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PubMed1996

Cloning, sequencing and functional analysis of a truncated cDNA encoding red deer prolactin receptor: an alternative tyrosine residue mediates beta-casein promoter activation.

This study reports the isolation and in vitro characterisation of a truncated cDNA encoding the red deer long form prolactin receptor. The cDNA sequence predicts a protein of 557 amino acids which differs from the rat sequence by a 3' truncation of the cytoplasmic domain located 34 residues before the stop codon. The deer sequence shares the regions of homology which are important for maintenance of structural and functional integrity, high affinity binding and signal transduction. However, the truncated deer receptor lacks the most C-terminal tyrosine residue in the intracellular domain which is believed to be essential for activation of the beta-casein promoter. Transfection studies of the cervine cDNA into human 293 fibroblast cells confirmed the expression of a receptor that has high affinity binding to ovine prolactin (Ka = 0.65 x 10(9)M(-1) and Bmax = 548.6 fmol/mg protein). Co-transfection of CHO cells with expression vector encoding the cervine prolactin receptor cDNA along with a fusion gene containing the promoter region of beta-casein followed by beta-luciferase coding sequence led to 8.13 +/- 0.13-fold induction of luciferase enzyme activity in the presence of 400 ng/ml ovine prolactin. This was comparable to fold induction observed with the wild type long form rat prolactin receptor (6.37 +/- 0.48); macaque growth hormone receptor was without effect. Western blot analysis demonstrated tyrosine phosphorylation of the cervine receptor and the associated kinase Jak2 following stimulation with prolactin. This confirms that the cervine cDNA although truncated is fully functional and that Jak2 and an alternative tyrosine residue in the intracellular domain are involved in the signalling pathway leading to activation of the beta-casein promoter. Northern blot analysis provides evidence that the prolactin receptor in the liver is encoded by transcripts of approximately 2.5 and 3.5 kb. Comparison of Northern blots of different deer species suggests that the receptor is conserved amongst the Cervidae. Northern blot analysis of red deer testis suggests that this species expresses a second form of the receptor, encoded by a transcript of 1.7 kb, which may correspond to a smaller receptor form or a binding protein.

Amino Acid Sequence↗

PubMed2001

Genetic and functional analysis of the tbc operons for catabolism of alkyl- and chloroaromatic compounds in Burkholderia sp. strain JS150.

Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated tbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that the tbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.

Benzene↗

PubMed2002

Functional analysis of the Bacillus subtilis Zur regulon.

The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an approximately 28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.

Bacillus subtilis↗

PubMed1980

Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type 1 immediate early protein VP175.

Herpes simplex virus (HSV)-specific proteins fall into at least three kinetic classes whose synthesis is sequentially and coordinaely regulated. Temperature-sensitive (ts) mutants of one complementation group (1-2) are defective in the transition from immediate early to early and late protein synthesis. To elucidate the function of the 1-2 gene product in the HSV type 1 replicative cycle, nine ts mutants in this group were mapped by fine-structure analysis and characterized members of the group lie within the terminally repeated sequences of the S region of the genome. Fine-structure genetic and physical mapping permitted the mutations to be ordered within these sequences. Because it has been shown that the message for VP175 and the DNA template specifying this protein extend beyond the limits of the physical map of the mutations, it follows that the mutations must lie within the structural gene for VP175. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that most members of the group overproduced the immediate early proteins VP175, -136, -110, and -63 and markedly underproduced early and late proteins at the nonpermissive temperature. In temperature shiftup experiments, it was fund that the synthesis of early and late proteins ceased, whereas the synthesis of immediate early proteins began again. Thus, it is postulated that VP175 is (i) involved in the transition from immediate early to early protein synthesis, (ii) requird continuously to maintain early protein synthesis, (iii) autoregulated, acting to inhibit immediate early protein synthesis.

Chromosome Mapping↗

PubMed2002

[Functional analysis of Na+/Ca2+ exchanger using novel drugs and genetically engineered mice].

The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on the ion gradients across the plasma membrane and the membrane potential. In heart, smooth muscle cells, neurons, and nephron cells, this exchanger is thought to play an important role in the regulation of intracellular Ca2+ concentration. We aim at clarifying the function and pathologic state of the exchanger by developing NCX inhibitors and NCX-engineered mice as experimental tools, and finally establishing a novel therapeutic method using the exchanger as a molecular target. First, we present the pharmacological profiles and the molecular mechanism of NCX inhibitors (KB-R7943, SEA0400, SN-6) recently developed. Our results suggest that these NCX inhibitors preferentially block the Ca2+ influx mode rather than the Ca2+ efflux mode. Further mutational analysis reveals that these NCX inhibitors have different interaction domains in the exchanger molecule. Secondly, we present that heterozygous NCX1-knockout mice are useful as a low NCX1-expressing animal model. From comparative experiments with heterozygous and wild-type mice and pharmacological experiments of the NCX inhibitors, we provided an evidence that NCX1 functionally contributes to maintaining the vascular tension.

Aniline Compounds↗

PubMed2004

Identification and functional analysis of dual-specific A kinase-anchoring protein-2.

Since the cloning of dual-specificity A kinase-anchoring protein 2 (D-AKAP2), there has been considerable progress in understanding the structural features of this AKAP and its interaction with protein kinase A (PKA). The domain organization of D-AKAP2 is quite unique, containing two tandem, putative RGS domains, a PKA-binding motif, and a PDZ (PSD95/Dlg/ZO1)-binding motif. Although the function of D-AKAP2 has remained elusive, several reports suggest that D-AKAP2 is targeted to cotransporters in the kidney and that it may play a role in regulating transporter activity. In addition, the finding that a single nucleotide polymorphism in the PKA-binding region of D-AKAP2 may contribute to increased morbidity and mortality emphasizes the potential importance of this protein in pathogenesis. The first part of this article focuses on initial efforts to identify and clone D-AKAP2, followed by tissue localization and expression profiles. The latter half of the article focuses on the domain organization of D-AKAP2 and its interaction with PKA. Finally, a comprehensive analysis of the PKA binding motif is described, which has led to the development of novel peptides derived from D-AKAP2 that can be useful tools in probing the function of this AKAP in cellular and animal models.

A Kinase Anchor Proteins↗

PubMed1986

Functional analysis of treatment of oral cavity cancer.

There are few studies that have addressed themselves to measuring the speech, swallowing results, and quality of life that patients are left with after the treatment of oral cavity cancer. We developed a test series to assess the oral cavity function and the general health of patients treated for oral cavity cancer. The results of 51 patients treated for oral cavity cancer were compared by site and stage of the lesion as well as by treatment modality. In addition, the functional results achieved by different reconstructive techniques in the oral cavity were evaluated. The series also identified site-specific speech and swallowing problems.

Combined Modality Therapy↗

PubMed2006

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity.

AIMS/HYPOTHESIS: Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. MATERIALS AND METHODS: Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. RESULTS: We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. CONCLUSIONS/INTERPRETATION: Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

Adaptor Proteins, Signal Transducing↗

PubMed2007

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Animals↗

PubMed1986

Differentiating DST suppressors from nonsuppressors: a discriminant function analysis of clinical symptoms.

Ninety-seven patients who presented with depressive complaints were studied in an attempt to cross-validate a statistical model that uses items from the Hamilton Rating Scale for Depression (HRSD) to differentiate dexamethasone suppressors from nonsuppressors. Two split-sample discriminant function analyses (DFA) of data from this sample showed little agreement with either the original model or with each other. Accuracy of classification was acceptable for the derivation samples but was no better than chance for the validation samples. No single HRSD item was identified by all three DFAs as distinguishing between groups. The HRSD does not appear to be a reliable instrument for clinically distinguishing dexamethasone suppressors from nonsuppressors.

Adult↗

PubMed1991

Cloning and characterization of the promoter of the murine lipoprotein lipase-encoding gene: structural and functional analysis.

The enzyme lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides into free fatty acids and glycerol. Its synthesis is induced as the murine bone marrow stromal cell clone, BMS2, undergoes adipocyte differentiation. The murine genomic LPL promoter has been cloned, sequenced, and characterized by functional and structural assays. The transcriptional start points have been mapped by S1 nuclease and primer extension techniques. Comparison of the 1.7-kb of LPL 5'-flanking sequence between mouse and man reveals 65% identity or greater with conservation of many potential protein-recognition motifs. Using constructs linking this region to the luciferase-encoding reporter gene, transient transfection experiments have documented the promoter function of this sequence in a number of cell lines. Based on a battery of restriction endonucleases, at least 260 bp immediately adjacent to and including the 5'-untranslated region of the first exon are hypersensitive to exogenous nuclease digestion, consistent with an altered chromatin structure. Protein-DNA interactions are detected within this area at the octamer binding protein 1 site and immediately 5' to the translation initiation site based on ExoIII footprinting and gel retention assays.

Animals↗

PubMed2001

Genetic and functional analysis of PARP, a DNA strand break-binding enzyme.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by binding to a single- or double-strand break of DNA and is one of the death substrates for caspase-3 in apoptosis. The nuclear function of PARP is well studied and recent PARP-knockout studies indicate that PARP takes part in chromosomal stability. To analyze the effect of PARP overexpression, or loss of function, we have cloned PARP cDNA and the gene from Drosophila melanogaster and studied its function in developmental stages. Organization of exons corresponds to the functional domains of PARP. An alternatively spliced form of PARP lacking exon 5, which encodes the auto-modification domain, is found in Drosophila. Expression of the PARP gene is at high levels in embryos at 0-6h after egg laying and gradually decreased. In situ mRNA hybridization indicates localization of PARP mRNA in cells along the central nervous system at a late stage of embryogenesis. Overexpression of the gene in the developing eye primordia of D. melanogaster is an excellent experimental model to analyze the cell cycle and programmed cell death. We introduced PARP expression vector overexpresses PARP in the eye discs of Drosophila, and established the PARP transgenic flies by P element-mediated germ line transformation. These flies showed mild roughening of the normally smooth ommatidial lattice involving tissue polarity disruption characterized by missrotation and incorrect chirality of ommatidia. Possible mechanisms of involvement of PARP in the development are discussed.

Animals↗

PubMed2002

Functional analysis of oocyte-expressed genes using transgenic models.

An oocyte's journey is highly distinct from the vast majority of cells in the body. As one of the largest and rarest cells, oocytes express unique genes required for the genesis of healthy and competent eggs. The function of only a handful of oocyte-specific genes is beginning to be unraveled. Transgenic mouse models have proven to be extremely valuable in studying the effects of gene deletions on oocytes and surrounding somatic cells. Growth differentiation factor 9 (Gdf9), bone morphogenetic protein 15 (Bmp15), zona pellucida genes (Zp1, Zp2 and Zp3), factor in the germline alpha (Figla), and the c-mos protooncogene (c-mos) are some of the genes preferentially expressed in oocytes which play important roles during folliculogenesis. In order to identify other novel genes preferentially expressed in oocytes, we have utilized subtractive hybridization and in silico subtraction. The combination of these identification approaches, coupled with the use of knockout mice, will lead to many future functional studies of genes uniquely devoted to oogenesis and folliculogenesis.

Animals↗

PubMed2003

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fya mAbs to K562 cells respectively, Anti-Fy3 binding was abolished by D58-D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti-Fy3 and CXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 binding was also abrogated by mutations of F22-E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1-4 participate in CXCL-8 binding; and (3) Fy3 is a conformation-dependent epitope involving all ECDs. We also showed that N-glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

Amino Acid Sequence↗

PubMed2003

Structure-function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis.

The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a cytopathic serine protease, which is prototypical of a large family of bacterial autotransporter toxins. To further elucidate the structure-function relationships of this toxin, we employed transposon-based scanning linker mutagenesis. A subset of insertions throughout the Pet mature toxin (passenger) domain reduced secretion to the extracellular space. Many of these mutants were undetectable, but secretion of a subset of mutants with insertions in the N-terminal half of the toxin could be restored to wild type secretion levels if cultured in the presence of 0.1% Triton X-100. Secretion of two mutants with insertions at the extreme C terminus was partially restored when co-expressed with a minimal clone of EspP, a related autotransporter protein. Several well secreted mutants with insertions in the N-terminal third of the molecule reduced protease activity over 20-fold, suggesting that the protease domain is located within this N-terminal region of Pet. We have also identified two insertional mutants in the middle of the passenger domain that were proteolytic but no longer cytopathic; these mutants displayed decreased binding and internalization upon incubation with HEp-2 cells. Our data suggest the existence of separate functional domains mediating Pet proteolysis, secretion, and cell interaction.

Bacterial Toxins↗

PubMed2002

A yeast model system for functional analysis of beta-catenin signaling.

We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of beta-catenin (beta-cat) signaling. Coexpression of mammalian beta-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both beta-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with beta-cat, reduces beta-cat binding to TCF4, and leads to increased cytoplasmic localization of beta-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to beta-cat and restores reporter gene transcription. Additional beta-cat signaling components, Axin and glycogen synthase kinase 3beta, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein beta-transducin repeat-containing protein reduces the stability of beta-cat and decreases reporter activation. Thus, we have reconstituted a functional beta-cat signal transduction pathway in yeast and show that beta-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.

Active Transport, Cell Nucleus↗

PubMed2002

Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension.

A wide range of mutations in the type II receptor for bone morphogenetic protein (BMPR-II) have been shown to underlie primary pulmonary hypertension. To determine the mechanism of altered BMPR-II function, we employed transient transfection studies in cell lines and primary cultures of pulmonary vascular smooth muscle cells using green fluorescent protein (GFP)-tagged wild-type and mutant BMPR2 constructs and confocal microscopy to localize receptors. Substitution of cysteine residues in the ligand binding or kinase domain prevented trafficking of BMPR-II to the cell surface, and reduced binding of (125)I-BMP4. In addition, transfection of cysteine-substituted BMPR-II markedly reduced basal and BMP4-stimulated transcriptional activity of a BMP/Smad responsive luciferase reporter gene (3GC2wt-Lux), compared with wild-type BMPR-II, suggesting a dominant-negative effect of these mutants on Smad signalling. In contrast, BMPR-II containing non-cysteine substitutions in the kinase domain were localized to the cell membrane, although these also suppressed the activity of 3GC2wt-Lux. Interestingly, BMPR-II mutations within the cytoplasmic tail trafficked to the cell surface, but retained the ability to activate 3GC2wt-Lux. Transfection of mutant, but not wild-type, constructs into a mouse epithelial cell line (NMuMG cells) led to activation of p38(MAPK) and increased serum-induced proliferation compared with the wild-type receptor, which was partly p38(MAPK)-dependent. We conclude that mutations in BMPR-II heterogeneously inhibit BMP/Smad-mediated signalling by diverse molecular mechanisms. However, all mutants studied demonstrate a gain of function involving upregulation of p38(MAPK)-dependent proproliferative pathways.

Animals↗

PubMed2001

Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis.

Cyclin-dependent kinase inhibitors, such as the mammalian p27(Kip1) protein, regulate correct cell cycle progression and the integration of developmental signals with the core cell cycle machinery. These inhibitors have been described in plants, but their function remains unresolved. We have isolated seven genes from Arabidopsis that encode proteins with distant sequence homology with p27(Kip1), designated Kip-related proteins (KRPs). The KRPs were characterized by their domain organization and transcript profiles. With the exception of KRP5, all presented the same cyclin-dependent kinase binding specificity. When overproduced, KRP2 dramatically inhibited cell cycle progression in leaf primordia cells without affecting the temporal pattern of cell division and differentiation. Mature transgenic leaves were serrated and consisted of enlarged cells. Although the ploidy levels in young leaves were unaffected, endoreduplication was suppressed in older leaves. We conclude that KRP2 exerts a plant growth inhibitory activity by reducing cell proliferation in leaves, but, in contrast to its mammalian counterparts, it may not control the timing of cell cycle exit and differentiation.

Amino Acid Sequence↗