Specific protein-protein interaction between basic helix-loop-helix transcription factors and homeoproteins of the Pitx family.

Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis.

The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum.

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.

Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling.

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.

Beta-mannanase of Streptomyces lividans 66: cloning and DNA sequence of the manA gene and characterization of the enzyme.

The gene coding for a beta-mannanase was cloned homologously from Streptomyces lividans and its DNA sequence was determined. The fully secreted enzyme was isolated and purified from culture filtrates of the hyperproducing clone S. lividans IAF36 grown in mineral salt media containing galactomannan as the main carbon source. It had a molecular mass of 36 kDa and a specific activity of 876 i.u./mg of protein. Under the assay conditions used, the optimal enzyme activity was obtained at 58 degrees C and a pH of 6.8. The pI was 3.5. The kinetic constants of this mannanase determined with galactomannan as substrate were a Vmax. of 205 i.u./mg of enzyme and a Km of 0.77 mg/ml. Data from SDS/PAGE and Western blotting show that the cloned enzyme was identical to that of the wild-type strain.

Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85.

The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles.

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.

Expression and secretion of beta-glucuronidase and Pertussis toxin S1 by Streptomyces lividans.

Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coli beta-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the beta-glucuronidase gene was fused with the leader sequence produced up to 30 mg beta-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/1. The disappearance of the B. pertussis toxin S1 and beta-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.

The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains.

The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen.