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In this report, we have examined the effect of aging on thyroid hormone regulated S14 gene expression by comparing levels of the mRNA in the liver of young (6-month) and aged (26-month) rats of various thyroid states. Although levels of mRNA-S14 were stimulated by L-triiodothyronine (T3) in both young and aged animals, net activity of the gene in response to the hormone was reduced approximately 2-fold in the aged rats. Next, we wondered whether the effect of age and T3 on levels of the mRNA correlated with changes in the DNA binding activity of two transcription factors, PS-1 and P1 that increase and decrease, respectively, S14 gene activity. The DNA binding activity of PS-1 correlated closely with both age-related and T3-induced S14 mRNA expression. Whereas the DNA binding activity of P1 was significantly reduced in aged rats, T3 failed to influence the activity of P1. Based on these observations, we postulated that age-related increases in S14 gene expression arise from combined changes in activity of the transcription factors, PS-1 and P1. However, T3 regulated S14 gene activity is more closely related to PS-1.

In parallel, we measured the receptor binding affinities for epidermal growth factor-urogastrone (EGF-URO) and transforming growth factor-alpha (TGF-alpha) in cultured smooth muscle (GCM) and epithelial (GPC) cells derived from guinea pig intestine. The relative order of binding affinities in the GCM cells was TGF-alpha > EGF-URO, in keeping with the relative order of biological potencies of these polypeptides in a guinea pig gastric circular muscle contractile bioassay. These data established by ligand binding criteria the presence of a TGF-alpha-preferring receptor in the guinea pig. In contrast, there was a reversed order of binding affinities (EGF-URO > TGF-alpha) for the polypeptides in GPC cells, in accord with an identical order of bioassay potencies previously observed in a guinea pig gastric longitudinal muscle contractile bioassay. Using a reverse transcription-polymerase chain reaction approach, we also cloned and sequenced putative EGF-URO receptor ligand binding domain III from each cell type. Although the binding specificity for TGF-alpha and EGF-URO differed in the GCM and GPC cells, the amino acid sequences of receptor domain III were identical in the two cell types. We conclude that the previously measured differences in biological potencies of EGF-URO and TGF-alpha in the contractile bioassay preparations are due to the distinct receptor binding affinities of EGF-URO and TGF-alpha that can be detected in different tissues. However, our data document that the distinct relative binding affinities for EGF-URO and TGF-alpha that can be observed in different cell types from the same species cannot be accounted for solely by the sequence of putative receptor ligand binding domain III.

Background: The known catabolic effects of glucocorticoid excess on protein metabolism prompted us to devise a method to assess this measure in reproductive-aged females with Cushing's disease. Since collagen protein is a major component of skin, decreased abundance of this protein should cause a reduction in skin-fold thickness. To determine whether skin-fold thickness is useful as an added tool in the diagnosis of Cushing's disease, we compared this value in female patients with Cushing's disease with those who presented with a similar set of symptoms., Methods: This open prospective study was conducted in an endocrinology clinic at a tertiary care center. The study population consisted of 88 females in the reproductive age group who presented to the clinic with hirsutism, oligomenorrhea, and/or obesity. Measurement of skin-fold thickness, body mass index, Ferriman-Gallwey index, and serum testosterone were performed in all patients., Results: Skin-fold thickness in the patients with Cushing's disease was 1.5 +/- 0.2 mm (range, 1.0 to 1.8 mm). This value was significantly (P < .01) lower than that in controls or subjects with other disorders that have a similar set of presenting symptoms., Conclusions: Bedside assessment of skin-fold thickness is an easy, low-cost, and noninvasive test for distinguishing Cushing's disease from disorders with similar presenting symptoms in females of reproductive age. Assessment of skin-fold thickness should be used as an adjunct to current physical and biochemical study of patients with symptoms suggestive of Cushing's disease.

We have achieved efficient electro-optic phase modulation at high frequencies in a resonant modulator cavity. We enhance modulation by matching the phase velocities of the optical and microwave fields in the modulator substrate and by placing the modulator inside an optical cavity that is resonant for the input optical beam and the generated sidebands. An optical frequency comb with a span of 3 THz and at a spacing of 17 GHz is generated with 1 W of microwave power. The terahertz comb is utilized to phase lock an optical parametric oscillator at a signal-idler difference frequency of 665 GHz.

Thyroid hormones may stimulate or repress transcriptional activity depending upon the specific gene. Whereas, a palindromic DNA sequence, TREpal, mediates positive regulation by thyroid hormone, the negative response element (nT3RE) remains undefined. Therefore, we have examined the DNA sequences that mediate the inhibitory effects of thyroid hormone on the transcription of the beta-subunit gene of rat thyrotropin (rTSH beta). In rat pituitary tumor cells (GH3), transient expression of plasmid constructs containing the putative nT3RE of rTSH beta mediated negative regulation by L-triiodothyronine (T3). Since this nT3RE contained sequences which resembled a half-site motif of the consensus T3RE and the idealized palindrome (TREpal), we tested a construct containing this half-site motif in the same cells. T3 decreased the activity of this plasmid. Cotransfection studies in T3-receptor (T3R)-deficient cells indicated that either alpha or beta isoforms of T3R were required for the inhibitory effects of the hormone. Both T3R isomers bind to DNA sequences containing the nT3RE from rTSH beta DNA or the half-site motif of TREpal. In summary, our results show that the repressive properties of T3 are mediated by a nT3RE from rTSH beta. Unexpectedly, this motif resembles a half-site component of TREpal which enhances promoter activity in response to T3.

In this report, we have measured the cadmium (Cd2+)-induced expression of all known metallothionein I (MT-I) mRNAs in a human hepatoma cell line, Hep G2. Among the human MT-I gene family promoters, marked sequence conservation exists; despite this, the mRNA accumulation level for each species was found to be quite unique. This differential Cd2+ induction of MT-I family members provides an ideal opportunity to assess whether the characteristic response results from subtle isoform-specific variations in promoter structure. Accordingly, we have examined the mechanism for differential expression of two isoforms, MT-IG and MT-IF, by transient transfection into Hep G2 cells. In the presence of Cd2+, MT-IG promoter activity and endogenous mRNA level were, respectively, 4.7- and 3-fold greater than those of MT-IF. This close correlation between promoter activity and mRNA accumulation strongly suggests that differential expression occurs at the level of transcription. The difference in Cd(2+)-stimulated activity was found to be conferred by 240- and 243-base pair promoter fragments spanning nucleotides -174 to +66 and -172 to +71 of the MT-IG and MT-IF genes, respectively. One of the most striking nonhomologies between the promoters is a single A (TATAAA) to C (TATCAA) transversion in the TATA motifs of MT-IG and MT-IF genes, respectively. To determine whether such a subtle change in the TATA motif could account for the marked differences in promoter function, we constructed MT-IG-TATCA and MT-IF-TATAA promoters and measured their activities in transient transfection and cell-free transcription assays. Results of both assays showed a profound difference between the two motifs that paralleled the difference in Cd(2+)-stimulated MT-IG and MT-IF mRNA levels. In summary, we have shown that differential regulation of two MT-I promoters is primarily due to a single base alteration in their TATA motifs.

P1 is a nuclear protein found exclusively in rat liver and binds to a motif that spans nucleotides -310 to -288 of the thyroid hormone responsive gene, S14. We expect P1 to play an important role in regulating gene expression because the binding motif for this factor is contained within a DNase I-hypersensitive site of S14 chromatin. In this report, we have attempted to define the function of P1 by correlating its DNA binding activity with levels of mRNA-S14 in response to aging and obesity. Results of all studies revealed inverse relationships between the activity of P1 and levels of mRNA-S14, thus suggesting that P1 may function as a repressor of S14 gene expression. Accordingly, we tested the repressor hypothesis using cell-free transcription and transient transfection assays to measure the activity of reporter constructs with and without the P1 binding motif. In the presence of the P1 motif, S14 promoter activity was repressed and the negative effect on gene transcription was further enhanced by thyroid hormone. These observations are consistent with P1 being a repressor of S14 gene transcription.

Apolipoprotein A1 (Apo A1) is the major protein component of high density lipoprotein (HDL) particles. HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo A1 will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo A1 promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo A1 gene.

We have previously identified a rat hepatonuclear factor, PS-1 that binds to the thyroid hormone responsive gene, S14. To determine whether PS-1 is involved in regulating tissue-specific expression of the S14 gene, we have correlated the DNA binding activity of PS-1 with mRNA-S14 expression in a variety of tissues. Gel retardation analysis revealed a pattern of binding to the recognition site that was characteristic of tissues with high levels of mRNA-S14, a different pattern was found in tissues which do not express the gene. Competition studies using mutant oligonucleotides showed that the first 4 nucleotides and the CAAT motif contained within the PS-1 recognition sequence are essential for protein binding. C/EBP, a CCAAT-transcription factor binds to the PS-1 recognition site thus raising the possibility that both C/EBP and PS-1 may belong to the same family of proteins. Next we used a cell-free transcription assay to measure activity of a template, pS14-GFC(-72), that contained the PS-1 sequence. The pS14-GFC(-72) template was active in hepatonuclear extracts but deletion of or competition with the PS-1 binding sequence rendered the construct inactive. A template containing three PS-1 binding sequences increased S14 promoter activity by 12- to 13-fold. In nuclear extracts from spleen and testis, relative S14 promoter activity was only 2% of that in the liver, this observation mimicked closely in vivo expression of the gene. Mixing together extracts from liver and spleen in varying proportions, prior to incubation with S14 template, yielded a linear increase in S14 promoter activity that correlated with the amount of liver extract in the reaction. This finding is consistent with the absence of an essential factor or factors in spleen that is/are required for S14 promoter activity in vitro. In summary, PS-1 binds to a DNA sequence that contains a CAAT motif and appears to play a critical role in determining tissue-specific activity of the S14 promoter in vitro.

Objective: To determine whether simultaneous ingestion of ferrous sulfate and thyroxine reduces the efficacy of thyroid hormone in patients with primary hypothyroidism., Design: Uncontrolled clinical trial., Setting: Outpatient research clinic of a tertiary care center., Patients: Fourteen patients with established primary hypothyroidism on stable thyroxine replacement., Intervention: All patients were instructed to ingest simultaneously, a 300-mg ferrous sulfate tablet and their usual thyroxine dose every day for 12 weeks., Results: After 12 weeks of ferrous sulfate ingestion with thyroxine, the mean level of serum thyrotropin (thyroid stimulating hormone, TSH) rose from 1.6 +/- 0.4 to 5.4 +/- 2.8 mU/L (P < 0.01), but the free thyroxine index did not change significantly. Subjective evaluation using a clinical score showed that nine patients had an increase in symptoms and signs of hypothyroidism; the mean score for the 14 patients changed from 0 to 1.3 +/- 0.4 (P = 0.011). When iron and thyroxine were mixed together in vitro, a poorly soluble purple complex appeared that indicated the binding of iron to thyroxine., Conclusions: Simultaneous ingestion of ferrous sulfate and thyroxine causes a variable reduction in thyroxine efficacy that is clinically significant in some patients. The interaction is probably caused by the binding of iron to thyroxine.

We have previously identified a 57-bp DNA fragment encompassing exon 1 of the beta-subunit gene of rat thyrotropin (rTSH beta) that mediates the negative response to L-triiodothyronine (T3). To determine the specific motif that confers this negative regulation, we tested the T3 sensitivity of various segments of this 57-bp gene fragment in transiently transfected pituitary tumor cells, GH3. The suppressive effects were mediated by a 17-bp motif (+11/+27, CGCCAGTGCAAAGTAAG) located at the 3' end of exon 1. The inhibitory effects mediated by the sequence were evident when a single copy of the motif was inserted 125 bp upstream or 11 bp downstream of the transcriptional start site. These findings indicate that the suppressive effect of T3 is an intrinsic property of the T3-responsive element and not dependent on position relative to the promoter. The T3 receptor (T3R) extracted from GH3 cells or expressed in vitro bound specifically to this sequence. Specific mutations introduced into this region result in a selective loss of nuclear protein binding and a corresponding loss of T3 sensitivity. Additional studies showed that the 17-bp sequence was not responsive to T3 in COS cells which lack endogenous T3R. Cotransfection of a T3R restored the T3 responsiveness of the TSH beta motif. In summary, we have identified an element in the rTSH beta gene that mediates negative regulation by T3 and binds to the T3R.

A method for measuring optical frequencies from the UV to the near IR relative to a microwave frequency standard is proposed. The concept is to measure the frequency difference of two known ratios ((1/2) and ?) of an optical frequency f relative to the cesium clock. By employing optical parametric oscillators and wideband modulators to link the ((1/2))f and (?)f frequencies, a precise and accurate optical frequency comb can be provided in the ~1-2-microm wavelength region. By using this comb, most optical frequencies from the UV to the near IR can be measured or synthesized. A configuration for measuring the frequency of the hydrogen 1S-2S transition is described.

The induction of rat hepatic mRNA S11 by L-T3 (T3) is a useful model for studying the mechanisms of thyroid hormone action. Although numerous reports have examined the response of mRNA S11 to various physiological and hormonal manipulations, the role of S11 protein in cellular metabolism remains unknown. In this study we show that mRNA S11 is abundantly expressed and regulated by T3 only in liver and small intestine. High levels of the mRNA are present at birth, but drop sharply between 30-60 days of age. These and other features of the S11 gene product were similar to those of rat apolipoprotein-A1 (Apo-A1). The sequence of S11 cDNA was identical to a portion of the Apo-A1 mRNA, thus confirming identity of the S11 mRNA. To examine whether DNA sequences immediately adjacent to the transcription start site mediate the effects of thyroid hormone, we measured the activity of an Apo-A1 gene fragment, U-1 (-474 to -7) using a transient transfection assay. The activity of the full-length U-1 DNA in HuH-7 hepatoma cells was 2- to 2.5-fold higher in the presence of thyroid hormone. This finding closely matched previous results using the in vitro nuclear run-on assay. Internal deletion of a motif that resembles a thyroid hormone response element from U-1 DNA not only abolished the induction by T3, but suppressed promoter activity by 3- to 4-fold in response to the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the experimental demonstration of a novel optical parametric oscillator approach to tunable optical frequency division. The beat frequency of the signal and idler subharmonic outputs of a tunable cw KTP optical parametric oscillator was phase locked to a microwave reference frequency source, which thus permitted precise determination of the output frequencies at approximately half the input pump frequency.

A linearized quantum analysis of an injection-seeded optical parametric amplifier is presented. When the amplifier is operated in a saturated gain regime, the amplified output intensity of the injected coherent-state signal becomes amplitude squeezed, and the signal-to-noise ratio improves. Above threshold, an injection-seeded parametric oscillator is found to generate more single-beam squeezing than an unseeded one. An amplitudesqueezed pump is shown to yield even better results.

We describe an experiment that generates squeezed states by means of forward four-wave mixing in sodium vapor with a single optical beam. The single-beam arrangement maximizes the pump-probe spatial overlap in the nonlinear medium. Self-focusing (or self-defocusing) is found to be the major limiting factor in achieving optimal squeezing.

Diabetes mellitus (DM) has been shown previously to decrease hepatic synthesis of both albumin and albumin mRNA. These findings prompted us to ask whether decreased albumin expression is due to changes in the rate of gene transcription. A cell-free in vitro transcription assay demonstrated that activity of the albumin promoter in hepatonuclear extracts was diminished 3- to 4-fold in diabetic compared with control animals. This decrease was abolished in extracts prepared from diabetic animals treated with insulin. The potential mechanisms for diminished activity were examined by mixing extracts from normal and diabetic animals prior to incubation with DNA templates. In the presence of limited amounts of albumin promoter DNA, addition of diabetic to control extract caused a reduction in promoter activity, suggesting that an inhibitor or inhibitors of albumin promoter activity is/are present in the liver of diabetic rats.

To determine the age-related alterations in tissue-responsiveness to thyroid hormone action, the mRNA levels and the enzyme activities of hepatic cytosolic malate dehydrogenase (ME) and mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) were determined in groups of euthyroid, hypothyroid and hyperthyroid male Fischer 344 rats at various ages. The basal alpha-GPD level (change in optical density/min per mg) in 2-month-old rats (0.163 +/- 0.003) (S.E.M.) was significantly (P less than 0.001) higher than that in 6-month-old and 26-month-old rats (0.116 +/- 0.012 and 0.098 +/- 0.013 respectively). The basal ME activity (mU/mg) was significantly (P less than 0.001) reduced in aged rats (9.2 +/- 0.89) compared with younger rats (54.2 +/- 3.4 and 17.1 +/- 2.3 for 2-month-old and 6-month-old rats respectively). The response of ME to tri-iodothyronine (T3) in aged rats compared with young rats was reduced approximately 50%. This age-related reduction in ME response to T3 could not be attributed to reduced food intake with ageing. Northern blot analysis showed that ME mRNA levels in hyperthyroid aged rats were 50% of those of young hyperthyroid rats. There were no significant differences in either basal or T3-stimulated alpha-GPD mRNA levels. The proportional reduction in steady-state ME mRNA levels and ME activity in aged rats indicates that this age-related change is modulated at a pretranslational level.

A novel method of frequency division based on optical parametric oscillation is proposed. This scheme converts with high efficiency an input signal into two intense, coherent subharmonic outputs whose frequencies are tunable and whose linewidths are essentially limited by the input pump linewidth. By locking their difference frequency to a microwave, a millimeter-wave, or an infrared reference source, the output frequencies are precisely determined. The proposed frequency dividers can be operated in series or in parallel to measure, compare, and synthesize frequencies from optical to microwave. A line-narrowing effect for the generation of ultrastable radiation is discussed.

Signal and idler beams from a type I phase-matched nondegenerate optical parametric oscillator are separated by a Mach-Zehnder interferometer. Broadband nonclassical intensity correlation is observed, in agreement with a theory that includes pump noise. The maximum observed correlation, which occurs at 1.1 MHz, yields a noise level 2.8 dB below the shot noise.

We present a linearized quantum analysis of the optical parametric oscillator that includes the effects of pump noise. We show that excess pump noise reduces the intensity correlation between the signal and idler at low frequencies, which explains the low-frequency spectrum of current experimental observations. Its dependence on the cavity loss mismatch permits the possibility of ultrasensitive intracavity absorption spectroscopy, even in the absence of nonclassical correlation observation.

The rat hepatic S14 gene is regulated by L-triiodothyronine (T3) and codes for a cytosolic protein (pI 4.9 and Mr 17,010) that is believed to be involved in lipogenesis. Recent studies have identified at least five DNase I hypersensitive sites (HS 1-5) in hepatic chromatin flanking the 5' region of the gene. The HS-1 site is situated immediately adjacent to the transcription initiation site. We have isolated a DNA fragment (USS-1) which contains a portion of the HS-1 site to examine the binding of nuclear proteins to S14 DNA. DNase I footprinting studies demonstrated that material extracted from hepatic nuclei with 0.42 M NaCl contained proteinase K-sensitive factors (presumed to be proteins), which bind to USS-1 DNA between positions -63 and -48 (PS-1) relative to the transcription initiation site. Examination of the binding activity with a synthetic oligonucleotide identical to the protected sequence indicated the formation of at least three protein-DNA complexes. The DNA binding activity of the PS-1 binding protein or proteins correlated with the T3 regulated expression of mRNA-S14. Although the nucleotide sequence of PS-1 closely resembles the binding site for the CCAAT transcription factor (CTF/NF-1), competition studies attempting to displace protein binding from the PS-1 sequence with DNA fragments containing the CTF/NF-1 binding motif were unsuccessful. In vitro transcriptional assay studies suggested that the DNA fragment (-441 to -2) containing the PS-1 site promotes the transcription of the S14 gene in an orientation fashion. The in vitro transcriptional activity of the S14 DNA containing the PS-1 sequence was significantly higher in hepatonuclear extracts from hyperthyroid compared with euthyroid or hypothyroid animals. In summary, our findings indicate that the DNA binding activity of proteins which bind to PS-1 site is influenced by the thyroid status of the animal.

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.

We have examined the role of rapidly turning over proteins in the T3 regulation of multiple rat hepatic genes. T3 induction of the rapidly responsive mRNA-S14 was markedly inhibited by cycloheximide (1 mg/100 g BW) or emetine (3 mg/100 g) injected ip 30 min before T3 (mRNA-S14 concentration was only 35% of that in T3-treated controls 8.5 h after administration of either protein synthesis inhibitor, P less than 0.01). Cycloheximide exhibited a similar effect on each of five other more slowly responsive T3 regulated genes. When cycloheximide was given 10 h after T3, the expected T3-induced rise of mRNA-S7 activity was completely prevented, and for mRNA-S4 activity the anticipated rise was blunted to 40% of T3-treated control (P less than 0.05). Cycloheximide caused sharp declines in the activity of two other mRNAs, S6 and S8, which because of shorter lag times of response to T3, had already risen when the drug was given. Values for both these mRNAs returned to the baseline hypothyroid level within 6 h of injection of the drug and remained low for a further 8 h (P less than 0.05). The expected deinduction of mRNA-S10 by T3 was also markedly modified. T3 lowered this mRNA to 11% of the hypothyroid control after 8 h, whereas cycloheximide given 30 min before the hormone blunted this fall to only 72% of control (P less than 0.01). Thus there appeared to be a 70% reduction in the rate of T3 induced fall of mRNA-S10. We did not find that cycloheximide caused a generalized decrease in poly (A)+ RNA mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated trout testis nuclei rapidly incorporate [alpha-32P]NAD+ into chromosomal proteins. Three proteins, very-lysine-rich histone (H1), a specific trout chromosomal protein (H6) and the sperm-specific protamines, incorporate the label as adenosine diphosphoribosyl (ADP-Rib) residues. No significant labeling of the nucleosomal 'core' histones H2A, H2B, H3 and H4 was observed. The linkage of the [32P](ADP-Rib) residues to each protein was very labile at pH values greater than 7.0 but by working at acidic pH and low temperatures the ADP-Rib label could be shown to be covalently bound to protein by gel electrophoresis and ion-exchange chromatography. The [32P]ADP-Rib chains could be removed by digestion with snake venom phosphodiesterase with the formation of AMP and phosphoribosyl-AMP.

The complete amino-acid sequence of 194 residues of trout testis histone H1 has been elucidated by automated Edman degradation of large peptides derived from specific cleavages at infrequent amino-acid residues. Trout testis histone H1 has a molecular weight of 19314, is only slighly microheterogeneous, and possesses pentapeptide sequences related to Ala-Ala-Ala-Lys-Lys repeated six times in the complete sequence. Although the N-terminus of histone H1 is known to be blocked, some 10% of intact trout testis H1 contains a free N-terminal alanine residue. Three seryl residues in the C-terminal half (97-194) of the molecule occur in the sequence Lys-Ser-Pro-Lys known to be phosphorylated in trout testis H1. The sequence has been compared to the known partial sequence of rabbit thymus H1. The results are consistent with a role for histone H1 in the maintenance of the higher-order structure of chromatin.

Previous studies from our laboratory have shown that ongoing protein synthesis is required for L-tri-iodothyronine (T3) regulation of rat hepatic genes. In this report we have examined the role of ongoing protein synthesis in T3 regulation of the growth hormone gene (GH) in rat pituitary and GC cells. T3 (200 micrograms/100 g body weight injected intraperitoneally or 10(-8) M added to the media) induced a 3-fold rise of GH mRNA concentration after 8 h in rat pituitary (from 5.9 +/- 1% to 17.8 +/- 1.4% of an internal standard (IS), p less than 0.01) and a 5-fold rise in GC cells after 6 h (from 0.1 +/- 0.01% to 0.54 +/- 0.03% of IS, p less than 0.01). Cycloheximide (1 mg/100 g body weight intraperitoneally or 25 microM added to the media) completely blocked the increase in GH mRNA when given before the hormone in rat pituitary (7.5 +/- 0.5% of IS after 8 h) and GC cells (0.095 +/- 0.01% of IS after 6 h). Similar results were observed when emetine and puromycin were used to block protein synthesis. GH transcription rate in pituitaries from hypothyroid animals was 20 +/- 8 ppm and increased to a value of 295 +/- 85 parts per million 2 h after the injection of T3, and a similar 8-fold increase was observed in GC cells after 2 h. However, when cycloheximide was given before the hormone, the T3-induced increase in transcription was completely blocked. We also demonstrate here that the differences observed in GH transcription rate between hypo- and euthyroid rat pituitaries fully account for the differences observed in mRNA concentration. We conclude that short-lived proteins are involved in T3 regulation of the GH gene in rat pituitary and GC cells.

Nuclei and chromatin from trout testis cells were digested with three different nucleases (DNase I, DNase II, and micrococcal nuclease), and the acid-soluble proteins that were solubilized and those remaining bound to the nuclease-resistant DNA were compared electrophoretically. With the conditions described by H. Weintraub and M Groudine [(1976) science, 193, 848-856], which we previously found to be selective in digesting actively transcribed regions in trout testis chromatin, a single chromosomal protein, H6, was solubilized. The nucleosomal histones and H1 remained insoluble, bound to the resistant DNA. In contrast, digestion with micrococcal nuclease led to a preferential solubilization of a second protein, HMG-T, together with the release of some nucleosomal histones and H1 into the soluble fraction. DNase II also discriminated between "active" and "inactive" chromatins; when a DNase II-solubilized "active" chromatin fraction was prepared, it too was enriched in H6 and HMG-T. Thus, both H6 and HMG-T, the two major low-salt extractable chromosomal nonhistone the two major low-salt extractable chromosomal nonhistone proteins from trout testis, are associated with chromatin regions selectively sensitive to nucleases. The preferential solubilization of HMG-T by micrococcal nuclease action suggests that it might be located at the internucleosomal "spacer" region.

Earlier studies from our laboratory have demonstrated that cycloheximide administration to hypothyroid rats inhibited the induction of the hepatic mRNA-S14 by T3. These results suggested a role of short-lived proteins in the hormonal regulation of this gene. To define the possible mechanism of the cycloheximide effect, we examined the influence of cycloheximide on the in vitro transcription rate of the gene and its chromatin structure. Forty-five minutes after injection of cycloheximide to euthyroid rats, the in vitro transcriptional rate fell by 60% and this effect persisted for 4 h. In the same euthyroid rats, cycloheximide caused the disappearance of all four DNase I-hypersensitive sites situated in the 5'-flanking region of the gene. However, cycloheximide given to hypothyroid rats affected neither the basal transcription rate nor the chromatin structure. When cycloheximide was administered 30 min after an acute injection of T3 (200 micrograms/100 g BW) to hypothyroid animals, it completely blocked the hormone induction of the transcriptional rate. These results suggest that one or more labile proteins are required for maintenance of S14 chromatin structure in a configuration which permits hormonal regulation of gene expression. The ability of cycloheximide to block mRNA-S14 induction by T3 appears to be mediated at least in part by an inhibition of T3-stimulated transcription.

The gene for S14 in the rat codes for an mRNA which in lipogenic tissues (liver, fat, mammary gland) responds both to L-triiodothyronine and a high-carbohydrate, fat-free diet. In an effort to understand the molecular basis for the tissue-specific regulation of mRNA-S14 expression, we have examined the organization of this gene in chromatin. Specifically, we examined the distribution of DNase I-hypersensitive sites and DNA methylation sites associated with a 25-kilobase chromosomal domain containing the S14 gene. Our results show that DNase I preferentially digests four regions of the DNA flanking the 5' end of the hepatic S14 gene which have characteristics of DNase I-hypersensitive sites. The sites, identified as HS-1, HS-2, HS-3, and HS-4, are located at or adjacent to the site of transcription initiation and at 1.2, 7, and 8 kilobases upstream from this site, respectively. In lactating mammary gland where the S14 gene is also highly expressed and regulated by L-triiodothyronine, sites HS-1, HS-2, and HS-4 are present, but HS-3 is absent. No hypersensitive sites were detected either within the gene or flanking the 3' end of the gene. In brain, kidney, and spleen, tissues in which mRNA-S14 is expressed at levels less than or equal to 10% of that found in euthyroid liver, sites HS-1 and HS-3 were absent. Despite the marked effect of thyroid state on the abundance of hepatic mRNA-S14, no significant alterations were observed in the DNase I sensitivity of hepatic chromatin containing the S14 gene. Analysis of the DNA methylation pattern at HpaII and HhaI sites showed a positive correlation of hypomethylation of the gene and the contiguous flanking regions with S14 gene expression. All HpaII/MspI sites and most HhaI sites either within or flanking the S14 gene were undermethylated in liver and lactating mammary gland. Although thyroid status had no generalized effect on the site-specific DNA methylation state of the hepatic S14 chromosomal domain, one site (H3) situated in the second exon close to the 3' terminus of the S14 gene appeared to undergo demethylation in the transition from hypo- to euthyroidism. In essence, our results show that the 5' DNA flanking region of the S14 gene contains a tissue-specific DNase I-hypersensitive site which, although not influenced by thyroid status, appears essential for the expression of S14 and its regulation by L-triiodothyronine.(ABSTRACT TRUNCATED AT 400 WORDS)

In contrast to the rapid response of mRNA-S14 which occurs within 20 min after L-triiodothyronine (T3) administration, the induction of other rat hepatic mRNA sequences exhibits a lag time of several hours. We have studied the induction of mRNA-S11 which codes for a protein of pI 6.1 and an Mr of 22,500 as a model of such a slowly responsive gene. In addition to T3, both glucocorticoids and growth hormone regulate the expression of this gene. For each of these stimuli, the response exhibits a lag time of approximately 6 h. Following this lag time, there is a linear increase in the level of mRNA-S11 induced by a single maximal dose of T3, dexamethasone, and growth hormone to levels of 15-, 6-, and 3-fold, respectively, in excess of the hypothyroid base-line levels. The similarity in the lag time and the differences in maximal responses effectively argues against the possibility that the effect of one hormone is mediated exclusively by a change in the secretion or metabolism of another. Support for a direct action of T3 and glucocorticoids on the hepatic cell comes from the observation that these hormones stimulate mRNA-S11 in rat hepatocytes under primary culture. The increases in in vitro nuclear transcription measured following T3 and dexamethasone administration were clearly insufficient to account for the observed increases in mRNA. Furthermore, the hormonal induction of mRNA-S11 was promptly abrogated by cycloheximide (10 mg/kg) injected 6 h after the administration of T3 or dexamethasone at the time of the expected mRNA increase. The post-transcriptional control of the mRNA-S11 and its sensitivity to cycloheximide are similar to previously documented responses of the rapidly induced mRNA-S14 and suggest for both sequences a requirement for ongoing synthesis of rapidly turning over proteins. We speculate that the lag time of response of a given gene to T3 or glucocorticoid may be an intrinsic characteristic of the gene and may represent a common set of molecular events involved in the activation of that gene by diverse stimuli.

A phagemid was adapted for use as the vector in the vector-primer-cloner-sequencer cloning system. The use of this new vector markedly expanded the utility of this technology for the construction of cDNA libraries. Technological advantages and new capabilities include: (1) a greater number of unique restriction sites within the polylinker region; (2) the ability to produce single-stranded templates for nucleotide sequencing, and (3) a convenient means to synthesize strand-specific hybridization probes. With the use of this cloning system, a rat liver cDNA library (8.56 x 10(5) recombinants from 1 microgram of poly(A)+ RNA) was rapidly (in two days) constructed.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].

The rat hepatic S14 gene has served as a model of thyroid hormone regulation of gene expression. Earlier studies of the S14-containing chromatin region demonstrated that a cytosine residue at position 625 (C-625) in the 3' untranslated exon was hypermethylated in hepatic DNA derived from hypothyroid animals. This observation was consistent with the markedly reduced level of expression of the S14 gene in these rats. The current studies have extended these observations to groups of rats in various thyroidal states. By using the restriction enzyme Hhal, the percent demethylation of this site was quantitated (hypothyroid, 9.3%; euthyroid, 19.2%; hyperthyroid, 66.6%). Moreover, the level of methylation was shown to be reversible as the thyroidal state was altered. Our data also indicate that these changes are probably independent of de novo DNA synthesis. Kinetic studies of the demethylation of this cytosine residue after T3 administration showed no change for at least 1 day and maximal change after about 4 days. This contrasts with the significant rise in S14 mRNA evident within 30 min and suggests that demethylation plays no role in the acute induction of this gene by T3. Carbohydrate feeding, another stimulus of S14 expression, similarly caused the demethylation of this cytosine residue. Earlier studies had demonstrated that mRNA S14 expression was not detectable in rat pups before about 20 days of age and continued to rise through the first year of life. Consistent with those findings, S-14 C-625 was fully methylated up to 15 days of age. Progressive demethylation then occurred up to 12 months of age. These results indicate that increased demethylation of a specific site in the 3' untranslated region of the S14 gene, possibly resulting from augmented excision repair processes, is correlated with increased expression of the gene.