Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Functional analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,531 records · Page 85Linked to original sources
PubMed2000

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

Base Sequence↗
PubMed1996

Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter.

A single point mutation in the insect gamma-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti. Promoter activity is contained within a 2.53-kb fragment immediately upstream from the rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals anumber of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis-acting control elements underlying ion-channel gene regulation are discussed.

Animals↗
PubMed2005

A new model for outcome prediction in intra-abdominal sepsis by the linear discriminant function analysis of IL-6 and IL-10 at different heart rates.

BACKGROUND: The inflammatory response plays an important role in the pathogenesis of sepsis, and the prognosis is known to be affected by the pro-inflammatory and anti-inflammatory cytokines. Therefore, a simple and reliable outcome prediction function that derived from IL-6 and IL-10 was hypothesized. MATERIAL AND METHODS: We used 27 Sprague-Dawley rats, weighing 250 to 300 g, underwent cecal ligation and puncture (CLP) procedures and femoral arterial catheterization. When the heart rates reached 1.15, 1.25, and 1.35 times that of the baseline heart rate, blood sampling was done for IL-6 and IL-10. The prognosis was followed up for 3 days after CLP, and all rats were then divided into survival or non-survival groups. Finally, the relationships among survival condition, heart rate, IL-6, and IL-10 were analyzed by two-way ANOVA and multiple comparison. A linear discriminant function for the prognosis of sepsis was developed by IL-6 and IL-10 at different heart rates. RESULTS: IL-6 elevated continuously with the increment of the heart rate in the non-survival group, but in the survival group, the IL-6 elevation started to fall when the heart rate reached 1.25 times; a similar trend was found in the change of IL-10. The prediction rate was 68.33% when the heart rate reached 1.15 times, and increased to 93.33% and 95.45% when heart rates reached 1.25 and 1.35 times, respectively. CONCLUSION: This linear discriminant function developed on the basis of IL-6 and IL-10 might be adopted as a simple and reliable indicator and further become a critical tool for the outcome prediction of sepsis.

Abdomen↗
PubMed1997

Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1, and HNF-3alpha transcription factors are necessary for lung cell-specific activation of SP-B gene transcription.

Surfactant protein B (SP-B) is essential for maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA expression is restricted to alveolar type II epithelial cells and bronchiolar epithelial cells (Clara cells) of adult lung. We previously (Margana, R. K., and Boggaram, V. (1996) Am. J. Physiol. 270, L601-L612) found that a minimal promoter region (-236 to +39) of rabbit SP-B gene is sufficient for high level expression of chloramphenicol acetyltransferase reporter gene in NCI-H441 cells, a cell line with characteristics of Clara cells. In the present study we used mutational analysis, electrophoretic mobility shift assays, and DNase I footprinting to identify cis-DNA regulatory elements and trans-acting protein factors required for lung cell-specific expression of SP-B gene. We found that in addition to thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3alpha (HNF-3alpha) binding sites, two spatially separate DNA sequences that bind Sp1 and Sp3 factors are necessary for the maintenance of SP-B promoter activity. Mutation of any one of the transcription factor binding sites caused a significant reduction in SP-B promoter activity suggesting that Sp1, Sp3, and TTF-1 and HNF-3alpha interact cooperatively with SP-B promoter to activate gene transcription.

Animals↗
PubMed1996

Structural, functional analysis and localization of the human CAP18 gene.

CAP18 is an antimicrobial protein found in specific granules of PMNs. The human CAP18 (HCAP18) gene was cloned from a human genomic phage library. Sequence analysis revealed the HCAP18 gene to have 4 exons spanning 3 kb, including 700 bp of upstream DNA. Using 3' RACE no homologs of human HCAP18 were found in human bone marrow or leukocyte populations. By PCR analysis of a somatic cell mapping panel and fluorescence in situ hybridization of a genomic clone to metaphase chromosomes the gene was mapped to chromosome band 3p21.3. Like several other genes expressed late in PMN development the CAP18 gene did not contain typical TATA box or CCAAT sequences. Expression in Cos 7 cells permitted limited mapping of the promoter function in upstream fragments of the HCAP18 gene. Western blot, Northern blot and RT-PCR analysis show HCAP18 to be produced specifically in granulocytes. This work forms the groundwork for future analysis of the genetic regulation of this antimicrobial protein during PMN differentiation.

Antimicrobial Cationic Peptides↗
PubMed1990

Structure function analysis of in vitro mutated CD4 and major histocompatibility complex class II gene products.

The Class II major histocompatibility molecules are implicated in the initiation of antigen-driven autoimmune responses by CD4-positive T cells. In order to study the structure and function of CD4 and MHC Class II molecules, strategies were developed with the intent of generating secreted forms of these molecules by in vitro mutagenesis of the respective genes. A full length cDNA encoding an expressible human CD4 molecule was mutagenized to introduce a premature stop codon corresponding to residue 367 located 8 amino acids amino terminal to the start of the predicted trans-membrane region. Following DNA-mediated gene transfer of the mutant gene, secreted CD4 was detected in the supernatant of transiently transfected COS-1 cells. Surface expression of the membrane-bound form of CD4 was detected under the same conditions. In an attempt to create a secreted form of the mouse Class II molecule I-Ak the exons encoding the connecting stalk, transmembrane and cytoplasmic domains of both the alpha and beta chains were replaced by the corresponding exons from the gene encoding a secreted Class I-like molecule, Q10b. Transfer of these genes into mouse L cells failed to generate detectable secreted I-A molecules. In view of the secretion of CD4 reported in other mutagenesis studies, it is concluded that very subtle differences in the structure of the COOH-terminus can influence the folding, solubility or transport of the CD4 molecule. In addition, the assembly of heterodimeric Class II molecules may require membrane anchorage of the separate chains or some other contribution from the COOH-terminal domains of the alpha and beta chains.

Animals↗
PubMed2001

Functional analysis of the p300 acetyltransferase domain: the PHD finger of p300 but not of CBP is dispensable for enzymatic activity.

Acetylation of nucleosomal histones is a major regulatory step during activation of eukaryotic gene expression. Among the known acetyltransferase (AT) families, the structure-function relationship of the GNAT superfamily is the most well understood. In contrast, less information is available regarding mechanistic and regulatory aspects of p300/CBP AT function. In this paper, we investigate in closer detail the structure and sequence requirements for p300/CBP enzymatic activity. Unexpectedly, we find that the PHD finger of p300, but not of CBP, is dispensable for AT activity. In order to identify residues involved in substrate or acetyl-coenzyme A (acetyl-CoA) recognition, we have introduced 19 different amino acid substitutions in segments that are highly conserved between animal and plant p300/CBP proteins. By performing acetylation reactions with histones, a p53 peptide or the AT domain itself, we define several residues required for histone and p53 substrate recruitment but not for acetyl-CoA binding. Finally, we show that identical mutations in the p300 and CBP AT domain impair AT activity differently. This latter result combined with the finding of a differential requirement for the PHD finger provides evidence for structural differences between p300 and CBP that may in part underlie a previously reported functional specialization of the two proteins.

Acetyl Coenzyme A↗
PubMed2000

Autocorrelation function analysis of ECG signals in 20 rabbits.

In this experiment, we measured ECG signals of lead I, respectively, during normal and arrhythmia after 20 rabbits were anaesthetized. These signals were recorded, then the information was put into a computer and it analyzed Autocorrelation (AC) Function, and Power Spectrum. The results make us know: (1) periodicity corresponding relationship of ECG in normal rabbits can derive reflects from changes of ECG AC function changes of ECG signals in arrhythmia rabbits can derive relatively sensitive reflection from changes of ECG function (attenuating area S and the value of k) in normal rabbits. (2) The Autocorrelation Function of Electrocardio change but ECG in the normal. All these provide another useful method for the diagnosis of arrhythmia.

Animals↗
PubMed1978

Allied health educators: a functions analysis and needs assessment survey.

Three hundred and Thirty-eight allied health educators, geographically and institutionally distributed throughout Kentucky, participated in a field survey conducted to gather background and employment information and to determine topics and teaching skills identified by practitioners both as functions performed in their work and as areas where the need for further teaching expertise is greatest. No stereotype "allied health educator" existed. Job descriptions, teaching qualifications and experience, and needs for further preparation differ according to the settings in which allied health professionals teach. A large audience exists for teacher-training efforts directed toward improvement of applied skills and day-to-day teaching activities. Those desiring further preparation and improvement of their teaching skills prefer to do so within their own communities rather than by traveling to major educational centers for traditional coursework. There is, however, a desire for CEU or academic credit.

Allied Health Personnel↗
PubMed1988

Resistance to tolerance induction to human gammaglobulin (HGG) in autoimmune BXSB/MpJ mice: functional analysis of antigen-presenting cells and HGG-specific T helper cells.

Autoimmune male, but not female, BXSB/MpJ mice resist the induction of tolerance to human gammaglobulin (HGG). To better define the cellular basis for this abnormality, we have compared in vitro the functional activity of antigen-presenting cells between tolerant-resistant male and susceptible female mice, and evaluated in vivo the immunological status of HGG-specific T helper cells in male BXSB mice after the treatment with monomeric deaggregated HGG (DHGG). Our results indicated that there were no significant differences in the ability of male and female antigen-presenting cells to present HGG to either male or female HGG-specific immune T blasts. Further, thymic cells from DHGG-treated male BXSB mice failed to support anti-HGG antibody responses when adoptively transferred with non-treated bone marrow cells, and that draining lymph node cells from male as well as female BXSB mice treated with DHGG prior to the challenge of immunogenic aggregated HGG (AHGG) were unable to exhibit the proliferation upon in vitro restimulation with AHGG. This indicates that HGG-specific T helper cells were indeed tolerized in tolerant-resistant male BXSB mice by the treatment with DHGG. In contrast, when the tolerance induction was inhibited by bacterial lipopolysaccharides, HGG-specific T helper cells from such mice exhibited a marked proliferation upon in vitro restimulation with AHGG. These results suggest that the cellular defect in the abnormal resistance to tolerance induction to HGG in male BXSB mice resides neither in the antigen-presenting cells nor in the HGG-specific T helper cells, and that the mechanism responsible for this abnormality in male BXSB mice is basically different from that involved when tolerance is overcome by lipopolysaccharide.

Animals↗
PubMed1995

A functional analysis of tumor suppressor activity for peripheral neuroepitheliomas by monochromosome transfer.

The microcell hybridization technique provides a powerful method for the identification and characterization of tumor suppressor genes. By introducing chromosomes from a normal human cell into a tumor cell, several studies have presented functional evidence for the presence of tumor suppressor activity. In order to map the location(s) of functional tumor suppressor gene(s) for peripheral neuroepithelioma (PNET) cells, we have used the microcell hybridization technique to transfer three individual human chromosomes into three different PNET cell lines, A673, SK-N-MC and TC32. We could not isolate microcell hybrids from one of the cell lines as the transferred chromosome tended to fragment upon transfer. Introduction of chromosome 13 into the remaining two cell lines caused a marked inhibition of in vitro and in vivo growth. Chromosome 11 appeared to harbor a functional tumor suppressor gene while transfer of chromosome 17 caused a suppression of growth in culture, presumably due to the presence of the p53 tumor suppressor gene. Thus, each cell line showed a different response to the introduction of normal genetic information suggesting diverse genetic abnormalities among these tumors of similar histological or origin.

Animals↗
PubMed1996

Structure-function analysis of the Escherichia coli GrpE heat shock protein.

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.

Adenosine Triphosphatases↗
PubMed1999

Mutational and functional analysis of HPV-16 URR derived from Korean cervical neoplasia.

OBJECTIVE: The YY1 mutation has been suggested as one of the indicators that explains development of cervical neoplasia by episomal-type HPV. To extend this hypothesis, we examined whether a mutation(s) in the YY1 site is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. METHODS: The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients and cloned into pUC18 for sequencing and into pBLCAT8+ for functional CAT assay. RESULTS: Our previous data classified HPV-infected patients into three groups: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women: G-->A transition at nt 7520 (100%, 27/27), A-->C transition at nt 7729 (70%; 19/27), and G-->A transition at nt 7841 (78%; 21/27). Selective mutations were observed at the YY1 binding sites of HPV-16 URR in the 3 patients with invasive cervical cancer who have the episomal forms of HPV-16 DNA: A-->C transition at nt 7484 and G-->A transition at nt 7488 (YY1-binding site 2; from 7481 to 7489). Additionally, C-->T transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) was found in 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine whether these mutations have effects on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episomal HPV-16 DNA of cervical cancers were two- to fourfold higher than that of the HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in CIN, where no mutation of the YY1 binding site was detected, showed similar levels of promoter activity to that of the URR prototype. CONCLUSIONS: Our results support the hypothesis that the mutation at the YY1 binding site is functionally related to the development of cervical neoplasia caused by episomal HPV-16 DNA in Korean cervical cancer patients. Thus, mutation in the YY1 site of episomal HPV-16 URR may play a corresponding role of HPV integration in the progression of cervical cancer.

DNA Mutational Analysis↗
PubMed2004

Functional analysis of genes involved in the synthesis of syringolin A by Pseudomonas syringae pv. syringae B301 D-R.

Strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae secrete a family of structurally closely related peptide derivatives dubbed syringolins, of which syringolin A is the major variant. The function of syringolins in the interaction of P. syringae pv. syringae with their host plants presently is unknown. It is hypothesized that they may constitute virulence factors. However, syringolins are determinants recognized and reacted to by nonhost plant species, and syringolin A has been shown to induce hypersensitive death of cells colonized by powdery mildew in wheat and, thus, to reprogram a compatible interaction into an incompatible one. Syringolin A is an unusual derivative of a tripeptide that contains a 12-membered ring consisting of the amino acids 5-methyl-4-amino-2-hexenoic acid and 3,4-dehydrolysine, two nonproteinogenic amino acids. Here we report the cloning, sequencing, and analysis of genes involved in the biosynthesis of syringolin A. The genes encode proteins consisting of modules typical for nonribosomal peptide synthetases and type I polyketide synthetases, as well as proteins likely involved in the transcriptional regulation of syringolin A biosynthesis and in syringolin A export. The structure and arrangement of the modules lead to the formulation of a model explaining the synthesis of the tripeptide, including the formation of the two nonproteinogenic amino acids in the ring structure of syringolin A.

Amino Acid Sequence↗
PubMed1998

Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4.

In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell. Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging. It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity. In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis. In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function. Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions. Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development. According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function. This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-H436 pair, which constitute the histidine-rich region near the C terminus of gp17. A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect. In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal ATP-binding site also resulted in a loss of gp17 function. We propose that a metal-binding motif involving the histidine residues within the sequence H411-X2-H414-X15-H430-X5-H436 is essential for gp17 function. Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases.

Adenosine Triphosphate↗