Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.
(9/2024)
Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain. (+info)
Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas.
(10/2024)
CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans. (+info)
Characterization and mutation analysis of human LEFTY A and LEFTY B, homologues of murine genes implicated in left-right axis development.
(11/2024)
Members of the transforming growth factor (TGF)-beta family of cell-signaling molecules have been implicated recently in mammalian left-right (LR) axis development, the process by which vertebrates lateralize unpaired organs (e.g., heart, stomach, and spleen). Two family members, Lefty1 and Lefty2, are expressed exclusively on the left side of the mouse embryo by 8.0 days post coitum. This asymmetry is lost or reversed in two murine models of abnormal LR-axis specification, inversus viscerum (iv) and inversion of embryonic turning (inv). Furthermore, mice homozygous for a Lefty1 null allele manifest LR malformations and misexpress Lefty2. We hypothesized that Lefty mutations may be associated with human LR-axis malformations. We now report characterization of two Lefty homologues, LEFTY A and LEFTY B, separated by approximately 50 kb on chromosome 1q42. Each comprises four exons spliced at identical positions. LEFTY A is identical to ebaf, a cDNA previously identified in a search for genes expressed in human endometrium. The deduced amino acid sequences of LEFTY A and LEFTY B are more similar to each other than to Lefty1 or Lefty2. Analysis of 126 human cases of LR-axis malformations showed one nonsense and one missense mutation in LEFTY A. Both mutations lie in the cysteine-knot region of the protein LEFTY A, and the phenotype of affected individuals is very similar to that typically seen in Lefty1-/- mice with LR-axis malformations. (+info)
Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons.
(12/2024)
The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB. (+info)
Structure and promoter region of the surface membrane protein HS9 gene expressed on the thymic epithelial cells.
(13/2024)
The HS9 gene encoding a surface membrane protein is expressed in thymic epithelial cells. We have isolated the mouse HS9 gene and determined the sequence of all exons. The mouse HS9 gene is composed of 14 exons spanning approx. 31 kb. Primer extension analysis identified two transcription initiation sites 33 bp and 179 bp upstream from the ATG start codon. DNA sequence analysis of the 5'-flanking region of the first exon revealed a number of consensus binding sites for known transcription factors such as GC box, Sp1, NFkappaB, gamma-IRE. Neither typical TATA nor CCAAT boxes were found in this region. These results and the analysis of the luciferase activity showed that transcription of the HS9 gene is regulated at a TATA-less promoter. (+info)
Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development.
(14/2024)
The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens. (+info)
Microsatellite loci in wild-type and inbred Strongylocentrotus purpuratus.
(15/2024)
Strongylocentrotus purpuratus, a major research model in developmental molecular biology, has been inbred through six generations of sibling matings. Though viability initially decreased, as described earlier, the inbred line now consists of healthy, fertile animals. These are intended to serve as a genomic resource in which the level of polymorphism is decreased with respect to wild S. purpuratus. To genotype the inbred animals eight simple sequence genomic repeats were isolated, in context, and PCR primers were generated against the flanking single-copy sequences. Distribution and polymorphism of these regions of the genome were studied in the genomes of 27 wild individuals and in a sample of the inbred animals at F2 and F3 generations. All eight regions were polymorphic, though to different extents, and their homozygosity was increased by inbreeding as expected. The eight markers suffice to identify unambiguously the cellular DNA of any wild or F3 S. purpuratus individual. (+info)
The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1.
(16/2024)
A mutant of Saccharomyces cerevisiae deficient in the lactate-proton symport was isolated. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 that restored lactate transport. Disruption of JEN1 abolished uptake of lactate. The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in S. cerevisiae. (+info)