protein known as TRA-2B, which is equivalent to the intracellular domain of TRA-2A (Okkema and Kimble 1991; Kuwabara et al. 1998). Genetic evidence suggests that TRA-2B may limit the extent of spermatogenesis in the hermaphrodite. Because TRA-2B includes the entire FEM-3-binding domain of TRA-2A, it is reasonable to expect that TRA-2B might also act by binding to and inhibiting FEM-3. Although the mRNA encoding TRA-2B is restricted to the hermaphrodite germ line, a similar soluble protein might be produced in other tissues by cleavage of TRA-2A. The tra-3 gene, which acts at the same level in the sex-determining pathway as tra-2, encodes an atypical calpain protease (Barnes and Hodgkin 1996). When the two proteins are expressed together in insect cells, TRA-3 can cleave TRA-2A to release a fragment that includes part of the intracellular domain of TRA-2A (Sokol and Kuwabara 2000). This observation led to a suggestion that tra-3 might fulfill its role in sex determination by producing a soluble, FEM-3-binding fragment of TRA-2A. In this paper, we will use TRA-2c to refer to the intracellular domain of TRA-2A, whether it originates as part of TRA-2A or as TRA-2B. Interaction with FEM-3 is the only effector function of TRA-2 that has been defined to date. FEM-3 binding does not require the C-terminal 200 amino acids of TRA-2c, but genetic evidence suggests that this region is nevertheless important for TRA-2 activity. Doniach (1986) identified tra-2 alleles that cause germ line feminization, a phenotype associated with increased tra-2 activity. Some of these alleles, now known as tra-2(mx) alleles, are unusual in that although they feminize the XX germ line, they appear to reduce tra-2 activity in somatic tissues. All of the tra-2(mx) alleles carry missense mutations that alter the sequence of a 22-amino acid region near the C-terminal of TRA-2c, outside the FEM-3-binding domain (Kuwabara et al. 1998). It has been suggested that this “MX region” might represent a domain required for interaction with a negative regulator of tra-2 in the germ line. Here we report that TRA-2 directly interacts with TRA-1A and that the MX region of TRA-2 is critical for the interaction. The somatic effects of tra-2(mx) mutations and the results of overexpression experiments lead us to suggest that TRA-2 may promote somatic female development not only by inhibiting FEM-3, but also by directly stimulating the activity of TRA-1A. Mutations that alter either TRA-2 or TRA-1A so as to disrupt their interaction feminize the germ line. This observation raises the conundrum of why an interaction that enhances the feminizing activity of both proteins in somatic tissues should appear to have a role in hermaphrodite spermatogenesis.

Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking.

Abstract: The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (approximately 350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.

A fatty acid desaturase modulates the activation of defense signaling pathways in plants.

Abstract: Salicylic acid (SA) plays an important role in activating various plant defense responses, including expression of the pathogenesis-related (PR) genes and systemic acquired resistance. A critical positive regulator of the SA signaling pathway in Arabidopsis is encoded by the NPR1 gene. However, there is growing evidence that NPR1-independent pathways can also activate PR expression and disease resistance. To elucidate the components associated with NPR1-independent defense signaling, we isolated a suppressor of the npr1–5 allele, designated ssi2. The recessive ssi2 mutation confers constitutive PR gene expression, spontaneous lesion formation, and enhanced resistance to Peronospora parasitica. In contrast, a subset of defense responses regulated by the jasmonic acid (JA) signaling pathway, including expression of the defensin gene PDF1.2 and resistance to Botrytis cinerea, is impaired in ssi2 plants. With the use of a map-based approach, the SSI2 gene was cloned and shown to encode a stearoyl-ACP desaturase (S-ACP DES). S-ACP DES is an archetypical member of a family of soluble fatty acid (FA) desaturases; these enzymes play an important role in regulating the overall level of desaturated FAs in the cell. The activity of mutant S-ACP DES enzyme was reduced 10-fold, resulting in elevation of the 18:0 FA content in ssi2 plants. Because reduced S-ACP DES activity leads to the induction of certain defense responses and the inhibition of others, we propose that a FA-derived signal modulates crosstalk between different defense signaling pathways.

Multiple C-Terminal Lysine Residues Target p53 for Ubiquitin-Proteasome-Mediated Degradation

Abstract: In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.