An Analysis of the Transcriptional Control Domains of the Human c-myc Proto-Oncogene

Transfection of mammalian cells with recombinant plasmid DNAs containing the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter has been used to analyse genomic sequences regulating the transcription of the human c-myc proto-oncogene. Several regulatory domains 5' to the c-myc coding region have been identified, and their locations defined by deletion analysis. Each of these sites encompass previously identified DNase I in vivo hypersensitive sites. Published data suggested that the c-myc gene may be regulated in vivo by a repressor. The mapping of translocation breakpoints for Burkitt's lymphoma and murine plasmacytoma which involve the c-myc gene, suggests that the cis-acting recognition sequence for this putative repressor is located within the 5' flanking region. I have identified a negative regulatory element (NRE-2) in the 5' flanking region of the gene and localised it to a region between -1052 and -607bp 5' to the PI start site of the c-myc mRNA, by deletion analysis. Subsequent competition experiments showed a 270bp sub-fragment to contain an essential component of the negative regulatory element. This element can function in an orientation independent manner, and has the ability to repress heterologous promoters (both viral and eukaryotic), but to a lesser degree than when acting in cis upon its homologous promoter. My data from both DNA titration and competition transfection analysis indicates that this repression is mediated by at least one trans-acting factor. Since the repression was observed in every cell line used as the transfection recipients, a certain promiscuity in the tissue- and species- specificity of the trans-acting repressor(s) is implied. In vivo footprint analysis tentatively identified two sequence-specific DNA-binding proteins which interact with this domain. Both the CCAAT-binding Transcription Factor (CTF) and Spl bind within the NRE-2 domain. This is the first time either of these DNA-binding factors have been implicated in the transcriptional repression of a gene. In addition, deletion analyses identified an Upstream Promoter Element (UPE), located between the NRE-2 and the c-myc mRNA major cap sites, which is responsible for activation of the high levels of CAT expression observed in cells transfected with the recombinant plasmids. This UPE appears to be a highly complex domain which was shown, by DNase I in vivo footprint analysis, to bind several Spl-like factors. In addition, the UPE is somehow involved in the control of the repression function, although it is not required for the repression of heterologous promoters by the NRE-2. Other data (assayed preliminary) suggested also that two other distal regulatory domains are involved in the control of c-myc expression. The more distal element (PRE) has an activating activity, and was localised to a region which showed sequence homology to enhancer elements. A second element (NRE-1) was tentatively identified which had a negative effect on CAT activity. I conclude that the removal, and/or the rearrangement, of these transcription regulatory domains may play a crucial role in the deregulation of the expression of c-myc that is observed in some neoplastic cells

Novel combination of CRISPR-based gene drives eliminates resistance and localises spread

Invasive species are among the major driving forces behind biodiversity loss. Gene drive technology may offer a humane, efficient and cost-effective method of control. For safe and effective deployment it is vital that a gene drive is both self-limiting and can overcome evolutionary resistance. We present HD-ClvR in this modelling study, a novel combination of CRISPR-based gene drives that eliminates resistance and localises spread. As a case study, we model HD-ClvR in the grey squirrel (Sciurus carolinensis), which is an invasive pest in the UK and responsible for both biodiversity and economic losses. HD-ClvR combats resistance allele formation by combining a homing gene drive with a cleave-and-rescue gene drive. The inclusion of a self-limiting daisyfield gene drive allows for controllable localisation based on animal supplementation. We use both randomly mating and spatial models to simulate this strategy. Our findings show that HD-ClvR could effectively control a targeted grey squirrel population, with little risk to other populations. HD-ClvR offers an efficient, self-limiting and controllable gene drive for managing invasive pests.</p

Smoking cessation interventions for patients with coronary heart disease and comorbidities:an observational cross-sectional study in primary care

Background: Little is known about how smoking cessation practices in primary care differ for patients with coronary heart disease (CHD) who have different comorbidities. Aim: To determine the association between different patterns of comorbidity and smoking rates and smoking cessation interventions in primary care for patients with CHD. Design: and setting Cross-sectional study of 81 456 adults with CHD in primary care in Scotland. Method: Details of eight concordant physical comorbidities, 23 discordant physical comorbidities, and eight mental health comorbidities were extracted from electronic health records between April 2006 and March 2007. Multilevel binary logistic regression models were constructed to determine the association between these patterns of comorbidity and smoking status, smoking cessation advice, and smoking cessation medication (nicotine replacement therapy) prescribed. Results: The most deprived quintile had nearly three times higher odds of being current smokers than the least deprived (odds ratio [OR] 2.76; 95% confidence interval [CI] = 2.49 to 3.05). People with CHD and two or more mental health comorbidities had more than twice the odds of being current smokers than those with no mental health conditions (OR 2.11; 95% CI = 1.99 to 2.24). Despite this, those with two or more mental health comorbidities (OR 0.77; 95% CI = 0.61 to 0.98) were less likely to receive smoking cessation advice, but absolute differences were small. Conclusion: Patterns of comorbidity are associated with variation in smoking status and the delivery of smoking cessation advice among people with CHD in primary care. Those from the most deprived areas and those with mental health problems are considerably more likely to be current smokers and require additional smoking cessation support

Comparative analysis on the structural features of the 5' flanking region of κ-casein genes from six different species

κ-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of κ-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human κ-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the κ-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the κ-casein gene promoter

Gene-edited meat: Disentangling consumers’ attitudes and potential purchase behaviour

Novel gene-editing (GE) technologies provide promising opportunities to increase livestock productivity and to tackle several global livestock production sustainability and food security challenges. However, these technologies, as with previous genetic modification technologies in food production, are very likely to generate social controversy and opposition toward their use in the meat industry. Here, we explored public attitudes and consumption predisposition toward gene-edited meat products and their potential added benefits to livestock farming. Our results show that societal perception currently comes as a package, where the use of gene-editing technology acts as an extrinsic cue of meat products quality, and is used to make a range of inferences about all quality facets at once. Although consumers with anti-GE attitudinal positions generally were not sensitive to price discounts or added benefits, added benefits increased the consumption predisposition of most moderate and pro-GE consumers, where benefits related to animal welfare had larger effects than those relating to the environment or human health issues.Publishe