(1/1277) High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.
Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species. (+info)
(2/1277) Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood.
Proteins containing amino acid repeats are widespread among protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be of primary importance. We have recently suggested that tandem repeats present in Trypanosoma cruzi trans-sialidase stabilize the catalytic activity in blood. Because the parasite releases trans-sialidase, this delayed clearance of the enzyme might have implications in vivo. In the present work, the ability of repetitive units from different T. cruzi molecules in stabilizing trans-sialidase activity in blood was evaluated. It is shown that repeats present on T. cruzi shed proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA]) increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and catalytic properties of both groups of chimeric enzymes show no substantial differences. Interestingly, antigens 13 and SAPA also increase the persistence in blood of chimeric glutathione S-transferases, thus suggesting that this effect is inherent to these repeats and independent of the carrier protein. Although the molecular basis of this phenomenon is still uncertain, its biotechnological potential can be envisaged. (+info)
(3/1277) Cooperation between SMAD and NF-kappaB in growth factor regulated type VII collagen gene expression.
We have previously demonstrated that transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, synergistically enhance the expression of type VII collagen gene (COL7A1) in human dermal fibroblasts in culture (Mauviel et al., 1994). Recently, we identified a SMAD-containing complex, rapidly induced by TGF-beta and binding the region [-496/-444] of the COL7A1 promoter, responsible for COL7A1 gene transactivation (Vindevoghel et al., 1998a). In this report, we demonstrate that TGF-beta and TNF-alpha response elements are distinct entities within the COL7A1 promoter. In particular, we demonstrate that the TNF-alpha effect is mediated by NF-kappaB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-kappaB complexes binding the TNF-alpha response element (TaRE) located in the region [-252/-230], with RelA acting as the transcriptional activator. Finally, we provide definitive evidence for the role of both TGF-beta and TNF-alpha response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-beta and TNF-alpha. This study provides the first identification of a functional interaction between the two immediate-early transcription factors, SMAD and NF-kappaB, to activate the expression of an extracellular matrix-related gene, COL7A1. (+info)
(4/1277) Target joining of duplicated insertion sequence IS21 is assisted by IstB protein in vitro.
Tandemly repeated insertion sequence IS21, located on a suicide plasmid, promoted replicon fusion with bacteriophage lambda in vitro in the presence of ATP. This reaction was catalyzed in a cell extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa IstB helper protein of IS21 after both proteins had been overproduced in Escherichia coli. Without IstB, replicon fusion was inefficient and did not produce the 4-bp target duplications typical of IS21. (+info)
(5/1277) Recognition of multiple patterns of DNA sites by Drosophila homeodomain protein Bicoid.
Our previous studies demonstrated that the Drosophila homeodomain protein, Bicoid (Bcd), binds DNA cooperatively. In this study, we determined the patterns of adjacent DNA sites required for cooperative recognition by Bcd. Our in vitro selection and biochemical experiments demonstrated that Bcd binds preferentially to both head-to-head and tail-to-tail symmetric sites that are separated by short spacing. An increase in the spacing reduces the strict requirement of symmetric patterns of adjacent sites, permitting Bcd to recognize tandem repeat sites cooperatively. Our further experiments in vivo showed that the only pair of optimally spaced symmetric Bcd sites in a hunchback (hb) enhancer element contributes the most to transcriptional activation by Bcd, demonstrating the biological importance of the binding site patterns revealed by our in vitro selection studies. (+info)
(6/1277) Structure and function of the prDNA and the genomic termini of the gamma2-herpesvirus bovine herpesvirus type 4.
The linear virion DNA of bovine herpesvirus type 4 (BHV-4) is flanked by tandem repeats designated polyrepetitive DNA (prDNA). To investigate the structure and functional role of the prDNA for cleavage/packaging of progeny viral DNA, the complete nucleotide sequence (2267 bp) of a cloned prDNA unit of BHV-4 was determined. Moreover, the terminal fragments of the genome and the junctions between prDNA and the central unique DNA were analysed. In order to characterize the function of the prDNA of BHV-4, a transient packaging assay was developed. The prDNA has a G+C content of 71.1%. Its structure is composed of numerous internal repeats and every unit contains the conserved sequence of the cleavage/packaging signal. A fragment of 443 bp comprising the cleavage/packaging signal was found to be sufficient for cleavage and encapsidation of replicated concatemeric viral DNA. These results suggest that prDNA is a functionally important region of the genome of BHV-4. (+info)
(7/1277) Prenatal diagnosis in factor XIII-A deficiency.
Congenital factor XIII deficiency is a severe bleeding disorder that is inherited as an autosomal recessive trait. The condition is commonly due to absence of the factor XIII-A subunit protein in the plasma. The case of a baby is reported who showed typical clinical features of factor XIII-A deficiency, including recurrent bleeding from the umbilical stump and a life threatening haemorrhage after circumcision. Family studies were performed and molecular analysis, using a Short Tandem Repeat (STR) marker closely linked to the A subunit gene, allowed antenatal exclusion diagnosis to be undertaken in a subsequent pregnancy. The case highlights the importance of seeking a family history of bleeding disorders before surgery in the neonatal period, particularly if the parents are consanguineous. (+info)
(8/1277) Tandem repeat recombination induced by replication fork defects in Escherichia coli requires a novel factor, RadC.
DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB+ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork. (+info)