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PubMed1995

Topographical characterization of the DNA polymerase from Thermus aquaticus. Defining groups of inhibitor mAbs by epitope mapping and functional analysis using surface plasmon resonance.

Among 24 unique monoclonal antibodies (mAb) generated against Taq polymerase (TaqPol) 13 are potent inhibitors of polymerase activity. These antibodies have been sorted into groups defined by their topographical or functional properties using surface plasmon resonance-based methods to examine three different types of interactions. An epitope map of all the pairwise interactions among all 24 antiTaqPol antibodies revealed the surface of TaqPol as a complex space populated by isolated antigenic domains with no evident relationship to each other. 11 discrete epitopes or epitope clusters were defined and potent inhibitors bound to sites within seven of them. The second method examined the ability of antiTaqPol mAbs to bind to recombinant forms of the constituent functional domains of TaqPol, the N-terminal 5'-nuclease domain and the C-terminal polymerase domain. Most of the antibodies demonstrated a clear specificity for one domain or the other. The third method measured the ability of each mAb to block the interaction of TaqPol with a preformed, immobilized primer:template complex (PTC). Some antibodies had no effect on this interaction while others effectively blocked it. Together these latter two methods resolved many of the antibodies into five distinct groups. In addition, antibodies that bound to overlapping epitopes in the pairwise interaction analysis were members of the same group by their interaction with the polymerase fragment and PTC. Three groups of polymerase inhibitors were clearly resolved by these analyses: (1) those that recognize an epitope on the 5'-nuclease domain and have no effect on the interaction of TaqPol with PTC; (2) those that recognize an epitope on the polymerase domain and block the interaction of TaqPol and PTC; and (3) those that recognize an epitope on the polymerase domain, but have no effect on the interaction of TaqPol with PTC. The surface of TaqPol bears at least three antigenic regions that are topographically and functionally distinct and may correspond to sites for inhibition of different steps in the enzymatic activity of TaqPol.

Antibodies, Monoclonal↗
PubMed1989

Functional analysis of monocyte-macrophages derived from nonadherent cord blood progenitor cells: correlation with the ontogeny of cell surface proteins.

In this study we examine some of the phenotypic and functional characteristics that accompany the differentiation of monocyte-macrophages from nonadherent precursors present in cord blood. Class II major histocompatibility complex (MHC) molecules identified by monoclonal antibody (mAb) L243 were the earliest monocyte-macrophage-associated antigens to be expressed. CD14 molecules, identified by mAb MO.2, and the transferrin receptor, identified by mAb OKT9, increased linearly over the 21-day culture period. In contrast, Fc gamma R were not expressed on these cells until 14 days of culture. Fc gamma RI was present on approximately 80% of the cells, whereas Fc gamma RII and Fc gamma RIII were present on 30% and 20% of the cells, respectively. Functional activity of the nonadherent cell-derived monocyte-macrophages paralleled the phenotypic maturation of these cells. The capacity to stimulate a mixed leukocyte reaction paralleled the expression of class II MHC molecules. Similarly, the capacity to phagocytose Escherichia coli and Staphylococcus aureus paralleled the appearance of CR3. Indeed, phagocytosis of these organisms was partially blocked by mAb OKM-1. These studies demonstrate that monocyte-macrophages derived from cord blood nonadherent mononuclear cells are a useful population with which to characterize human macrophage differentiation and functional and phenotypic heterogeneity.

Cell Differentiation↗
PubMed2001

Functional analysis of mutant human carnitine acylcarnitine translocases in yeast.

Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (<1%) in the milder patient. Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient approximately 27% residual activity was found. Sequencing of the CACT cDNAs revealed a c.241G>A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast. Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive.

Amino Acid Sequence↗
PubMed1996

Functional analysis of the promoter and first intron of the human lysyl oxidase gene.

Alterations in the synthesis and activity of lysyl oxidase occur concomitant with developmental changes in collagen and elastin deposition and with the pathogenesis of several acquired and heritable connective tissue disorders. To begin to unravel the mechanisms that control lysyloxidase gene expression, we have previously reported the complete exon-intron structure of the human lysyl oxidase gene. We have now sequenced this entire gene, including all six introns and 4 kb of DNA 5' of exon 1. Analysis of over 13 kb of intervening sequence and 5' flanking sequence revealed a concentration of conserved consensus sequence elements within the first intron and 1 kb immediately 5' of exon 1. Analysis of intron 1 and the 5' flanking domain, using recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) reporter gene, identified functional DNA sequence elements within these non-coding domains responsible for inhibition and up-regulation of CAT activity in primary cultures of human skin fibroblasts, in smooth muscle cells, revertant cells derived from an osteosarcoma cell line and malignant c-Ha-ras-transformed osteosarcoma cells. DNA sequence elements within intron 1, in particular, resulted in a marked increase in CAT reporter activity in cultured fibroblasts, smooth muscle cells and osteosarcoma cells. In c-Ha-ras-transformed osteosarcoma cells, however, no such enhancer activity of intron 1 sequence was observed. Ras-transformed osteosarcoma cells exhibited reduced steady-state levels of lysyl oxidase mRNA that was primarily controlled through reduced transcription of the lysyl oxidase gene. The lack of any up-regulation of CAT activity in these ras-transformed cells by sequence elements within intron 1 suggests a complex interaction between cis-acting domains and trans-acting transcriptional factors in the 5' promoter domain and the first intron of the lysyl oxidase gene.

Animals↗
PubMed1997

Functional analysis of third ventriculostomy patency with phase-contrast MRI velocity measurements.

Our purpose was to explore the utility of cine phase-contrast MRI velocity measurements in determining the functional status of third ventriculostomies, and to correlate the quantitative velocity data with clinical follow-up. We examined six patients with third ventriculostomies and 12 normal subjects by phase-contrast MRI. The maximum craniocaudal to maximum caudocranial velocity range was measured at regions of interest near the third ventricular floor, and in cerebrospinal fluid anterior to the upper pons and spinal cord on midline sagittal images. Ratios of the velocities of both the third ventricle and prepontine space to the space anterior to the spinal cord were obtained. The velocities near the third ventricular floor and in the pontine cistern were significantly higher in patients than in normal subjects, but the velocity anterior to the spinal cord was similar between the groups. The velocity ratios, used to normalize individual differences, were also higher in patients than in controls. Two patients had lower velocity ratios than their fellows at the third ventricular floor and in the pontine cistern; one required a shunt 11 months later, while in the other, who had a third ventricular/thalamic tumor, the lower values probably reflect distortion of the third ventricular floor. We conclude that phase-contrast MR velocity measurements, specifically the velocity ratio between the high pontine cistern and the space anterior to the spinal cord, can help determine the functional status of third ventriculostomies.

Adolescent↗
PubMed1995

Functional analysis of aryl hydrocarbon receptor nuclear translocator interactions with aryl hydrocarbon receptor in the yeast two-hybrid system.

The aryl hydrocarbon receptor (AHR) mediates dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced transcriptional activation of a battery of genes by interaction with a cofactor, called aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Both AHR and ARNT belong to a family of proteins that includes the Drosophila circadian-rhythm protein and "single-minded" protein. These proteins share a domain called the PAS domain. In addition to the PAS domain, both AHR and ARNT contain basic helix-loop-helix (bHLH) and glutamine (Q)-rich domains. The roles of these domains in the receptor-mediated transcriptional activation are not understood completely. By using the yeast two-hybrid system with the N-terminal half of AHR as a probe, which contains the bHLH and PAS regions, to screen cDNA libraries prepared from human lymphocytes and C57BL mouse liver for clones encoding proteins capable of binding to these regions, we isolated a partial ARNT cDNA clone. These results demonstrated that the N-terminal half of AHR is capable of interacting with ARNT in yeast (probably through the bHLH motif). A fusion protein containing the GAL4 DNA binding domain (DB) linked to the full-length AHR was not capable of activating expression of a reporter gene containing the GAL4 DNA binding site, suggesting that ligand-free AHR alone has no transactivating properties in yeast. However, the C-terminal portion (amino acid residues 580-797) of the AHR, including the Q-rich domain, could confer transactivation of the reporter gene expression in the same system, suggesting that the N-terminal portion of the AHR contains transcription repression properties. In contrast, GAL4(DB)-ARNT fusion protein was able to activate expression of the same reporter gene. Deletion analysis of ARNT revealed that the C-terminal 75 amino acids, including the Q-rich domain, exhibited full transactivation function in yeast and mammalian cells. These results revealed different structural organizations for the transactivation properties between AHR and ARNT, although both contained transactivation domains at the C-termini.

Animals↗
PubMed1989

Structural and functional analysis of raphe neurone implants into denervated rat spinal cord.

The ability of grafts of embryonic raphe cells to the adult rat spinal cord to reverse the morphological, neurochemical and functional deficits caused by ablation of the serotoninergic afferents has been studied. After grafting, extensive reinnervation was observed by 5HT-immunoreactive fibres, many of which appeared to make contacts with host motoneurones. The neurotransmitter complement was apparently normal. There was also a reinstatement of 5HT levels in the denervated cord after transplantation, amounting to some 40% of normal at the level of the graft. Similarly, Na+-dependent uptake of [3H]-5HT into P3 fractions was over 40% of that recorded in unlesioned animals. Antidromic stimulation of the ventral roots of the spinal cord was used to assess the degree of motoneurone excitability. The field potential in the ventral horn of grafted animals was increased by electrical stimulation of discrete regions along the cord, probably corresponding to the graft loci. It is concluded that serotoninergic neurones transplanted to the denervated spinal cord survive and develop normally, reinnervating the host tissue extensively. Furthermore, the graft/host connections appear to be functionally viable.

Action Potentials↗
PubMed2006

Structural and functional analysis of the sarcoglycan-sarcospan subcomplex.

Sarcospan is a component of the dystrophin-glycoprotein complex that forms a tight subcomplex with the sarcoglycans. The sarcoglycan-sarcospan subcomplex functions to stabilize alpha-dystroglycan at the plasma membrane and perturbations of this subcomplex are associated with autosomal recessive limb-girdle muscular dystrophy. In order to characterize protein interactions within this subcomplex, we first demonstrate that sarcospan forms homo-oligomers within the membrane. Experiments with a panel of site-directed mutants reveal that proper structure of the large extracellular loop is an important determinant of oligo formation. Furthermore, the intracellular N- and C-termini contribute to stability of sarcospan-mediated webs. Point mutation of each cysteine residue reveals that Cys 162 and Cys 164 within the large extracellular loop form disulfide bridges, which are critical for proper sarcospan structure. The extracellular domain of sarcospan also forms the main binding site for the sarcoglycans. We propose a model whereby sarcospan forms homo-oligomers that cluster the components of the dystrophin-glycoprotein complex within the membrane.

Animals↗
PubMed2000

Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in viral hepatitis.

In the present studies, we report the cloning and structural characterization of the HFGL2 gene and its functional role in human fulminant hepatitis. The HFGL2 gene is approximately 7 kb in length with 2 exons. The putative promoter contains cis element consensus sequences that strongly suggest the inducibility of its expression. From the nucleotide sequence of the human gene, a 439-amino acid long protein is predicted. The overall identity between the murine fgl2 and hfgl2 coded proteins is over 70%. About 225 amino acids at the carboxyl end of these molecules are almost 90% identical, and correspond to a well-conserved fibrinogen-related domain. Both HFGL2 and FGL2 encode a type II transmembrane protein with a predicted catalytic domain toward the amino terminus of the protein. Transient transfection of Chinese hamster ovary (CHO) cells with a full-length cDNA of HFGL2 coding region resulted in high levels of prothrombinase activity. Livers from 8 patients transplanted for fulminant viral hepatitis were examined for extent of necrosis, inflammation, fibrin deposition, and HFGL2 induction. In situ hybridization showed positive staining of macrophages in areas of active hepatocellular necrosis. Fibrin stained positively in these areas and was confirmed by electron microscopy. These studies define a unique prothrombinase gene (HFGL2) and implicate its importance in the pathogenesis of fulminant viral hepatitis.

Adolescent↗
PubMed2004

Functional analysis of bacterial artificial chromosomes in mammalian cells: mouse Cdc6 is associated with the mitotic spindle apparatus.

Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the functional organization of genes and other large chromosomal domains. To facilitate functional studies in cell cultures, we have developed a simple approach for generating stable cell lines with variable copy numbers of any BAC. Here we describe hamster cell lines with BAC transgenes that express mouse Cdc6 at levels that correlate with BAC copy number; show that mouse Cdc6 is regulated normally during the cell cycle, binds chromatin, and is degraded during apoptosis; and report a novel fraction of Cdc6 that associates with the spindle apparatus during mitosis. With RNA interference to assess genetic complementation by BAC alleles, this system will facilitate functional studies on large chromosomal domains at variable copy number in mammalian cell models.

Animals↗
PubMed2001

Functional analysis and intracellular localization of p53 modified by SUMO-1.

p53 tumor suppressor is a subject of several post-translational modifications, including phosphorylation, ubiquitination and acetylation, which regulate p53 function. A new covalent modification of p53 at lysine 386 by SUMO-1 was recently identified. To elucidate the function of sumoylated p53, we compared the properties of wild type p53 and sumoylation-deficient p53 mutant, K386R. No differences were found between wild type p53 and K386R mutant of p53 in transactivation or growth suppression assays. Moreover, overexpression of SUMO-1 has no effect on p53-regulated transcription. Biochemical fractionation showed that sumoylated p53 is localized in the nucleus and is tightly bound to chromatin structures. p53 and SUMO-1 co-localized in PML nuclear bodies in 293 cells and the nucleoli in MCF7 and HT1080 cells. However, sumoylation-deficient p53 mutant showed a similar pattern of intranuclear localization, suggesting that SUMO-1 does not target p53 to subnuclear structures. These data indicate that SUMO-1 modification of p53 at lysine 386 may not be essential for p53's cellular localization, transcriptional activation, or growth regulation.

Amino Acid Sequence↗
PubMed2000

Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast.

Mammalian phosducins are known to bind G protein betagamma subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gbetagamma. Binding was enhanced by pheromone stimulation and by the addition of GTPgammaS, conditions that favor dissociation of Gbetagamma from Galpha. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Delta mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1Delta mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70-80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana.

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.

Arabidopsis↗
PubMed2003

Functional analysis of PKD1 transgenic lines reveals a direct role for polycystin-1 in mediating cell-cell adhesion.

The PKD1 protein, polycystin-1, is a large transmembrane protein of uncertain function and topology. To study the putative functions of polycystin-1, conditionally immortalized kidney cells transgenic for PKD1 were generated and an interaction between transgenic polycystin-1 and endogenous polycystin-2 has been recently demonstrated in these cells. This study provides the first functional evidence that transgenic polycystin-1 directly mediates cell-cell adhesion. In non-permeabilized cells, polycystin-1 localized to the lateral cell borders with N-terminal antibodies but not with a C-terminal antibody; there was a clear difference in surface intensity between transgenic and non-transgenic cells. Compared with non-transgenic cells, transgenic cells showed a dramatic increase in resistance to the disruptive effect of a polycystin-1 antibody raised to the PKD domains of polycystin-1 (IgPKD) in both cell adhesion and cell aggregation assays. The differential effect on cell adhesion between transgenic and non-transgenic cells could be reproduced using recombinant fusion proteins encoding non-overlapping regions of the IgPKD domains. In contrast, antibodies raised to other extracellular domains of polycystin-1 had no effect on cell adhesion. Finally, the specificity of this finding was confirmed by the lack of effect of IgPKD antibody on cell adhesion in a PKD1 cystic cell line deficient in polycystin-1. These results demonstrate that one of the primary functions of polycystin-1 is to mediate cell-cell adhesion in renal epithelial cells, probably via homophilic or heterophilic interactions of the PKD domains. Disruption of cell-cell adhesion during tubular morphogenesis may be an early initiating event for cyst formation in ADPKD.

Animals↗
PubMed2006

Functional analysis of EID1, an F-box protein involved in phytochrome A-dependent light signal transduction.

Empfindlicher im Dunkelroten Licht 1 (EID1) is an F-box protein that functions as a negative regulator in phytochrome A (phyA)-specific light signalling. F-box proteins are components of SCF ubiquitin ligase complexes that target proteins for degradation in the proteasome. Here we present further characterization of EID1 at the expression level, and show that it regulates photomorphogenesis in seedlings, rosette leaf development and flowering. Data on transcript expression patterns indicate that EID1 is expressed during all stages of Arabidopsis development and exhibits no light response. Microscope studies demonstrate that EID1 is localized to the nucleus, where it can form speckles under continuous far-red light that resemble clastosomes. To characterize the composition and formation of SCF(EID1) complexes further, we used two-hybrid and bridge assays in yeast and in planta. EID1 interacts specifically with several Arabidopsis Skp1-like (ASK) proteins and Cullin1 to form stable dimeric and trimeric complexes. Our results support a two-step association process in which the F-box protein binds first to the ASK adaptor, forming a unit which then associates with the catalytic core of the SCF complex. Finally, our data indicate that the EID1 target interaction domain is composed of two independent modules.

Alleles↗
PubMed2002

Functional analysis of the Escherichia coli zinc transporter ZitB.

The membrane transporter ZitB responsible for Zn(II) efflux in Escherichia coli was studied by site-directed mutagenesis to elucidate the function of individual amino acid residues. Substitutions of several charged or polar residues, H53, H159, D163 and D186, located in predicted transmembrane domains resulted in loss of ZitB function. In contrast, neither the amino-terminal nor the carboxy-terminal regions, both histidine-rich, were required for function.

Amino Acid Sequence↗
PubMed1997

Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130.

We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth. All mutant proteins were stably expressed in vivo. The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth. Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype. Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant. By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM. Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

Amino Acid Sequence↗
PubMed2005

Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments.

The cytochrome P450 CYP153 family is thought to mediate the terminal hydroxylation reactions of n-alkanes. We isolated 16 new P450 CYP153A genes (central region) from various environments such as petroleum-contaminated soil and groundwater, as well as one from the n-alkane-degrading bacterium Alcanivorax borkumensis SK2 (designated P450balk). The sequences of the new P450 genes were extended by PCR to generate full-length chimeric P450 genes, using the N- and C-terminal domains of P450balk. A differential CO-reduced P450 spectral analysis indicated that 8 P450 genes among the 16 chimeric genes were expressed in Escherichia coli to generate a soluble and functional enzyme. The several functional chimeric P450s and P450balk were further fused to the reductase domain of the self-sufficient P450 monooxygenase (P450RhF) at the C-terminus. E. coli cells expressing these self-sufficient P450 chimeric genes converted n-alkanes, cyclohexane, 1-octene, n-butylbenzene, and 4-phenyl-1-butene into 1-alkanols, cyclohexanol, 1,2-epoxyoctane, 1-phenyl-4-butanol, and 2-phenethyl-oxirane, respectively.

Alkanes↗