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At least 793 records · Page 44Linked to original sources
PubMed1993

Functional analysis of hepatitis B virus transactivator X: implication of the leucine zipper-like region and C-terminal seven conserved amino acids in functional regions.

X protein of hepatitis B virus (HBV-X) is a transactivator to a wide variety of viral and cellular transcriptional regulatory elements. Since HBV-X does not act on a common cis-regulatory element of a wide variety of regulatory elements nor does it bind to DNA directly, it has been proposed that HBV-X acts indirectly through protein-protein interactions with other transcription factors or signal transducing pathway. In order to determine the functional domain of HBV-X, we have constructed and analyzed a number of deletion and site specific mutants. Our results showed that leucine zipper-like sequences were found in the C-terminal region of HBV-X and were very important for its transactivating activity. In the analysis of deletion mutants, seven conserved and strong basic amino acids (amino acids 135-141) were essential for the transactivating activity of HBV-X.

Amino Acid Sequence↗
PubMed2004

Functional analysis of CDK inhibitor p21WAF1.

p21WAF1 was originally identified as a protein that binds and inhibits cyclin-dependent kinases (CDKs). p21WAF1 is recognized to have at least two separate roles-first as a CDK inhibitor, and second as an inhibitor of PCNA, an accessory protein of DNA polymerase delta. p21WAF1 plays a critical role in the cellular response to DNA damage. Additionally, p21WAF1 plays a role in DNA repair, apoptosis, cellular senescence, terminal differentiation, and cell cycle arrest upon extracellular signaling. p21WAF1 protein levels are regulated both by transcriptional control by p53 and by factors other than p53, as well as by posttranscriptional regulation. Although the role of p21WAF1 has been explained so far only by its interaction with CDKs and with PCNA, it has several other binding partners. The ability of p21WAF1 to participate in several cellular functions has been widely studied by transfection of cells with p21WAF1 vectors. We describe here procedures for analysis of p21WAF1 function in mammalian cells after transfection of p21 plasmids. The procedures include inhibition of DNA synthesis, cellular localization, association with binding partners, and half-life measurements.

Amino Acid Sequence↗
PubMed2026

Genome-Wide Identification of the LdARF Gene Family in Lilium davidii var. unicolor and Transient Functional Analysis of LdARF17 in Bulblet Regeneration.

Auxin response factors (ARFs) are key transcriptional regulators of the auxin signaling pathway and play important roles in plant organogenesis and regeneration. However, the functions of ARF family genes in lily scale-derived bulblet regeneration remain largely unclear. In this study, 24 LdARF genes were identified from the genome of Lilium davidii var. unicolor. Phylogenetic analysis revealed that LdARF proteins showed evolutionary conservation with ARF homologs from other monocot species. Genome-wide identification, phylogenetic analysis, and expression profiling revealed functional divergence among LdARF genes during scale-derived bulblet regeneration. Among them, LdARF17 exhibited a distinct regeneration-associated expression pattern, characterized by rapid induction after scale excision and sustained high expression during subsequent bulblet initiation and formation. Subcellular localization analysis demonstrated that LdARF17 is localized in the nucleus. Transient overexpression of LdARF17 significantly promoted bulblet regeneration and was associated with increased expression of auxin-responsive and regeneration-related genes, including IAA14, LBD16, and LBD29. These findings suggest that LdARF17 acts as a positive regulator of lily scale regeneration and may influence auxin-responsive transcriptional processes associated with early cell proliferation, providing new insights into the molecular mechanisms underlying vegetative regeneration in lilies.

Auxin response factor↗
PubMed1993

Functional analysis of the tyrosinase gene and brown-locus protein gene promoters.

Tyrosinase is a rate-limiting enzyme of melanin biosynthesis and the brown (b)-locus protein is responsible for the formation of black melanin rather than brown. To identify the cis-acting element(s) required for pigment cell-specific gene transcription, we analyzed the promoter function of two pigment cell-specific genes encoding mouse tyrosinase and b-locus protein using a cell-free transcription system prepared from mouse melanoma cells. Functional and structural analysis of both gene promoters reveals that three elements are conserved in both genes at equivalent positions, suggesting that these elements may be responsible for pigment cell-specific transcription. We discuss possible mechanisms for pigment cell-specific expression of the tyrosinase and b-locus protein genes.

Animals↗
PubMed2002

Functional analysis of the copper-dependent quercetin 2,3-dioxygenase. 2. X-ray absorption studies of native enzyme and anaerobic complexes with the substrates quercetin and myricetin.

Quercetin 2,3-dioxygenase (2,3QD) is a mononuclear copper-dependent dioxygenase which catalyzes the cleavage of the heterocyclic ring of the flavonol quercetin (5,7,3',4'-tetrahydroxy flavonol) to produce 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide. In this study, X-ray absorption spectroscopy has been used to characterize the local structural environment of the Cu(2+) center of Aspergillus japonicus 2,3QD. Analysis of the EXAFS region of native 2,3QD at functionally relevant pH (pH 6.0) indicates an active site equally well-described by either four or five ligands (3N(His) + 1-2O) at an average distance of 2.00 A. Bond valence sum analysis confirms that the best model is somewhere between the two. When, however, 2,3QD is anaerobically complexed with its natural substrate quercetin, the copper environment undergoes a transition to a five-coordinated cage, which is also best modeled by a single shell of N/O scatterers at the average distance of 2.00 A. This coordination is independently confirmed by the anaerobic complex with myricetin (5'-hydroxy quercetin). XANES analysis confirms that substrate binding does not reduce the Cu(2+) ion. The present study gives the first direct insights into the coordination chemistry of the enzyme complexed with its substrates. It suggests that activation for O(2) attack is achieved by monodentate substrate complexation to the copper ion through the 3-hydroxyl group. In addition, monodentate carboxylate ligation by the Glu73 side chain is likely to play a role in the fine-tuning of the equilibrium leading to the formation of the activated E.S complex.

Anaerobiosis↗
PubMed2002

Functional analysis of the murine sarcoma virus RNA packaging sequence.

We investigated the features of the Moloney murine sarcoma virus leader sequence necessary for RNA packaging function by using a deletion analysis approach. We found that sequences that extend beyond those characterized genetically in previous reports are important for optimal packaging efficiency. A fragment covering a minimum of four potential stem-loop structures is required for the shortest packaging element compatible with gene transfer. Our results reveal the extent to which each of the segments of the packaging sequence contribute to packaging efficiency.

5' Untranslated Regions↗
PubMed2006

Expression and functional analysis of Tgif during mouse midline development.

The Tgif gene encodes a homeodomain protein that functions as a transforming growth factor beta (TGF-beta) repressor by binding to Smad2. Mutations in the TGIF gene are associated with human holoprosencephaly, a common birth defect caused by the failure of anterior ventral midline formation. However, Smad2-mediated TGF-beta signaling in the axial mesendoderm has been demonstrated to be essential for ventral midline formation, and loss of a Smad2 antagonist should in principle promote rather than inhibit ventral midline formation. This suggests a more complex mechanism for the function of TGIF in controlling ventral midline formation. To explore the role of TGIF in ventral forebrain formation and patterning, we investigated Tgif expression and function during mouse development by in situ hybridization and gene targeting. We found that Tgif is highly expressed in the anterior neural plate, consistent with the proposed neural differentiation model in which TGF-beta suppression is required for normal neural differentiation. This result suggests a possible role for Tgif in anterior neural differentiation and patterning. However, targeted disruption of the Tgif gene during mouse development does not cause any detectable defects in development and growth. Both histological examination and gene expression analysis showed that Tgif-/- embryos have a normal ventral specification in the central nervous system, including the forebrain region. One interpretation of these results is that the loss of TGIF function is compensated by other TGF-beta antagonists such as c-Ski and SnoN during vertebrate anterior neural development.

Animals↗
PubMed1990

Structural and functional analysis of the insulin receptor promoter.

The insulin receptor plays a critical role in the maintenance of glucose homeostasis. Regulation of this key function must be under stringent controls. In order to study the regulation of insulin receptor gene expression, we have cloned, sequenced and characterized its promoter. The first exon of the insulin receptor gene is embedded in an unusual segment of DNA composed of Alu repeats. The promoter has the characteristics typical of a housekeeping gene. It is GC-rich and has multiple start sites of transcription. A 574 base pair fragment immediately upstream of the translation initiation site contains promoter activity when transfected into eukaryotic cell lines. Deletion analysis was performed to study promoter function. These studies showed that only 150 base pairs of promoter sequence were necessary for promoter function. This region contains three potential binding sites for the transcription factor, Sp1 and a TC box sequence. Furthermore, the fragment functions equally well in either orientation. We have defined an element in this region with enhancer function for both its homologous and a heterologous promoter. In addition, this region seems to contribute some degree of tissue specificity to insulin receptor gene expression.

Base Sequence↗
PubMed1997

Functional analysis of a 38 kilobase region on chromosome XVI in Saccharomyces cerevisiae.

In this report we assess the functional importance of 16 open reading frames (ORFs) contained within a 38 780 base-pair region immediately adjacent to the centromere on the right arm of chromosome XVI in Saccharomyces cerevisiae. This analysis involved replacing one copy of each ORF in a diploid strain with a cassette encoding the green fluorescent protein from the jellyfish Aequorea victoria and HIS3. Each replacement cassette was generated by PCR using oligonucleotide pairs with 45-base extensions complementary to sequences immediately upstream and downstream of the target gene's coding region. After replacement of the targeted genes, each gene-replacement strain was subjected to a series of genetic and phenotypic tests to assess the functional importance of the deleted gene. This analysis showed that two ORFs were essential, one for spores to germinate and another for vegetative growth. A third gene encoded a copper-fist-like transcription factor that was required for proper bud-site selection. One of the 16 ORFs was duplicated, a situation not observed in the strain used to sequence the yeast genome (S288C). RNA analysis showed 11 of the 16 ORFs in this region expressed steady-state poly(A+) RNA levels that were greater than or equal to 2% of the level expressed from the yeast actin gene, ACT1.

Blotting, Northern↗
PubMed1997

Genetic and functional analysis of neuronatin in mice with maternal or paternal duplication of distal Chr 2.

Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.

Animals↗
PubMed1996

Functional analysis of the c-myb proto-oncogene.

Targeted mutagenesis studies were initiated to determine the normal biological function of the c-myb proto-oncogene. While heterozygous mice are phenotypically indistinguishable from their wild-type littermates, homozygous mutant fetuses die at approximately 15.5 days of gestation apparently due to anemia, which results from an inability to switch from embryonic yolk sac to fetal liver erythropoiesis. Studies are currently being done to determine the extent of hematopoietic abnormalities in the homozygous mutant fetuses. In vitro assays for hematopoietic colony-forming cells have been used to determine the frequency of both erythroid and myeloid progenitors in the fetal livers of wild-type, heterozygous, and homozygous mutant c-myb fetuses. The reduced number of erythroid progenitors was not unexpected considering the mutant fetus's pale color and reduced hematocrit. The dramatically reduced number of colonies derived from myeloid progenitors in the mutant fetuses in comparison to the number detected in phenotypically normal littermates suggests that expression of the c-myb proto-oncogene is critical for the proliferation and/or differentiation of early hematopoietic progenitors and possibly hematopoietic stem cells. Other possible explanations would include a hematopoietic progenitor migration problem from the yolk sac to the fetal liver or a defect in the microenvironment of the liver. Whether the lymphoid lineage is also adversely affected by the lack of c-myb expression remains to be determined. RT-PCR and Northern blot analyses were used in an attempt to identify downstream genes which may be directly or indirectly regulated by the Myb gene product. While the levels of expression of several genes involved in erythropoiesis (GATA-1, NF-E2, SCL, and EpoR) were reduced in the livers of homozygous mutant fetuses in comparison to phenotypically normal littermates and one gene, Kit ligand (KL), was expressed at higher levels in the mutant livers, these results must be viewed with caution. The livers of the mutant fetuses have been shown to be hypocellular in comparison to those of phenotypically normal littermates (35). It is possible that the Myb gene product is directly or indirectly modulating the expression of these genes. Conversely, the alteration in expression may be due to the reduced number or absence of specific hematopoietic lineages in the livers of the mutant fetuses. Differential display has also been used to identify putative novel genes that are involved in hematopoiesis. Preliminary studies suggest that this may be a powerful methodology to compare the expression pattern of genes in the fetal liver of wild-type, heterozygous, and homozygous mutant littermates at 14.5 days of gestation. To date nearly 60% of the partial cDNAs subcloned analyzed have been shown to be differentially expressed. More importantly, 75% of the differentially expressed cDNAs that have been sequenced appear to encode novel genes. Whether any of these novel genes are involved in the c-myb transcriptional cascade remains to be determined. Overall, analysis of the c-myb mutant fetuses have provided valuable insight into the biological function of this interesting proto-oncogene. The continued analysis of this resource will undoubtedly provide additional information concerning the role of the c-myb gene in hematopoiesis.

Animals↗
PubMed1991

Basal forebrain cholinergic system: a functional analysis.

This chapter has been organized empirically, focusing on the types of approaches that have been taken to understand BFCS function. This approach reflects the state of our knowledge about the behavioral and psychological functions of the BFCS. Considerable information has been gathered in the very short time that the BFCS has been the object of intense investigation. The results from the neurotoxic lesions and from the HACU studies provide some points of consistency and some puzzling differences. Both approaches to the study of basal forebrain function suggest that the MSA is involved in tasks that require spatial working memory; MSA lesions impaired choice accuracy, and HACU in the HIP was increased after performance. The pattern of results in simpler tasks is more difficult to interpret. In a left-right reference memory discrimination in a T-maze, MSA lesions did not impair acquisition or performance, whereas HACU in the HIP was activated during performance. This pattern of results suggests that although the MSA is engaged during this type of task, its activity is not necessary for normal performance. These, and other comparisons indicate the need for a systematic analysis of task demand (Olton, 1989b). Parametric manipulations of different task demands in a systematic fashion can indicate the extent to which the BFCS is involved in the function associated with each parametric manipulation. Ultimately, of course, the organization of this material should focus on particular psychological functions, rather than the techniques and procedures used to gather the information. Achieving this goal is going to require careful attention to the design of behavioral experiments so that definitive conclusions can be made about the extent to which the BFCS is involved in a given psychological function. A systematic application of task analysis can achieve this goal (Olton, 1986, 1989a, 1989b). For example, BFCS lesions in rats impair choice accuracy in spatial working memory tasks, and performance in these tasks engages the HACU system, at least in the HIP. If the spatial functions of this task involve the BFCS, then a nonspatial version of the task should produce a different pattern of results. If the spatial nature of the task is unimportant for BFCS function, then a nonspatial version of the task should produce the same results. By systematically changing one characteristic of the task at a time, the contribution of each component can be assessed.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed2004

Structure-function analysis of the BEACH protein LvsA.

Most eukaryotes have several members of the BEACH family of proteins but the molecular function of these large proteins remains unknown. The Dictyostelium BEACH protein LvsA is essential for cytokinesis and contractile vacuole activity. The functional contribution of different portions of LvsA was tested here by deletion analysis. The C-terminal WD domain was important for protein stability and C-terminal deletions resulted in loss of LvsA function. In contrast, N-terminal deletions yielded abundant protein expression that could be assayed for function. Despite very low sequence conservation of the N-terminal portion of LvsA, this region is important for its function in vivo. Deletion of 689 N-terminal amino acids produced a protein that was functional in cytokinesis but partially functional in osmoregulation. Further deletions resulted in the complete loss of LvsA function. Using in vitro fractionation assays we found that LvsA sedimented with membranes but that this association does not require the N-terminal portion of LvsA. Interestingly, the association of LvsA with the contractile vacuole was perturbed by the loss of drainin, a protein important for vacuole function. In drainin-null cells, LvsA bound irreversibly to engorged contractile vacuoles that fail to expel water. These experiments help delineate the biochemical and physiological requirements for function of one important BEACH protein, LvsA.

Animals↗
PubMed2003

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells.

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.

Amino Acid Sequence↗
PubMed2004

Functional analysis of each blue light receptor, cry1, cry2, phot1, and phot2, by using combinatorial multiple mutants in Arabidopsis.

Blue light receptors in Arabidopsis include two types of proteins, cryptochromes and phototropins. Previous studies have suggested that the cryptochromes cry1 and cry2 function mainly in photomorphogenic responses and that the phototropins phot1 and phot2 mainly regulate photo-induced movements. Receptors in the same family have redundant functions, although their responses to the fluence rate of blue light differ. To uncover functions of blue light receptors that may be concealed by their functional redundancy, we conducted analyses of combinatorial multiple mutants of blue light receptors. Comparison of the responses of the quadruple mutant cry1 cry2 phot1 phot2 to blue light with those of related triple mutants revealed that cryptochromes function in blue light-dependent, random hypocotyl-bending and that phototropins function in one photomorphogenic response, cotyledon expansion. Microarray analysis suggested that cry1 and cry2 independently function as key regulators of early blue light-induced genes, whereas phot1 and phot2 play subsidiary roles in transcriptional regulation by blue light.

Arabidopsis↗
PubMed1998

Structure-function analysis of Lymantria testis ecdysiotropin: a search for the active core.

A structure-function study was performed on the synthetic 21 residue neuropeptide, Lymantria testis ecdysiotropin (LTE), originally isolated from brains of Lymantria dispar pupae. The peptide induces ecdysteroid synthesis by testis sheaths of various lepidopteran species. LTE, as well as synthetic LTE 1-11, 11-21, and 11-15, stimulated synthesis in larval and pupal testes of Lymantria dispar at concentrations of 10(-9) to 10(-15) M; LTE 16-21 was weakly active, and an elongated LEU-LTE was inhibitory to synthesis at all but extremely low concentrations (10(-15) M). Since the sequence and polarity of residues in LTE 1-11, 11-15, and 11-21 are quite different, several parts of the molecule must activate receptors which initiate the cascade, resulting in ecdysiogenesis in Lepidopteran testes.

Amino Acid Sequence↗
PubMed2004

Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe.

Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1alpha-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg(2+) is not required for GDP binding to eRF3c. Mg(2+) at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.

Amino Acid Motifs↗
PubMed1990

Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector.

Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker. Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.

Chromosome Deletion↗