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Messenger RNA in dormant cells of Sterkiella histriomuscorum (Oxytrichiade): indentification of putative regulatory gene transcripts.

In the absence of food, the oxytrichid Sterkiella histriomuscorum, like many ciliates, enters into dormancy and transforms into a round and walled encysted cell. When transferred back into a feeding medium, the cyst re-transforms into a vegetative cell in a few hours. This encystment-excystment pathway, which is common to many free-living and parasitic protists, is still poorly understood at the molecular level. In order to identify potential dormant transcripts in the cysts of Sterkiella, we have constructed cDNA libraries from mature cysts. Transcripts have been isolated confirming the presence of a mRNA pool in the dormant cells. The sequence analysis of two cDNA indicates open reading frames which show significant similarities to known proteins involved in mechanisms of regulation: 1) nifR3, an element of the nitrogen regulatory system in bacteria and 2) CROC-1, a newly identified human transcription factor. The two corresponding macronuclear genes represent the first putative regulatory genes isolated in ciliates. From a differential screening of the cDNA library against vegetative cDNA, one cyst-specific (and very abundant) transcript has been isolated but the product has not yet been identified. The possible involvment of these new ciliate genes in the excystment process is discussed.

Amino Acid Sequence↗

Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants.

Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2-fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.

Journal Article↗

Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation.

Changes in the relative sizes of pools of transcripts of organelle genes during plastid development are common in flowering plants, but technical difficulties have prevented direct determinations of the effects of changes in rates of transcription and degradation on such fluctuations. It has been possible to follow both rates in Chlamydomonas reinhardtii. In synchronous or asynchronous cultures of cells grown in 12 h light/12 h dark periods, sizes of pools of transcripts of the chloroplast genes atpA, atpB, tufA, and psaB fluctuate. Differences in chloroplast transcript abundances in light/dark cycles were found to be cell cycle-independent but controlled by either an endogenous rhythm (atpA, atpB, and tufA) or by light (psaB). In vivo labeling experiments showed that transcriptional regulation and light/dark-regulated degradation both contribute, in gene-specific manners, to the level of transcripts of individual C. reinhardtii chloroplast genes in cells grown in alternating light/dark cycles.

Animals↗

The wheat peptidyl prolyl cis-trans-isomerase FKBP77 is heat induced and developmentally regulated.

We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37 degreesC than at 25 degreesC. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37 degreesC, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25 degreesC after recovery of the embryos for 90 min at 25 degreesC. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts.

Amino Acid Sequence↗

Aedes aegypti phosphohexomutases and uridine diphosphate-hexose pyrophosphorylases: comparison of primary sequences, substrate specificities and temporal transcription.

Phosphohexomutases reversibly catalyse the transfer of the phosphate group of a glycosyl phosphate between the C6 and C1 positions, and uridine diphosphate (UDP)-hexose pyrophosphorylases catalyse the synthesis of UDP-hexose from uridine triphosphate (UTP) and hexose-1-phosphate. Both enzyme families are essential for nucleoside diphosphate hexose biosynthesis and are therefore critical for various physiological functions in the midgut of mosquitoes after a blood meal. We cloned and sequenced three phosphohexomutase and two UDP-hexose pyrophosphorylase cDNAs from Aedes aegypti. The products of the cDNAs were expressed and substrate specificities were examined. Herein we describe Ae. aegypti phosphoglucomutase 1, phosphoglucomutase 2, phosphoacetylglucosamine mutase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase. Transcripts of the genes expressing the enzymes are constitutively present in all life stages and blood-feeding does not seem to influence transcript abundance.

Aedes↗

Molecular biology of P2Y purinoceptors: expression in rat heart.

1. Application of molecular biology to the study of P2Y purinoceptors has led to the identification of seven such receptors. Here we briefly review their properties and investigate qualitatively the expression of four rat receptor transcripts in heart. 2. The reverse transcriptase-polymerase chain reaction was used to ascertain whether the rat P2Y1, P2Y2, P2Y4 and P2Y6 receptor transcripts were expressed in whole heart, neonatal cardiac fibroblasts, neonatal cardiac myocytes and adult cardiac myocytes. 3. All receptor sequences could be amplified from neonatal rat whole heart, with P2Y6 appearing the most abundant transcript of the four. P2Y1 is expressed at higher levels in comparison to P2Y2, P2Y4 and P2Y6 in the neonatal myocyte. In the adult myocyte P2Y1, P2Y2 and P2Y6 could be amplified but P2Y4 could not be detected. In the neonatal fibroblast, P2Y1 and P2Y6 appear to be expressed at higher levels than P2Y2 and P2Y4. 4. In summary, it is concluded that multiple P2Y receptor subtypes are expressed in heart and that the expression in myocytes changes from the neonate to the adult.

Animals↗

A molecular analysis of mouse development from 8 to 10 days post coitum detects changes only in embryonic globin expression.

The pattern of protein synthesis in 8-, 9- and 10-day post coitum (p.c.) mouse embryos was examined by 2-D gel electrophoresis of [35S]methionine-labelled proteins. Of the 600-800 polypeptides detected only one, a 14 X 10(3) Mr (14K) protein, was found to accumulate over this period. To isolate cDNA clones that potentially encode this protein, 32P-labelled cDNA was synthesized from 9 and 10 days p.c. embryo poly(A) +RNA, and used for the differential screening of an 8.5-day p.c. mouse embryo cDNA library cloned in lambda gt10. Six clones that hybridized strongly to the 10-day probe were purified and their inserts subcloned into plasmid vectors. Cross hybridization and restriction mapping of these inserts indicate that they fall into four distinct groups. Each of these hybridize with transcripts of approximately 600 nucleotides, which accumulate in the embryo from 9 to 10 days p.c. Expression was barely detectable in adult tissues and restricted to liver and spleen. Expression of one of these clones, 10.1, was examined by in situ hybridization of 35S-labelled RNA probes to 8.5-12.5 day p.c. embryo sections. Strong hybridization was observed in yolk sac blood islands, fetal liver and embryonic erythrocytes, suggesting that 10.1A encodes an erythrocyte-specific protein. DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the alpha 1, zeta, beta h1 and epsilon globin genes. Labelling of 10-day p.c. erythrocyte proteins with [35S]methionine, followed by 2-D gel electrophoresis, clearly demonstrates that the most abundant polypeptide migrates to the same position as the 14K protein which accumulates from 8 to 10 days p.c. Thus the only abundant transcripts and corresponding proteins that change over a period of profound morphogenetic change correspond to globins of the newly established blood system.

Amino Acid Sequence↗

The SinR·SlrR Heteromer Attenuates Transcription of a Long Operon of Flagellar Genes in Bacillus subtilis.

During growth, Bacillus subtilis differentiates into subpopulations of motile individuals and non-motile chains, associated with dispersal and biofilm formation, respectively. The two cell types are dictated by the activity of the alternative sigma factor SigD encoded as the penultimate gene of the 27-kb long fla/che flagellar operon. The frequency of SigD-ON motile cells is increased by the heteromeric transcription factor SwrA·DegU that activates the fla/che promoter. Conversely, the frequency of motile cells is decreased by the heteromeric transcription factor SinR·SlrR, but the mechanism and location of inhibition is poorly understood. Here, using ChIP-Seq analysis, we determine the binding sites of the SinR·SlrR heteromer on the genome. We identified two sites within the fla/che operon that were necessary and sufficient to attenuate transcript abundance by causing premature termination upstream of the gene that encodes SigD. Thus, cell motility and the transition to biofilm formation depend on the expression of a long operon governed by two opposing heteromeric transcription factors that operate at two different stages of the transcription cycle. More broadly, our study serves as a model for transcription factors that control transcriptional elongation and the regulation of long operons in bacteria.

Bacillus subtilis↗

Duplication of growth hormone receptor (GHR) in fish genome: gene organization and transcriptional regulation of GHR type I and II in gilthead sea bream (Sparus aurata).

Nucleotide sequences encoding for functional growth hormone receptors (GHR) are now available in salmonids (coho and masu salmon) and other fish orders. Several authors have hypothesized a divergent evolution of salmonid GHRs, but the recent finding that a trout cDNA is related to non-salmonid (GHR type I) rather than to salmonid GHRs (GHR type II) points out a possible duplication of actively transcribed GHR genes. To address this issue, we search by RT-PCR for GHR type II in trout, gilthead sea bream, European sea bass, and turbot. Both in trout and gilthead sea bream, a cDNA sequence with all the characteristic features of masu and coho salmon GHRs (GHR type II) was found. Similarly, in European sea bass, a cDNA encoding for the intracellular domain of GHR type II was reported. No positive results were found in turbot, but searches in genome databases of fugu and zebrafish identified DNA sequences with a significant similarity to fish GHR type I and II, which are more related each other than to GHRs of tetrapods. Gene organization is, however, highly conserved through the evolution of vertebrates, and eight exons homologous to exons 2 and 4-10 of mammals were found in fish GHRs. Transcriptional regulation of GHR type I and II was also addressed by means of real-time PCR assays in gilthead sea bream. In liver and adipose tissue, GHR type I was the most abundant transcript, but the expression of GHR type I and II was practically equal in skeletal muscle. Both in liver and adipose tissue, the two GHRs were down-regulated by fasting. In skeletal muscle, the expression of GHR type I remained unaltered in fasted fish, whereas a 2- to 3-fold increase was found for GHR type II. All this provides suitable evidence for a tissue-specific regulation of fish GHRs, but further research is needed to determine whether these duplicated genes are evolved in a new or redundant fashion.

Amino Acid Sequence↗

Differential expression profiles and gene structure of two tumor necrosis factor-alpha variants in Atlantic salmon (Salmo salar L.).

Here we present the gene structure and expression data on two Atlantic salmon TNF-alpha genes. Both genes are approximately 2.0kb in length and organized into four exons and three introns. The open reading frame of both genes translates into 246 amino acid putative peptides, being 91.5% identical at the amino acid level. The upstream regulatory region of both genes were amplified by inverse PCR and analyzed for putative regulatory binding sites. Variant specific gene-expression both in vitro and in vivo revealed that the two variants are differentially regulated. TNF-alpha2 is the dominant variant throughout the experimental period following LPS stimulation of TO-cells. The picture was different in head kidney tissue following vaccination of Atlantic salmon with an experimental multivalent oil-based vaccine. TNF-alpha2 was the most dominant transcript in the non-stimulated controls and in the early phase. A shift from TNF-alpha2 to TNF-alpha1 was however seen, and from day 12 the TNF-alpha1 was most abundant transcript. Implications of variant specific gene-expression are discussed.

Amino Acid Sequence↗

Genetics and genomics of Drosophila mating behavior.

The first steps of animal speciation are thought to be the development of sexual isolating mechanisms. In contrast to recent progress in understanding the genetic basis of postzygotic isolating mechanisms, little is known about the genetic architecture of sexual isolation. Here, we have subjected Drosophila melanogaster to 29 generations of replicated divergent artificial selection for mating speed. The phenotypic response to selection was highly asymmetrical in the direction of reduced mating speed, with estimates of realized heritability averaging 7%. The selection response was largely attributable to a reduction in female receptivity. We assessed the whole genome transcriptional response to selection for mating speed using Affymetrix GeneChips and a rigorous statistical analysis. Remarkably, >3,700 probe sets (21% of the array elements) exhibited a divergence in message levels between the Fast and Slow replicate lines. Genes with altered transcriptional abundance in response to selection fell into many different biological process and molecular function Gene Ontology categories, indicating substantial pleiotropy for this complex behavior. Future functional studies are necessary to test the extent to which transcript profiling of divergent selection lines accurately predicts genes that directly affect the selected trait.

Animals↗

Heat shock interferes with steroidogenesis by reducing transcription of the steroidogenic acute regulatory protein gene.

A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.

Animals↗

Circadian regulation of rice (Oryza sativa L.) CONSTANS-like gene transcripts.

We identified three rice cDNA clones showing amino acid similarity to the Arabidopsis CONSTANS-like proteins from a database search (S12569, S3574, and C60910), to examine if their transcript abundances were under circadian control. Unlike the other two proteins, the protein encoded by the S12569 cDNA contains only one CONSTANS-like zinc finger B box, and a CCT region. We found that the transcript levels of these rice CONSTANS-like (COL) genes were under circadian control. The oscillation phase of the S12569 gene transcript was more or less opposite to those of OsGI (rice GIGANTEA homolog) and Hd1 (rice COSTANS homolog), whereas the phases of the other two gene transcripts were similar to that of the Hd1 transcript. S12569 mRNA started to increase about 3 h after the onset of the dark period, with a peak about 3 h after its end. The S3574 and C60910 genes were expressed to similar extents during the vegetative and reproductive phases, like OsGI. Higher levels of S12569 transcripts, however, like those of Hd1, were detected in the earlier stages of panicle development. Unlike Hd1 transcripts, S12569, S3574, and C60910 transcripts were present at similar levels in the aerial parts of plants and in their roots during the vegetative phase. In conclusion, the rice COL genes showed distinctive expression patterns from the CO and COL genes, as well as Hd1, a rice CO homolog.

Amino Acid Sequence↗

Regulation of senescence-related gene expression in carnation flower petals by ethylene.

Ethylene plays a regulatory role in carnation (Dianthus caryophyllus L.) flower senescence. Petal senescence coincides with a burst of ethylene production, is induced prematurely in response to exogenous ethylene, and is delayed by inhibitors of ethylene biosynthesis or action. We have investigated the role of ethylene in the regulation of three senescence-related cDNA clones isolated from a senescent carnation petal library (KA Lawton et al. [1989] Plant Physiol 90: 690-696). Expression of two of the cloned mRNAs in response to ethylene is floral specific, while the expression of another mRNA can be induced in both leaves and flowers exposed to ethylene. Although ethylene induces expression of these mRNAs in petals, message abundance decreases when flowers are removed from ethylene unless an autoenhancement of ethylene production is induced. This indicates continued perception of ethylene is required for their expression. Interruption of ethylene action following the onset of natural senescence results in a substantial decrease in transcript abundance of two of these mRNAs. However, the abundance of another mRNA remains unaffected, indicating this gene responds to temporal cues as well as to ethylene. As flowers age the dosage of exogenous ethylene required to induce expression of the cloned mRNAs decreases, indicating sensitivity to ethylene changes as the tissue matures. Nuclear run-on transcription experiments indicate that relative transcription rates of cloned mRNAs increase in response to exogenous ethylene.

Journal Article↗

Juvenile hormone regulates de novo isoprenoid aggregation pheromone biosynthesis in pine bark beetles, Ips spp., through transcriptional control of HMG-CoA reductase.

Evidence is presented for transcriptional regulation of de novo pheromone biosynthesis in Ips spp. bark beetles, but the comparative biochemical and molecular approach reveals a dichotomy between species in the pini and grandicollis subgeneric groups. Radiotracer studies with 14C-acetate demonstrate that feeding on host phloem stimulates biosynthesis in males of three Ips spp. However, treatment with juvenile hormone III (JH III) stimulates biosynthesis only in Ips pini. Thus, two species in the grandicollis subgeneric group (I. grandicollis and I. paraconfusus) appear to have a different mode of regulation related to JH III than does I. pini. Between 16 and 20 hr after feeding has commenced, pheromone production, as measured by accumulation in abdominal tissue, is stimulated about 150- (I. pini) and 350-times (I. paraconfusus) above the control level of 1-10 ng/male measured at 0 hr. Treatment with JH III results in accumulation in I. pini that is 3-4 times more than in phloem-fed males, whereas the identical treatment results in only weak accumulation in I. paraconfusus (45-times less than phloem-fed males). Comparative studies of gene expression and enzyme activity related to biosynthesis also support different modes of JH III-related regulation in I. pini and I. paraconfusus. In males of both species, feeding on host phloem results in increased transcript abundance and increased activity for the key de novo isoprenoid pathway enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). However, while JH III treatment results in comparable maximal increases in HMG-R transcript levels in both species (similar to feeding), the activity of HMG-R in crude extracts from JH III-treated male I. paraconfusus is low in comparison with male I. pini. Hypothetical explanations for the interspecific dichotomy in the regulation of pheromone biosynthesis include a second hormone or factor in grandicollis group species that functions either alone or with JH III; in both cases acting after HMG-R has been transcribed.

Animals↗

Ex vivo analysis of aberrant splicing induced by two donor site mutations in PKLR of a patient with severe pyruvate kinase deficiency.

Two single-nucleotide substitutions in PKLR constituted the molecular basis underlying pyruvate kinase (PK) deficiency in a patient with severe haemolytic anaemia. One novel mutation, IVS5+1G>A, abolished the intron 5 donor splice site. The other mutation, c.1436G>A, altered the intron 10 donor splice site consensus sequence and, moreover, encoded an R479H substitution. We studied the effects on PKLR pre-mRNA processing, using ex vivo-produced nucleated erythroid cells from the patient. Abolition of the intron 5 splice site initiated two events in the majority of transcripts: skipping of exon 5 or, surprisingly, simultaneous skipping of exon 5 and 6 (Delta5,6). Subcellular localization of transcripts suggested that no functional protein was produced by the IVS5+1A allele. The unusual Delta5,6 transcript suggests that efficient inclusion of exon 6 in wild-type PKLR mRNA depends on the presence of splice-enhancing elements in exon 5. The c.1436G>A mutation caused skipping of exon 10 but was mainly associated with a severe reduction in transcripts although these were, in general, normally processed. Accordingly, low amounts of PK were detected in nucleated erythroid cells of the patient, thus correlating with the patient's PK-deficient phenotype. Finally, several low-abundant transcripts were detected that represent the first examples of "leaky-splicing" in PKLR.

Adult↗

Differential modulation of E. coli mRNA abundance by inhibitory proteins that alter the composition of the degradosome.

In Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitor-specific changes in degradosome composition--and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.

Endoribonucleases↗

Effect of an anti-HLA class I monoclonal antibody on the antigenic and transcriptional expression of HLA class I genes in U937 cells.

The phenomenon of antigenic modulation was studied in the histiocytic lymphoma line U937. A redistribution of cell surface HLA antigen after incubation of U937 cells with the monomorphic anti-HLA class I monoclonal antibody W6/32 was demonstrated by immunofluorescence analysis. As assessed by hybridization of RNA obtained from W6/32-treated U937 cells with a probe corresponding to the alpha 3 domain of HLA Cw3, prolonged W6/32 incubation (24 to 72 hours) induced a decrease in HLA class I transcript abundance. This decrease was about 25% as compared with untreated control cells. These data indicate that W6/32 incubation can induce changes in HLA class I gene expression not only at the antigenic but also at the transcriptional level. Possible implications for the molecular basis of antigenic modulation are discussed.

Antibodies, Monoclonal↗