FBP1 Expression Research Articles

Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex.

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family.

Molecular characterization has been accomplished for five members of the peroxidase gene family in French bean. The most important of these, designated FBPI, corresponds to the isoform believed to be responsible for the apoplastic oxidative burst demonstrated by suspension-cultured cells in response to fungal elicitor. Identification was made by a complete match of six peptide sequences derived from the native protein to the translated sequence of the cDNA. Modelling of the surface structure in comparison with two other members of the peroxidase family did not reveal any unusual features which might account for its role in the oxidative burst. However, FBP1 when expressed in Pichia pastoris generated H2O2 using cysteine at pH 7.2, a specific property of the native protein when isolated from suspension-cultured cells. FBP1, together with other members of the family, were all induced in cell cultures by elicitor action although they all showed some expression in non-induced cultured cells. They were also expressed in all tissues examined with varying levels of intensity of detection in northern blots. This was confirmed by in situ hybridization and FBP1 expression was confirmed in tissues where it has been previously detected by immunolocalization methods. Assigning roles to individual peroxidases is an important goal and molecular identification of the oxidative burst peroxidase allows further exploration of the relative roles of the different systems involved in generating reactive oxygen species.

Ecdysone-regulation of synthesis and processing of fat body protein 1, the larval serum protein receptor of Drosophila melanogaster.

At the end of the third larval instar of Drosophila melanogaster, larval serum proteins 1 and 2 (LSP-1 and -2) are taken up by cells of the fat body. Here, we show that the product of the ecdysteroid-inducible gene Fbp-1 (Fat Body Protein 1) is the receptor that binds LSP-1. Transcription and translation of Fbp-1 is stage-specifically restricted to the end of the third larval instar, starting around 99 h after egg laying. Expression of Fbp-1 is induced by a low level of 20-hydroxy-ecdysone (>/= 10-7 m). After translation, the FBP-1 protein is thought to be proteolytically cleaved in three subsequent steps. The final cleavage step is delayed by 6 h and relies on a higher concentration of ecdysone (>/= 10-5 m). Therefore, 20-hydroxy-ecdysone regulates Fbp-1 expression and function at two different levels. To the best of our knowledge, this study is the first to date to demonstrate two distinct functions for different concentrations of a steroid hormone on a single biochemical process.

Expression Patterns of Folate Binding Proteins One and Two in the Developing Mouse Embryo

Expression patterns of mRNAs coding for the murine folate binding proteins one and two (FBP1 and FBP2) were determined by ribonuclease protection assay (RPA) in highly inbred SWV/Fnn mouse embryos. Tissue samples for RPA were collected from the anterior neural tube throughout the period of embryonic development, as well as from maternal- and fetal-derived term placenta. The peak in expression of FBP1 occurred in term placental tissue compared to neural tissue from any time point. This relative increase in FBP1 expression occurred in placental tissue of embryonic, as opposed to maternal, origin. The expression of FBP2 did not differ statistically between any timepoints or tissues examined. Expression of both FBP1 and FBP2 was slightly elevated throughout the period of neural tube closure (Gestational Days 8 through 10), although not significantly. These data fit the anticipated expression patterns of the homologues of human folate receptors alpha and beta, thus helping to resolve some of the confusion secondary to the nomenclature associated with this gene family. Furthermore, the expression of these two genes in the neural tube closure stage of embryological development supports their involvement in regulatory events related to normal neural tube morphogenesis.

The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast.

The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family. Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock. It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants. We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation. Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants. In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase. We show here that wis1 function is required for mei2 induction following nitrogen starvation. Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function. Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function. These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets. The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1 delta genetic backgrounds.

Schizosaccharomyces pombe pcr1+ Encodes a CREB/ATF Protein Involved in Regulation of Gene Expression for Sexual Development

The Schizosaccharomyces pombe pcr1 gene encodes a bZIP protein that apparently belongs to the cyclic AMP response element (CRE)-binding protein/activating transcription factor family. The deduced pcr1 gene product consists of 171 amino acid residues and is most similar to the mammalian CRE-BP1. A glutathione S-transferase-Pcr1 fusion protein produced in Escherichia coli was able to bind specifically to the CRE motif in vitro. Analysis with anti-Pcr1 serum suggested that Pcr1 is included in the major CRE-binding factors present in the S. pombe cell extract. Disruption of the pcr1 gene was not lethal, but the disruptant showed cold-sensitive growth on rich medium. The disruptant was also inefficient in mating and sporulation, though it was not completely sterile. Expression of the ste11 gene, which encodes a key transcription factor for sexual development, was greatly reduced in the disruptant, and overexpression of ste11+ suppressed the deficiency of the pcr1 disruptant in sexual development. It has been shown that expression of ste11 is negatively regulated by cyclic AMP-dependent protein kinase (PKA) and that the loss of PKA activity results in ectopic sexual development. Disruption of pcr1 blocked ectopic sexual development. Furthermore, disruption of pcr1 reduced expression of fbp1, a glucose-repressible gene negatively regulated by PKA. These results suggest that Pcr1 is a putative transcriptional regulator whose activity may be controlled by PKA. Alternatively, its activity may be independent of PKA, and full induction of ste11 and fbp1 expression requires the function of Pcr1 in addition to elimination of the repression by PKA.

Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibit binding of [ 3H]jfolic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6 S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6 R) diastereoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

Petal and stamen formation in petunia is regulated by the homeotic gene fbp1

For Arabidopsis and Antirrhinum, the so-called ABC model has been developed, which postulates that the determination of floral organ primordia is controlled by the action of three classes of homeotic genes. A number of these ABC genes encode putative transcription factors with the MADS box DNA binding motif. This paper reports on the functional analysis of the petunia MADS box gene fbp1. The temporal and spatial expression of fbp1 has been investigated in detail in transgenic plants containing the beta-glucuronidase (GUS) reporter gene fused to an fbp1 promoter fragment. fbp1-driven GUS activity was specifically detected in emerging petal and stamen primordia, suggesting a function of fbp1 in the control of second and third floral whorl identity. To test this hypothesis, transgenic petunia plants were generated in which fbp1 expression was inhibited by a co-suppression approach. The flowers of such plants exhibited homeotic conversions of petals towards sepals and stamens towards carpels. Occasionally, the third whorl carpels are fused forming a pentalocular gynoecium. This dominant fbp1 mutation acted as a single Mendelian trait in genetic crosses. These results strongly indicate that fbp1 is a petunia class B homeotic gene which is required for the correct initiation and determination of petals and stamens.

Insertion of an intracisternal A particle within the 5'-regulatory region of a gene encoding folate-binding protein in L1210 leukemia cells in response to low folate selection. Association with increased protein expression.

We have previously described a subline of L1210 murine leukemia cells (LL1) selected in a low level of 5-formyltetrahydrofolate which overexpresses a membrane-bound folate-binding protein (FBP1) and exhibits a rearrangement at the locus encoding this protein. Genomic clones containing the entire FBP1-encoding DNA from both L1210 and LL1 were isolated and characterized. Sequence analysis indicates that, with exception of the 5'-region, the FBP1-encoding locus in both cell lines is identical. The rearrangement in LL1 results from the insertion of an intracisternal A particle (IAP) in the head-to-head (antisense) orientation 72 base pairs (bp) upstream of the FBP1 ATG start codon. The IAP likely provides an alternative promoter for FBP1 expression which may produce a novel transcript with enhanced stability. Presence of the IAP appears to inactivate or relocate normal cis-acting regulatory sequences as expression of the FBP1 transcript in LL1 is not regulated by the folate status of the cell.

Identification of UAS elements and binding proteins necessary for derepression of Saccharomyces cerevisiae fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase is a key enzyme in gluconeogenesis and the FBP1 gene is not transcribed during growth with glucose. Genetic analysis indicated a positive regulation of FBP1 expression after exhaustion of glucose. By linker-deletion analysis, two upstream activation sites (UAS1 and UAS2) were localized and the respective UAS-binding factors (DAP I and DAP II for derepression activating protein) were identified by gel retardation. UAS1 and UAS2 span about 30 bp each, and are separated by approximately 30 bp. Both UAS sites act synergistically. Although UAS1 showed some similarities to the DNA-binding consensus for the general yeast activator Rap1, competition experiments and DEAE-chromatography proved that DAP I and Rap1 correspond to different proteins. Gel retardation by DAP I depended on carbon sources and did not occur in cells growing logarithmically with glucose, whereas a strong retardation signal was obtained with ethanol-grown cells. The present results suggest that DAP I and DAP II are the final regulatory elements for glucose derepression.

Differential expression of two MADS box genes in wild-type and mutant petunia flowers.

We isolated and characterized two flower-specific genes from petunia. The protein products of these genes, designated floral binding protein 1 (FBP1) and 2 (FBP2), are putative transcription factors with the MADS box DNA binding domain. RNA gel blot analysis showed that the fbp1 gene is exclusively expressed in petals and stamen of petunia flowers. In contrast, the FBP1 protein was only detectable in petals and not in stamens, suggesting post-transcriptional regulation of the fbp1 gene in these tissues. The fbp2 gene is expressed in petals, stamen, carpels, and at a very low level in sepals but not in vegetative tissues. We analyzed the spatial expression of these fbp genes in floral organs of two homeotic flower mutants. In the blind mutant, whose flower limbs are transformed into antheroid structures on top of normal tubes, identical expression levels of both genes were observed in the antheroid structures as in normal anthers. In the homeotic mutant green petals, the petals are replaced by sepaloid organs in which the expression of fbp1 is strongly reduced but not completely abolished. Our results suggest a regulation of the fbp1 gene expression by the green petals (gp) gene. Expression of the fbp2 gene was not affected in the green petals mutant. In contrast to the proposed models describing floral morphogenesis, our data indicated that homeotic genes can be functional in one whorl only.