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A purine/pyrimidine mirror repeat element (M-PMR3) in the MUC1 promoter has been shown to form H-DNA under in vitro conditions. We investigated this element for biological function in the regulation of transcription of this gene. Chloramphenicol acetyltransferase reporter-promoter constructs were prepared in which the mirror repeat element (PMR3) was intact, deleted, or modified, and their activities were evaluated by transient transfection assays into the cell lines Capan-2, PANC1, and HT-29. Deletion or modification of M-PMR3 increased expression of chloramphenicol acetyltransferase activity in MUC1-expressing cells; however, a role for an H-DNA structure in this activity was not supported by the results.

Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A approximately 90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.

We have identified two regions of non-random purine/pyrimidine strand asymmetry that were nearly identical in sequence in the 5' flanking (promoter) regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene and the human MUC1 gene. These regions contain perfect mirror repeat elements, a sequence motif previously found to be associated with the formation of H-DNA conformations. In this report we demonstrate that a single-stranded non-B DNA conformation exists at low pH in supercoiled plasmids containing the similar mirror repeat elements, and that S1 nuclease digestion maps the single-stranded region to the position of the mirror repeats. In addition, we identify a nuclear protein of approximately 27 kD that binds to single-stranded oligonucleotides corresponding to the purine-rich strand of this region, but not to the pyrimidine-rich strands or to double-stranded oligonucleotides with corresponding purine/pyrimidine strand asymmetry.

Concentration of prolactin was measured by radioimmunoassay for 11 primiparous and 16 multiparous buffalo. Prolactin concentration of blood plasma on day of insemination was about 200 ng/ml for primiparous and about 315 ng/ml for multiparous animals and fluctuated between 130 and 200 ng/ml for primiparous and between 250 and 345 ng/ml for multiparous pregnant animals. For the nonpregnant group prolactin fluctuated between 145 and 240 ng/ml for primiparous and between 210 and 310 ng/ml for multiparous animals with minor elevations 1 to 2 days before estrus. Concentration of prolactin of milk was not significantly different from that of plasma and was positively correlated .68 for nonpregnant and .93 for pregnant animals.

Radioimmunological measurements of prostaglandin F2 alpha, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), estradiol-17 beta, progesterone, prolactin and luteinizing hormone (LH) were carried out in peripheral plasma around parturition in Murrah buffaloes. The PGF2 alpha concentration fluctuated before parturition and a peak was observed 1 day prior to parturition. PGFM, estradiol-17 beta and prolactin concentration increased gradually over the last 7 days with a significant peak (P less than 0.001) 1 day before parturition. The progesterone level declined gradually and an abrupt fall occurred 1-2 days before parturition. The estradiol-17 beta and prolactin concentrations declined to basal concentrations 1-2 days post partum, whereas PGF2 alpha and PGFM remained higher than basal concentrations. The LH concentration remained low without any significant variation. The progesterone concentration was negatively correlated with PGF2 alpha (r = 0.7039; P less than 0.05), PGFM (r = -0.8322; P less than 0.01) and estradiol-17 beta (r = -0.8896; P less than 0.001) before parturition.