Three types of iodothyronine deiodinase have been identified in vertebrate tissues. cDNAs for the types I and III have been cloned and shown to contain an inframe TGA that codes for selenocysteine at the active site of the enzyme. We now report the cloning of a cDNA for a type II deiodinase using a reverse transcription/ polymerase chain reaction strategy and RNA obtained from Rana catesbeiana tissues. This cDNA (RC5DII) manifests limited but significant homology with other deiodinase cDNAs and contains a conserved in-frame TGA codon. Injection of capped in vitro synthesized transcripts of the cDNA into Xenopus laevis oocytes results in the induction of deiodinase activity with characteristics typical of a type II deiodinase. The levels of RC5DII transcripts in R. catesbeiana tadpole tail and liver mRNA at stages XII and XXIII correspond well with that of type II deiodinase activity but not that of the type III activity in these tissues. These findings indicate that the amphibian type II 5-deiodinase is a structurally unique member of the family of selenocysteine-containing deiodinases.Intracellular concentrations of the thyroid hormones, T 4 1 and T 3 , are profoundly influenced by the activity of three iodothyronine deiodinases, classified as types I, II, and III (1). In mammals, the type I enzyme (5DI) catalyzes 5Ј-deiodination (5ЈD), the removal of iodine from the 5Ј (or 3Ј) positions of T 4 and its derivatives. The enzyme can also catalyze 5-deiodination (5D), the removal of an iodine located at either the 5 (or 3) positions of iodothyronines, but does so efficiently only with sulfated iodothyronine substrates (2). The type II enzyme (5DII) also catalyzes 5ЈD, but it is readily distinguished from the 5DI by its kinetics, substrate specificity, sensitivity to propylthiouracil (PTU) and aurothioglucose (AThG) (1, 3), and response to thyroid status (1). The type III enzyme (5DIII) catalyzes primarily 5D activity (1), a process that results in derivatives with little or no thyromimetic activity (1).The primary function of the types I and II deiodinases is to convert T 4 to its metabolically more active derivative, T 3 . However, the tissue distribution and physiological roles of the two enzymes are very different. The principal role of the 5DI in mammals is to provide a source of plasma T 3 by deiodination of T 4 in peripheral tissues such as liver and kidney. In contrast, the 5DII is responsible for the majority of the intracellular T 3 in tissues such as the pituitary, brain, and brown fat by mediating local deiodination of T 4 and is considered to be of major importance in regulating thyroid hormone action in these tissues (1, 3). The 5DII also plays a major role during development. 5DII is the principal 5Ј-deiodinase expressed in the mammalian fetus, and it is notable that 5DII activity in brain peaks in the neonatal period, the time that is critical for thyroid hormone-dependent development in this tissue (4). Moreover, 5DII is the only 5Ј-deiodinase present in the developing frog in which the orderly prog...

Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.

Accurate quantification of gene transfer (or gene correction) is a universal challenge in the field of gene therapy. In developing a clinical trial of lymphocyte gene therapy for Hunter syndrome (mucopolysaccharidosis type II), methods using Southern blot or automated DNA sequencing technology were employed, but found to be laborious and subject to considerable variation. As an alternative approach, we explored a real-time kinetic PCR assay appropriate to new instrumentation (PE Biosystems model 7700). A TaqMan probe was designed to hybridize directly across the exon 2-exon 3 junction of the iduronate-2-sulfatase transgene cDNA. In this assay system, cDNA from the retroviral vector L2SN generates a PCR product that is 84 nucleotides long and readily quantified by TaqMan probe binding and subsequent cleavage. Evaluation of this method demonstrated sensitivity over at least 5 logs with respect to the standard (vector plasmid pL2SN). There was no detectable signal from genomic DNA from nontransduced cells, thus indicating the specificity of this assay. The sample preparation method used to prepare specimens was a relatively simple cell lysis procedure, without DNA extraction, and represents a significant advancement over the more complex methods of DNA extraction that are typically used for such assays. This specific assay, and comparison to previous methods, illustrates the utility of a new method that is readily generalized to many gene therapy studies, and that has the potential to be extended to measure gene expression by means of quantitative RT-PCR.

In vivo and in vitro (static incubation and perifusion) procedures were used to examine the role of insulin-like growth factors (IGFs) in growth hormone (GH) feedback. An α₂-adrenergic agonist, clonidine (CLON; 2 × 10^-8M in vitro or 30 µg/ml/kg body weight i.v. in vivo), which mimics the hypothalamic mechanism triggering GH release, was injected to induce a GH surge. Feedback was initiated by human GH (hGH; 2 × 10-⁶M) in vitro or ovine GH (oGH) (20 µg/2 µl intraventricularly) in vivo. GH-releasing factor (GRF; 1 × 10^–8M) was added at the end of in vitro experiments to test pituitary responsiveness. The involvement of somatostatin (SRIF), GRF and IGFs in mediating GH feedback was evaluated in hypothalamic-pituitary coperifusion. CLON-induced GH release in this system was associated with increased GRF and decreased SRIF release, and the pattern was reversed by hGH. The influence of hGH was mimicked by IGF-I (1.5 × 10^–8 M), except that the GH release was depressed below baseline levels, suggesting a direct effect of IGF-I on the pituitary. Furthermore, the inhibitory effect of hGH on the CLON-induced GH surge and hypothalamic releasing factors (increased SRIF and decreased GRF) was reversed by antisera to IGF-I (1:100), IGF-II (1:100), or both. To determine whether IGF-I is released from hypothalamus or pituitary in response to GH, tissues were tested separately in static incubation. As compared with basal levels, incubation of hypothalami with hGH increased IGF-I and SRIF and decreased GRF release. Because GH and IGF-I release remained unchanged when pituitaries were incubated alone with hGH, the site of IGF-I release and GH feedback is most likely at the hypothalamic level. To evaluate the role of IGFs on GH feedback in vivo, male rats were prepared with permanently implanted 3rd-ventricular and jugular cannulae. CLON was administered intravenously, and oGH, IGF-I (0.5 µg/2 µl), and IGF-I and -II antisera (1:100) were injected intraventricularly. In this as in in vitro studies, IGF-I mimicked the inhibitory feedback effect of GH on the CLON-induced GH surge, and IGF antisera blocked GH feedback. We propose that these studies suggest that endogenous hypothalamic IGF-I mediates the influence of GH in the feedback mechanism by increasing SRIF and depressing GRF release.

From studies with their cDNAs, the types 1 and 3 deiodinases (D1 and D3) have been shown unequivocally to be selenoproteins. Studies with recently cloned cDNAs for the mammalian type 2 deiodinase (D2) indicate that they also code for selenoproteins. However, these D2 cDNAs are not full length and they do not contain an essential selenocysteine insertion sequence (SECIS) in their 3'UTR; a heterologous SECIS had to be ligated to the coding region before expression of the D2 could be achieved. Thus their role as cDNAs for the native D2 is open to question. We now report the cloning of a 5.8 kb cDNA for the mouse D2. This cDNA contains a SECIS in its 3'UTR located more than 4.5 kb from the coding region. When the mRNA transcribed in vitro from this cDNA is injected into X. laevis oocytes, a deiodinase with characteristics of D2 is expressed.