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At least 757 records · Page 42Linked to original sources
PubMed1997

Insertion mutagenesis of the lac repressor and its implications for structure-function analysis.

We recently developed a simple technique for the generation of relatively large (31-codon) insertion mutations in cloned genes. To test whether the analysis of such mutations could provide insight into structure-function relationships in proteins, we examined a set of insertion mutants of the Escherichia coli lac repressor (LacI). Representatives of several LacI mutant classes were recovered, including mutants which exhibit fully active, inducer-insensitive, or weak dominant-negative phenotypes. The various properties of the recovered mutants agree with previous biophysical, biochemical, and genetic data for the protein. In particular, the results support the prior designation of mutationally tolerant spacer regions of LacI as well as proposed differences in dimerization interactions among regions of the protein core domain. These findings suggest that the analysis of 31-codon insertion mutations may provide a simple approach for characterizing structure-function relationships in proteins for which high-resolution structures are not available.

Bacterial Proteins↗
PubMed1991

Structural and functional analysis of Trichoderma reesei endoglucanase I expressed in yeast Saccharomyces cerevisiae.

The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied. Truncated EGI proteins were expressed from the 3'-end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADC1 expression cassette. EGI protein was detected by monoclonal antibody EI-2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR). The results showed that the The-Ser-rich hinge region and the conserved 'tail' are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.

Blotting, Western↗
PubMed2004

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana.

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.

Amino Acid Sequence↗
PubMed2002

Design and functional analysis of actomyosin motor domain chimera proteins.

To gain more structural and functional information on the actomyosin complexes, we have engineered chimera proteins carrying the entire Dictyostelium actin in the loop 2 sequence of the motor domain of Dictyostelium myosin II. Although the chimera proteins were unable to polymerize by themselves, addition of skeletal actin promoted polymerization. Electron microscopic observation demonstrated that the chimera proteins were incorporated into actin filaments, when copolymerized with skeletal actin. Copolymerization with skeletal actin greatly enhanced the MgATPase, while the chimera proteins without added skeletal actin hydrolyzed ATP at a very low rate. These results indicate that the actin part and the motor domain part of the chimera proteins are correctly folded, but the chimera proteins are structurally stressed so that efficient polymerization is inhibited.

Actin Cytoskeleton↗
PubMed1979

Functional analysis of neutrophil granulocytes from healthy, infected, and stressed neonates.

Neutrophil granulocyte function was assessed in 17 well term infants, 14 stressed infants, and eight infants with group B streptococcal infection. Chemiluminescence production elicited by opsonized zymosan or by a wild strain of type III group B streptococci, as well as phagocytosis and killing of streptococci, were measured. Chemiluminescence production by PMNs of term neonates in response to opsonized zymosan or group B streptococci was equal to that of adult controls. In contrast, six of nine stressed or infected neonates had depressed CL responses upon zymosan challenge. When opsonized type III group B streptococci were used to elicit CL, seven of ten stressed or infected infants had markedly depressed responses. Phagocytosis, as determined by a radiolabeled bacterial uptake technique, was normal in the healthy and stressed neonates. Depressed CL production by the PMNs of stressed or infected neonates was associated with impaired intracellular bactericidal activity, however. These studies indicate that stressed or infected neonates have impaired leukocyte metabolic activation that may be associated with depressed bactericidal activity. Such impairment may contribute to the morbidity and mortality observed in serious neonatal infections.

Blood Bactericidal Activity↗
PubMed1999

The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Amino Acid Sequence↗
PubMed1993

Immunological characteristics of umbilical cord blood cells: phenotypic and functional analysis.

The purpose of this study was to phenotype and assay immunological function on cord blood from 70 samples. Immunophenotyping indicated similar numbers of CD2, 3, and 8-positive cells as in adult bone marrow. CD4-positive cells were increased and CD19, 20, and CD56-positive cells were decreased in numbers. Proliferative responses to nonspecific mitogens were lower in cord blood cells than in adult cells, as was NK activity and spontaneous secretion of IL-6 and TNF-alpha. This study confirms the relative immaturity of cord blood cells even when some functional assays appear normal.

Adult↗
PubMed2006

Functional analysis of the five melon necrotic spot virus genome-encoded proteins.

Function of the melon necrotic spot virus (MNSV) genome-encoded proteins (p29, p89, p7A, p7B and p42) has been studied. Protein-expression mutants of an infectious, full-length cDNA clone of a Spanish MNSV-Al isolate and a recombinant green fluorescent protein (GFP)-expressing virus were used in infection bioassays on melon plants. Results revealed that p29 and p89 are both essential for virus replication, whereas small proteins p7A and p7B are sufficient to support viral movement between adjacent cells operating in trans. It is also demonstrated that, in addition to its structural role as coat protein, p42 is an important factor controlling symptoms and is required for systemic transport. Moreover, both p42 and p7B, among all of the MNSV-encoded proteins, were able to delay RNA silencing in transient-expression assays on GFP-transgenic Nicotiana benthamiana plants. Finally, the presence of p42 also produced an enhancing effect on local spread similar to that of potyviral helper component proteinase (HC-Pro), probably due to its RNA silencing-suppression ability.

Carmovirus↗
PubMed2006

Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis.

We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCF(SLY1) E3 ubiquitin ligase that targets the DELLA protein for degradation.

Alkyl and Aryl Transferases↗
PubMed1996

Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis.

We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.

Cross Reactions↗
PubMed1976

The anterior cruciate ligament. A functional analysis based on postmortem studies.

In forty fresh human cadaver knees the function of the anterior cruciate ligament and of its two component parts, the posterolateral part and the anteromedial band, were studied by cutting these ligaments and others in different sequences and combinations and then manually stressing the knees. The anterior drawer sign cannot be obtained unless the anteromedial band is severed. The postolateral part and the medial collateral ligament are, respectively, the secondary and tertiary restraints limiting the anterior drawer sign. Both internal and external rotation are limited by the anterior cruciate ligament, especially when the knee is in extension. The anterior cruciate ligament also limits hyperextension.

Autopsy↗
PubMed2005

[Cloning and functional analysis of melanocortin 4 receptor mutation gene F261S].

OBJECTIVE: To evaluate the function change of the melanocortin 4 receptor (MC4R) protein with mutation of F261S. METHODS: Human embryonic cells of the HEK293 line were cultured. Wild-type genomic DNA and F261S mutation human melanocortin 4 receptor genes from the genomic DNA of aproband of homozygotic F612 mutation were amplified and cloned into a topo-TA eukaryotic expression plasmid vector. After the wild-type and F261S mutated proteins were expressed in HEK293 cells, alpha-MSH (10(-11) approximately 10(-5) mmol/L) was added, then the intracellular cAMP was detected with dual luciferase reporter assay system. RESULTS: When the concentration of alpha-MSH added was 10(-9) approximately 10(-8) mmol/L, the intracellular alpha-MSH concentration of the cells transfected with wild-type MC4R gene was significantly higher than that of the cells transfected with F261S mutation gene (P < 0.05). When the concentration of alpha-MSH added went to 10(-7) approximately 10(-5) mmol/L, the differences became even more significant (all P < 0.01). CONCLUSION: The novel MC4R mutation F261S undermines the signal transduction. It may be the possible reason leading to monogenic mutation obesity in Chinese.

Adult↗
PubMed1988

The portal hypertensive gastric mucosa: histologic, ultrastructural, and functional analysis after aspirin-induced damage.

We assessed macroscopic, histologic, ultrastructural, and functional features of aspirin-induced gastric mucosal injury in portal hypertensive and sham-operated rats. Portal hypertension was produced by staged portal vein ligation. Four hours after intragastric acidified aspirin administration, intraluminal pH in portal hypertensive rats was 6.6 +/- 0.2 and 4.3 +/- 0.5 in sham-operated controls (p less than 0.01). Gross mucosal damage was significantly greater in portal hypertensive rats compared with controls (18 +/- 2 versus 7 +/- 1% of total mucosal area). Histologic deep necrosis involved 22 +/- 2% of mucosal section lengths in portal hypertensive rats compared with 7 +/- 1% in sham-operated rats (p less than 0.01). In portal hypertensive rats, histologic and ultrastructural evaluation demonstrated capillary endothelial abnormalities, arterialization of submucosal veins, and markedly greater severity of microvascular damage than in sham-operated controls. Neutralized aspirin (pH, 7.0) did not produce any significant damage detectable grossly, histologically, or by transmission electron microscopy in portal hypertensive rats. We conclude that acid-dependent aspirin-induced gastric mucosal damage is significantly increased in portal hypertension.

Animals↗
PubMed1993

Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival.

The bcl-2 gene can potentially encode 26- and 22-kDa proteins that differ only in their carboxyl tails because of an alternative splicing mechanism. The larger of these proteins contains a hydrophobic transmembrane domain within its carboxyl terminus, resides (at least in part) in mitochondrial membranes and has been shown to prolong cell survival by blocking programmed cell death (also termed "apoptosis"). To explore the function of the shorter 22-kDa Bcl-2 protein that lacks a transmembrane domain, DNAs encoding p26-Bcl-2-alpha or p22-Bcl-2-beta were expressed in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D. In contrast to p26-Bcl-2 alpha that markedly prolonged cell survival, p22-Bcl-2-beta did not extend the survival of 32D cells when cultured in the absence of IL-3. Expression in 32D cells of a chimeric DNA that fused portions of the open reading frame common to Bcl-2-alpha and Bcl-2-beta (amino-acids 1-195) with sequences encoding the transmembrane and cytosolic domains of the IL-2 receptor-alpha protein resulted in production of a Bcl-2/IL-2R fusion protein that was capable of prolonging 32D cell survival in the setting of IL-3 withdrawal. Based on fractionation of cells to produce crude heavy membrane, light membrane, nuclei, and cytosolic preparations, much of the p22-Bcl-2-beta protein appeared to reside in the cytosol, whereas Bcl-2-alpha and the Bcl-2/IL-2R chimeric proteins were found exclusively in fractions that also contained the inner mitochondrial membrane protein F1-beta-ATPase. Taken together, these findings demonstrate the importance of membrane association for the function and intracellular targeting of the apoptosis-blocking Bcl-2 protein. Furthermore, despite the strong evolutionary conservation of the carboxyl regions of Bcl-2-alpha proteins observed previously for mammalian and avian species, these data suggest that a heterologous transmembrane domain can be substituted without loss of function.

Amino Acid Sequence↗
PubMed1997

Sensitivity theory and mental retardation: why functional analysis is not enough.

Sensitivity theory holds that aberrant behavior is a function of aberrant contingencies (direct reinforcement of maladaptive behavior), aberrant motivation (a desire for an excessive amount of reinforcement), and aberrant environments (situations in which most people cannot satiate their fundamental desires by behaving appropriately). Applied behavior analysts are almost exclusively concerned with aberrant contingencies and, to a lesser extent, aberrant environments. Because they do not treat people for aberrant motivation, behaviorists tend to treat the same people repeatedly. When a person craves an excessive amount of attention, this desire must be reduced for a durable and generalized treatment outcome, but behaviorists do not even try to do this. Implications for communication theory, assessment, and treatment strategies were discussed.

Behavior Therapy↗
PubMed1990

Cloning and functional analysis of the rat ornithine decarboxylase-encoding gene.

We have isolated a functional gene (ODC) encoding rat ornithine decarboxylase (ODC; EC 4.1.1.17) from a partial rat liver genomic DNA bank. The entire gene is located on a 7776-bp BamHI fragment and was shown to comprise twelve exons, of which ten encode the ODC protein (exons III-XII). Introduction of the BamHI fragment into an ODC-deficient hamster cell line restores ODC activity, indicating that the gene is functional. Comparison of the structure and nucleotide (nt) sequence of the rat ODC gene with recently reported mouse ODC genes, reveals that the gene is highly conserved. Primer extension analysis and RNA sequencing demonstrates that the transcription start point of rat ODC mRNA is located 303 nt upstream from the A residue in the start codon. Compared with our previously published sequence of the rat ODC cDNA, this indicates that a short sequence at the extreme 5' end of our cDNA clone represents a cloning artefact. The correct 5' leader of ODC mRNA, which is very G + C rich (62%), can be folded into a highly stable secondary structure, which may play a role in the translational control of ODC activity. Like in mouse, the promoter region of rat ODC is also extremely rich in G + C, and contains a TATA box and several putative SP1-binding sites. Possible binding sites for other transcription factors, like AP-1, AP-2 and CREB, can also be observed in the promoter region and, moreover, in the first intron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Structure-function analysis of the feline herpesvirus virulence factors gE and gI.

The biosynthesis and function of the gE-gI complex of feline herpesvirus (FHV) was studied by heterologous expression of the cloned genes in the vaccinia virus-based vTF7-3 expression system and by analysis of FHV recombinants. This work has led to the identification of domains in gI involved in complex formation with gE, and to a model for the disulfide-bonded structure of gI. The effects of mutations in gI on gE-gI-dependent cell-to-cell transmission are discussed.

Animals↗