Some studies have shown that the excessive metabolic heat production is the primary cause for dead chicken embryos during late embryonic development. Increasing heat shock protein (HSP) expression and adjusting metabolism are important ways to maintain body homeostasis under heat stress. This study was conducted to investigate the effects of in ovo injection (IOI) of vitamin C (VC) at embryonic age 11th day (E11) on HSP and metabolic genes expression. A total of 320 breeder eggs were randomly divided into normal saline and VC injection groups. We detected plasma VC content and rectal temperature at chick’s age 1st day, and the mRNA levels of HSP and metabolic genes in embryonic livers at E14, 16 and 18, analysed the promoter methylation levels of differentially expressed genes and predicted transcription factors at the promoter regions. The results showed that IOI of VC significantly increased plasma VC content and decreased rectal temperature (P < 0.05). In ovo injection of VC significantly increased heat shock protein 60 (HSP60) and pyruvate dehydrogenase kinase 4 (PDK4) genes expression at E16 and PDK4 and secreted frizzled related protein 1 (SFRP1) at E18 (P < 0.05). At E16, IOI of VC significantly decreased the methylation levels of total CpG sites and −336 CpG site in HSP60 promoter and −1137 CpG site in PDK4 promoter (P < 0.05). Potential binding sites for nuclear factor-1 were found around −389 and −336 CpG sites in HSP60 promoter and potential binding site for specificity protein 1 was found around −1137 CpG site in PDK4 promoter. Our results suggested that IOI of VC increased HSP60, PDK4 and SFRP1 genes expression at E16 and 18, which may be associated with the demethylation in gene promoters. Whether IOI of VC could improve hatchability needs to be further verified by setting uninjection group.

Transgenic Nicotiana tabacum with tolerance to 2,4-D has previously been produced using a bacterial 2,4-D-dioxygenase gene (tfdA) driven by the 35S promoter of cauliflower mosaic virus. Using promoters from the Pisum sativum plastocyanin gene (petE) and an Arabidopsis thaliana histone gene (H4A), we demonstrate that similar protection from 2,4-D can be obtained in transgenic N. tabacum by targeting expression of tfdA to either meristematic tissues or chloroplast-containing tissues. As with the 35S promoter constructs, the plants are tolerant but not completely resistant; very young seedlings in particular are only slightly protected. However, the levels of tolerance observed could offer a useful degree of protection from accidental spray drift.

Identification of individuals within pairs of monozygotic (MZ) twins remains unresolved using common forensic DNA typing technology. For some criminal cases involving MZ twins as suspects, the twins had to be released due to inability to identify which of the pair was the perpetrator. In this study, we performed a genome-wide scan on whole blood-derived DNA from four pairs of healthy phenotypically concordant MZ twins using the methylated DNA immunoprecipitation sequencing technology to identify candidate DNA methylation markers with capacity to distinguish MZ twins within a pair. We identified 38 differential methylation regions showing within-pair methylation differences in all four MZ pairs. These are all located in CpG islands, 17 of which are promoter-associated, 17 are intergenic islands, and four are intragenic islands. Genes associated with these markers are related with cell proliferation, differentiation, and growth and development, including zinc finger proteins, PRRX2, RBBP9, or are involved in G-protein signaling, such as the regulator of G-protein signaling 16. Further validation studies on additional MZ twins are now required to evaluate the broader utility of these 38 markers for forensic use.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder caused by heterozygotic inactivation of the NF1 tumor suppressor gene at 17q11.2. The associated phenotypes are highly variable, and modifying genes have been proposed to explain at least in part the intriguing expressivity. Given that haploinsufficiency of the NF1 gene product neurofibromin is responsible for some of the clinical manifestations, variations in expression of the wildtype NF1 allele might modify the phenotype. We therefore investigated epigenetic molecular modifications that could result in variable expression of the normal NF1 allele. To exclude confounding by DNA sequence variations, we analyzed monozygotic twin pairs with NF1 who presented with several discordant features. We fine-mapped the methylation pattern of a nearly 1 kb NF1 promoter region in lymphocytes of 8 twin pairs. All twin pairs showed significant intra-pair differences in methylation, especially of specific promoter subregions such as 5'UTR, exon 1 and intron 1 (+7 to +622), transcription factor binding sites and promoter elements like NF1HCS. Furthermore, we detected significant intra-pair differences in cytosine methylation for the region from -249 to -234 with regard to discordance for optic glioma with a higher grade of methylation in glioma cases. In conclusion, our findings of epigenetic differences of the NF1 promoter in leukocytes within mono zygotic twin pairs may serve as a proof of principle for other tissues. The results point towards a role of methylation patterns of the normal NF1 allele for expression differences and for modification of the NF1 phenotype.

Protein disulphide isomerase (PDI) catalyses the formation, reduction and isomerization of disulphide bonds in the newly synthesized secretory proteins. Plant PDIs have been shown to be involved in the folding and deposition of seed storage proteins, which makes this enzyme particularly interesting in wheat, as flour quality is strongly affected by composition and structure of seed storage proteins. In hexaploid wheat cultivar (AABBDD) Chinese Spring (CS), the genomic, complementary DNA and promoter sequences of the three homoeologous gene encoding PDI had been isolated and characterized in a previous study revealing high levels of sequence conservation. In this study, we report the isolation and sequencing of a ~700 bp region, comprising ~600 bp of the putative promoter region and 88 bp of the first exon of the typical PDI gene, in five accessions each from Triticum urartu (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD). Sequence analysis indicated large variation among sequences belonging to the different genomes, while close similarity was found within each species and with the corresponding homoeologous PDI sequences of Triticum aestivum cv. CS (AABBDD) resulting in an overall high conservation of the regulatory motifs conferring endosperm-specific expression.

Milk protein genes are among the most intensively expressed and they are active only in epithelial mammary cells of lactating animals. They code for proteins which represent 30% of the proteins consumed by humans in developed countries. Mammary gland development occurs essentially during each pregnancy. This offers experimenters attractive models to study the expression mechanisms of genes controlled by known hormones and factors (prolactin, glucocorticoids, progesterone, insulin-like growth factor-1 and others) as well as extracellular matrix. In the mid-1970s, it became possible to identify and quantify mRNAs from higher living organisms using translation in reticulocyte lysate. A few years later, the use of radioactive cDNAs as probes made it possible for the quantification of mRNA in various physiological situations using hybridisation in the liquid phase. Gene cloning offered additional tools to measure milk protein mRNAs and also to identify transcription factors. Gene transfer in cultured mammary cells and in animals contributed greatly to these studies. It is now well established that most if not all genes of higher eukaryotes are under the control of multiple distal regulatory elements and that local modifications of the chromatin structure play an essential role in the mechanisms of differentiation from embryos to adults. The technique, known as ChIP (chromatin immunoprecipitation), is being implemented to identify the factors that modify chromatin structure at the milk protein gene level during embryo development, mammogenesis and lactogenesis, including the action of hormones and extracellular matrix. Transgenesis is not just a tool to study gene regulation and function, it is also currently used for various biotechnological applications including the preparation of pharmaceutical proteins in milk. This implies the design of efficient vectors capable of directing the secretion of recombinant proteins in milk at a high concentration. Milk protein gene promoters and long genomic-DNA fragments containing essentially all the regulatory elements of milk protein genes are used to optimise recombinant protein production in milk.

Draft genome sequences for Schistosoma mansoni and Schistosoma japonicum are now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6–18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, ≥1×107 infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1×107–1×103 iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8+ T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1×107 iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression.

Numerous genetic variants of the Echinococcus antigen B (AgB) are encountered within a single metacestode. This could be a reflection of gene redundancy or the result of a somatic hypermutation process. We evaluate the complexity of the AgB multigene family by characterizing the upstream promoter regions of the 4 already known genes (EgAgB1-EgAgB4) and evaluating their redundancy in the genome of 3 Echinococcus species (E. granulosus, E. ortleppi and E. multilocularis) using PCR-based approaches. We have ascertained that the number of AgB gene copies is quite variable, both within and between species. The most repetitive gene seems to be AgB3, of which there are more than 110 copies in E. ortleppi. For E. granulosus, we have cloned and characterized 10 distinct upstream promoter regions of AgB3 from a single metacestode. Our sequences suggest that AgB1 and AgB3 are involved in gene conversion. These results are discussed in light of the role of gene redundancy and recombination in parasite evasion mechanisms of host immunity, which at present are known for protozoan organisms, but virtually unknown for multicellular parasites.

Recently we showed that de novo ribosome biosynthesis is transcriptionally regulated in Coccidia, depending on their life-cycle stage. Since the expression of ribosomal protein genes is likely coordinated, the transcriptional control of all Toxoplasma gondii ribosomal protein (RP) genes was analysed. Therefore, the complete set of all cytoplasmic RPs was defined, containing 79 different RPs in T. gondii. RP genes were randomly distributed over the genome, each with a unique upstream region with the exception of 8 RP genes which were paired in a head-to-head orientation. To study if the RP genes share conserved promoter elements, a database was made containing upstream sequences of all T. gondii RP genes. Promoter activity was confirmed for the upstream sequences of 8 RP genes, some of which are comparable in strength to the alpha-tubulin promoter. In the complete set of RP upstream sequences 2 novel and highly conserved elements were identified, named Toxoplasma Ribosomal Protein (TRP)-1 (consensus: TCGGCTTATATTCGG) and TRP-2 ([T/C]GCATGC[G/A]). TRP-1 and/or TRP-2 were present in 95% of all RP upstream sequences and moreover, were specifically localized in a small region near the presumptive transcriptional start site (10–330 bp upstream). Although TRP elements were mostly absent in known T. gondii promoters, they are present elsewhere in the T. gondii genome suggesting that they operate not only in RP genes but in a larger set of genes. The identification of TRP elements creates a basis to further study the underlying mechanism by which RP transcription is controlled in T. gondii.The nucleotide sequence data reported in this paper are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank databases under the Accession numbers TPA: BK004896-BK004974.

β-lactoglobulin polymorphisms have been reported in the milk of different goat breeds, although no genetic variants affecting the protein have been characterized. In the present study, we amplified and sequenced the proximal promoter and the first six exons containing the entire coding region for the β-lactoglobulin gene in eleven goat breeds from Spain, France, Italy, Switzerland, Senegal and Asia to identify genetic variants. Fifteen polymorphisms were detected, nine in the promoter region and six in the exons of the β-lactoglobulin gene. All polymorphisms were single nucleotide substitutions with the exception of one deletion/insertion in the promoter region. The polymorphisms in the coding region did not produce any amino acid change. In addition, pyrosequencing technology was used to genotype four polymorphisms in the promoter region in 200 goats belonging to eleven breeds. Differences in allelic frequencies for these polymorphisms between breeds are described and a specific polymorphism for the Italian populations was identified. Finally, the analysis of association between these four promoter point mutations was investigated resulting in five haplotypes, GCGC being the most frequent haplotype in all breeds analysed.

A sigmoidal transcriptional response (STR) is thought to act as a molecular switch to control gene expression. This nonlinear behaviour arises as a result of the cooperative recognition of a promoter/enhancer by transcription factors (TFs) and/or their synergy to attract the basal transcriptional machinery (BTM). Although this cooperation between TFs is additive in terms of energy, it leads to an exponential increase in affinity between the BTM and the pre-initiation complexes. This exponential increase in the strength of interactions is the principle that governs synergistic systems. Here, I propose a minimalist quasi-equilibrium model to explore qualitatively the STR taking into account cooperative recognition of the promoter/enhancer and synergy. Although the focus is on the effect of activators, a similar treatment can be applied to inhibitors. One of the main insights obtained from the model is that generation of a sigmoidal threshold is possible even in the absence of cooperative DNA binding provided the TFs synergistically interact with the BTM. On the contrary, when there is cooperative binding, the impact of synergy diminishes. It will also be shown that a sigmoidal response to a morphogenetic gradient can be used to generate a nested gradient of another morphogen. Previously, I had proposed that halving the amounts of TFs involved in sigmoidal transcriptional switches could account for the abnormal dominant phenotypes associated with some of these genes. This phenomenon, called haploinsufficiency (HI), has been recognised as the basis of many human diseases. Although a formal proof linking HI and a sigmoidal response is lacking, it is tempting to explore the model from the perspective of dosage effects.