Regulatory Gene Products Research Articles

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants.

The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.

Isolation of IFAPa-400 cDNAs: evidence for a transient cytostructural gene activity common to the precursor cells of the myogenic and the neurogenic cell lineages

Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195–204; Cossette and Vincent (1991) J. Cell Sci. 98, 251–260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAP1-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.

Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sjögren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.

The Role of MHC Gene Products in Immune Regulation and its Relevance to Alloreactivity

Scandinavian Journal of ImmunologyVolume 35, Issue 4 p. 376-396 The Role of MHC Gene Products in Immune Regulation and its Relevance to Alloreactivity BARUJ BENACERRAF, BARUJ BENACERRAFSearch for more papers by this author BARUJ BENACERRAF, BARUJ BENACERRAFSearch for more papers by this author First published: April 1992 https://doi.org/10.1111/j.1365-3083.1992.tb02872.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume35, Issue4April 1992Pages 376-396 RelatedInformation

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.

Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli

Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli

Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome [formula omitted] oxidase ( cyoABCDE and cydAB) genes in Escherichia coli

The individual and the combined effect of the fnr and arcA regulatory gene products on cytochrome o oxidase and cytochrome d oxidase gene expression in Escherichia coli were evaluated using lacZ reporter fusions to the cyoABCDE and cydAB operons. Fnr repressed cyolacZ and cyd-lacZ expression during anaerobic growth but not during aerobic growth conditions. ArcA functioned as an anaerobic repressor of cyo-lacZ expression while, in contrast, it activated cydAB expression during both aerobic and anaerobic growth. ArcA and Fnr appear to function independently of each other to control cyoABCDE operon expression. In contrast, FNR repression of cydAB expression was dependent on arcA +, as indicated by the inability of fnr + plasmids to repress cyd-lacZ expression in an arcA strain. Under no conditions tested did Fnr activate cydAB expression. Most, but not all, of the observed aerobic/anaerobic regulation of cyo and cyd was accounted for by the two transcriptional regulators. These data suggest the existence of additional levels of anaerobic gene control in E. coli. Additionally, the expression of the fnr regulatory gene, and regulation of the anaerobic respiratory genes, narGHJI, dmsABC and frdABCD, was found to be independent of ArcA.

Quantitation of HIV: Correlation with clinical, virological, and immunological status

A quantitative assay has been used to measure titres of infectious HIV in peripheral blood of symptomatic and asymptomatic patients. Viral titres were assessed in conjunction with virological and immunological status of patients including measurement of p24 antigen, antibody responses to structural (gp41, p24) and regulatory gene products (NEF, REV, TAT, and VIF), determination of beta 2 microglobulin levels and enumeration of lymphocyte subsets. Titres of HIV were significantly higher among symptomatic than asymptomatic patients. Viral load was closely associated with the number of CD4+ cells, the proportion of these cells harbouring HIV increasing with disease progression. Higher titres of infectious HIV among symptomatic patients was also associated with p24 antigenaemia and decreased antibody responses to NEF.

Genetic competence in Bacillus subtilis

Genetic competence may be defined as a physiological state enabling a bacterial culture to bind and take up high-molecular-weight exogenous DNA (transformation). In Bacillus subtilis, competence develops postexponentially and only in certain media. In addition, only a minority of the cells in a competent culture become competent, and these are physiologically distinct. Thus, competence is subject to three regulatory modalities: growth stage specific, nutritionally responsive, and cell type specific. This review summarizes the present state of knowledge concerning competence in B. subtilis. The study of genes required for transformability has permitted their classification into two broad categories. Late competence genes are expressed under competence control and specify products required for the binding, uptake, and processing of transforming DNA. Regulatory genes specify products that are needed for the expression of the late genes. Several of the late competence gene products have been shown to be membrane localized, and others are predicted to be membrane associated on the basis of amino acid sequence data. Several of these predicted protein sequences show a striking resemblance to gene products that are involved in the export and/or assembly of extracellular proteins and structures in gram-negative organisms. This observation is consistent with the idea that the late products are directly involved in transport of DNA and is equally consistent with the notion that they play a morphogenetic role in the assembly of a transport apparatus. The competence regulatory apparatus constitutes an elaborate signal transduction system that senses and interprets environmental information and passes this information to the competence-specific transcriptional machinery. Many of the regulatory gene products have been identified and partially characterized, and their interactions have been studied genetically and in some cases biochemically as well. These include several histidine kinase and response regulator members of the bacterial two-component signal transduction machinery, as well as a number of known transcriptionally active proteins. Results of genetic studies are consistent with the notion that the regulatory proteins interact in a hierarchical way to make up a regulatory pathway, and it is possible to propose a provisional scheme for the organization of this pathway. It is remarkable that almost all of the regulatory gene products appear to play roles in the control of various forms of postexponential expression in addition to competence, e.g., sporulation, degradative-enzyme production, motility, and antibiotic production. This has led to the notion of a signal transduction network which transduces environmental information to determine the levels and timing of expression of the ultimate products characteristic of each of these systems.

C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites.

Tissue-specific expression of the maize anthocyanin Bronze-1 (Bz1) gene is controlled by the products of several regulatory genes. These include C1 or Pl and R or B that share homology to the myb proto-oncogenes and myc-like genes, respectively. Bz1 expression in embryo tissues is dependent on C1 and an R-sc allele of R. Transient expression from mutated and deleted versions of the Bz1 promoter fused to a luciferase reporter gene was measured in C1, Rscm2 embryos after gene transfer by microprojectiles. This analysis revealed that the sequences between -76 base pairs (bp) and -45 bp and a 9-bp AT-rich block between -88 bp and -80 bp were critical for Bz1 expression. The -76 bp to -45 bp region includes two short sequences that are homologous to the consensus binding sites of the myb- and myc-like proteins. Site-specific mutations of these "myb" and "myc" sequences reduced Bz1 expression to 10% and 1% of normal, respectively. Additionally, a trimer of a 38-bp oligonucleotide containing these myb and myc sites increased the expression of a cauliflower mosaic virus 35S minimal promoter by 26-fold. This enhancement was dependent on both C1 and R. Because the sites critical for Bz1 expression are homologous to the myb and myc consensus binding sequences and the C1 and R proteins share homology with the myb and myc products, respectively, we propose that C1 and R interact with the Bz1 promoter at these sites.

The regulation of genetic competence in Bacillus subtilis

Genetic competence develops as a global response of Bacillus subtilis to the onset of stationary phase, in glucose-minimal salts-based media. The onset of competence is accompanied by the expression of several late gene products that are required for the binding, processing and uptake of transforming DNA. A number of regulatory genes have been identified that are needed for the appropriate synthesis of the late gene products. The regulatory gene products include a number of known transcription factors, as well as several members of the bacterial two-component regulatory system. Genetic analysis has suggested a scheme for the flow of regulatory information signalling the onset of competence. Most of these regulatory products appear to be involved in the response to nutritional status, while the components responsible for growth stage and cell-type-specific control remain unknown. The general implications of this scheme for post-exponential expression are discussed.

Genetic competence in Bacillus subtilis.

Genetic competence may be defined as a physiological state enabling a bacterial culture to bind and take up high-molecular-weight exogenous DNA (transformation). In Bacillus subtilis, competence develops postexponentially and only in certain media. In addition, only a minority of the cells in a competent culture become competent, and these are physiologically distinct. Thus, competence is subject to three regulatory modalities: growth stage specific, nutritionally responsive, and cell type specific. This review summarizes the present state of knowledge concerning competence in B. subtilis. The study of genes required for transformability has permitted their classification into two broad categories. Late competence genes are expressed under competence control and specify products required for the binding, uptake, and processing of transforming DNA. Regulatory genes specify products that are needed for the expression of the late genes. Several of the late competence gene products have been shown to be membrane localized, and others are predicted to be membrane associated on the basis of amino acid sequence data. Several of these predicted protein sequences show a striking resemblance to gene products that are involved in the export and/or assembly of extracellular proteins and structures in gram-negative organisms. This observation is consistent with the idea that the late products are directly involved in transport of DNA and is equally consistent with the notion that they play a morphogenetic role in the assembly of a transport apparatus. The competence regulatory apparatus constitutes an elaborate signal transduction system that senses and interprets environmental information and passes this information to the competence-specific transcriptional machinery. Many of the regulatory gene products have been identified and partially characterized, and their interactions have been studied genetically and in some cases biochemically as well. These include several histidine kinase and response regulator members of the bacterial two-component signal transduction machinery, as well as a number of known transcriptionally active proteins. Results of genetic studies are consistent with the notion that the regulatory proteins interact in a hierarchical way to make up a regulatory pathway, and it is possible to propose a provisional scheme for the organization of this pathway. It is remarkable that almost all of the regulatory gene products appear to play roles in the control of various forms of postexponential expression in addition to competence, e.g., sporulation, degradative-enzyme production, motility, and antibiotic production. This has led to the notion of a signal transduction network which transduces environmental information to determine the levels and timing of expression of the ultimate products characteristic of each of these systems.

Multiple factors bind the upstream activation sites of the yeast enolase genes ENO1 and ENO2: ABFI protein, like repressor activator protein RAP1, binds cis-acting sequences which modulate repression or activation of transcription.

Binding sites for three distinct proteins were mapped within the upstream activation sites (UAS) of the yeast enolase genes ENO1 and ENO2. Sequences that overlapped the UAS1 elements of both enolase genes bound a protein which was identified as the product of the RAP1 regulatory gene. Sequences within the UAS2 element of the ENO2 gene bound a second protein which corresponded to the ABFI (autonomously replicating sequence-binding factor) protein. A protein designated EBF1 (enolase-binding factor) bound to sequences which overlapped the UAS2 element in ENO1. There was a good correlation among all of the factor-binding sites and the location of sequences required for UAS activity identified by deletion mapping analysis. This observation suggested that the three factors play a role in transcriptional activation of the enolase genes. UAS elements which bound the RAP1 protein or the ABFI protein modulated glucose-dependent induction of ENO1 and ENO2 expression. The ABFI-binding site in ENO2 overlapped sequences required for UAS2 activity in wild-type strains and for repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1. These latter results showed that the ABFI protein, like the RAP1 protein, bound sequences required for positive as well as negative regulation of gene expression. These observations strongly suggest that the biological functions of the RAP1 and ABFI proteins are similar.

Multiple Factors Bind the Upstream Activation Sites of the Yeast Enolase Genes ENO1 and EN02: ABFI Protein, like Repressor Activator Protein RAP1, Binds cis-Acting Sequences Which Modulate Repression or Activation of Transcription

Binding sites for three distinct proteins were mapped within the upstream activation sites (UAS) of the yeast enolase genes ENO1 and ENO2. Sequences that overlapped the UAS1 elements of both enolase genes bound a protein which was identified as the product of the RAP1 regulatory gene. Sequences within the UAS2 element of the ENO2 gene bound a second protein which corresponded to the ABFI (autonomously replicating sequence-binding factor) protein. A protein designated EBF1 (enolase-binding factor) bound to sequences which overlapped the UAS2 element in ENO1. There was a good correlation among all of the factor-binding sites and the location of sequences required for UAS activity identified by deletion mapping analysis. This observation suggested that the three factors play a role in transcriptional activation of the enolase genes. UAS elements which bound the RAP1 protein or the ABFI protein modulated glucose-dependent induction of ENO1 and ENO2 expression. The ABFI-binding site in ENO2 overlapped sequences required for UAS2 activity in wild-type strains and for repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1. These latter results showed that the ABFI protein, like the RAP1 protein, bound sequences required for positive as well as negative regulation of gene expression. These observations strongly suggest that the biological functions of the RAP1 and ABFI proteins are similar.

Monitoring of naphthalene catabolism by bioluminescence with nah-lux transcriptional fusions

We have demonstrated the efficacy of a light-generating genetic construction in describing the induction of a nah operon for the catabolism of naphthalene. A fragment from plasmid NAH7, which contains the promoter for the upper pathway of degradation, was transcriptionally fused to the lux genes of Vibrio fischeri. A Pseudomonas strain containing this construction is inducible to high levels of light production in the presence of a suitable substrate and the nahR regulatory gene product. This system was used to examine catabolic activity in a unique manner under a variety of growth conditions. Induction of bioluminescence was demonstrated to coincide with naphthalene degradation in all cases through the use of mineralization assays. A significant delay in bioluminescence and biodegradation was observed when naphthalene was added to batch cultures that were growing exponentially. These results suggest that the metabolism of naphthalene by this Pseudomonas strain is optimal when the growth rate of the culture is slow and is greatly reduced during exponential growth.

A yeast H2A-H2B promoter can be regulated by changes in histone gene copy number.

The two divergently transcribed H2A-H2B gene pairs in yeast are differentially regulated as a function of the copy number of histone genes. Transcription of an HTA2-lacZ reporter gene is independent of histone gene copy number. Transcription of an HTA1-lacZ gene can be repressed or derepressed, depending on the number of HTA plus HTB genes in cells. Regulation by histone gene dosage is dependent on a negative site in the HTA1-HTB1 promoter and the products of regulatory genes that act through this site. The level of H2A plus H2B protein in the cell may signal the response to histone gene copy number, suggesting that transcription of the HTA1-HTB1 locus can be autogenously regulated. This phenomenon may be used, in part, to maintain the balanced synthesis of histones, a critical parameter in nucleosome assembly.

A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems.

A Bacillus subtilis gene, required for genetic competence, was identified immediately upstream from the previously characterized gene comA. The comA gene product has been found to exhibit amino acid sequence similarity to the so-called effector class of signal-transduction proteins. DNA sequencing of the new determinant, named comP, revealed that the carboxy-terminal domain of the predicted ComP protein is similar in amino acid sequence to that of several sensor members of the bacterial two-component signal-transduction systems. The predicted amino-terminal domain contains several hydrophobic segments, postulated to be membrane-spanning. In vitro-derived comP disruptions are epistatic on the expression of all late competence genes tested, including comG, comC, comD, and comE, but not on expression of the early gene comB. Although comA has its own promoter, some transcription of comA, especially later in growth, occurs via readthrough from comP sequences. A roughly twofold epistatic effect of a comP disruption was noted on the downstream comA determinant, possibly due to interruption of readthrough transcription from comP to comA. Overexpression of comA fully restored competence to a comP mutant, providing evidence that ComA acts after ComP, and consistent with a role for the latter protein in activation of the former, possibly by phosphorylation. ComP probably is involved in transmitting information concerning the nutritional status of the medium, particularly the presence of nitrogen- and carbon-containing nutrients. ComP was also shown to play a role in sporulation, at least partly interchangeable with that of SpoIIJ, another putative sensor protein.

Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients

Kaposi's sarcoma (KS) is frequently associated with human immunodeficiency virus-1 (HIV-1) infection. Supernatants from HIV-1-infected T cells carrying the CD4 antigen promote the growth of cells derived from KS lesions of AIDS patients (AIDS-KS cells), and the HIV-1 tat gene, introduced into the germ line of mice, induces skin lesions closely resembling KS. Here we report that the tat gene product (Tat) is released from both HIV-1-acutely infected H9 cells and tat-transfected COS-1 cells. These Tat-containing supernatants specifically promote growth of AIDS-KS cells which are inhibited by anti-Tat antibodies; recombinant Tat has the same growth-promoting properties. Therefore a viral regulatory gene product can be released as a biologically active protein and directly act as a growth stimulator. These and previous data indicate that extracellular Tat could be involved in the development or progression, or both, of KS in HIV-1-infected individuals.

Regulation of DdrasG gene expression during Dictyostelium development.

DdrasG gene expression during the early development of Dictyostelium discoideum has been examined in detail. The amount of DdrasG-specific mRNA increased approximately twofold during the first 2 to 3 h of development and then declined rapidly, reaching negligible levels by the aggregation stage. The increase in mRNA levels that occurred during the first 2 to 3 h of development also occurred during differentiation in cell suspensions and was enhanced when cells were shaken rapidly. This initial increase was unaffected by cell density. When cells were set up to differentiate on filters, the addition of a glucose-amino acid mixture slightly delayed differentiation and had a similar effect on the expression of the gene. The decline in DdrasG expression during development did not occur when cells were treated with cycloheximide, suggesting that the expression of a developmentally regulated gene product is essential for the reduction of DdrasG gene mRNA. There was no decrease in DdrasG mRNA level during differentiation in shake suspension, but the decrease did occur upon application of pulses of cyclic AMP to shaking cultures. The application of a continuously high level of cyclic AMP delayed the increase in expression of the gene and did not result in the subsequent decline. These results suggest that the induction of a functional cyclic AMP relay system is important in reducing DdrasG gene mRNA levels.

Regulation of DdrasG Gene Expression during Dictyostelium Development

DdrasG gene expression during the early development of Dictyostelium discoideum has been examined in detail. The amount of DdrasG-specific mRNA increased approximately twofold during the first 2 to 3 h of development and then declined rapidly, reaching negligible levels by the aggregation stage. The increase in mRNA levels that occurred during the first 2 to 3 h of development also occurred during differentiation in cell suspensions and was enhanced when cells were shaken rapidly. This initial increase was unaffected by cell density. When cells were set up to differentiate on filters, the addition of a glucose-amino acid mixture slightly delayed differentiation and had a similar effect on the expression of the gene. The decline in DdrasG expression during development did not occur when cells were treated with cycloheximide, suggesting that the expression of a developmentally regulated gene product is essential for the reduction of DdrasG gene mRNA. There was no decrease in DdrasG mRNA level during differentiation in shake suspension, but the decrease did occur upon application of pulses of cyclic AMP to shaking cultures. The application of a continuously high level of cyclic AMP delayed the increase in expression of the gene and did not result in the subsequent decline. These results suggest that the induction of a functional cyclic AMP relay system is important in reducing DdrasG gene mRNA levels.