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PubMed1999

Selectivity of polyamines on the stability of RNA-DNA hybrids containing phosphodiester and phosphorothioate oligodeoxyribonucleotides.

RNA-DNA hybrid stabilization is an important factor in the efficacy of oligonucleotide-based antisense gene therapy. We studied the ability of natural polyamines, putrescine, spermidine, and spermine, and a series of their structural analogues to stabilize RNA-DNA hybrids using melting temperature (Tm) measurements, circular dichroism (CD) spectroscopy, and the ethidium bromide (EB) displacement assay. Phosphodiester (PO) and phosphorothioate (PS) oligodeoxyribonucleotides (ODNs) (21-mer) targeted to the initiation codon region of c-myc mRNA and the corresponding complementary RNA oligomer were used for this study. In the absence of polyamines, the Tm values of RNA-PODNA and RNA-PSDNA helices were 41 +/- 1 and 35 +/- 1 degrees C, respectively, in 10 mM sodium cacodylate buffer. In the presence of a hexamine analogue of spermine at a concentration of 25 microM, the hybrids were stabilized with Tm values of 80 and 78 degrees C, for RNA-PODNA and RNA-PSDNA, respectively. The d(Tm)/d(log[polyamine]) values, representing the concentration-dependent stabilization of hybrid helices by polyamines, increased from 10 to 24 for both the RNA-PODNA and RNA-PSDNA helices. Bisethyl substitution of the primary amino groups of the polyamines reduced the hybrid stabilizing potential of the polyamines. Among the homologues of spermidine [H2N(CH2)3NH(CH2)nNH2, where n = 2-8; n = 4 for spermidine] and spermine [H)N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-8; n = 4 for spermine], spermidine and spermine were the most effective agents for stabilizing the hybrid helices. At a physiologically compatible concentration of 150 mM NaCl, the hybrid helix formed from PODNA was more stable than that formed from PSDNA in the presence of polyamines. CD spectroscopic studies showed that the hybrids were stabilized in a conformation close to A-DNA in the presence of polyamines. The relative binding affinity of the polyamine homologues for the hybrid helices, as measured by the EB displacement assay, followed the same order in which they stabilized the hybrids. These results are important in the antisense context and in the general context of polyamine-nucleic acid interactions, and suggest that pentamine and hexamine analogues of spermine might be useful in improving the efficacy of therapeutic ODNs.

Binding, Competitive↗

PubMed2006

Comparison between properties of 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) phosphorothioate oligonucleotides and N3'-P5' thiophosphoramidate oligonucleotides.

Synthesis and properties of an oligonucleotide uniformly modified with 2'-O,4-C-ethylene-bridged nucleic acid (ENA) units were compared with those of GRN163, which is modified with N3'-P5' thiophosphoramidates, with the sequence targeting human telomerase RNA subunit. Although an ENA phosphorothioate oligonucleotide, ENA-13, could be synthesized using ENA phosphoramidites on a 100-mg scale, synthesis of GRN163 was very hard even on a 1-micomol scale. In view of both stability of the duplex formation with complementary RNA and the efficiency of cellular uptake by endocytosis, ENA-13 was superior to GRN163. These findings suggest that ENA-13 has useful properties for antisense therapeutic application.

Chromatography, High Pressure Liquid↗

PubMed2003

Controlled expression of an rpoS antisense RNA can inhibit RpoS function in Escherichia coli.

We show that an inducible rpoS antisense RNA complementary to the rpoS message can inhibit expression of RpoS in both exponential and stationary phases and can attenuate expression of the rpoS regulon in Escherichia coli. Plasmids containing rpoS antisense DNA expressed under the control of the T7lac promoter and T7 RNA polymerase were constructed, and expression of the rpoS antisense RNA was optimized in the pET expression system. rpoS antisense RNA levels could be manipulated to effectively control the expression of RpoS and RpoS-dependent genes. RpoS expression was inhibited by the expression of rpoS antisense RNA in both exponential and stationary phases in E. coli. RpoS-dependent catalase HPII was also downregulated, as determined by catalase activity assays and with native polyacrylamide gels stained for catalase. Induced RpoS antisense expression also reduced the level of RpoS-dependent glycogen synthesis. These results demonstrate that controlled expression of antisense RNA can be used to attenuate expression of a regulator required for the expression of host adaptation functions and may offer a basis for designing effective antimicrobial agents.

Bacterial Proteins↗

PubMed2005

The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.

Base Sequence↗

PubMed1974

Regulation of herpesvirus macromolecular synthesis: nuclear retention of nontranslated viral RNA sequences.

We report two instances of selective accumulation of herpes simplex 1 RNA transcripts in different compartments of infected HEp-2 cells. In the first, transcripts derived from about 50% of the viral DNA accumulated in the nuclei of cells 8 hr after infection. However, only 40-42% of the DNA was represented in transcripts accumulating in both cytoplasm and polyribosomes. A more striking disparity in the distribution of transcripts between nuclei and cytoplasm occurred when viral infection was initiated and maintained for several hours in the absence of protein synthesis. RNA complementary to about 50% of the viral DNA accumulated in the nuclei, while transcripts derived from only about 10% of the DNA were detectable in the cytoplasm. The transcripts that were selectively transported in the presence of cycloheximide seem to be functional messenger RNA molecules, since they were found on polysomes immediately after cycloheximide reversal. In contrast, RNA retained in the nuclei during the period of cycloheximide treatment was not mobilized when protein synthesis subsequently resumed. The two instances of selective RNA transport observed during herpesvirus infection suggest that only viral transcripts competent to function in translation are exported from the nucleus.

Base Sequence↗

PubMed1985

Dysdifferentiative nature of aging: age-dependent expression of MuLV and globin genes in thymus, liver and brain in the AKR mouse strain.

The amount and sequence complexity of RNA transcribed by the murine leukemia virus (MuLV) genome and to the alpha- and beta-globin genes in thymus, brain and liver were measured throughout the life span of AKR mice using a cDNA X RNA hybridization technique. RNA complementary to the complete sequence of specific cDNA probes for both MuLV and globin was found in nuclei and cytoplasm of thymus, brain and liver at all ages studied. No significant age-dependent change in the amount or sequence complexity of globin RNA was detected. The amount of MuLV RNA in both nuclei and cytoplasm of thymus increased about five times from 2 to 5 months of age, but no significant change was observed over this age range in MuLV RNA from liver and brain. By the age of 10 months, most of the mice had developed leukemia and the thymus showed a further increase in the amount of MuLV RNA. Nuclear RNA from liver and brain then showed a significant increase in the amount of MuLV, but no change in the amount of MuLV was found in the cytoplasm. No qualitative age-dependent changes in the MuLV RNA sequence complexity in thymus, liver and brain were detected. These results suggest that in the AKR mouse strain for the three tissues studied, an age-dependent relaxation occurs in the repression of the MuLV genome but not for alpha- and beta-globin genes. The rate of the depression of MuLV genes in the short-lived AKR mouse strain appears similar to that of the long-lived C57BL/6J mouse strain. This age-dependent relaxation of MuLV genes appears to be limited to the nuclei. Thus, the presence of a specific regulatory system for the transport of MuLV RNA through the nuclear membrane which does not deteriorate with age is indicated.

Aging↗

PubMed1978

Identification and analysis of Dictyostelium actin genes, a family of moderately repeated genes.

Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostelium actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAse I agarose. The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15--20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated. When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.

Actins↗

PubMed1996

Aromatase activity and CYP19 gene expression in breast cancers.

The aromatase enzyme complex is responsible for the conversion of C19 androgens to oestrogens. Aromatase expression in oestrogen-responsive breast cancers may be an important mechanism of autocrine regulation in tumour growth. To evaluate whether aromatase cytochrome P450 (P450arom) transcript levels within breast tumours were correlated to the enzyme activity, a specific competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed. In this reaction, a 32 base-deleted complementary RNA was used as internal standard. In vitro aromatase activity was measured by either the tritium release assay or characterization of oestrogen fractions. Results indicate that there is a positive correlation between P450arom transcript levels and enzyme activity, but the relationship does not reach statistical significance. Therefore, whereas aromatase mRNA quantification may be an option by which to monitor the potential of tumour to synthesize oestrogens, it will not accurately reflect enzyme activity in a minority of tumours. Preliminary evidence was obtained in a tumour with low enzyme activity and a high P450arom transcript level for the presence of an endogenous aromatase inhibitor. This study highlights the necessity to characterize factors involved in the regulation of aromatase activity in such tumours.

Aromatase↗

PubMed2006

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling.

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.

Amino Acid Sequence↗

PubMed2001

[Experimental study of inhibition of hepatitis B by dual-target antisense RNA].

OBJECTIVE: To investigate the effect of retroviral vector expressing the dual--target antisense RNA of hepatitis B virus (HBV) on the replication and expression of HBV. METHODS: Recombinant retroviral vector plasmids expressing dual-target antisense RNA complementary to HBV X (1400 - 1430) and P (2375 - 2405) were constructed and transduced into 2.2.15 cells. The experimental cells were divided into PLXSN + X, PLXSN + P, PLXSN + X/P, and PLXSN + Xpos/P pos groups. The HBV expression was tested by ELISA method. HBV DNA and RNA were tested by FQ-PCR. RESULTS: The inhibition rates of 2.2.15 cells transduced with recombinant vector plasmids (PLXSN + X group, PLXSN + P group, especially the PLXSN + X/P group) on expression of HBV DNA and RNA were higher than those of blank control group, PLXSN group, and PLXSN + Xpos/Ppos group. The expression of the three ORFs, S, C, and P were significantly inhibited. CONCLUSION: The expression of HBV antisense RNA in 2.2.15 cells can inhibit the replication and expression of HBV. The inhibitory effect of dual antisense-RNA group is higher than that of single antisense-RNA group. Dual antisense RNA has the potentiality in anti-HBV gene therapy.

3T3 Cells↗

PubMed1988

A study of myc-related gene expression in small cell lung cancer by in situ hybridization.

The expression of myc-related genes (c-myc, N-myc, and L-myc) in small cell lung cancer (SCLC) was studied by RNA-RNA tissue in situ hybridization. The tissues investigated included cytospins of ten cell lines derived from patients with SCLC, four corresponding nude mouse xenografts from cell lines, and metastatic tumor tissue obtained by surgical biopsy and at autopsy. The probes were prepared as 35S labeled complementary RNA. The expression of each gene was demonstrated specifically by autoradiography in the cytoplasm of the neoplastic cell samples. The average levels of oncogene expression in each specimen corroborated previous data obtained by Northern blot assays. In addition, heterogeneity in gene expression from cell to cell in each sample was noted. This study represents the first attempt to demonstrate oncogene expression in lung cancer cell lines and tissues in situ, and confirms that the expression of these myc related genes can be seen in the primary tumor. The technique of RNA-RNA tissue in situ hybridization has great potential in answering fundamental questions of tumor cell heterogeneity and progression in SCLC. It should be useful in both prospective and retrospective studies.

Carcinoma, Small Cell↗

PubMed1990

NMR studies of the active site of DNA polymerase I and of a 50-residue peptide fragment of the enzyme.

Transferred nuclear Overhauser effects (NOEs) and selective T1 measurements were used to determine interproton distances in the substrates Mg2+dATP and Mg2+TTP bound to the large fragment of DNA polymerase I (Pol I). The distances are consistent with high anti, O1' endo conformations for the enzyme-bound substrates, similar to nucleotides of B-DNA. These substrate conformations show little or no change when the complementary RNA templates (rU)57 or (rA)50 are bound. In contrast, multiple conformations, including syn and anti species, are required to fit the interproton distances measured on the enzyme-bound guanine nucleotide substrates Mg2+dGTP and Mg2+ddGTP. These multiple substrate conformations simplify to a single high anti, O1' endo conformation when the complementary template (rC)37 is bound, possibly due to base-pairing with the template, as in the active complex. In the presence of both template and primer, enzyme-bound Mg2+ddGTP reverts to multiple conformations. This ability of Pol I to decrease the fraction of bound substrate which is appropriate for primer elongation may be an error-preventing mechanism. In all cases, the conformations of the average nucleotide of the enzyme-bound RNA templates are also B-like. Transferred NOEs from protons of the enzyme to those of bound dNTP substrates suggest hydrophobic (Ile, Leu) and an aromatic amino acid (Tyr) at the substrate binding site. Peptide I, a synthetic 50-residue peptide based on residues 728 to 777 of the Pol I sequence, containing the conserved sequence L-I-Y-G, retains significant secondary and tertiary structure in solution as found by circular dichroism (CD) and 2D NMR. While the X-ray structure shows 48% helix in this region, the sequence specific NOESY analysis suggests 18% helix, and the preservation of two of the three beta turns. Peptide I shows tight binding of dNTP substrates, the substrate analog 2',3'-trinitrophenyl-ATP, and duplex DNA, providing direct evidence that the active site for polymerization lies in this region of the enzyme, with the substrate binding along the O-helix near Leu-764, Ile-765, and Tyr-766. Another synthetic peptide, peptide II, based on residues 840 to 888 of the Pol I sequence also retains much secondary structure as detected by CD but does not bind the substrate analog TNP-ATP.

Adenosine Triphosphate↗

PubMed1987

Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212) we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive- and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive- and negative-strand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg++, was inhibited by Mn++, and was unaffected by the addition of Zn++. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and alpha-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNA-dependent RNA synthesis in differentiated cells and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.

Animals↗

PubMed1991

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA). In vitro translation of transcripts derived from cDNA clones representing regions of each ORF was used to identify the respective proteins. A ca. 22,000 Mr protein was translated from the 591-nt ORF transcript. This protein comigrated in SDS-PAGE with a protein produced by in vitro translation of MStV RNA and is referred to as the NS3 protein. The protein from the 948-nt ORF transcript was specifically immunoprecipitated by antiserum to the MStV nucleocapsid protein (N protein). RNA hybridization analyses identified two subgenomic RNAs in total RNA extracts from MStV-infected Zea mays plants. These RNAs are ca. 650 and 1350 nt in size, are of opposite polarities, and correspond to regions of RNA3 containing the two ORFs. These data suggest that MStV RNA3 has an ambisense coding strategy similar to that found for the S RNA of the vertebrate-infecting phleboviruses, uukuviruses, and arenaviruses, as for tomato spotted wilt virus.

Amino Acid Sequence↗

PubMed2000

Synthesis and biophysical properties of arabinonucleic acids (ANA): circular dichroic spectra, melting temperatures, and ribonuclease H susceptibility of ANA.RNA hybrid duplexes.

Arabinonucleic acid (ANA), the 2'-epimer of RNA, was synthesized from arabinonucleoside building blocks by conventional solid-phase phosphoramidite synthesis. In addition, the biochemical and physicochemical properties of ANA strands of mixed base composition were evaluated for the first time. ANA exhibit certain characteristics desirable for use as antisense agents. They form duplexes with complementary RNA, direct RNase H degradation of target RNA molecules, and display resistance to 3'-exonucleases. Since RNA does not elicit RNase H activity, our findings establish that the stereochemistry at C2' (ANA versus RNA) is a key determinant in the activation of the enzyme RNase H. Inversion of stereochemistry at C2' is most likely accompanied by a conformational change in the furanose sugar pucker from C3'-endo (RNA) to C2'-endo ("DNA-like") pucker (ANA) [Noronha and Damha (1998) Nucleic Acids Res. 26, 2665-2671; Venkateswarlu and Ferguson (1999) J. Am. Chem. Soc. 121, 5609-5610]. This produces ANA/RNA hybrids whose CD spectra (i.e., helical conformation) are more similar to the native DNA/RNA substrates than to those of the pure RNA/RNA duplex. These features, combined with the fact that ara-2'OH groups project into the major groove of the helix (where they should not interfere with RNase H binding), help to explain the RNase H activity of ANA/RNA hybrids.

Arabinonucleosides↗

PubMed1978

Nucleotide sequences at the 5' termini of influenza virus RNAs and their transcripts.

The results of analyses of the 5'-terminal sequences of Fowl Plague virus RNAs are presented. The first 13 residues of each of the eight RNA molecules which constitute the genome are in the identical sequence 5'AGUAGAAAUUAGG- and this conservation of sequence is shown to extend to other influenza viruses. The 5'-terminal sequences of virion RNA transcripts produced in vitro are also reported and again the first 12 nucleotides of these are identical for all influenza type A transcripts examined in the sequence 5'AGCAAAAGCAGG-. In addition the results of attempts to determine the sequence relationship between vRNAs and the two classes of complementary RNA synthesized in influenza infected cells are described which support the conclusion that influenza messenger RNAs are incomplete transcripts.

Base Sequence↗

PubMed1996

Regulation of corticotropin-releasing factor receptor messenger ribonucleic acid in rat anterior pituitary.

Adrenalectomy (ADX) leads to a decrease in the number of CRF-binding sites in the rat anterior pituitary (AP). However, the molecular mechanisms of CRF receptor (CRF-R) regulation are unknown. In the present study, we analyzed the effects of ADX on pituitary CRF-R1 messenger RNA (mRNA) levels in vivo and the direct effects of CRF, arginine vasopressin (AVP), and glucocorticoid, the levels of which are altered by ADX, on CRF-R1 mRNA levels in vitro. The mRNA level was determined by Northern blot analysis using a rat brain CRF-R1 complementary RNA probe. The CRF-R1 level in AP fell to 20% of the sham level 1 day after ADX and returned to the sham level after 14 days. In cultured rat AP cells, treatment with CRF, AVP, and dexamethasone led to significant reductions in CRF-R1 mRNA, with maximal inhibition to 32%, 22%, and 37% of control levels, respectively. The time course of CRF-R1 mRNA reduction varied depending on the drug, with effects detectable as early as 1 h after treatment. These findings indicate that elevated portal CRF and AVP levels may contribute to the decrease in CRF-R1 mRNA soon after ADX. A decrease in mRNA levels, in turn, may lead to a decrease in CRF-R1 protein on corticotrophs.

Adrenalectomy↗

PubMed1992

Relative expression of aromatase cytochrome P450 in human fetal tissues as determined by competitive polymerase chain reaction amplification.

The aromatase enzyme complex is responsible for the conversion of C19 steroids to estrogens. Aromatase activities ranging from moderate to very low have been measured in human fetal tissues. The inability to demonstrate aromatase cytochrome P450 (P450AROM) messenger RNA (mRNA) in several fetal tissues by northern blotting has been attributed to low levels of specific message. In order to identify and compare P450AROM mRNA levels in fetal tissues, we developed a specific competitive polymerase chain reaction amplification technique. This reaction uses coamplification of a rat P450AROM complementary RNA to normalize differences in amplification efficiencies. Using this technique, P450AROM mRNA was identified in all fetal tissues studied including; liver, lung, brain, skin, intestine, kidney, spleen, and heart. Fetal liver contained far more P450AROM mRNA per total RNA than any other tissues studied. Fetal brain and intestine also tended to have slightly higher levels than other tissues.

Aromatase↗