Carter Bancroft

Hiding messages in DNA microdots

The microdot is a means of concealing messages (steganography) that was developed by Professor Zapp and used by German spies in the Second World War to transmit secret information. A microdot (“the enemys masterpiece of espionage”) was a greatly reduced photograph of a typewritten page that was pasted over a full stop in an innocuous letter. We have taken the microdot a step further and developed a DNA-based, doubly steganographic technique for sending secret messages. A DNA-encoded message is first camouflaged within the enormous complexity of human genomic DNA and then further concealed by confining this sample to a microdot.

Making DNA Add

Recent studies have demonstrated the feasibility of using DNA-based experiments to compute solutions to combinatorial problems. However, a prerequisite for designing a computer useful in a wide range of applications is the ability to perform mathematical calculations. The development of a DNA-based algorithm for addition is presented. The DNA representation of two nonnegative binary numbers is presented in a form permitting a chain of primer extension reactions to carry out the addition operation. To demonstrate the feasibility of this algorithm, a simple example was executed biochemically.

Utilization of Never-Medicated Bipolar Disorder Patients towards Development and Validation of a Peripheral Biomarker Profile

There are currently no biological tests that differentiate patients with bipolar disorder (BPD) from healthy controls. While there is evidence that peripheral gene expression differences between patients and controls can be utilized as biomarkers for psychiatric illness, it is unclear whether current use or residual effects of antipsychotic and mood stabilizer medication drives much of the differential transcription. We therefore tested whether expression changes in first-episode, never-medicated BPD patients, can contribute to a biological classifier that is less influenced by medication and could potentially form a practicable biomarker assay for BPD. We employed microarray technology to measure global leukocyte gene expression in first-episode (n=3) and currently medicated BPD patients (n=26), and matched healthy controls (n=25). Following an initial feature selection of the microarray data, we developed a cross-validated 10-gene model that was able to correctly predict the diagnostic group of the training sample (26 medicated patients and 12 controls), with 89% sensitivity and 75% specificity (p<0.001). The 10-gene predictor was further explored via testing on an independent cohort consisting of three pairs of monozygotic twins discordant for BPD, plus the original enrichment sample cohort (the three never-medicated BPD patients and 13 matched control subjects), and a sample of experimental replicates (n=34). 83% of the independent test sample was correctly predicted, with a sensitivity of 67% and specificity of 100% (although this result did not reach statistical significance). Additionally, 88% of sample diagnostic classes were classified correctly for both the enrichment (p=0.015) and the replicate samples (p<0.001). We have developed a peripheral gene expression biomarker profile, that can classify healthy controls from patients with BPD receiving antipsychotic or mood stabilizing medication, which has both high sensitivity and specificity. Moreover, assay of three first-episode patients who had never received such medications, to first enrich the expression dataset for disease-related genes independent of medication effects, and then to test the 10-gene predictor, validates the peripheral biomarker approach for BPD.

[49] Ca2+/Calmodulin regulation of prolactin gene expression

Despite the extensive literature on the biological actions of Ca2+ and calmodulin, very little is known about their involvement in nuclear functions, e.g., regulation of specific gene expression. To date, the only genes other than prolactin and growth hormone shown to be regulated by perturbations in cell Ca2+ are those coding for two glucose-regulated proteins.20 However, there is a growing body of indirect evidence for nuclear functions of Ca2+ and calmodulin (reviewed in Ref. 1), and we suspect that other examples of Ca2+-regulated genes will emerge. We have described in this chapter several different experimental approaches which we have employed to examine first whether prolactin gene expression is regulated by changes in cell Ca2+ content, and then to begin searching for the components of the mechanism by which Ca2+ exerts its effects on the prolactin gene. The tentative identification of 56-kDa nuclear matrix protein as both a calmodulin-binding protein and a substrate of a Ca2+-calmodulin-dependent protein kinase suggests that NMP 56 may be a subunit of a multifunctional Ca2+-calmodulin-protein kinase. This enzyme was recently detected in the nuclear matrix fraction of neuronal nuclei, and was shown to phosphorylate a chromatin protein similar to high mobility group protein 17 (HMG 17).21 Since HMG 17 is associated with actively transcribed chromatin,22 its phosphotylation in GH3 cells might play a role in the Ca2+-calmodulin-dependent regulation of prolactin gene expression by hormones and growth factors.

Epidermal growth factor and phorbol ester regulate prolactin gene expression via distinct pathways.

Previous studies, involving phosphorylation of cytoplasmic proteins and localization of DNA regulatory elements, have suggested that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) have similar actions on prolactin (PRL) gene expression by pituitary (GH) cells. However, little is presently known about whether the actions of these two factors involve common gene-distal intermediates. In the present study, we have employed two approaches to examine this question. Chronic exposure of GH3 cells to TPA, which strongly down-regulates protein kinase C activity, completely inhibited acute TPA stimulation of transient expression of a transfected PRL promoter construct ((-187)PRL-CAT), but did not inhibit EGF stimulation of either accumulation of endogenous PRL mRNA or of expression of (-187)PRL-CAT. Furthermore, the acute stimulatory effects of EGF and TPA on expression of (-187)PRL-CAT were additive. Each of these observations implies that EGF and TPA have gene-distal actions on PRL gene expression that are at least partially non-overlapping.

Nicotine Acts Directly on Pituitary GH3 Cells to Inhibit Prolactin Promoter Activity

We have employed the GH3 rat pituitary cell line to investigate whether nicotine can regulate prolactin (PRL) gene expression. Nicotine strongly inhibited (45%) transient expression of a construct containing the first 187 base‐pairs of the rat PRL promoter cloned upstream of the chloramphenicol acetyl transferase (CAT) gene. This implies that nicotine acts directly on the GH3 cells to inhibit transcription directed by the PRL promoter. Expression of a control reporter construct containing the CAT gene under the control of the RSV promoter was not affected by exposure of the cells to nicotine, demonstrating that the effect of nicotine is promoter‐specific. The inhibition by nicotine of PRL promoter activity was not blocked by hexamethonium, suggesting that this effect of nicotine may be mediated by a novel type of nicotine receptor previously described in frog pituitary cells. Nicotine was also observed to yield a concentration‐dependent inhibition of the stimulation by thyrotrophin‐releasing hormone (TRH) of PRL promoter activity, implying that nicotine can also interfere with hormonal regulation of the PRL gene. These results suggest that the reduced serum PRL levels that result from smoking may originate in part from decreased transcription of the PRL gene resulting from a direct effect of nicotine on pituitary PRL‐secreting cells.

The D2 receptor: blocked transcription in GH3 cells and cellular pathways employed by D2A to regulate prolactin promoter activity.

Although the GH3 line of somatolactotropic rat pituitary cells has proven useful for many regulation studies, the absence of functional D2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH3 cells expressing recombinant D2 receptors for such investigations. We have investigated both the level at which expression of functional D2 receptors in GH3 cells is blocked, and the cellular pathways employed by the major pituitary D2 receptor isoform, D2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from either parental GH3 cells or a GH3 cell line stably expressing a D2A expression vector, Pit-1 gene transcription was detectable in either cell line, but only the latter cell line yielded detectable D2 receptor transcription, implying that the block in D2 receptor expression by GH3 cells is transcriptional. Further investigations employed GH3 cells transiently co-transfected with a D2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D2 agonist, of PRL-CAT activity, demonstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to raise intracellular Ca2+, Bay K8644 or thyrotropin-releasing hormone, prevented quinpirole repression of PRL-CAT activity, and that the repressive effects on this construct of quinpirole and the Ca2+ channel antagonist were independent, suggested that regulation of intracellular Ca2+ levels does not play a major role in D2A-mediated repression of the PRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutively active Gq-α stimulates prolactin promoter activity via a pathway involving Raf activity

We have investigated the ability of a constitutively active Gq-alpha mutant, Q209L-alpha q, to regulate target gene expression. Transient expression in GH3 pituitary cells of a rat proximal prolactin promoter-chloramphenicol acetyltransferase construct (-187)PRL-CAT, was stimulated by co-expression of Q209L alpha q, but not by wild-type alpha q. Q209L-alpha q stimulated expression of constructs driven by promoters for either rat prolactin or growth hormone, but not of a control construct driven by the thymidine kinase promoter. Thus, transcriptional effects of alpha q are specific both for the activated state of this G-alpha subunit and the promoter examined. Since both the prolactin and growth hormone promoters are activated by the pituitary cell-specific transcription factor Pit-1, we examined whether a Pit-1 binding site could direct a response to Q209L-alpha q. Two copies of prolactin promoter Pit-1 binding site 1P conferred upon a heterologous metallothionein promoter a response to Q209L-alpha q, implying an involvement of this site in the transcriptional action of Q209L-alpha q on the prolactin promoter. The phorbol ester activator of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate, stimulated (-187)PRL-CAT activity, but opposed the action of Q209L-alpha q on activity of this PRL-CAT construct. Q209L-alpha q stimulation of (-187)PRL-CAT activity was inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, but not by a point mutant of Raf-C4 with reduced inhibitory properties. These results imply that activated alpha q subunits can stimulate prolactin promoter activity via a pathway that involves a Pit-1 DNA binding site(s), is opposed by protein kinase C, and is mediated by a pathway in which Raf-1 kinase plays a role.

USE OF A SELECTABLE REPORTER FOR THE ISOLATION OF MAMMALIAN REGULATORY GENES

A general method is described for isolating the genes encoding differentiation-specific activators of transcription using genetic selection. Employing regulation of the prolactin encoding gene (PRL) as a model, we have shown that the hamster dihydrofolate reductase-encoding gene (dhfr) is an effective dominant selectable reporter in this methodology. The dhfr coding region was ligated to the rat PRL promoter, and the resultant construct was stably transfected into DHFR- Chinese hamster ovary (CHO) cells, where it had little or no activity. Transfection of these cells with plasmid DNA, containing the coding region of a pituitary-specific transcription factor (Pit-1/GHF-1) in a eukaryotic expression vector, resulted in transfectants in which activation of the chimeric construct, pPRLdhfr, had occurred, enabling these cells to be selected on the basis of their DHFR+ phenotype. Our results suggest that this strategy could be used to isolate unknown genes that regulate a variety of differentiated functions.