The role of thyroid hormone in the regulation of hepatic carbamyl phosphate synthetase activity in Rana catesbeiana.

Both spontaneous and thyroid hormone (TH)-induced metamorphosis of Rana catesbeiana are accompanied by a marked increase in the activity of the urea cycle enzyme carbamyl phosphate synthetase (CPS). The increase induced by exogenous TH is de novo synthesis of enzyme and appears to be secondary to an increase in the CPS mRNA level resulting from the elevated plasma TH. Since endogenous TH levels rise sharply during spontaneous metamorphosis, a similar sequence of events would be anticipated. However, after midclimax, CPS activity continues to increase, while plasma TH levels steadily decline, suggesting that other factors are involved. To obtain insight into this problem, the changes in CPS mRNA level during spontaneous development were determined using a mammalian CPS cDNA probe and correlated with changes in CPS activity and plasma T3 concentration. CPS mRNA level and CPS activity were barely detectable until midprometamorphosis, but both increased rapidly during the latter half of this phase. CPS activity continued to rise, reaching a maximum in the adult frog. The CPS mRNA level, however, was highest during the first half of climax, but declined after midclimax and was relatively low in the adult frog. Studies were also performed in which the rise and fall in the plasma T3 concentration typical of metamorphic climax were induced by exposure of premetamorphic tadpoles to T3, followed by its withdrawal. Both CPS activity and CPS mRNA level were induced by T3, but when plasma T3 levels fell after removal of the exogenous T3, CPS mRNA level, but not CPS activity, also decreased. Additional studies indicated that the TH-induced increase in CPS mRNA was evident within 24 h, could be prevented by simultaneous injection of actinomycin-D, and could not be induced in tadpoles undergoing climax; in this phase the T3 receptors are fully occupied with endogenous TH. When premetamorphic tadpoles were immersed in T3-containing water (0-500 nM) for 6 days, CPS mRNA, CPS activity, and plasma T3 concentration increased in parallel, reaching a maximum at 50 nM. At 50 nM T3, the plasma T3 level was sufficient to saturate the receptors, and no additional increase in CPS mRNA level or CPS activity was obtained at higher concentrations of T3. These studies indicate that the CPS mRNA level during spontaneous development correlates with the plasma T3 concentration and suggest that it is a function of T3 receptor occupancy. The data are also consistent with an effect of TH on transcription of the CPS gene and with a relatively long half-life of its protein product, the CPS enzyme.

Regulatory elements near the Drosophila hsp 22 gene required for ecdysterone and heat shock induction.

A transient expression assay was used to localize cis-acting DNA regulatory elements near the Drosophila heat shock protein (hsp) 22 gene, that are involved in heat shock expression and in ecdysterone-induced expression. The results identify a region between positions -320 and -232 that is essential for ecdysterone control, but not for heat-induced expression, and a sequence between -199 and -156, which, when deleted, leads to the loss of heat shock induction. To investigate the function of these DNA sequences, transfection-competition experiments were carried out. The evidence suggests that the DNA regulatory sequences identified by transient expression studies contain binding sites for transacting transcription factors.

Molecular cloning by hybrid arrest of translation in Xenopus laevis oocytes. Identification of a cDNA encoding the type I iodothyronine 5'-deiodinase from rat liver.

The enzyme(s) catalyzing the 5'-monodeiodination of thyroxine and 3,3',5'-triiodothyronine has not yet been purified, and antibodies of demonstrated specificity are not available. Thus, molecular cloning strategies which rely on the traditional screening techniques of using cDNA probes or monospecific antibodies are problematic. We previously reported that expression of type I 5'-deiodinase can be induced in Xenopus laevis oocytes by the injection of poly (A)+ RNA prepared from rat liver (St. Germain, D.L., and Morganelli, C.M. (1989) J. Biol. Chem. 264, 3054-3056). Using this expression system, we developed a hybrid arrest assay, and with it identified a 241-base pair cDNA which encodes part of this enzyme. The cDNA inhibits translation of 5'-deiodinase activity in oocytes by greater than 99% and 5'-deiodinase mRNA from rat liver poly(A)+ RNA in hybrid selection experiments. The cDNA hybridizes to a 1.9-kilobase RNA species on Northern analysis and demonstrates no significant homology to any previously cloned protein. The application of this hybrid arrest strategy for molecular cloning may prove useful for the isolation of cDNAs for proteins that are low in abundance, difficult to purify, or are subunits of a polymeric functional unit.

Expression of type I iodothyronine 5'-deiodinase in Xenopus laevis oocytes.

The enzymes mediating the conversion of thyroxine to 3,5,3'-triiodothyronine have proved difficult to purify and characterize biochemically. In this report we describe the successful expression in Xenopus laevis oocytes of type I iodothyronine 5'-deiodinase. Oocytes injected with rat liver mRNA and then incubated for 5 days demonstrated a progressive increase in 5'-deiodinase activity. This activity: (a) manifested a Km for 3,3',5'-triiodothyronine of 0.24 microM, (b) was sensitive to inhibition by 6-n-propyl-2-thiouracil, and (c) was associated with the membrane fraction of oocyte homogenates. Size fractionation of the mRNA by agarose gel electrophoresis under non-denaturing conditions resulted in the identification of a 1.5-2.0-kilobase fraction capable of inducing type I 5'-deiodinase activity. This oocyte assay system should provide a mechanism for identifying cDNA(s) encoding the enzymes involved in the peripheral metabolism of thyroid hormones.

Natural and synthetic heat shock protein gene promoters assayed in Drosophila cells.

Hybrid genes containing mRNA encoding sequences for herpes virus thymidine kinase (tk), chloramphenicol acetyltransferase (CAT), or Drosophila alcohol dehydrogenase (Adh), ligated to truncated Drosophila melanogaster heat-shock protein 70 (hsp 70) gene promoters or to synthetic sequences containing one or several copies of a previously defined heat-shock consensus sequence, were transfected into cultured Drosophila line S3 cells. Each construction was then assayed for gene expression at 25 degrees C and 37 degrees C, using a CAT enzyme assay, slot blot hybridization, or S1 nuclease protection analysis. In the Drosophila cell transient expression assay system, we found that deletions extending beyond position -97, or synthetic constructions containing a single heat shock consensus sequence, were not induced by high-temperature shock. In constructions containing deletions extending to position -186, -130, or -97, in the hsp 70 promoter, and in synthetic constructions containing tandemly spaced heat-shock consensus sequences mRNA transcription was greatly induced by high temperature.

Deletion polymorphism in a Drosophila melanogaster heat shock gene.

We have continued the transcriptional analysis of the region of cytological locus 67B that contains the four small heat shock genes and other genes. Transcription from one of the heat shock genes in the region, hsp 26, takes place during high temperature treatment and at certain developmental stages, without heat shock, in several tissues, such as imaginal discs and adult ovaries. Observations of unexpected products after nuclease protection experiments provided the first indication of what genomic blot experiments showed to be small deletions. The alleles containing the deletion are expressed at the same level as the wild type allele. The deletion shortens the protein product, implying that it is in the coding region. Furthermore, flies homozygous for one of the deletion alleles are viable.

Transcript length heterogeneity at the small heat shock protein genes of Drosophila.

Expression of the small heat shock protein (hsp) genes can be induced in cultured Drosophila cells by high temperature shock and by exposure to physiological doses of the insect molting hormone ecdysterone. Northern blot analysis was performed in order to compare the size of small hsp transcripts synthesized in response to these two stimuli. Transcripts from several other genes were also examined. Two types of length heterogeneity were observed for the small hsp gene transcripts. One involved the synthesis of what are designated as long form transcripts during heat shock; small hsp messenger RNAs extended at the 3' end by some 1.5 X 10(3) base-pairs. The second type of size heterogeneity observed is based on differences in the length of the poly(A) tail. The results of S1 nuclease protection analysis provided evidence that different initiation sites are not used for hsp 22 mRNA transcription in response to the two stimuli.

Transcription of Drosophila small hsp-tk hybrid genes is induced by heat shock and by ecdysterone in transfected Drosophila cells.

Hybrid genes containing the 5' region of Drosophila heat shock protein (hsp) genes, ligated to the herpesvirus thymidine kinase (tk) gene, were transfected into Drosophila line S3 cells. Constructions containing sequences upstream from hsp70, or from any of the small hsp genes, showed heat-inducible tk expression. Ecdysterone-inducible tk expression was seen only in transfections with small hsp-tk hybrid genes. Plasmids containing a synthetic "heat shock consensus sequence" or a deletion of the hsp23 upstream region were totally inactive in transfection studies.

Drosophila cells and ecdysterone: a model system for gene regulation.

When Drosophila cell lines are exposed to physiological doses of the steroid molting hormone, ecdysterone, they enter mitotic arrest and differentiate morphologically. These responses are accompanied by specific changes in gene expression. Several enzyme activities (acetylcholinesterase, beta-galactosidase, dopa decarboxylase, and catalase) are induced and the synthesis of a cytoplasmic actin and the four small heat-shock proteins is initiated. Several of these ecdysterone inducible genes have been physically isolated and characterized, in several cases by DNA sequencing. Current studies focus on introducing cloned ecdysterone inducible genes into responsive cells by DNA mediated transfection. Once it is clear that these introduced genes acquire the normal pattern of hormone-regulated gene expression in the cell, in vitro mutagenesis can be used before transfection to modify their structure. Transient expression, then, can become a functional assay to define regions of DNA flanking the coding region of inducible genes that are needed for proper gene expression and regulation in cultured cells.

Transient expression of homologous genes in Drosophila cells.

A cloned Drosophila heat shock protein 22 gene was transfected into two independently established Drosophila cell lines. Each line carried a different heat shock protein 22 allele, distinguishable by electrophoresis of the protein. The transfected gene was not expressed at 25 degrees C but could be induced at 36 degrees C. In one line, two heat shock protein 22 electromorphs were synthesized.

Stimulation of cytoplasmic actin gene transcription and translation in cultured Drosophila cells by ecdysterone.

When cultured Drosophila line S3 cells are incubated in the presence of the steroid hormone, ecdysterone, they flatten, elongate, and become motile. We show here that accompanying this morphological transformation there is a 5-fold increase in the rate of actin synthesis and a 2-fold increase in actin content. These increases, in turn, are primarily based on a 9-fold increase in the level of mRNA transcribed from the cytoplasmic actin A3 gene. We also find that during hormone treatment the cells go into proliferative arrest, accumulating in the G2 phase of the cell cycle. During this period the level of histone mRNA in the cells decreases significantly.