Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells

A rat thyroid peroxidase cDNA has been isolated from a FRTL-5 thyroid cell library and sequenced. The cDNA is 2776 base pairs long with an open reading frame of 770 amino acids. By comparison to full-length human thyroid peroxidase cDNA and based on its identification of a 3.2 kilobase mRNA in rat thyroid FRTL-5 cell Northern blots, the rat peroxidase cDNA appears to lack 400-500 base pairs at the 5'-end of the mRNA. It exhibits only a 74% nucleotide and 77% amino acid sequence similarity to human thyroid peroxidase cDNA within the total aligned sequences, although the predicted active site regions are highly conserved (greater than 90-100%). The cDNA has been used to map the thyroid peroxidase gene in mice to chromosome 12 and to compare thyroid peroxidase and thyroglobulin gene expression in FRTL-5 rat thyroid cells. Despite the fact TSH action in both cases is duplicated, and presumably mediated, by cAMP, TSH-induced increases in thyroid peroxidase and thyroglobulin mRNA levels differ. Differences exist with respect to hormone concentration and time. The ability of TSH to increase thyroglobulin, but not thyroid peroxidase mRNA levels, requires insulin, 5% serum, or insulin-like growth factor-I. Insulin or insulin-like growth factor-I alone can increase thyroglobulin mRNA levels as well as or better than TSH but have only a small effect on thyroid peroxidase mRNA levels by comparison to TSH. The ability of TSH to increase thyroglobulin gene expression is readily detected in nuclear run-on assays but not the ability of TSH to increase thyroid peroxidase gene expression. Cycloheximide inhibits TSH-increased thyroglobulin but not peroxidase mRNA levels. Finally, methimazole and phorbol 12-myristate 13-acetate show different effects on TSH-induced increases in thyroglobulin and thyroid peroxidase mRNA levels.     

Expression of an enhancer-binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus IE1 gene.

The baculovirus Autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. To begin to identify proteins that interact with hr5, DNA-protein interactions were analyzed by using extracts from Spodoptera frugiperda cells and a fragment of DNA containing the left half of the hr5 enhancer. This 252-bp DNA fragment contains two copies of a 30-bp direct repeat (DR30) and two copies of a 24-bp imperfect palindrome contained within a 60-bp direct repeat (DR60). Extracts prepared from normal S. frugiperda cells and cells transfected with pUC8 lacked enhancer-binding proteins. However, when gel shift assays were performed with extracts from cells transfected with a plasmid containing the viral trans-activator IE1 gene, two DNA-protein complexes were formed. Both DNA-protein complexes were specifically inhibited by competition with a 60-bp oligonucleotide corresponding to DR60 but not by competition with a different oligonucleotide corresponding to DR30. Formation of the two complexes did not appear to involve cooperative interactions between binding proteins. When DR60 was used as a probe, a single complex was formed. To measure the enhancer activity of DR60, a reporter plasmid was constructed that contained DR60 cloned upstream of the reporter chloramphenicol acetyltransferase gene under the control of the delayed-early 39K promoter. Transient expression analysis indicated that the oligonucleotide increased expression of this gene 300-fold over the level obtained in the absence of any enhancer sequences.     

Numerous nuclear proteins bind the long control region of human papillomavirus type 16: a subset of 6 of 23 DNase I-protected segments coincides with the location of the cell-type-specific enhancer.

The long control region of the human papillomavirus type 16 genome is 856 base pairs (bp) long. It contains a cell-type-specific enhancer, a glucocorticoid response element, and sequences mediating the response to the viral gene products of open reading frame E2; all three regulate the promoter P97. We mapped binding sites of trans-acting proteins relevant for the cell-type-specific enhancer and other cis-acting elements by DNase I footprint experiments with nuclear extracts from HeLa cells. Throughout the human papillomavirus type 16 long control region 23 footprints protect 557 of 900 bp. Nine footprints fall into a 400-bp segment that was previously identified to contain the cell-type-specific enhancer. Variations of the protein concentration in the footprint reaction do not affect six of these nine footprints. At high protein concentrations, three footprints fuse to a 106-bp protected region, suggesting that this segment specifically binds several proteins of lower affinity or abundance. Unexpectedly, extracts from human MCF7 and mouse 3T3 cells, in which the enhancer is inactive, give footprints identical to those obtained with HeLa extracts. Seven footprints contain the sequence 5'-TTGGC-3'. Footprint competition experiments suggest that factor NFI binds to these seven motifs. Competition with cloned oligonucleotides in transfections suggests that these elements contribute to the enhancer function. Subcloning identifies a 232-bp fragment between positions 7524 and 7755 as sufficient for full enhancer activity. Several of the six footprinted elements on this segment may cooperate functionally, since subclones of this region show decreased or no cell-type-specific enhancer function.     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.