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The asyaloglycoprotein receptor is a dimer formed by two polypeptide chains abundantly expressed in the liver (RHL-1 and RHL-2). Using specific primers for the two polypeptide chains we measured, by semiquantitative reverse PCR (RT-PCR), the corresponding mRNAs in different rat tissues. We found that both RHL-1 and RHL-2 mRNAs are expressed in the liver, kidney, brain and thyroid. Under the same conditions we did not detect any specific mRNA in the spleen. In the brain these sequences are expressed along a posterior-anterior gradient. Cerebellum and brainstem display the highest expression of the brain RHL-1 and RHL-2 mRNAs. Tissues and regional distribution of this receptor suggest that other body districts besides liver may participate in the clearance of serum glycoproteins.

The expression of thyroglobulin and other thyroid-specific markers depends upon the activation of protein kinase A (PKA) by cyclic AMP. A rat thyroid cell line dedifferentiates when transformed with Ki-ras oncogene. The decrease in thyroglobulin gene expression parallels a reduction in the level of PKA nuclear catalytic subunit. We find that the activity of cAMP-responsive elements and thyroglobulin promoters is down-regulated in Ras-transformed cells. Transcription of a third cAMP-regulated gene, H-ferritin, is similarly reduced. cAMP-responsive element and H-ferritin expression were stimulated when intracellular cAMP levels were increased. Reactivation of the thyroglobulin promoter required depletion of PKC in addition to increased cAMP. We also find that v-Ras activation leads to a significant increase in membrane-bound PKC. These data support the idea that v-Ras via PKC inhibits the transmission of cAMP-PKA signals to the nucleus. We suggest that the thyroglobulin promoter is more sensitive than other cAMP-dependent promoters to reduced nuclear levels of PKA catalytic subunit.

Hyperfunctioning thyroid adenomas are clonal neoplasms with the intrinsic capacity of growing and differentiate independently of thyroid-stimulating hormone (TSH). We analysed the mRNA encoding thyrotropin receptor of 11 adenomas obtained by fine needle aspiration biopsy (FNAB) and found 7 mutants all located in three aminoacids clustered in the sixth transmembrane domain of the receptor. These mutations were somatic and specifically present in the tumour tissue. DNA sequence revealed that 80 to 90% of the mutations can be rapidly screened and identified by restriction enzyme analysis of the amplified cDNA obtained from the FNABs. The mutation Thr->Ile was introduced in the wild type receptor and expressed in mouse fibroblasts. These cells constitutively activate the transcription of a reporter gene under the control of cyclic AMP responsive element.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

We have studied the expression of cell polarity in hybrids between two rat thyroid epithelial cells: FRT and FRTL-5. FRT cells are polarized but do not express tissue-specific properties, FRTL-5 are unpolarized and express many thyroid-specific genes. A and express many thyroid-specific genes. A pool of 170 hybrid clones and five independent clones were characterized. The chromosome complement was that expected from 1:1 fusion of the parental cells. No chromosome loss was observed for several generations. All hybrids were polarized as judged from: (1) morphology, (2) transepithelial resistance, (3) preferential secretion of several proteins either through the apical (e.g. thyroglobulin) or through the basolateral pole, and (4) basolateral trapping of iodide. On the other hand, the expression of thyroid-specific markers: thyroglobulin synthesis and secretion, trapping of iodide, thyrotropin-dependent growth and expression of specific membrane antigens, were greatly reduced or inhibited in the pool and in the isolated clones. We also found that reduction of thyroglobulin synthesis was correlated with the loss of activity of the trans-acting factor TgTF1. We conclude that cell polarity, a property of FRT cells, is dominant in the hybrids whereas thyroid differentiation is recessive.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.

A series of overlapping recombinant clones, which cover the alpha 2 (type I) collagen gene, have been isolated by stepwise screening of two libraries of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned cDNA containing alpha 2 collagen DNA sequences as hybridization probe. The other clones were obtained by a sequence of screenings using defined fragments of the successive genomic clones as hybridization probes. Several types of experiments indicated that the DNA of these clones are truly overlapping and span 55 kilobase pairs of contiguous DNA sequences in the chicken genome. Sequence analysis of small DNA segments of some of these clones confirm that they contain coding sequences which specify alpha 2 collagen. Electron microscopic analysis of hybrids between type I alpha 2 collagen mRNA and the overlapping genomic clones indicates that the chicken alpha 2 collagen gene has a length of at least 37 kilobases, about 7.4 times longer than the corresponding translatable cytoplasmic mRNA. The coding information for alpha 2 collagen is distributed in more than 50 coding sequences which are interrupted by intervening sequences of various sizes. The structure of the gene implies that the conversion of precursor RNA to mature mRNA for alpha 2 collagen includes at least 50 splicing events.

Sixty-four unrelated healthy subjects were studied for the detection of a DNA polymorphism at the 5' end of the insulin gene. No significant difference between the groups was found in blood glucose values at fasting and after an oral glucose load. A significant association was found between fasting (P less than 0.05) and after load plasma C-peptide levels (P less than 0.01) and the presence of a 1.6 Kb insertion at the 5' end of the insulin gene. A gene dose-dependent effect was noted, class 3/3 individuals having the lowest after-load C-peptide concentration and class 1/3 an intermediate level (F for the linear trend: P = 0.007). This might suggest that insulin gene polymorphism affects insulin secretion in healthy individuals. In order to confirm this, a subgroup of six class 3/3 and eight class 1/1 individuals subsequently underwent a hyperglycaemic clamp. The tissue sensitivity to insulin was similar in the two groups but glucose-stimulated insulin secretion was markedly impaired in homozygotes for the class 3 allele. In this group, insulin secretion was, on average, only one-third of that in class 1/1 individuals (P less than 0.02). Similarly impaired in class 3/3 persons was the glucose + arginine-stimulated insulin secretion (P less than 0.05). We conclude that the polymorphism at the 5' end of the insulin gene is associated with variations in insulin secretion in healthy humans.

Purified pig thyroid chromatin has been transcribed in vitro with Escherichia coli RNA polymerase. The transcript, analyzed by DNA-RNA hybridization, shows two major kinetic components: 40% of the transcript is copied by repetitive sequences present 100 times per haploid genome; another 25% anneals to DNA with a rate constant Kh 10-4 M - S-1, typical of single-copy sequences. The transcript annealed at cot = 40 M - S to fractions of 2000-nucleotide DNA, when banded in neutral CsCl gradient only hybridizes to the heavy side of the main band. At cot = 3000 M - S, another hybridizing fraction appears on the light side of the main band of the gradient. The reassociation properties of these fractions show that the heavy DNA fraction is reiterated about 100 times per haploid genome, whereas the light DNA appears as a unique sequence, associated to small repetitive elements. The transcript, analyzed by formamide/sucrose gradient, shows two peaks with sedimentation coefficients of 10 S and 4 S, respectively. The 10-S RNA, hybridized to native 2000-nucleotide-length DNA, has a Kh of 10-4 M - S-1 and a cot1/2 of 10(3) M - S, typical of single-copy sequences.

The entire rat thyroglobulin mRNA sequence (about 8500 nucleotides) has been cloned in five recombinant plasmids containing overlapping cDNA inserts. The 3' end of the mRNA is precisely defined by the poly (A) tail found in the furthest 3' end clone. Evidence that most of the 5' end is cloned come from size considerations and from a primer extension experiment. At the 3' end of the mRNA only one long open reading frame is present in the sequence of 3018 nucleotides that has been established. In the deduced protein sequence we have localized two thyroxine-forming sites in a region containing a high concentration of tyrosine residues.

We have measured the synthesis of thyroglobulin in two differentiated cell lines, FRTL-5 and FRTL-424, and two transformed thyroid cell lines, FRA and 1-5G. The untransformed cells actively synthesized and exported thyroglobulin in the medium: however, the FRTL-5 cell line synthesized seven times less thyroglobulin than the FRTL-424 cell line, even though both cell types contained equal amounts of functional thyroglobulin mRNA. In contrast the transformed cells expressed extremely low levels of thyroglobulin mRNA, even though there was no detectable change in gene structure or copy number as determined by Southern blot analysis. On the basis of these data we conclude that (a) the different levels of thyroglobulin synthesis in the two untransformed cell lines are due to stable post-transcriptional alterations in the biosynthesis of thyroglobulin and (b) the transformation of thyroid cells results in a substantial reduction in thyroglobulin gene expression.

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.

A partially purified mRNA preparation enriched for chick collagen messenger RNA activity was used as template for the synthesis of double stranded cDNA. The cDNA was ligated into the HindIII site of the plasmid vector pBR322 and used to transform Escherichia coli x1776. One plasmid with an 800-base pair insert was shown to contain DNA sequences corresponding to Type I pro-alpha 1 collagen.

We report the structural organization of a segment of the human thyroglobulin gene, located 70kb from the 3' end of the gene, containing the exons 8 and 9 starting from the 3' end. Selected probes from this region have been used for the chromosomal mapping of the thyroglobulin gene by in situ hybridization techniques. Only one site in the human haploid karyotype is labeled with the genomic DNA probes. Twenty percent of the grains are localized on the long arm of chromosome 8, mostly in the subregion q-2-23 q-2-24 of the long arm of chromosome 8. The localization of the autoradiographic grains suggests a subregional assignment of the human thyroglobulin gene locus to 8q 2-23 or 8q 2-24.

We have determined the nucleotide sequence of two short introns (respectively 215 and 90 nucleotides) in the chick alpha 2-collagen (type I) gene as well as parts of the adjacent exons. For one of these introns we find that the 5' end of U1 RNA is complementary not only to the two ends of the intron but also to one end of the intron and sequences inside this intron. These complementarities predict three potential internal splicing sites. By S1 mapping experiments we find three discrete RNA precursors in which different portions of this intron have been deleted. The sizes of the deleted segments are in good agreement with the location of the predicted splicing points inside the intron. The DNA sequence indicates that removal of one portion of the intron should still allow the subsequent elimination of the rest of the intron and the correct splicing of the coding segments located at each end of the intron. The new introns created by the first splicing events contain sequences at each end which are also complementary to U1 RNA. Our data indicate that in the intron which we have examined the sequences at the 3' end of the intron are removed before those at the 5' end.

We have determined the size and sequence of 8 exons in the gene that specifies chick type 1 alpha 2 collagen. These 8 exons represent three different segments of the gene, but all encode information for the helical portion of the protein. Seven exons have a length of 54 bp, and the 8th has a size of 99 bp. The sequences within these exons vary except for th glycine codons, which occur every third triplet. Each exon begins with a glycine codon and ends with a triplet that precedes a glycine codon. The size and the sequences of the introns do not show any homology except at their ends. Of 7 introns examined the first six bases at the 5' end of 5 of these are identical. The sequences at the 3' end of the introns also show homologies. Our results imply that the ancestral gene for collagen arose by multiple duplications of a single genetic unit containing a 54 bp condig segment. The sequences within these exons drifted by successive point mutations and in some cases by additions or deletions of 9 bp or multiples of 9 bp.

We have studied the effect of thyrotropin on the intracellular concentration of thyroglobulin and of its mRNA both in a differentiated rat thyroid cell line and in the rat thyroid in vivo. Our data demonstrate that the absence of thyrotropin causes a two fold decrease of both thyroglobulin and of its mRNA suggesting that thyroglobulin synthesis in thyroid cells is 50% constitutive.

The sequence organization of porcine DNA isolated from thyroid has been analyzed by hydroxylapatite (HAP) chromatography. The reassociation of 0.4 kilobase (Kb) DNA fragments shows, besides the presence of 5% inverted repeat sequences (foldback DNA), that 45% of the genome is represented by high (10%) and intermediate (35%) repetitive components, whereas the remaining 50% is unique sequences. 30% of the unique sequences consists of 1,000 nucleotide fragments interspersed with repetitive elements 400 nucleotides in length. The remaining 20% is longer unique sequences (10,000 nucleotides) apparently not linked to repetitive elements.

A recombinant plasmid containing chick pro-alpha2 collagen gene sequences has been constructed and cloned in Escherichia coli. Using partially purified collagen mRNA as template, we synthesized double-stranded DNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) from avian myeloblastosis virus and the Klenow A fragment of E. coli DNA polymerase I. From this complex mixture of double-stranded DNAs, a specific 200-base-pair restriction fragment was generated by cleavage with the restriction endonucleases BamHI and EcoRI. These enzymes also make unique cuts in the plasmid vector pBR322. The restriction fragment was inserted into pBR322 via these BamHI and EcoRI sites and cloned in E. coli chi1776. The cloned recombinant plasmid was shown to contain pro-alpha2 collagen DNA by its specific hybridization to chick pro-alpha2 collagen mRNA, as assayed in an in vitro translation system. Thus, a clone containing pro-alpha2 collagen DNA was constructed without first obtaining highly purified collagen mRNA.

Thyroglobulin (Tg) and its specific mRNA were analyzed in the thyroid gland of two cases of familial goiter. A three-fold increase in the level of stable protein and functional Tg mRNA was found in the goiter of one of the two patients in whom a desiodase defect was demonstrated. Normal concentrations of both Tg and its mRNA were found in the other goiter in which no enzymatic defect could be shown. Our results suggest that the increase of Tg mRNA levels found in the goiter with a desiodase defect was due to a chronic stimulation of the gland by TSH.

The expression of rat thyroglobulin gene is repressed following the transformation of rat thyroid cells with Kirsten murine sarcoma virus. The expression of a reporter gene fused to the thyroglobulin promoter is down-regulated in transformed thyroid cells in transient or in stable transfection assays. DNase and exonuclease III cleavage-protection analysis reveals that a promoter binding activity located at -60 base pairs from the transcription start site is substantially reduced in transformed thyroid cells. The repression in the transformed cells of the reporter gene joined to the thyroglobulin promoter can be reversed by fusion with normal differentiated thyroid cells. Fusion of transformed thyroid cells to liver cells does not reactivate the reporter under control of the thyroglobulin promoter.

We report the structural organization of an 80 Kb segment of rat DNA, which encodes for about 40% of Thyroglobulin mRNA at the 3' end. The codogenic information included in this segment is splitted in 17 exons of homogeneous size (about 200 bp). The seven exons at the extreme 3' end have been precisely defined by DNA sequence analysis. No clear sequence homology is found among the exons, even though their coding capacity is quite similar, from 55 to 63 aminoacids residues. We located 2 hormonogenic (T4 forming) sites on the extreme 3' end of the gene in different exons. The DNA sequence coding for these functional sites shows a 70% homology in a 50 nucleotides segment. In addition we found a remnant of this sequence in other exons of the gene. Two large introns have been found on the 3' end of the gene: one is 17 Kb and the other one is more than 30 Kb long. On the basis of these findings and of preliminary studies on the remaining 5' end of the gene, we can predict that the minimum length of the rat TGB gene will be 150 Kb, which makes this gene the largest so far identified eukaryotic gene. We propose in addition that the 3' end exons arose by duplication of a common ancestor.

In vitro, transcription by thyroid chromatin has been compared to that carried out by deproteinized DNA prepared from the same tissue. The chromatin-primed transcripts are represented by 4S and 10S RNAs, whereas the DNA-primed transcript is heterogenous and polydisperse. The sequences expressed in vitro have been analyzed by hydridization of 4S and 10S RNA to the total native thyroid DNA. Most of the sequences transcribed are represented by intermediate repetitive-associated an unique sequence. Furthermore, they are expressed only by chromatin and not by DNA. Thus, thyroid chromatin very likely holds in vitro the same fonctional restriction exhibited in vivo, whereas the transcriptional behaviour of thyroid DNA is totally aspecific.

Transformation of rat thyroid epithelial cells by Kirsten murine sarcoma virus results in the block of certain thyroid differentiated functions, such as synthesis and secretion of thyroglobulin. Our studies, performed by a run-on assay, demonstrate that this block occurs at the transcriptional level. We also demonstrate the de novo methylation of two methylation-sensitive sites, located within the 5' end regulatory sequences of the thyroglobulin gene, in transformed cells, in the absence of any rearrangement of the gene. These two methylation-sensitive sites were methylated also in a rat thyroid cell line transformed by another retrovirus and in two normal cell lines which do not express the thyroglobulin gene.