Transfection of Schistosoma mansoni by electroporation and the description of a new promoter sequence for transgene expression

We sought to investigate the efficacy of electroporation for the introduction of plasmid-based DNA constructs into Schistosoma mansoni, and expanded our study to examine parameters governing transgene expression, including requirements of a 5′ and 3′ flanking sequence, as well as parasite developmental effects on transgene expression. We used luciferase as a reporter gene for this application. Our data show that electroporation allows the transfection of immature schistosomes, and defines 5′ promoter sequence from the schistosome actin gene (SmAct1.1), coupled promiscuously with various 3′ terminator sequences, as a powerful promoter of transgene expression in growing, but not early non-growing, schistosomula. The methodology described herein will facilitate ectopic expression of genes of interest in schistosomes.     

Binding of a nuclear factor to a consensus sequence in the 5' flanking region of zein genes from maize

The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.     

Hoxa5 gene regulation: A gradient of binding activity to a brachial spinal cord element

The Hox genes cooperate in providing positional information needed for spatial and temporal patterning of the vertebrate body axis. However, the biological mechanisms behind spatial Hox expression are largely unknown. In transgenic mice, gene fusions between Hoxa5 (previously called Hox-1.3) 5' flanking regions and the lacZ reporter gene show tissue- and time-specific expression in the brachial spinal cord in day 11-13 embryos. A 604-bp regulatory region with enhancer properties directs this spatially specific expression. Fine-detail mapping of the enhancer has identified several elements involved in region-specific expression, including an element required for expression in the brachial spinal cord. Factors in embryonic day 12.5 nuclear extracts bind this element in electrophoretic mobility shift assays (EMSA) and protect three regions from DNase digestion. All three sites contain an AAATAA sequence and mutations at these sites reduce or abolish binding. Furthermore, this element binds specific individual embryonic proteins on a protein blot. The binding activity appears as a gradient along the anterior-posterior axis with two- to threefold higher levels observed in extracts from anterior regions than from posterior regions. In parallel with the EMSA, the proteins on the protein blot also show reduced binding to probes with mutations at the AAATAA sites. Most importantly, transgenic mice carrying Hoxa5/lacZ fusions with the three AAATAA sites mutated either do not express the transgene or have altered transgene expression. The brachial spinal cord element and its binding proteins are likely to be involved in spatial expression of Hoxa5 during development.     

Interaction of tobacco nuclear protein with an elicitor-responsive element in the promoter of a basic class I chitinase gene

The expression of tobacco class I chitinase genes is effectively induced by a fungal elicitor in suspension-cultured cells. A putative cis-acting elicitor-responsive element (ElRE) was identified previously in the promoter of the class I chitinase gene, CHN50. To confirm that the ElRE sequence directly mediates the regulation of gene expression by the elicitor, I constructed a deleted promoter that controlled a reporter gene for -glucuronidase (gus) and examined expression of the construct in transgenic tobacco calli. Both expression and responsiveness to the elicitor disappeared, when the region of the promoter that included the ElRE sequence had been deleted. To define the specific sequence within the ElRE that interacts with nuclear factor(s), a gel mobility shift assay was performed with wild-type and mutated elements. Results of binding and competition experiments revealed that the nuclear factor(s) bound specifically to the sequence motif, ^-534GGTCANNNAGTC^-523, and that both of the repeated sites were involved in the binding of the nuclear factors. Moreover, the binding was influenced by the distance between the two repeated sites. In addition, the elicitor-inducible activity of the binding to this motif was reduced in nuclear extracts prepared from the cells that had been treated with cycloheximide or staurosporine.     

Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene.

The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis.     

Identification of the binding sites for potential regulatory proteins in the upstream enhancer element of the Drosophila fushi tarazu gene.

With a view to identifying proteins that regulate the expression of the Drosophila ftz gene we have sequenced its enhancer-like upstream element (USE) and determined the binding sites for embryonic nuclear proteins within this region by in vitro DNAaseI footprinting. We find that greater than 50% of this element is bound by nuclear protein. By footprinting and gel-retardation studies in embryonic extracts from different developmental stages, we have characterised a number of USE/protein complexes whose nature alters in concert with changes in the ftz expression pattern, suggesting that these USE-binding proteins may be involved in the regulation of gene activity. In some cases this suggestion is substantiated by the observation that the protected DNA sequences show homology to the binding sites for ftz regulating DNA-binding proteins such as the pair-rule gene product even-skipped.     

Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs.

Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5' and 3' ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3' MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S-phase and the control of gene expression and pattern formation in embryogenesis.     

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.     

The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis -elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii , we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis -acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6–8 bp resolution and these minimal cis -elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis -elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.