Sequence of the tumor necrosis factor/cachectin (TNF) gene from Peromyscus leucopus (family Cricetidae).
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Amino Acid Sequence↗
Biomedical subjects
M D Crew
Publications and source records attributed to M D Crew.
Positive and negative thyroid hormone response elements are composed of strong and weak half-sites 10 nucleotides in length.
The steroid-thyroid hormone receptors bind to imperfect repeats of two or more half-sites. It is generally accepted that a T3 response element (TRE) half-site consists of a six-nucleotide core motif (5'-AGGT(C/A)A-3'). It is less widely appreciated that the nucleotides flanking this core motif also have a major influence on the affinity of T3 receptor (TR) for its response element. We analyzed TR-DNA interactions under conditions in which the affinity of receptor monomers for individual TRE half-sites of the rat GH (rGH) gene was measured. These studies avoided the effects of half-site spacing and orientation on receptor binding. Variations in the nucleotides flanking the core sequence can modulate receptor binding by more than 15-fold. Systematic mutational analysis of TRE half-site structure demonstrated that at least two nucleotides flanking either side of the half-site core motif strongly influence TR binding affinity and activity, indicating that half-sites are approximately 10 nucleotides long. Thus, the half-sites of most TREs overlap, and mutations in one half-site may affect the activity of its partner. The TRE half-site sequence 5'-CTGAGGTAACG-3' was bound with highest affinity by TRs. The negatively T3-responsive promoter of the rGH gene was used to investigate the functional significance of the nucleotides flanking the core motif in vivo. A promoter consisting of only 22 rGH nucleotides, containing two functional TRE half-sites which overlap the rGH TATA box, directed T3-inhibited transcription. Mutation of nucleotides flanking the core sequence of the weaker half-site dramatically reduced the activity of the element, demonstrating that the flanking sequences of the half-sites can profoundly affect TRE activity.
Animals↗
Transmembrane domain length variation in the evolution of major histocompatibility complex class I genes.
The fifth exons of major histocompatibility complex (MHC) class I genes encode a transmembrane domain (TM) that is largely responsible for class I antigen cell-surface expression usually through conventional hydrophobic amino acid-membrane interactions or, less often, through phosphatidylinositol linkage. In this report we show that Peromyscus leucopus, a Cricetidae rodent, has MHC class I genes (Pele-A genes) encoding three distinct sizes of TMs. Increases in TM lengths were due to tandem duplications of sequences similar to human hypervariable minisatellite repeats and the lambda chi site. We discerned remnants of a similar duplication event in comparable rodent and primate MHC class I genes. Furthermore, several duplications and deletions appear to have occurred independently in H-2, RT1, Pele-A, and ChLA genes in near-identical positions. Accumulated data suggests that sequences in the fifth exon of MHC class I genes may, therefore, constitute a mutational or recombinational hot spot that is mediated by minisatellite- and chi-like sequences imbedded within the coding region. The MHC class I genes may thus have recruited "selfish" DNA in their evolution to encode cell surface proteins. Expression of Pele-A genes was examined by the polymerase chain reaction (PCR) using oligonucleotide primers specific for exon 4 and 5 sequences. The PCR product sizes indicated that genes encoding each TM domain length are ubiquitously transcribed.
Animals↗
Major histocompatibility complex class I genes of Peromyscus leucopus.
Class I genes of the Peromyscus leucopus major histocompatibility complex (MhcPele) were examined by Southern blot hybridization, genomic cloning, and DNA sequencing. At least three distinct subtypes of Pele class I genes were discerned, which we have designated Pele-A, B, and C. The nucleotide sequences of exon 5-containing regions (encoding the transmembrane domain) suggested that Pele-A genes are homologs of mouse H-2K, D, L, and Q genes and that Pele-B genes correspond to mouse Tla genes. The Pele-C genes appeared similar to mouse M1 genes. The number of unique genes in each subtype cloned from an individual P. leucopus were 20 for Pele-A, 13 for Pele-B, and 2 for Pele-C. Three genomic clones showed cross-hybridization to both Pele-A and Pele-B gene-specific probes. Six genomic clones remained unclassified as they did not cross-hybridize to exon 5-containing probes from Pele-A, B, or C genes. The homology between the transmembrane domains of Pele class I gene subtypes was found to be similar to that observed between the transmembrane domains of H-2 subtypes (or groups). Interspecific similarity of exon 5 was found to be 81%-88% between Pele class I genes and their H-2 counterparts.
Animals↗
Dietary energy restriction in mice reduces hepatic expression of glucose-regulated protein 78 (BiP) and 94 mRNA.
The influence of life span-prolonging dietary energy restriction on hepatic expression of glucose-regulated protein 78 and 94 (GRP78 and GRP94) RNA was investigated in female C3B10RF1 mice. Mice were either fed ad libitum or fed diets reduced 20 or 40% in energy but containing approximately equivalent amounts of protein, fats, vitamins and minerals. Aging produced no changes in GRP mRNA. However, GRP78 and GRP94 mRNA levels were reduced approximately 50 and 40%, respectively, by 40% energy restriction. This level of energy restriction produced a 43% reduction in the mean plasma glucose levels of young and old mice. The changes in GRP mRNA expression appear to be specific, because the levels of these RNAs were normalized to the level of polyadenylated RNA, and no changes were detected in the levels of a number of other mRNAs. Although extreme glucose deprivation increases GRP mRNA levels in cultured cell lines, physiologically relevant reductions in blood glucose had the opposite effect in the liver, in vivo. The regulatory pathway responsible for these effects is not known. GRP mRNA levels are elevated by agents that increase the level of malfolded proteins in the endoplasmic reticulum. Thus, energy restriction may act to reduce malfolded proteins in the endoplasmic reticulum of hepatic cells.
Aging↗
Polymorphism in the major histocompatibility complex class II genes of Peromyscus leucopus.
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Animals↗
Thyroid hormone transcriptional regulatory region of the growth hormone gene.
By deletion-transfection analysis, a region of the rat growth hormone gene has been identified which directs accurate, thyroid hormone responsive transcriptional initiation in vivo. In addition, a thyroid hormone-responsive DNase I hypersensitive domain containing three discrete hypersensitive sites has been identified near the GH promoter. One site is coincident with the TATA homology, and the others lie approximately 150 nucleotides 5' and 3' of this sequence. The TATA and 5' flanking DNA hypersensitive sites are located in the region of the gene which promotes hormone-responsive gene transcription. Based on these results, it is possible that the molecular basis for thyroid hormone induction of GH gene transcription includes binding of the occupied receptor to chromatin sites flanking the TATA homology, promoting binding of the TATA activating protein to this sequence. Together, these events may enhance the rate of RNA polymerase II initiation at the promoter.
Animals↗
Sequences essential for activity of the thyroid hormone responsive transcription stimulatory element of the rat growth hormone gene.
Thyroid hormone dependent transcription stimulatory and inhibitory elements exist at the 5'-end of the rat GH (rGH) gene (TSE and TIE, respectively). In this study, the location of the sequences essential for TSE activity was examined using stably transfected GC cells. Because the TIE may influence TSE activity, we investigated TSE activity both on the rGH promoter, in the presence of the TIE, and on the viral thymidine kinase promoter, with the TIE deleted. The results of these studies indicate that the minimum sequences essential for TSE activity exist between positions -194 and -169 of the rGH gene.
Animals↗
Discrete positive and negative thyroid hormone-responsive transcription regulatory elements of the rat growth hormone gene.
We have recently shown that a thyroid hormone-responsive transcription stimulatory element exists in the 5'-flanking DNA near the rat growth hormone (rGH) gene (Crew, M. D., and Spindler, S. R. (1986) J. Biol. Chem. 261, 5018-5022). Progressive deletion-transfection analysis of the 5' end of the gene has led to the identification of two genetic elements responsive to thyroid hormone. The first of these is a thyroid hormone-responsive transcription stimulatory element, or TSE. The TSE induced a thyroid hormone-dependent induction-attenuation transcription cycle similar to that of the natural rGH gene. Deletion of sequences between positions -254 and -241 in the rGH 5'-flanking DNA eliminated TSE activity. The second regulatory element is a thyroid hormone-responsive transcription inhibitory element (TIE). When this element was active, thyroid hormone strongly but transiently inhibited rGH promoter utilization. Deletion of sequences between nucleotides -46 and -21 abolished the effects of the TIE. To determine whether the TSE and TIE are enhancer-like, we ligated various regions of rat growth hormone 5'-flanking DNA containing these elements to a chimeric test gene containing the Herpes simplex virus thymidine kinase promoter. Thyroid hormone activated heterologous promoter utilization when a rat growth hormone 5'-flanking DNA fragment containing the TSE (-520 to -115) was linked in cis, regardless of the distance or orientation of the TSE with respect to the promoter. These data suggest that the TSE is a thyroid hormone-dependent enhancer. In contrast, when the TIE was placed immediately 5' to the thymidine kinase promoter, transcription was not effected by 3,5,3'-L-triiodothyronine, suggesting that the TIE is not enhancer-like.
Animals↗
Age-related decrease of growth hormone and prolactin gene expression in the mouse pituitary.
The effects of aging on pituitary GH, PRL, and alpha-tubulin messenger RNA (mRNA) levels were measured in 3-, 12-, and 27-month-old male C57BL/6J mice by dot-blot hybridization. The amount of GH and PRL mRNA in the pituitary deceased dramatically with age. However, total poly(A+) RNA (mRNA), as measured by hybridization with radioactively labeled oligo-(dT), was not altered during aging. In addition, there were no age-related changes in the level of alpha-tubulin mRNA. Thus, the effects of aging on GH and PRL mRNA levels are specific; the levels of the majority of cellular mRNAs are not altered with age. GH and mRNA levels decreased 35% between 3 and 12 months (P less than 0.05) and a total of 75% after 27 months (P less than 0.01). PRL mRNA levels decreased 65% between 12 and 27 months (P less than 0.01), although there was no significant decrease before 12 months. Whereas T3 is the most potent regulator of GH gene expression, we did not detect any significant age-related change in serum T3 levels. These results suggest that factors other than T3 play a role in the age-related decline in GH and PRL gene expression.
Aging↗
Thyroid hormone regulation of the transfected rat growth hormone promoter.
A region of the rat growth hormone gene and 5' flanking DNA has been identified which promotes accurate, thyroid hormone-regulated transcriptional initiation. GC rat pituitary tumor cells were transfected with chimaeric plasmids containing various lengths of rat growth hormone gene and 5' flanking DNA fused to the coding region of the dominant selectable marker gene neo. Thyroid hormone induction of rGH-neo RNA was observed by Northern and dot blot analysis of cells transfected with rGH-neo chimaeric genes sharing the rat growth hormone gene and upstream regions from -235 to +11. Initiation of rGH-neo transcription was mapped by S1 nuclease protection to the in vivo initiation site of the natural growth hormone gene. Transcription of the most deleted thyroid hormone responsive construct involved an induction-attenuation cycle qualitatively similar to the response of the natural gene. However, the 3,5,3'-triiodo-L-thyronine responsiveness of this deleted construct was approximately 2- to 3-fold less than that of less deleted rGH-neo genes tested. These results suggest that, at a minimum, the sequences required for the cyclic 3,5,3'-triiodo-L-thyronine transcriptional response are located within the region of the gene from -235 to +11. Other sequences essential for full responsiveness appear to be located elsewhere in the 5'-flanking DNA. Rat growth hormone promoter utilization appears to be strongly cell-type dependent. We obtained stable transfectants with rGH-neo constructs only in GC cells.
Animals↗