Increment In mRNA Levels Research Articles

The Integration of Multiple Nuclear-Encoded Transgenes in the Green Alga Chlamydomonas reinhardtii Results in Higher Transcription Levels

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines ‘the position-effect’, which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerase-chain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copy-number variation on heterologous expression.

Expression of Avian UCP and ANT in Skeletal Muscle of Cold-exposed Laying and Meat-type Chickens

Most bird species studied thus far have no distinct brown adipose tissue (BAT) or a related type of thermogenic tissue ; however, our recent studies on cold (4- 6°C)-acclimatized chickens suggest that simultaneous increments in mRNA levels of the mitochondrial anion carriers, avian adenine nucleotide translocator (avANT) and avian uncoupling protein (avUCP), may be involved in the regulation of thermogenesis in avian skeletal muscle (FEBS Lett. 529 : 313-318, 2002). The present experiments were conducted to compare the responses of laying- and meat-type chickens in regard to the expression of avUCP and avANT when they undergo acute (24-48h) mild cold (8°C) exposure. Twenty-four male laying- and twelve meat-type chickens (3-wk old) were used. Groups from each species were exposed to mild cold for 24 and 48h, while the control group remained at 23°C. In laying-type chickens, weight gain and feed efficiency were both compensated in some degree after 48h of cold exposure. In contrast, meat-type chickens exhibited a linear decrease in weight gain corresponding to the duration of cold exposure. There were differences in the expression of avANT mRNA in Pectoralis superficialis muscle, between laying- and meat-type chickens, i.e. expression of avANT mRNA was significantly increased in laying-type chickens exposed to cold for 48h, but not in meat-type chickens. However, no differences were observed in the expression of avUCP mRNA in pectoral muscle between control and mild cold groups for both type of chickens, showing that acute mild cold exposure was not sufficient to induce increased expression of avUCP mRNA in skeletal muscle of either laying- or meat-type chickens. Up-regulation of avANT mRNA, but not avUCP mRNA, in laying-type chickens exposed to acute mild cold suggested that avANT might be involved in thermogenesis and adaptation to acute mild cold exposure. These findings will contribute to our understanding of potential differences in the responses of laying- and meat-type chickens to cold exposure at the molecular level.

Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B

The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.