Where, when and how much: regulation of myelin proteolipid protein gene expression.

The myelin proteolipid protein (PLP) gene ( Plp) encodes the most abundant protein found in myelin from the central nervous system (CNS). Expression of the gene is regulated in a spatiotemporal manner with maximal levels of expression occurring in oligodendrocytes during the active myelination period of CNS development, although other cell types in the CNS as well as in the periphery can express the gene to a much lower degree. In oligodendrocytes, Plp gene expression is tightly regulated. Underexpression or overexpression of the gene has been shown to have adverse effects in humans and other vertebrates. In light of this strict control, this review provides an overview of the current knowledge of Plp gene regulation.

Functional characterization of a cis-acting DNA antisilencer region that modulates myelin proteolipid protein gene expression.

Regulation of myelin proteolipid protein (PLP:) gene expression is tightly controlled, both spatially and temporally. Previously, we have shown with transgenic mice that a PLP:-lacZ fusion gene (which includes the entire sequence for PLP: intron 1 DNA) is regulated in a similar manner to endogenous PLP: gene expression. Furthermore, by deletion-transfection analyses using assorted PLP:-lacZ constructs with partial deletion of PLP: intron 1 sequences, we have shown that the first intron possesses an antisilencer region that is capable of over-coming repression mediated by two distinct regions located elsewhere within intron 1 DNA. Here, we report the ability of various fragments encompassing the antisilencer region to restore beta-galactosidase activity when inserted into PLP:-lacZ constructs, which originally exhibited low levels of beta-galactosidase activity. Additional constructs were generated to test the effects of these antisilencer-containing fragments in constructs that are missing either one or both of the negative regulatory regions that are overridden during antisilencing. Transfection analyses, in conjunction with protein-DNA binding assays, suggest that several nuclear factors are necessary for derepression of PLP: gene activity in an oligodendroglial cell line. Moreover, either the "core" or complete antisilencing region can act in an additive or synergistic fashion when multiple copies are inserted into the Plp-lacZ constructs.

Antisilencing: myelin proteolipid protein gene expression in oligodendrocytes is regulated via derepression.

Antisilencer or antirepressor elements have been described, thus far, for only a few eukaryotic genes and were identified by their ability not to augment gene expression per se but to override repression mediated via negative transcription regulatory elements. Here we report the first case of antisilencing for a neural-specific gene, the myelin proteolipid protein (PLP) gene (Plp). PLP is the most abundant protein found in CNS myelin. The protein is synthesized in oligodendrocytes, and its expression is regulated developmentally. Previously we have shown that a PLP-lacZ transgene (which includes the entire sequence for Plp intron 1) is regulated in mice, in a manner consistent with the spatial and temporal expression of the endogenous Plp gene. In the present report, we demonstrate by transfection analyses, using various PLP-lacZ deletion constructs, that Plp intron 1 DNA contains multiple elements that collectively regulate Plp gene expression in oligodendrocytes. One of these regulatory elements functions as an antisilencer element, which acts to override repression mediated by at least two negative regulatory elements located elsewhere within Plp intron 1 DNA. The mechanism for antisilencing appears to be complex as the intragenic region that mediates this function binds multiple nuclear factors specifically.

Myelin proteolipid protein intron 1 sequences do not appear to enhance myelin proteolipid protein gene transcription.

Sequences from the first intron of the mouse myelin proteolipid protein (PLP) gene were examined for their ability to modulate PLP gene expression. Glial (N20.1) or nonglial (NIH 3T3) cells were transiently transfected with constructs that contained 1.4 kb of PLP promoter sequence driving luciferase reporter gene expression, as well as various portions of PLP intron 1 DNA. Although these same PLP intron 1 fragments enhanced reporter gene expression from a heterologous basal promoter in a previous study, the results reported here demonstrate that they do not augment PLP promoter activity. Thus, the regulation of PLP cell-type-specific expression, conferred by the first intron, appears to be mediated by an enhancer-independent mechanism.

The first intron of the myelin proteolipid protein gene confers cell type-specific expression by a transcriptional repression mechanism in non-expressing cell types.

Chimeric genes containing portions of the mouse myelin proteolipid protein (PLP) gene fused to the lacZ reporter gene were used to detect the effect of PLP intron 1 sequences on cell type-specific expression. A transfected fusion gene containing PLP intron 1 sequences was expressed in an oligodendrocyte cell line but not in a liver cell line, consistent with endogenous PLP gene expression. However, an analogous fusion gene missing the first intron was expressed in either oligodendrocyte or liver transfected cells. These studies suggest that transcriptional repressor element(s) located in PLP intron 1 are important in extinguishing expression in non-glial cell types and that the promoter alone functions in an indiscriminate manner. This moderately large intron (>8 kb) was sequenced to aid in future fine mapping of these cell-specific regulatory element(s).

Regulation of murine myelin proteolipid protein gene expression.

To identify putative sequences that direct cell type-specific expression and/or enhance proteolipid protein (PLP) gene expression, glial or nonglial cells were transfected with various PLP-luciferase constructs that collectively span the entire mouse PLP-specific DNA present in a transgene known to direct cell type specificity in transgenic mice. These constructs were transfected into murine oligodendrocyte cell lines that transcribe the PLP gene and, hence, should contain the requisite trans-acting factors necessary for PLP gene expression. Mouse NIH/3T3 fibroblasts were used as a nonglial model. We have finely mapped the PLP promoter region for transcriptional regulatory elements and demonstrate both positive and negative elements, none of which appear to extinguish expression in nonglial cells. The 5'-flanking PLP DNA tested did not enhance the basal herpes simplex-1 virus thymidine kinase (TK) promoter, nor did PLP sequences present in the distal half of intron 1. The 5' portion of intron 1 did enhance TK promoter activity, suggesting that this region of the gene may contain enhancer elements that modulate PLP gene expression; however, the enhancement did not appear to be cell type-specific. Intriguingly, a 541 bp region of the intron that significantly enhanced TK promoter activity contains multiple JC virus repeated elements and other elements known to be important in restricting the virus to oligodendrocytes. These results suggest that intron 1 sequences may modulate expression of the PLP gene.

Dual luminescence-based reporter gene assay for luciferase and beta-galactosidase.

A unique combined luminescence assay for firefly (Photinus pyralis) luciferase and beta-galactosidase (beta-gal) reporter gene products is described. Luciferase and beta-gal activities are determined with the same aliquot of cell lysate prepared from cells contransfected with both reporter genes, thereby reducing manual labor and increasing experimental accuracy. With the Dual-Light assay system, luciferase activity is measured first with an enhanced luciferase assay, followed by quantitation of beta-gal with Galacton-Plus chemiluminescent substrate and Sapphire-II enhancer. Highly sensitive detection of luciferase (2 fg) and beta-gal (8 fg) is achieved with a dynamic range over seven orders of magnitude of enzyme concentration. Comparative analysis of both independent and combined (Dual-Light) detection methods for cells contransfected with luciferase and beta-gal reporter genes is also described.

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice.

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.

Mutations in the myelin proteolipid protein gene alter oligodendrocyte gene expression in jimpy and jimpymsd mice.

The mouse myelin proteolipid protein (PLP) gene has been studied in normal and jimpymsd mice. Potential upstream regulatory regions of the normal gene have been cloned and mapped, but when these regions were studied in jimpymsd mice by Southern blots, no alterations were observed, relative to the normal gene. To assess whether the low ratio of PLP to DM20 proteins in this mutant reflected an altered PLP/DM20 ratio mRNAs, S1 nuclease analyses were undertaken, which demonstrated that at all ages studied in both jimpy and jimpymsd mice, PLP mRNA was elevated above DM20 mRNA. When exon 3 (the site of the alternative splice signal for DM20 mRNA) of the jimpymsd PLP gene was sequenced, no mutation was identified. The transcription of the PLP gene in normal and mutant animals was studied. The transcription rate increases in normal animals with development, and in very young jimpymsd or jimpy mice, the transcription rate of the PLP gene was close to that of age-matched normal animals. However, by 10 days of age, the transcription rate of this gene in both mutants was significantly below that of age-matched controls. The transcription rate of the myelin basic protein (MBP) gene was also reduced, indicating that expression of both genes is affected by this mutation. In contrast, the transcription rate of the glycerol phosphate dehydrogenase (GPDH) gene, an early marker of oligodendrocytes, is equal to or greater than normal in both mutants. We have confirmed an earlier report of a point mutation in exon 6 of the jimpymsd PLP gene, which converts an alanine to a valine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Role of glucosinolates in the causation of liver haemorrhages in laying hens fed water-extracted or heat-treated rapeseed cakes.

Glucosinolates were removed from whole rapeseed by a hot-water extraction procedure or depleted by heat treatment. When laying hens were maintained for three months on diets containing about 300 g kg-1 of these rapeseed cakes, the incidence of liver haemorrhages detected at post mortem examination was similar to that in birds maintained on 300 g kg-1 commercial rapeseed meal and significantly greater than in control birds fed soya-based diets. The effectiveness of glucosinolate extraction or depletion was determined by chemical analysis and by histological examination of the thyroid glands. Histologically the haemorrhages were similar after feeding extracted and commercial rapeseed meals. Diets containing mixtures of nitriles and glucosinolates severely depressed food intake and egg production but did not cause a greater incidence of haemorrhages than the other rapeseed products tested. Mortality from causes other than liver haemorrhage was higher with the diets containing rapeseed and this suggests that rapeseed has a more generalised effect on the body's defence mechanisms. These observations suggest that other factors in rapeseed meal, alone or acting with glucosinolates, may be responsible for inducing liver haemorrhages in laying hens.

Experimental systems which modify and simulate rapeseed-induced liver haemorrhages in in-lay hens.

The occurrence of liver haemorrhages was compared when diets containing 30 or 40 per cent rapeseed meal (RSM) or 30 per cent soybean meal (SBM), with and without experimental additives, were fed to in-lay hens of a commercial egg-producing strain for 12 weeks. The incidence of haemorrhages was significantly greater when the birds were maintained on the basal (unsupplemented) RSM diet than on the equivalent SBM diet. Haemorrhages were either small and infrequent, minute and multiple as in peliosis hepatis, or large enough to rupture the liver capsule. They might be recent or old and encapsulated, sometimes both varieties affecting the same specimen, and they occurred in any part of the liver. Histologically, hepatocyte necrosis and reticulin derangement were not detected in livers without gross haemorrhages and even in those with haemorrhages these abnormalities were only seen closely adjacent to haemorrhages or to foci of eosinophilic fibrinoid. In some instances there was sinusoidal ectasia. Separate additions of 50 g dried skimmed milk powder, 0.5 g zinc oxide, 0.25 g ferrous sulphate or 2.0 mg selenium (as sodium selenite) kg-1 to the basal RSM diet did not significantly modify the incidence of haemorrhage. Ferrous sulphate slightly reduced goitrogenicity. Supplements of 2.2 mg menadione and 1.0 g sodium phenobarbital kg-1 RSM diet induced slight reductions in the number of cases of liver haemorrhage or their severity, indicating that the multifunction oxidase system may be involved in rapeseed hepatotoxicity. The addition of 0.5 g methimazole kg-1 to the basal SBM diet induced severe colloid goitre but did not induce liver haemorrhage. Both thiouracil (0.5 g kg-1 diet) and beta-aminopropionitrile (0.5 g and 2.5 g kg-1 diet) when added to the basal SBM diet induced liver haemorrhages which did not differ in incidence or histological appearance from those induced by RSM. Hyperplastic goitre was caused by thiouracil. Intrahepatic cholestasis induced by sodium taurolithocholate, bilirubin and alpha-naphthylisothiocyanate and extrahepatic cholestasis induced by bile duct ligation resulted in hepatocyte necrosis but not gross liver haemorrhages. Spontaneous deaths due to conditions other than liver haemorrhages were significantly more numerous in RSM-fed than SBM-fed hens.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Liver haemorrhages induced by rapeseed meal: incidence in adult male and female fowls.

Adult cockerels of a commercial egg-producing strain did not develop liver haemorrhages when maintained for 12 weeks on a diet containing 400 g rapeseed meal/kg food. Haemorrhages occurred in 43% of laying hens of the same strain as the cockerels and 33% of laying hens of another strain maintained on the same diet for the same period. Oestrogenisation of the male birds did not influence the incidence of haemorrhage.

Hemiagenesis of the thyroid gland in the quail (Coturnix coturnix japonica).

Hemiagenesis of the thyroid occurred in a closed population of Japanese quails. The unilateral gland was histologically normal, occurred most frequently in females, was heavier than individual pairs of glands and the birds showed no clinical effects. Comparative aspects are discussed.

Zinc toxicity in the fowl: ultrastructural pathology and relationship to selenium, lead and copper.

Some dietary, enzymic and morphological relationships of selenium, lead and copper to the toxicity of zinc in adult female fowls have been investigated. The anorexia, pancreatic and gizzard lesions which resulted from incorporating 20,000 and 25,000 mg zinc as zinc oxide per kg diet were not prevented by concurrent daily injections of 0.3 mg selenium or the addition of 500 and 2500 mg lead per kg diet. Anorexia and gizzard lesions were also produced by feeding copper sulphate at levels of 2000 and 4000 mg per kg of diet but the pancreas was unaffected. This suggests that zinc may act directly on the pancreas rather than by interfering with the protective function of the selenium. Glutathione peroxidase activity significantly decreased in the blood when zinc-containing diets were fed and selenium injection prevented this. Activity in the liver was also reduced but in the pancreas it was increased by both zinc and zinc plus selenium. Electron microscopy showed that there was an infiltration of heterophils in the gizzard of birds given excess zinc but otherwise there was little qualitative difference in surface cell ultrastructure from the degenerative processes of the normal desquamatory sequence. Pancreatic exocrine cells had reduced numbers of zymogen bodies, distended rough endoplasmic reticulum, many cytoplasmic small, electron-dense bodies and large autophagic vacuoles, sometimes leading to cell death. The acinar lumina were dilated and there was periacinar fibrosis. The significance of these lesions as an indication of zinc-induced interference with pancreatic protein synthesis and membrane integrity is discussed.

Elevated bile acids in the plasma of laying hens fed rapeseed meal.

A simple procedure was developed for the estimation of bile acid taurine conjugates in fowl plasma. Laying hens fed a diet containing rapeseed meal (RSM) (400 g kg-1) for 12 weeks had higher bile acid levels (154 mumol litre-1) than hens fed a control soyabean diet (116 mumol litre-1) (P less than 0.01). The incidence of liver haemorrhages was higher (34.8 per cent) in RSM-fed hens than in controls (21 per cent), but the severity of the lesions did not correlate with the bile acid concentration in affected birds. Histological examination of sections from livers of RSM-fed birds did not reveal significant hepatocyte degeneration outside the immediate vicinity of the haemorrhage. Canalicular bile plugs were never seen. The incidence of liver haemorrhages (13 per cent) and plasma bile acids (85 mumol litre-1) were lower in hens fed a diet containing beta-aminopropionitrile (0.5 g kg-1), a known lathyrogen. Acute treatment of hens with the hepatotoxin alpha-naphthyl isothiocyanate over four days induced necrosis of hepatocytes and resulted in elevated bile acid concentrations (262 mumol litre-1) compared with controls (73 mumol litre-1) given arachis oil. It was concluded that laying hens fed high levels of RSM develop cholestasis but the toxic principle is not known.

Ectopic pulmonary cartilage and bone in domestic fowl.

The occurrence and histological appearance of ectopic pulmonary cartilage and bone in domestic fowl was investigated. Its incidence was shown to vary in different strains of birds. The structures may occur in newly hatched chicks and are similarly present in birds maintained on diets containing bonemeal and diets without it. They could not be experimentally induced by bronchial insufflation of fine bone particles. Previous theories of their genesis are discussed and it is concluded that they do not originate from either inhaled dietary bonemeal or disease processes but are probably abnormal embryonic induction of mesenchyme or cartilaginous germ cells displaced from adjacent bronchi.

The morphology of the thyroid glands of quails and fowls maintained on diets containing rapeseed.

Maintaining adult quails and fowls on diets containing rapeseed meal resulted in increased thyroid weight; this increase was greatest when ground, full-fat rapeseed was included in the ration. Thyroids of quails showed a relatively much greater enlargement than those of fowls. A biometric method indicated that the increase in hens and quails was associated with follicle enlargement. Light- and electron-microscopy showed that the thyroid epithelium of quails had frequently become tall columnar, due mainly to extensive dilation of the granular endoplasmic reticulum, and that electron-dense bodies and microvilli were increased in size and number. Vascularity increased and microhaemorrhages occurred. There was sometimes epithelial proliferation (hyperplastic goitre), particularly when the diet contained ground whole rapeseed. If smaller amounts of glucosinolates were offered for a longer period, electron-dense bodies were very enlarged. There were similar cytological reactions in fowls but these, particularly cisternal distension, were less prominent and the enlarged, colloid-filled follicles were usually lined by flat or cuboidal epithelium (colloid goitre). It was concluded that the morphological reaction of the gland is modified by amount of glucosinolate hydrolysis products, period of exposure and species of bird. The changes were discussed in relation to the toxicity of rapeseed to fowls.

A computer method for scanning and analysis of dot blots.

A system is described for computer analysis of autoradiograms produced from dot blots. The autoradiograms are first digitized on a rotating drum densitometer. The system automatically locates, integrates, and corrects the intensity of each spot for background. A linear range of exposures for each series of dilutions on the dot blot is calculated, and the amount of specific RNA is reported relative to other samples or to an internal standard. Without operator intervention, the system can directly scan and analyze all possible spots produced by the commercial filtration devices which use the geometry of a 96-well microtiter plate.