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At least 1,711 records · Page 95Linked to original sources
PubMed1989

Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.

The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences. In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae. When expressed in the yeast, the bovine papilloma virus E2 trans-activator could stimulate transcription from a yeast promoter having E2 DNA-binding sites present in cis. Whereas a single E2 DNA-binding site was sufficient for trans-activation, a strong cooperative effect was observed with two E2 DNA-binding sites. The level of trans-activation was dependent on the position of the E2 DNA-binding sites in relation to the yeast promoter, with the maximal effect demonstrated when the binding sites were positioned upstream. Deleted E2 proteins, lacking part of the trans-activation or DNA-binding domains, failed to activate transcription in yeast, similar to their behavior in mammalian cells. Replacement of the amino-terminal region of the E2 trans-activation domain with a synthetic amphipathic helix partially restored the trans-activation function; however, it did not result in a molecule that exhibited cooperativity between neighboring E2 DNA-binding sites.

DNA-Binding Proteins↗
PubMed2003

Functional analysis of basic transcription element binding protein by gene targeting technology.

Basic transcription element binding protein (BTEB) is a transcription factor with a characteristic zinc finger motif and is most remarkably enhanced by thyroid hormone T(3) treatment (R. J. Denver et al., J. Biol. Chem. 272:8179-8188, 1997). To investigate the function of BTEB per se and to touch on the effects of T(3) (3,5,3'-triiodothyronine) on mouse development, we generated BTEB-deficient mice by gene knockout technology. Homologous BTEB(-/-) mutant mice were bred according to apparently normal Mendelian genetics, matured normally, and were fertile. Mutant mice could survive for at least 2 years without evident pathological defects. From the expression of lacZ, which was inserted into the reading frame of the BTEB gene, BTEB showed a characteristic tissue-specific expression profile during the developmental process of brain and bone. Dramatically increased expression of BTEB was observed in Purkinje cells of the cerebellum and pyramidal cell layers of the hippocampus at P7 when synapses start to form in the brain. Although general behavioral activities such as locomotion, rearing, and speed of movement were not so much affected in the BTEB(-/-) mutant mice, they showed clearly reduced activity levels in rotorod and contextual fear-conditioning tests; this finding was probably due to defective functions of the cerebellum, hippocampus, and amygdala.

Amygdala↗
PubMed2006

Functional analysis of Sox8 during neural crest development in Xenopus.

Among the families of transcription factors expressed at the neural plate border, Sox proteins have been shown to regulate multiple aspects of neural crest development. Sox8, Sox9 and Sox10, exhibit overlapping expression domains in neural crest progenitors, and studies in mouse suggest that Sox8 functions redundantly with Sox9 and Sox10 during neural crest development. Here, we show that in Xenopus, Sox8 accumulates at the lateral edges of the neural plate at the mid-gastrula stage; in contrast to its mouse and chick orthologs, Sox8 expression precedes that of Sox9 and Sox10 in neural crest progenitors. Later in development, Sox8 expression persists in migrating cranial crest cells as they populate the pharyngeal arches and in trunk neural crest cells, in a pattern that recapitulates both Sox9 and Sox10 expression domains. Although morpholino-mediated knockdown of Sox8 protein did not prevent the formation of neural crest progenitors, the timing of their induction was severely affected. This delay in neural crest specification had dramatic consequences on the development of multiple lineages of the neural crest. We demonstrate that these defects are due to the inability of neural crest cells to migrate into the periphery, rather than to a deficiency in neural crest progenitors specification and survival. These results indicate that the control of Sox8 expression at the neural plate border is a key process in initiating neural crest formation in Xenopus, and highlight species-specific differences in the relative importance of SoxE proteins during neural crest development.

Animals↗
PubMed2003

Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors.

Mutations in the B-Raf gene have been reported in a number of human cancers, including colorectal carcinoma. More than 80% of the B-Raf mutations were V599E. Although other mutations have been reported, their functional consequences were unclear. Here, we examined the effect of colon tumor-associated B-Raf mutations within the kinase activation segment, including V599E, on extracellular signal-regulated kinase (Erk) and nuclear factor kappaB (NFkappaB) signaling, and on the transformation of NIH3T3 fibroblasts. Among the six mutations examined, only the B-Raf V599E and K600E mutations greatly increased Erk and NFkappaB signaling, and the transformation of NIH3T3 cells. The B-Raf F594L mutation moderately elevated Erk signaling and NIH3T3 transformation, but did not significantly increase NFkappaB signaling. Although the basal kinase activity of the B-Raf T598I mutant was comparable with that of wild-type, its oncogenic Ras-induced kinase activity was decreased to 60% of wild-type activity. The B-Raf D593V and G595R mutants showed severely reduced kinase activity and affected neither NFkappaB signaling nor NIH3T3 transforming activity. These results suggest that the B-Raf activation segment mutations other than V599E reported in colorectal tumors do not necessarily contribute to carcinogenesis by increasing kinase and transforming activities.

14-3-3 Proteins↗
PubMed1991

Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. I. Role in the anti-S. mansoni antibody response.

Previous data have shown that from an antiparasitic IgE mAb (mAb1), antianti-Id IgG and IgE antibodies (Ab3) could be prepared. These Ab3 demonstrated the same functional properties as the Ab1, such as in vitro cytotoxic activity toward schistosomula and in vivo a protective effect against Schistosoma mansoni infection. To study the possible interactions between the idiotypic network and the regulation of isotypic expression, we focused on Id-specific T cells obtained by immunization with Ab2. Both Ab2 idiotopes and native schistosomula Ag were able to stimulate the proliferation of anti-Ab2 T cells in vitro. The activation of anti-Ab2 T cells by Ab2 shared the classic characteristics of Th cells, namely, it was MHC-restricted and required APC. A T cell line could be maintained in long term culture by stimulation with schistosomula Ag. The adoptive transfer of cells from this line to 26-kDa Ag-immunized or S. mansoni-infected rats led to a dramatic increase in the specific humoral response. This effect was restricted to antibodies specific for 26- and 56-kDa Ag (the targets of the mAb1) and was observed for the two isotypes tested, i.e., IgG and IgE. Finally, the helper effect on the antibody response could be further amplified by cooperation of anti-Ab2 T cells with Id-specific cells of the first generation (anti-Ab1 cells). Together with Ag-specific Th cells, the Id-specific T cells may, due to their specificity and their functional properties, play a major role in the induction and more importantly, in the maintenance of the immune response.

Animals↗
PubMed1985

On the problem of measuring the distance between distribution functions: analysis of hospitalization versus mortality.

The relationship between hospitalization, as a measure of morbidity, and mortality is examined. The difference between age at hospitalization in a general medical department and age at death in Oslo, Norway, is studied. The problem is transferred to the one of examining the difference between two cumulative distribution functions F and G. For this purpose, a quantile distance function based on the inverses of the distribution functions is applied. We give the natural estimate of the quantile distance function, and some asymptotic properties of the corresponding empirical process. For the particular situation where one of the distribution functions is known, a confidence band for the quantile distance function is derived. Applying these results, we show that there are reasons to believe that age at hospitalization and age at death are equally distributed, apart from a constant shift.

Adult↗
PubMed1993

Lymphoid V(D)J recombination. Functional analysis of the spacer sequence within the recombination signal.

The V(D)J recombination reaction is directed by a pair of signal sequences, each consisting of a palindromic heptamer, an A/T-rich nonamer, and an intervening spacer sequence of 12 or 23 base pairs. The spacer sequence previously has not been analyzed for a functional role. In this study, numerous simultaneous sequence changes have been made in the spacer of each signal to test their functional importance. All of the AT base pairs in each signal were changed to GC base pairs. This particular change is of interest because it markedly increases the energy that would be required to melt out the two strands of each signal to permit the intersignal base pairing proposed in a commonly invoked model for signal-signal interaction in V(D)J recombination. We find that changing 6 to 12 AT base pairs in the 12-signal to GC does not affect V(D)J recombination, nor does changing 11 of 23 AT base pairs in the 23-signal. Substrates with all-GC spacer sequences in both the 12- and the 23-signal also recombine at efficiencies that are not significantly reduced. These studies demonstrate that the sequences at these particular positions are not recognized by the recombinase. In addition, the data do not support models invoking signal-signal base pairing.

Animals↗
PubMed2004

TCIRG1-dependent recessive osteopetrosis: mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA.

Human malignant infantile osteopetrosis (arOP) is a genetically heterogeneous autosomal recessive disorder of bone metabolism. The TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, is responsible for more than one-half of the arOP patients. We performed genetic analysis of TCIRG1 in 55 arOP patients including 25 new cases and identified nine novel mutations. The two most frequent mutations, c.1674-1G>A (aberrant splicing: r.1674_1884del) and c.2005C>T (protein variation: p.Arg669X), found in 17 and 16 alleles, respectively, constituted 30% of all TCIRG1 abnormalities. They both originated in Northern Europe, p.Arg669X quite recently from West Flanders, Belgium. As substitutions in splicing regulatory sequences represented a large portion (40%; 44 alleles) of the TCIRG1 variations, we developed a functional splicing assay to distinguish between polymorphic variants and disease-causing mutations. Three intronic nucleotide substitutions flanking the splice sites (c.117+4A>T; c.1673+5G>A; and c.504-8G>A) were studied using hybrid minigenes and an abnormal processing of the transcripts was demonstrated in all cases. Cotransfection experiments with complementary U1 snRNAs performed in c.117+4A>T and c.1673+5G>A mutations showed that only in the first case was the defect at the 5' splice site corrected, indicating that mutations near the invariant GT donor sites are mechanistically different. These findings indicate the feasibility of the hybrid minigene approach to detect splicing defects, particularly in patients in whom the RNA is not available. In addition, the present results suggest that modified U1 snRNAs may represent a new therapeutic strategy for arOP patients with a U1 snRNP-dependent splicing defect.

Alternative Splicing↗
PubMed2003

Structure-function analysis of the streptokinase amino terminus (residues 1-59).

Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the alphabeta1 strand (residues 17-26) of SK completely prevented nonproteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by alpha2-antiplasmin. However, when fibrin-bound, the activities of alphabeta1 strand mutants were similar to that of wild-type (WT) SK and resistant to alpha2-antiplasmin. Mutation of Ile1 of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike alphabeta1 strand mutants, the functional defect of Ile1 mutants was not relieved by fibrin, and complexes of Ile1 mutants and plasmin were resistant to alpha2-antiplasmin. Plasmin enhanced the activities of alphabeta1 strand and Ile1 mutants, suggesting that SK-plasmin complexes activated mutant SK.Pg complexes by hydrolyzing the Pg Arg561-Val562 bond. Mutational analysis of Glu39 of SK suggested that a salt bridge between Glu39 and Arg719 of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK alpha domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to alpha2-antiplasmin, and controls fibrin-independent Pg activation.

Catalytic Domain↗
PubMed2001

The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase.

Glycogen storage disease type 1a is caused by a deficiency in glucose-6-phosphatase (G6Pase), a nine-helical endoplasmic reticulum transmembrane protein required for maintenance of glucose homeostasis. To date, 75 G6Pase mutations have been identified, including 48 mutations resulting in single-amino acid substitutions. However, only 19 missense mutations have been functionally characterized. Here, we report the results of structure and function studies of the 48 missense mutations and the DeltaF327 codon deletion mutation, grouped as active site, helical, and nonhelical mutations. The 5 active site mutations and 22 of the 31 helical mutations completely abolished G6Pase activity, but only 5 of the 13 nonhelical mutants were devoid of activity. Whereas the active site and nonhelical mutants supported the synthesis of G6Pase protein in a manner similar to that of the wild-type enzyme, immunoblot analysis showed that the majority (64.5%) of helical mutations destabilized G6Pase. Furthermore, we show that degradation of both wild-type and mutant G6Pase is inhibited by lactacystin, a potent proteasome inhibitor. Taken together, we have generated a data base of residual G6Pase activity retained by G6Pase mutants, established the critical roles of transmembrane helices in the stability and activity of this phosphatase, and shown that G6Pase is a substrate for proteasome-mediated degradation.

Acetylcysteine↗
PubMed2001

Purification and functional analysis of a novel leucine-zipper/nucleotide-fold protein, BZAP45, stimulating cell cycle regulated histone H4 gene transcription.

Regulation of histone gene transcription at the G1/S phase transition via the Site II cell cycle control element is distinct from E2F-dependent mechanisms operative at the growth factor-related restriction point. E2F-independent activation of histone H4 gene expression combines contributions of several promoter factors, including HiNF-M/IRF2 and the HiNF-D/CDP-cut complex which contains pRB, CDK1, and cyclin A as non-DNA binding subunits. Mutational analyses suggest additional rate-limiting factors for Site II function. Using sequence-specific Site II DNA affinity chromatography, we identified a 45 kDa protein (KIAA0005 or BZAP45) that is embryonically expressed and phylogenetically conserved. Based on amino acid sequence analysis, BZAP45 contains a unique decapeptide that is part of a putative leucine-zipper protein with a nucleotide (ATP or GTP) binding fold. Bacterial expression of a full-length cDNA produces a 45 kDa protein. Binding studies reveal that highly purified BZAP45 does not interact with Site II, suggesting that BZAP45 function may require partner proteins. Forced expression of BZAP45 strongly stimulates H4 promoter (nt -215 to -1)/CAT reporter gene activity. Deletion analyses and point mutations indicate that BZAP45 enhances H4 gene transcription through Site II. Thus, BZAP45 is a novel regulatory factor that contributes to transcriptional control at the G1/S phase transition.

Binding Sites↗
PubMed2004

Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations.

Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of a Caenorhabditis elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of Saccharomyces cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.

Alleles↗
PubMed1993

Functional analysis of the herpes simplex virus UL42 protein.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.

Base Sequence↗
PubMed1991

Functional analysis of the Streptomyces ambofaciens element pSAM2.

pSAM2 is an 11-kb element integrated in the Streptomyces ambofaciens ATCC23877 genome and found additionally as a free replicon present at several copies per chromosome in strain JI3212, the derivative of ATCC23877 isolated after uv irradiation. In spite of its small size, this element specifies numerous functions including maintenance, site-specific integration, self-transmissibility, pock formation, and mobilization of chromosomal markers. After transfer of the free form of pSAM2 to Streptomyces lividans, the free and the integrated forms coexist. A functional map of pSAM2 was deduced from phenotypes exhibited in S. lividans by numerous deletion or insertion derivatives. In addition to the previously characterized regions sufficient for site-specific integration we have shown that separate regions are involved in either plasmid maintenance as a free molecule, plasmid transfer, and pock formation. Transfer of pSAM2 could depend on its ability to be maintained in a free form, since plasmids deficient in this function are transferred at very low frequency. Deletions of some regions of the plasmid are lethal for the plasmid or the host, but if some other regions are deleted simultaneously, transformants can be obtained.

Blotting, Southern↗
PubMed2004

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins.

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.

Amino Acid Motifs↗