Imprinted and DNA methyltransferase gene expression in the endometrium during the pre- and peri-implantation period in cattle.

The endometrium plays a key role in providing an optimal environment for attachment of the preimplantation embryo during the early stages of pregnancy. Investigations over the past 2 decades have demonstrated that vital epigenetic processes occur in the embryo during the preimplantation stages of development. However, few studies have investigated the potential role of imprinted genes and their associated modulators, the DNA methyltransferases (DNMTs), in the bovine endometrium during the pre- and peri-implantation period. Therefore, in the present study we examined the expression profiles of the DNMT genes (3A, 3A2 and 3B) and a panel of the most comprehensively studied imprinted genes in the endometrium of cyclic and pregnant animals. Intercaruncular (Days 5, 7, 13, 16 and 20) and caruncular (Days 16 and 20) regions were analysed for gene expression changes, with protein analysis also performed for DNMT3A, DNMT3A2 and DNMT3B on Days 16 and 20. An overall effect of day was observed for expression of several of the imprinted genes. Tissue-dependent gene expression was detected for all genes at Day 20. Differences in DNMT protein abundance were mostly observed in the intercaruncular regions of pregnant heifers at Day 16 when DNMT3A, DNMT3A2 and DNMT3B were all lower when compared with cyclic controls. At Day 20, DNMT3A2 expression was lower in the pregnant caruncular samples compared with cyclic animals. This study provides evidence that epigenetic mechanisms in the endometrium may be involved with implantation of the embryo during the early stages of pregnancy in cattle.

Expression, regulation, and function of progesterone receptors in bovine cumulus oocyte complexes during in vitro maturation.

Progesterone (P4) exerts its effects by binding to specific genomic (nPR-A/B) and non-genomic (mPRalpha/beta, PGRMC1/2) receptors. P4 has a role in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Therefore, the aim of the present study was to characterize the protein expression of P4 receptors (PRs) in bovine oocytes and cumulus cells during in vitro maturation (IVM) and to study the effect of P4 and its receptors on oocyte developmental competence. Cumulus-oocyte complexes (COCs) were subjected to IVM, in vitro fertilization, and in vitro culture. IVM was performed for 24 h in the presence or absence of P4, luteinizing hormone (LH), follicle-stimulating hormone (FSH), trilostane, promegestone (R5020), mifepristone (RU 486), or antibodies against mPRalpha or mPRbeta. Protein expression of PRs was studied by Western blotting and immunofluorescence. The results demonstrate the presence of both genomic and nongenomic PRs in bovine COCs. The dynamic changes observed in the protein expression of PRs following IVM or in response to supplementation with LH, FSH, or P4 suggest an important role during bovine oocyte maturation. Inhibition of P4 synthesis by cumulus cells or blocking of nPR and mPR alpha activity produced a decrease in bovine embryo development, indicating that P4 intracellular signaling is mediated by its interaction with nuclear and membrane PRs and is important for oocyte developmental competence.

Effect of 5-fluorouracil substitution on the self-splicing activity of Tetrahymena ribosomal RNA.

Recent studies from several laboratories have suggested that the anticancer drug-5-fluorouracil (FUra) promotes abnormal splicing of precursor RNA molecules. In order to determine the effects of FUra on the chemistry of RNA splicing, we studied the splicing reaction of FUra-containing Tetrahymena rRNA [(FUra) RNA], a Group I self-splicing system having one intron [intervening sequence (IVS)] and two exons. When subjected to splicing conditions, the (FUra) precursor RNA gave all of the normal splicing products, ligated exons, IVS, circulation IVS (C-IVS), and the hydrolyzed circle (L-19 IVS) as well as other hydrolysis side products. No abnormal products indicative of missplicing were observed at pH 7.5. However, the presence of FUra in the RNA decreased the rates and extents of formation of all of the product species. At pH 7.5, the rate of ligated exon formation of (FUra) RNA was inhibited 3-fold and the maximum yield of ligated exons was 50% of normal. Substitution with FUra inhibited the rate of formation of C-IVS about 2-fold, while the extent of formation of this product was decreased by more than 3-fold compared to uracil-containing RNA [(Ura) RNA]. The circularization of (Ura) IVS remained constant to pH 9 and then increased, while that of (FUra) IVS declined abruptly after pH 7.3, indicating that ionization of the FUra residues of (FUra) RNA abolishes its catalytic activity. A temperature dependence experiment showed that the circularization activity of (FUra) IVS was lost at a temperature 15 degrees C lower than that of the (Ura) IVS. The labile phosphodiester bond of the (FUra) C-IVS was more stable to hydrolysis than was that of the (Ura) C-IVS at all pH values. The data suggest that a major effect of FUra substitution is to destabilize the active conformation of RNA because of weaker base pairing between FUra and adenine owing to partial ionization of the FUra residues.

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.

Characterization of the mode of binding of substrates to the active site of Tetrahymena self-splicing RNA using 5-fluorouracil-substituted mini-exons.

Splice-site selection specificity in Tetrahymena self-splicing RNA is thought to be mediated by a base-paired complex between a CUCUCU sequence on the end of the 5' exon and a GGGAGG guide sequence in the intron. The substitution of uracil (U) in oligonucleotide mini-exons with 5-fluorouracil (UF), an analogue bearing a much more acidic N-3 proton, allowed us to test the role of hydrogen bonding between complementary bases in the splice-site selection process. The affinities of (U) and (UF) mini-exons for the ribozyme active site were similar and several orders of magnitude greater than expected from base pairing alone. In contrast to CUCU, the CUFCUF mini-exon lost substrate activity with increasing pH, presumably due to ionization of the UF residues. However, the apparent pK values of these residues were several pK units above that of free UF, indicating that the mini-exon is shielded from the solvent by an active site of low polarity. Loss of the pyrimidine N-3 hydrogen bond by selective ionization of the UF residues decreased the binding of CUFCUF to the ribozyme only 3-fold but did prevent its ligation to the 3' exon. Temperature dependence of substrate activity was identical for both (U) and (UF) mini-exons, whereas the UF-substituted ribozyme lost activity at a considerably lower temperature than did the natural (U) ribozyme. These observations indicate that hydrogen-bonded base pairs involving the U residues contribute little to the total binding energy of the 5' splice site with the active site of the ribozyme, but probably help to align the splice sites properly for ligation.

Inactivation of Tetrahymena rRNA self-splicing by cis-platin proceeds through dissociable complexes.

The anti-cancer drug cis-diamminedichloroplatinum (II) (cis-DDP) reacted with Tetrahymena self-splicing rRNA ribozyme, causing loss of self-splicing activity and formation of a number of platinated RNA species. The formation of one distinct platinated product, migrating at an apparent size of 2400 nt, was closely associated with ribozyme inactivation. This platinated RNA was resistant to T1 ribonuclease digestion, suggesting the presence of inter-strand Pt cross-links. The reaction rate of cis-DDP with the ribozyme followed first order kinetics and showed a saturation effect with increasing cis-DDP concentration, characteristic of an affinity-label type of interaction rather than bimolecular collision. The apparent KI for binding of cis-DDP to the ribozyme was 62 microM. Ribozyme treated with urea was not inactivated by cis-DDP, indicating that the native structure of the RNA is required for reaction with cis-DDP. Mg++, which binds to the ribozyme and causes conformational changes in the molecule, protected the ribozyme from inactivation by cis-DDP and also prevented the formation of platinated RNA. These results suggest that binding of cis-DDP to sites formed by certain secondary or tertiary structural elements of the RNA enhance the rate and the specificity of reaction of the reagent with the ribozyme.

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.