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S L Gomes

Publications and source records attributed to S L Gomes.

At least 37 records · Page 2Linked to original sources
PubMed1995

A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42 degrees C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point (tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Sp1 and two metal-responsive elements.

Amino Acid Sequence↗
PubMed1995

Regulation of the Caulobacter crescentus dnaKJ operon.

The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.

Bacterial Proteins↗
PubMed1994

Cloning and characterization of the gene for the catalytic subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C-subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino-terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70-amino-acid extension at the amino terminus, when aligned to the mouse C alpha subunit, being one of the largest C subunits already characterized. The B. emersonii C-gene-coding region is interrupted by three introns, ranging in size over 57-69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5'-flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene.

Amino Acid Sequence↗
PubMed1992

Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene.

Amino Acid Sequence↗
PubMed1992

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.

Blastocladiella↗
PubMed1991

Genetic analysis of a temporally transcribed chemotaxis gene cluster in Caulobacter crescentus.

Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. Tn5 insertions causing a general chemotaxis phenotype, an inability to reverse swimming direction and to form large swarm colonies, have been mapped to an 8-kb region of the C. crescentus genome. These Tn5 mutations had different effects on the methyl-accepting chemotaxis proteins (MCP), and the activities of methyltransferase and methylesterase. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Using a cosmid library we isolated a clone that complemented SC1130. Complementation studies of the Tn5 mutants using derivatives of the cosmid clone showed that all the Tn5 insertions lie within a single operon that appears to encode many chemotaxis genes. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying beta-galactosidase protein fusions to portions of the operon. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The transcription of the che operon occurred at a defined time in the cell cycle, prior to cell division.

Bacterial Proteins↗
PubMed1990

Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures.

Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.

Bacteria↗
PubMed1989

Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii.

A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.

Blastocladiella↗
PubMed1987

Differential expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii.

The heat-shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. In contrast, heat-shock-related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70,000-Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation.

Blastocladiella↗
PubMed1986

Differential localization of membrane receptor chemotaxis proteins in the Caulobacter predivisional cell.

The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Both the flagellum and the MCPs are partitioned to only one daughter cell, the swarmer cell, upon division. We report the results of experiments designed to determine the distribution of these MCPs within swarmer cells and predivisional cells. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum.

Autoradiography↗
PubMed1986

General nonchemotactic mutants of Caulobacter crescentus.

We have examined 35 mutants that have defects in general chemotaxis. Genetic analysis of these mutants resulted in the identification of at least eight che genes located at six different positions on the Caulobacter crescentus chromosome. The cheR, cheB and cheT genes appeared to be located in a three-gene cluster. Mutations in these three genes resulted in the inability of the flagellum to reverse the direction of rotation. Defects in the cheR gene resulted in a loss of the ability to methylate the methyl-accepting chemotaxis proteins. In vitro experiments showed that the lack of in vivo methylation in cheR mutants was due to the absence of methyltransferase activity. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. The specific defects responsible for the lack of a chemotactic response have not been determined for the other identified che genes.

Bacteria↗
PubMed1986

Heat shock protein synthesis during development in Caulobacter crescentus.

Caulobacter crescentus cells respond to a sudden increase in temperature by transiently inducing the synthesis of several polypeptides. Two of the proteins induced, Hsp62 and Hsp70, were shown to be analogous to the heat shock proteins of Escherichia coli, GroEL and DnaK, respectively, by immunological cross-reactivity with antibodies raised against the E. coli proteins. Two-dimensional gel electrophoretic resolution of extracts of cells labeled with [35S]methionine during heat shock led to the identification of 20 distinct Hsps in C. crescentus which are coordinately expressed, in response to heat, at the various stages of the cell division cycle. Thus, a developmental control does not seem to be superimposed on the transient activation of the heat shock genes. Nonetheless, under normal temperature conditions, four Hsps (Hsp70, Hsp62, Hsp24b, and Hsp23a) were shown to be synthesized, and their synthesis was cell cycle regulated.

Bacterial Proteins↗
PubMed1985

Temporal and spatial control of flagellar and chemotaxis gene expression during Caulobacter cell differentiation.

Each Caulobacter cell division yields daughter cells that differ from one another both structurally and functionally. By focusing on the biogenesis of the polar flagellum and the proteins of the chemosensory system, several laboratories have now defined an extensive network of genes whose temporal expression is controlled in the predivisional cell. The differential turn-on of these genes contributes to the generation of asymmetry in the predivisional cell in that the products of these genes are targeted to specific cellular locations. To define the mechanisms that mediate this temporal and spatial control, fla genes whose products are not known were accessed by the insertion of transposon-carried drug resistance markers. The transposons were altered so that upon insertion into the chromosome, transcription fusions are formed in which the promoter regions of fla genes drive the expression of the downstream promoter-less drug resistance genes. Assays of the differential placement of the promoter-less drug resistance proteins (encoded within the interrupted fla genes) allow us to determine whether the positioning of the fla gene products is controlled by signal sequences in their proteins, by specific mRNA-targeting sequences in the 5'-regulatory regions of these genes, or by specific transcription from only one of the two newly replicated chromosomes in the predivisional cell.

Cell Cycle↗
PubMed1984

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter.

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.

Autoradiography↗
PubMed1984

Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in Caulobacter crescentus by using plasmid complementation.

The biosynthesis of the single polar flagellum and the proteins that comprise the chemotaxis methylation machinery are both temporally and spacially regulated during the Caulobacter crescentus cell-division cycle. The genes involved in these processes are widely separated on the chromosome. The region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. Chromosomal deletions that extend beyond the cloned region were not complemented by this plasmid. Plasmids containing small deletions in the flaY region failed to restore to any flaY or flaE mutants the ability to swim or to assemble a flagellar filament. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. The pleiotropic regulation of flagellin synthesis, assembly, and chemotaxis methylation functions exhibited by both the flaY and flaE genes suggest that their gene products function in a regulatory hierarchy that controls both flagellar and chemotaxis gene expression.

Bacteria↗
PubMed1983

Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores.

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.

Affinity Labels↗
PubMed1983

Methylation involved in chemotaxis is regulated during Caulobacter differentiation.

Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We have asked if the biochemical machinery that mediates chemotaxis exists coincident with the cell's structural ability to respond to a chemotactic signal. We first demonstrated that one function of the chemotaxis machinery, the ability to methylate the carboxyl side chains of a specific set of membrane proteins (methyl-accepting chemotaxis proteins, MCPs), is present in C. crescentus. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from S-adenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. In vivo methylation reappeared coincident with the biogenesis of the flagellum just prior to cell division. In vitro reconstitution experiments with heterologous cell fractions from different cell types showed that swarmer cells contain methyltransferase and their membranes can be methylated. However, newly differentiated stalked cells lack methyltransferase activity and membranes from these cells cannot accept methyl groups. These results demonstrate that MCP methylation is confined to that portion of the cell cycle when flagella are present.

Bacteria↗