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PubMed1998

In vivo protein binding and functional analysis of cis-acting elements in the U3 region of the bovine leukemia virus long terminal repeat.

Bovine leukemia virus (BLV) is a member of the human T-cell leukemia virus (HTLV)/BLV group of retroviruses. These viruses regulate their own transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR). To explore the molecular mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function by site- directed mutagenesis. We used in vivo dimethyl sulfate footprinting by ligation-mediated PCR to detect constitutive in vivo protein-DNA interactions in a BLV-producing cell line, Bat2Cl6. The U3 region and part of the R region of the LTR were footprinted. In addition to the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediated activation of the BLV LTR, we found footprints in regions flanking these elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mutagenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborate those of others showing that the CREs are necessary for transactivation of the LTR, and they identify two new functional sites not previously reported by others. We show that the middle region of the BLV U3 contains multiple dual-functioning cis-acting elements which act as either positive or negative regulatory elements depending on the cell type tested. This is the first report of a functional mapping of the cis-acting elements of a virus of the HTLV/BLV group.

Animals↗
PubMed2002

[Functional analysis of yeast homologue gene associated with human DNA helicase causative syndromes].

Proteins having DNA helicase activity play very important roles in many processes involving DNA workings such as replication, repair, and recombination. In this decade, many DNA helicase genes have been cloned as the causative genes of human recessive heredity diseases. These are the causative genes for Xeroderma pigmentosum (XPB and XPD), Cockayne syndrome (CSB), diffuse collagen disease (Ku80), alpha-thalassmia (ATR-X), Bloom syndrome (BLM), Werner syndrome (WRN) and Rothmund-Thomson syndrome (RTS). The yeast homologue genes of these human DNA helicase genes exist. S. cerevisiae RAD25/SSL2, RAD3, RAD26, YKU80/HDF2 and RAD54 are the homologue for XPB/ERCC3, XPD/ERCC2, CSB/ERCC6, Ku80/XRCC5 and ATR-X/HX2, respectively. E coli. recQ gene and S. cerevisiae SGS1 are the homologue for all BLM, WRN and RTS. A search of whole genome of S. cerevisiae revealed that SGS1 is the sole homologue of recQ in S. cerevisiae. Thus it seems likely that SGS1 is a functional homologue of one or several human RecQ family genes. Many basic or essential functions are well conserved in the cells from lower eukaryotic to higher mammalian. The functional analysis in yeast could make an useful insight for the human homologue. To clarify the functions of S. cerevisiae Sgs1 and to get an insight into the functions of Blm, Wrn and Rts, in this study, we analyzed the phenotype of sgs1 disruptant and in detail the cause of the poor sporulation phenotype of sgs1 disruptants in relation to meiotic processes including meiotic recombination. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate (MMS) and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants. The N-terminal 1-45 amino acid region and 698-1195 amino acid region of Sgs1, which including helicase domain and C-terminal RecQ conserved region with helicase activity, were required for complementation of MMS sensitivity and suppression of hyperrecombination of sgs1 disruptants in mitotic growth. The 126-400 and 596-1195 amino acid regions of Sgs1 were required for complementation of poor sporulation and of reduced meiotic functions. These regions required for the mitotic or meiotic functions of Sgs1 were well overlapped with the interaction regions of Top3 and Top2. Some of these results might explain the mechanism of the symptom of RecQ-related syndromes.

Adenosine Triphosphatases↗
PubMed1992

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.

Acetolactate Synthase↗
PubMed2004

Purification and functional analysis of Ric-8A: a guanine nucleotide exchange factor for G-protein alpha subunits.

Ric-8A (synembryn) has been shown to accelerate the in vitro guanine nucleotide exchange activities of most G-protein alpha subunits (with the exception of Galphas). Methods are presented in this article for the purification of Ric-8A and functional analysis of the effects Ric-8A has on G-protein alpha subunit guanine nucleotide-binding activities. The use of Ric-8A to prepare GTPgammaS-Galpha and nucleotide-free Galpha (in complex with Ric-8A) is described.

Animals↗
PubMed2002

Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.

BACKGROUND & AIMS: Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult. To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae. The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system. METHODS: The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms I219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH1 Delta 9/10) were cloned. On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay. RESULTS: Transfection was successful for all hMLH1 constructs. As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts. The V185G, G244D, and Delta 9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the I219V, R265H, R265C, R217C, and V326A mutations were MMR competent. CONCLUSIONS: These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products in a homologous expression system.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species.

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.

Aspartate Carbamoyltransferase↗
PubMed2002

Improving the prediction of outcome in severe acute closed head injury by using discriminant function analysis of normal auditory brainstem response latencies and amplitudes.

OBJECT: The auditory brainstem response (ABR) is a useful addition to standard medical measures for predicting outcome in patients with severe acute closed head injury (ACHI). Limiting this success, however, is the poor predictive value of a so-called "normal" ABR. In this study the authors used discriminant function analysis (DFA) of ABR Wave I, III, and V latencies and amplitudes to improve the predictive accuracy of the normal ABR, both as a single measure and in combination with other standard medical measures. METHODS: The DFAs were conducted using the ABR and medical results in 68 patients with severe ACHI (30 who died [ACHI-died], and 38 who survived [ACHI-lived]) who presented with normal ABR responses in the neurosurgical intensive care unit of the authors' hospital in Johannesburg. All patients had undergone surgery to remove an intracranial hematoma. Correct predictions of outcome by ABR DFA measures were 83% for the ACHI-died group (48% at > or = 90% confidence level) and 87% for the ACHI-lived group (71% at > or = 90% confidence level); by medical DFA measures the correct predictions were 83% for the ACHI-died group (96% at >; or = 90% confidence level) and 95% for the ACHI-lived group (94% at > or = 90% confidence level); and by combined ABR and medical DFA measures correct predictions were 100% for the ACHI-died group (100% at > or = 90% confidence level) and 97% for the ACHI-lived group (100% at > or = 90% confidence level). CONCLUSIONS: The DFA of ABR Wave I, III, and V latencies and amplitudes improved the predictive ability of normal ABR results to rates similar to those obtained using DFA for the medical measures, although at lower confidence levels. The DFA of the combined ABR and medical measures improved correct predictions to rates significantly higher than for either of the measures on its own.

Acute Disease↗
PubMed1978

Toward a biochemical classification of depressive disorders. II. Application of multivariate discriminant function analysis to data on urinary catecholamines and metabolites.

The previous article in this series reported on the differences in urinary excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG) in patients with various clinically defined subtypes of depressive disorders. We now report that further biochemical discrimination among depressive subtypes is provided by the following equation, derived empirically by applying multivariate discriminant function analysis to data on urinary catecholamine metabolits: Depression-type (D-type) score = C1(MHPG) + C2(VMA) + C3(NE) +C4(NMN + MN)/VMA + C0. In the original derivation of this equation, low scores were related to bipolar manic-depressive depressions, and high scores were related to unipolar nonendogenous (chronic characterological) depressions. Findings from a series of depressed patients whose biochemical data had not been used to derive this equation confirmed these differences in D-type scores among subtypes of depressions. The findings presented in this report further suggest that we can discriminate three biochemically discrete subgroups of depressive disorders.

Adult↗
PubMed1996

Discriminant function analysis of depressive symptoms in binge eating disorder, bulimia nervosa, and major depression.

OBJECTIVES: To examine the frequency and distribution of depressive symptoms among subjects with binge eating disorder (BED), bulimia nervosa (BN), and major depression. METHODS: This study examined depressive symptoms from the Hamilton Depression Scale in 122 BED, 142 BN, and 200 major depression subjects using discriminant function analysis. RESULTS: All three groups differed significantly on the Hamilton Depression Scale totals with major depressive disorder (MDD) subjects having the highest and BED subjects the lowest totals. Eighteen items differentiated MDD from the eating disorder groups. Three items-gastrointestinal (GI) somatic symptoms, paranoid symptoms, and obsessional symptoms-distinguished BED and BN. In each case these symptoms were more common in BN subjects. DISCUSSION: This study attempted to differentiate BN from BED on a basis other than eating behavior. The results provide limited support for the hypothesis that BN and BED can be distinguished on the basis of depressive symptoms.

Adult↗
PubMed2004

Transcriptional response to glucose starvation and functional analysis of a glucose transporter of Neurospora crassa.

The response to glucose availability in Neurospora crassa was characterized by measuring transcript levels of 1335 genes represented on a cDNA microarray. We found that 19% of the 1335 genes were regulated at least twofold in response to glucose deprivation. Several sugar transporter homologs were found to be regulated by glucose. Functional analysis of one of these, hgt-1, indicates that it encodes a high-affinity glucose transporter. Comparing N. crassa profiling data with the published diauxic shift data from Saccharomyces cerevisiae revealed that the two fungi share similar, but not identical, transcriptional response patterns. Regulation of transcript levels measured by microarray analysis is consistent with previous measurements of central metabolism enzyme activities for N. crassa. Transcripts of genes for ethanol fermentation and the tricarboxylic acid cycle are abundant in glucose-rich medium, consistent with N. crassa carrying out a high rate of both fermentation and respiration simultaneously.

Adaptation, Physiological↗
PubMed2006

Functional analysis of eubacterial ent-copalyl diphosphate synthase and pimara-9(11),15-diene synthase with unique primary sequences.

We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35 degrees C. The Km value was calculated to be 13.7 +/- 1.0 microM for GGDP and the kcat value was 3.3 x 10(-2)/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30 degrees C. The Km value for ent-CDP was estimated to be 2.6 +/- 0.2 microM and the kcat value was 1.4 x 10(-3)/sec.

Alkyl and Aryl Transferases↗
PubMed1995

Molecular cloning and functional analysis of the rat mu opioid receptor gene promoter.

The rat mu opioid receptor gene promoter was cloned and characterized. It has a few features in common with the mouse gene, e.g. the lack of a classical TATA box and the fact that several transcriptional start sites are used. The overall homology between the two species is greater than 85%. Functional analysis of the promoter was performed using transient expression of rat mu opioid receptor-reporter gene constructs in the mu opioid receptor expressing cell line SH SY5Y and in non mu opioid receptor expressing cell lines. A promoter region was defined which confers both high basal and TPA and forskolin stimulated reporter gene expression in SH SY5Y cells.

Animals↗
PubMed1990

Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants.

To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.

Amino Acid Sequence↗
PubMed1994

Separating Parkinson's disease from normality. Discriminant function analysis of fluorodopa F 18 positron emission tomography data.

OBJECTIVE: To explore the relationship between normal and parkinsonian fluorodopa F 18 (18F-6-L-fluorodopa [18F-dopa]) uptake data to identify clinically normal subjects who may have preclinical Parkinson's disease. DESIGN: A statistical comparison of striatal fluorodopa F 18 positron emission tomography scan data from patients with Parkinson's disease and normal controls. SETTING: Positron emission tomography unit within a postgraduate teaching hospital. MAIN OUTCOME MEASURES: Discriminant function analysis used to compare the pattern of striatal (left and right caudate and putamen) fluorodopa F 18 uptake in normal subjects and patients with Parkinson's disease. RESULTS: The discriminant score that best separates patients with Parkinson's disease from normal controls is a function of the lowest putamen influx constant minus a function of the caudate influx constant values. Borderline low normal subjects have slightly low fluorodopa F 18 uptake throughout the striatum, whereas patients with early Parkinson's disease have low fluorodopa F 18 uptake in one putamen with preserved uptake in the caudate (for normal subjects, subtracting the caudate influx constants from a function of the lowest putamen value lowers the discriminant score, although it remains positive; for patients, subtracting a larger caudate value from a function of the putamen uptake value leads to a negative score). One control subject had a borderline low discriminant score, compatible with focal nigral pathological changes as expected in preclinical Parkinson's disease. A repeated scan taken 3 years later showed a marked reduction in fluorodopa F 18 uptake, suggesting progressive nigral dysfunction. CONCLUSION: Normal and parkinsonian fluorodopa F 18 uptake data differ both in the overall level of tracer uptake and in its spatial distribution. Subjects whose overall striatal fluorodopa F 18 uptake falls at the borderline of normal and parkinsonian values are likely to be normal if they have uniformly low uptake, but may have early or preclinical Parkinson's disease if uptake into putamen is very much lower than uptake into caudate.

Aged↗
PubMed2003

Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein.

Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor. The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor. We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family. The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.

Amino Acid Sequence↗
PubMed1992

Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.

Animals↗
PubMed2005

A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis.

Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms.

Animals↗
PubMed2005

Functional analysis of fungal drug efflux transporters by heterologous expression in Saccharomyces cerevisiae.

Clinically important resistance of fungal pathogens to azole antifungal drugs is most frequently caused by the over-expression of energy-dependent drug efflux pumps. These pumps usually belong to either the ATP-binding cassette (ABC) family or the Major Facilitator Superfamily (MFS) class of membrane transporter. Little is known about how these pumps work and there is an urgent need to develop pump antagonists that circumvent azole resistance. We have developed a protein hyper-expression system to facilitate functional analysis of efflux pumps based on a Saccharomyces cerevisiae host which has been deleted in seven major ABC transporters to reduce the background of endogenous efflux activity. Plasmid pABC3 was engineered to allow functional hyper-expression of foreign proteins in this host. The main advantages of the system include its ease of directional cloning and the use of homologous recombination to stably integrate single copy constructs into the host genome under the control of a highly active transcriptional regulator. The system has been used to clone and functionally hyper-express genes encoding drug efflux pumps from several pathogenic fungi. Furthermore, the protein hyper-expression system has been used to screen for pump inhibitors and study the structure and function of heterologous membrane proteins.

Antifungal Agents↗