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A global analysis of developmentally regulated genes in Myxococcus xanthus.

Tn5 lac is a transposon that fuses the transcription of lacZ to exogenous promoters. We generated 2374 Tn5 lac insertion-containing strains of Myxococcus xanthus, a soil bacterium that undergoes multicellular development which culminates in the formation of spores. Thirty-six strains were identified that specifically increase beta-galactosidase expression at some particular time during development and these expression times range from minutes after starvation initiates development to 24 hr, when sporulation begins. Different maximum levels of beta-galactosidase expression were also observed and the maximum for many strains that begin beta-galactosidase expression late in development was observed only if spores were disrupted. Seven of the 36 strains display mild to severe defects in aggregation and/or sporulation, as did an additional five strains whose beta-galactosidase expression was not developmentally regulated. Restriction maps of the DNA adjacent to the Tn5 lac insertions that are developmentally regulated and/or cause developmental defects show that most of the 41 insertions are in different regions of the Myxococcus genome. The developmentally regulated Tn5 lac insertions described here provide a set of at least 29 new developmental markers for Myxococcus.

Bacterial Proteins↗

Characterization of a novel developmentally regulated gene from Trypanosoma brucei encoding a potential phosphoprotein.

We have isolated a cDNA clone corresponding to a single-copy nuclear gene that is upregulated at the mRNA level during in vitro differentiation of bloodstream trypomastigotes of strains of both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense to procyclic forms. Transcript levels begin to increase within minutes of introduction of bloodstream forms into culture and peak well before cultures exhibit a procyclic morphology. This increase in transcript levels was found to occur both in the absence of protein synthesis and in a nontransforming strain blocked very early in the developmental program, both conditions under which accumulation of procyclic acidic repetitive protein (PARP) transcripts did not occur in control experiments. DNA sequence analysis reveals an open reading frame sufficient to encode a protein of approximately 50 kDa within the cDNA, but data base searches for homology at either the amino acid or nucleotide level revealed no related sequences. A high density of kinase consensus target sites in the deduced amino acid sequence suggests that the gene product may be a phosphoprotein.

Amino Acid Sequence↗

Expression of beta-amyloid precursor protein gene is developmentally regulated in human muscle fibers in vivo and in vitro.

Regenerating muscle fibers in 25 human muscle biopsies, obtained from patients with a variety of neuromuscular diseases, manifested increased mRNA for the beta-amyloid precursor protein (beta APP) that contains the Kunitz-type protease inhibitor (KPI) motif, whereas adult human muscle fibers were negative in their vast non-extrajunctional region. Aneurally cultured normal human muscle fibers also expressed strong KPI-beta APP mRNA signal, which became significantly down-regulated during muscle differentiation. Our study demonstrates that in human muscle KPI-beta APP mRNA is developmentally regulated, and it suggests that beta APP may play a role in human muscle development.

Adolescent↗

Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA.

This study investigated the effects of a semi-defined culture system on the temporal pattern of expression of RNA from genes involved in compaction and cavitation: gap junction protein connexin43 (Cx43), desmosomal glycoproteins desmoglein 1 (Dg 1), desmocollins I, II and III (Dc I, Dc II, Dc III), desmosomal protein plakophilin (Plako); metabolism glucosetransporter-1 (Glut-1); RNA processing poly(A)polymerase (PolyA); heat shock protein 70.1 (HSP); and trophoblastic function trophoblast protein (TP) in bovine oocytes and embryos generated in vitro using TCM199 supplemented with BSA as the culture medium. Morulae and blastocysts derived in vivo were collected from superovulated heifers and also used for this study. Poly(A)+ RNA was extracted from pools of 20-50 oocytes or embryos, analysed by reverse transcription-polymerase chain reaction and the amplified fragments were verified by sequencing. Assays were repeated at least three times for each developmental stage and provided consistent results in all replicates. In bovine embryos produced in vitro, mRNA encoding Cx43 was detectable up to the morula stage, whereas blastocysts and hatched blastocysts did not express this gene. No transcripts were found for Dg 1 and Dc I throughout the tested preimplantation stages. Dc II and Dc III transcripts were found from 2-4-cell embryos up to the hatched blastocyst stage. mRNA encoding Plako was detected in immature and mature oocytes and zygotes, while no transcripts were seen in 2-4-cell and 8-16-cell embryos. The gene was expressed again from the morulae to the hatched blastocyst stage. Oocytes and bovine embryos produced in vitro showed transcripts for Glut-1, PolyA and HSP throughout preimplantation development up to the hatched blastocyst stage. The gene encoding TP was transcribed only in blastocysts and hatched blastocysts. Morulae and blastocysts produced in vivo showed the same expression as their in vitro counterparts, with one exception: the in vivo embryos transcribed Cx43. The results of this study reveal for the first time the transcriptional pattern of a set of 'marker' genes involved in various processes in early bovine embryonic development. Transferable morulae and blastocysts produced in vitro expressed most genes similar to their in vivo counterparts. These data contribute to the molecular characterization of this widely used in vitro culture system for bovine embryos and provide a major advance towards production of 'physiologically normal' embryos.

Animals↗

All-trans-retinoic acid upregulates the expression of COUP-TFI in early-somite mouse embryos cultured in vitro.

Exposure of embryos to an excess of retinoic acid (RA) modifies the spatio-temporal pattern of expression of developmental genes. RA regulates the expression of target genes through binding of the retinoid nuclear receptors (RARs and RXRs), as heterodimers, to regulatory cis-acting elements. COUP-TF factors, which are able to dimerize with the RXRs and to compete with the retinoid receptors for their DNA binding sites, are suspected to modulate the retinoid signal transduction pathway. Therefore, COUP-TF factors may be involved in the regulation of the expression of developmental genes and/or in the modifications induced by an excess of RA in the expression of these genes. The aim of this work is to assess whether RA-induced modifications in the expression of Krox-20 and Hox genes correlate with alterations of the expression of COUP-TF genes. In addition to spatial modifications in the expression patterns of Krox-20 and Hox genes, we report here an upregulation of the expression level of COUP-TFI after RA exposure. However, this abnormality did not spatially overlap with the modifications observed in the expression of Krox-20 and Hox genes. These data suggest an involvement of COUP-TFI in the generation of RA-induced abnormalities, but do not support the hypothesis of an involvement of this factor in the regulation of the expression of Hox or Krox-20 genes.

Animals↗

Organization of a gene family developmentally regulated during Dictyostelium discoideum spore germination.

mRNA specific to cDNA clone pLK109 is present in Dictyostelium discoideum spores, increases about two- to threefold at 0.5 to 1 h during spore germination, and then rapidly decreases. The mRNA is not detectable in vegetative cells or in early multicellular development on filters, but is present late during development, approximately at the time of sporulation. 109 mRNA in spores is 700 nucleotides in length but this is processed during germination by shortening of the poly(A) tail to about 600 nucleotides at 1 to 1.5 hours. pLK109 is a member of a multigene family containing three separate genes, and we have isolated and sequenced all of them. All three sequences code for deduced proteins of 127 amino acid residues, with only a few amino acid differences among them. Gene 1 represents the "transcribed" gene, since all 33 cDNAs we isolated are identical with the cDNA pLK109 and the coding region of this gene. Other open reading frames are in close proximity to each of the 109 sequences. About 200 base-pairs 3' to the gene 1 109 sequence is an open reading frame in the opposite orientation. Gene 2 fragment contains a sequence that codes for a protein similar to trypanosome alpha-tubulin 728 base-pairs 5' to the 109 sequence. Gene 3 fragment possesses two additional putative coding regions, one 5' and another 3' to the 109 gene. There is a remarkable similarity between the 5' upstream regions of all three genes. Each possesses a normal Dictyostelium TATA box and the usual T stretch. In addition, there are many other portions of about 400 to 500 base-pairs of the 5' regions that are either identical for long stretches or very similar.

Amino Acid Sequence↗

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.

Amino Acid Sequence↗

Protein L-isoaspartyl methyltransferase: developmentally regulated gene expression and protein localization in the central nervous system of aged rat.

We have cloned a cDNA encoding protein L-isoaspartyl methyltransferase (PIMT) and characterized gene expression in the development, maturation, and the aging process of the central nervous system by RNA blot analysis, western blot analysis, and immunohistochemistry. PIMT transcript was detected in rat embryonic brain and showed a linear up-regulation during the maturation of the brain and maintained its level in aged rat brain. Immunoblot analysis also supported a linear increase in the amount of PIMT in the maturation process of rat brains. An immunohistochemical study showed that PIMT is strongly expressed in neurons and weakly but definitively in glial cells and oligodendrocytes. These immunoreactivities significantly increased in some neurons of the hippocampus, cerebral cortex, and the brain stem of aged rat brain. The present results suggest that the expression of PIMT is associated with the amount of racemized/isomerized proteins accumulated during the developmental and aging process of the central nervous system.

Age Factors↗

Myosin light chain enhancer activates muscle-specific, developmentally regulated gene expression in transgenic mice.

The rat myosin light chain (MLC)1/3 gene locus contains a potent muscle-specific enhancer, located downstream of the coding region, greater than 24 kilobases away from the MLC1 transcription start site. To assess the role of this enhancer in the activation of MLC expression during development, transgenic mice were generated carrying multiple copies of a MLC1 promoter-chloramphenicol acetyltransferase (CAT) transcription unit linked to a genomic fragment including the enhancer. CAT expression was detected in four mouse lines, up to 1000-fold higher in skeletal muscles than in other tissues. Activation of endogenous MLC1 transcription in these animals 4 days before birth was reflected in the onset of CAT transgene expression. This study identifies the transcriptional control elements necessary to activate the 21-kilobase MLC1/3 locus at the appropriate fetal stage and indicates that the MLC enhancer is sufficient to induce developmentally regulated expression from the MLC1 promoter exclusively in skeletal muscle cells.

Aging↗

FruA, a putative transcription factor essential for the development of Myxococcus xanthus.

A new developmental gene, fruA, of Myxococcus xanthus was cloned using a one-step cloning vector, TnV. DNA sequencing of the wild-type allele of the fruA gene indicated that the fruA gene encodes a protein of 229 amino acid residues with a calculated molecular weight of 24672. The deduced amino acid sequence of FruA protein showed similarity to those of many bacterial regulatory proteins carrying a DNA-binding helix-turn-helix motif. The transcription-initiation site of the fruA gene was determined by a primer-extension experiment. Development of M. xanthus cells with a disrupted fruA gene stopped at the stage of mound formation. Although cells were able to aggregate to form mounds, myxospores were not formed. By Northern and Western blot analysis, it was found that the fruA expression was not detected during vegetative growth but initiated at around 6 h and reached the highest level at 12 h after the onset of development. Expression of the fruA gene was dependent on the expression of asg, bsg, csg, dsg, and esg genes, indicating that a series of intercellular signalling is necessary for the expression of the fruA gene. The effects of the fruA mutation on beta-galactosidase expression of various developmentally regulated genes fused with the lacZ gene were analysed; three developmental lacZ fusions (omega 4469, omega 4273 and omega 4500) were either poorly induced or not induced at all, while three other lacZ fusions (omega 4408, omega 4521 and omega 4455) expressed at the early stage of development were normally induced but were unable to be repressed at a later stage of development as in the wild-type strain. Interestingly, in the fruA mutant, tps (the gene for protein S) was not activated. From these results together with analysis of the amino acid sequence of FruA, we propose that FruA is a putative transcription factor required for the development of M.xanthus.

Amino Acid Sequence↗

Expression of Oryza sativa MAP kinase gene is developmentally regulated and stress-responsive.

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, there is evidence for MAPKs playing a role in the signalling of abiotic stresses, pathogens, plant hormones, and cell cycle cues. The large number and divergence of plant MAPKs indicates that this ancient mechanism of signal transduction is extensively used in plants. However, there have been no reports of classical MAPK module in rice. In this report, we have isolated a MAPK from rice (Oryza sativa) termed OsMAPK2. The cloned cDNA is 1457 nucleotides long and the deduced amino acid sequence comprised 369 amino acid residues. Sequence analysis revealed that the predicted amino acid sequence is 72% identical to tobacco wound-induced protein kinase (WIPK). Southern analysis suggested a single OsMAPK2 gene in rice. Analysis at the mRNA level has shown that OsMAPK2 is expressed in all plant organs and high relative amounts of OsMAPK2 were detected in the mature panicles in comparison with in the immature panicles. In suspension-cultured cells, the OsMAPK2 mRNA transcript increased markedly upon temperature downshift from 26 degrees C to 4 degrees C and sucrose starvation. In contrast, the OsMAPK2 mRNA level rapidly declined in rice cell challenged by high temperature. A similarly rapid response of OsMAPK2 was observed in stress-treated seedlings, demonstrating that response of the MAPK pathway occurs also in intact plants. These results suggest that this OsMAPK2 may function in the stress-signalling pathway as well as panicle development in rice.

Journal Article↗

Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells.

During growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79-87, 1987; Clarke et al., Dev Genet 9: 315-326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low-density cells grown in the presence of buffer conditioned by high-density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other "early developmental" genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.

Biological Factors↗

Translocation of an unusual cAMP receptor to the nucleus during development of Dictyostelium discoideum.

cAMP has been implicated in the control of the expression of developmental genes in Dictyostelium discoideum. To determine the potential role of cAMP receptors as regulators of gene expression, we have used immunocytochemical and immunoblotting techniques to reveal the subcellular localization of a cAMP binding protein CABP1. Most of the CABP1 antigen in early developing cells is localized near the cell periphery, with a small amount found in the nucleus. The level of CABP1 in the nucleus increases approximately 30-fold during development. Moreover, immunofluorescence studies showed that CABP1 can also be detected on the cell surface. Binding of anti-CABP1 to intact cells followed by reaction with 125I-labeled secondary antibody revealed that the cell-surface CABP1 activity peaks during aggregation and culmination. In addition, several proteins related to CABP1 are found mainly in the nuclear fraction of developing cells. The possible role of these proteins in the regulation of developmental gene activity is discussed.

Cell Nucleus↗

Gorlin syndrome: the PTCH gene links ocular developmental defects and tumour formation.

AIM: To identify a gene linking microphthalmia with cyst with early onset medulloblastoma. METHODS: Mutation analysis of the PTCH gene. RESULTS: A mutation in exon 10 of the PTCH gene was identified, confirming a diagnosis of Gorlin syndrome. CONCLUSIONS: This is the first genetically identified mutation giving rise to microphthalmia with cyst and provides a valuable link in the eye developmental gene pathway.

Basal Cell Nevus Syndrome↗

A second CYP26 P450 in humans and zebrafish: CYP26B1.

A new P450 gene has been found in humans. It has 44% sequence identity to CYP26A1 from human and mouse, which places it in a new subfamily, CYP26B. There is only one human EST from a retinal library (AA012833) that matches the coding region. No homologous ESTs are found in mouse. A zebrafish EST AI721901 shows 68% identity to the human protein. This zebrafish EST is only 41% identical to the zebrafish CYP26A1 protein sequence, so it represents the homolog of the human CYP26B1 sequence. It is not known if this gene product will act on all-trans-retinoic acid like the CYP26A1 protein or if it might hydroxylate the 9-cis- or 13-cis-retinoic acid isoforms not recognized by CYP26A1. The importance of the CYP26A1 P450 in mouse and zebrafish development flags the CYP26B1 gene as a potential developmental gene.

Amino Acid Sequence↗

Differential display identifies developmentally regulated genes during somatic embryogenesis in eggplant (Solanum melongena L.).

Changes in gene expression patterns during early somatic embryogenesis of eggplant (Solanum melongena L.) were characterized by means of differential display. Eight products of polymerase chain reaction (PCR) derived from newly expressed mRNAs after four days of culture were identified and cloned. One clone, pTM002, was found to contain a 416 bp insert and identical to the proximal arbitrary primer at both ends. Based on the terminal sequences, reverse-transcription PCR (RT-PCR) was carried out to monitor the expression level of pTM002. The results indicated that the message of pTM002 was increased to correlate with the intensities obtained in the differential display. The significance of this method in investigation of differentially expressed genes is also discussed.

Base Sequence↗