Upstream Regulatory Gene Research Articles

Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease.

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida.

A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine gamma-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and H. Tanaka. J. Biochem. (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex. A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of alpha-keto acid dehydrogenase complex were not found. When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for alpha-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important role in the metabolism of alpha-ketobutyrate produced by MdeA from L-methionine. Accordingly, mdeB encodes a novel E1 component, alpha-ketobutyrate dehydrogenase E1 component, of an unknown alpha-keto acid dehydrogenase complex in P. putida. In addition, we found that the mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucine-responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.

A 53-base-pair inverted repeat negatively regulates expression of the adjacent and divergently oriented cytochrome P450(BM-1) gene and its regulatory gene, bm1P1, in Bacillus megaterium.

To study the role of the cis-acting element(s) in controlling the expression of the cytochrome P450(BM-1) gene and its upstream regulatory gene, bm1P1, in Bacillus megaterium, various deletion derivatives were constructed. A 53-bp inverted repeat located midway between the P450(BM-1) gene and bm1P1 gene was found in vivo to negatively regulate the expression of both genes, the regulation of which may occur at the transcriptional level. The promoter of the P450(BM-1), gene was also identified and found to be similar to those recognized by the sigmaA RNA polymerase of Bacillus subtilis. Possible mechanisms by which the 53-bp inverted repeat regulates the gene expression are discussed.

The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development.

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

The Aspergillus Nidulans abaA Gene Encodes a Transcriptional Activator That Acts as a Genetic Switch To Control Development

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase.

A 5417-base pair (bp) region of Pseudomonas aeruginosa PAO chromosomal DNA containing the mmsAB operon and an upstream regulatory gene (mmsR) has been cloned and characterized. The operon contains two structural genes involved in valine metabolism: mmsA, which encodes methylmalonate-semialdehyde dehydrogenase; and mmsB, which encodes 3-hydroxyisobutyrate dehydrogenase. mmsA and mmsB share the same orientation and are separated by a 16-bp noncoding region. The transcriptional start site for the operon has been pinpointed to a cytidine residue located 77 bp from the translational start site of the operon. mmsR is located on the opposite strand and begins 134 bp from the translational start site of mmsA. MmsR has been identified as a member of the XylS/AraC family of transcriptional regulators and appears to act as a positive regulator of the mmsAB operon. Sequence comparison of MmsA to other proteins in the data bases revealed that MmsA belongs to the aldehyde dehydrogenase (NAD+) superfamily. MmsB shares a 44% amino acid identity with 3-hydroxyisobutyrate dehydrogenase from rat liver. Mutants with insertionally inactivated mmsR, mmsA, and mmsB grow slowly on valine/isoleucine medium and exhibit reduced enzyme activity in cell-free extracts compared to P. aeruginosa PAO.

Nucleotide sequence of the putative regulatory gene and major promoter region of the Streptomyces griseus glycerol operon

Nucleotide sequencing of the deduced major promoter region of the glycerol utilization operon and an upstream regulatory gene of Streptomyces griseus reveals extensive similarity to the previously sequenced homologous S. coelicolor region [Smith and Chater, J. Mol. Biol. 204 (1988) 569–580]. However, regions showing divergence are found in the noncoding parts of the sequence. These may help to evaluate the significance of various sequence features in realation to promoter activity.