Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression.

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.

Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.

Immunocytochemical localization of seleno-glutathione peroxidase in the adult mouse brain.

Cytoplasmic seleno-glutathione peroxidase, by reducing hydrogen peroxide and fatty acid hydroperoxides, may be a major protective enzyme against oxidative damage in the brain. Oxidative damage is strongly suspected to contribute to normal aging and neurodegenerative process of Alzheimer's and Parkinson's diseases. We report here an immunocytochemical analysis of the localization of glutathione peroxidase in the adult mouse brain, carried out with an affinity-purified polyclonal antibody. Most of the brain areas analysed showed weak to strong glutathione peroxidase immunoreactivity, expressed in both neurons and glial cells. The strongest immunoreactivity was found in the reticular thalamic and red nuclei. Highly immunoreactive neurons were observed in the cerebral cortex (layer II), the CA1, dentate gyrus and pontine nucleus. Other regions, such as the caudate-putamen, septum nuclei, diagonal band of Broca, hippocampus, thalamus and hypothalamus, showed moderate staining. This study provides original information about the wide distribution of glutathione peroxidase in the mouse brain. Double-staining experiments indicated that specific subsets of cholinergic neurons in septal and diagonal band nuclei were negative for this antigen. Similarly, many dopaminergic neurons of the substantia nigra pars compacta expressed low levels of glutathione peroxidase antigen, in contrast to the ventral tegmental area, wherein most catecholaminergic cells were strongly positive. A lack of glutathione peroxidase in subsets of dopaminergic or cholinergic neurons may thus confer a relative sensitivity of these cells to oxidative injury of various origins, including catecholamine oxidation, neurotoxins and excitotoxicity.

Immunocytochemical evidence that amyloid beta (1-42) impairs endogenous antioxidant systems in vivo.

Amyloid beta, the major constituent of the senile plaques in the brains of patients with Alzheimer's disease, is cytotoxic to neurons and has a central role in the pathogenesis of the disease. We have previously demonstrated that potent antioxidants idebenone and alpha-tocopherol prevent learning and memory impairment in rats which received a continuous intracerebroventricular infusion of amyloid beta, suggesting a role for oxidative stress in amyloid beta-induced learning and memory impairment. To test the hypothesis, in the present study, we investigated alterations in the immunoreactivity of endogenous antioxidant systems such as mitochondrial Mn-superoxide dismutase, glutathione, glutathione peroxidase and glutathione-S-transferase following the continuous intracerebroventricular infusion of amyloid beta for 2 weeks. The infusion of amyloid beta (1-42) resulted in a significant reduction of the immunoreactivity of these antioxidant substances in such brain areas as the hippocampus, parietal cortex, piriform cortex, substantia nigra and thalamus although the same treatment with amyloid beta (40-1) had little effect. The alterations induced by amyloid beta (1-42) were not uniform, but rather specific for each immunoreactive substance in a brain region-dependent manner. These results demonstrate a cytological effect of oxidative stress induced by amyloid beta (1-42) infusion. Furthermore, our findings may indicate a heterogeneous susceptibility to the oxidative stress produced by amyloid beta.

Effects of antioxidant enzyme modulations on interleukin-1-induced nuclear factor kappa B activation.

Nuclear factor kappa B (NF-kappa B) is a potent and pleiotropic transcription factor that can be activated by a wide variety of inducers, including interleukin-1 (IL-1). Although the detailed activation mechanism of NF-kappa B is still under investigation, it requires both phosphorylation and degradation of its inhibitory subunit I kappa B and the presence of an oxidative environment. In this study, we systematically evaluated the influence of glutathione peroxidase, glutathione reductase and catalase on IL-1-induced NF-kappa B activation by analysing the effect of specific inhibitors of these enzymes. For the three antioxidant enzymes mentioned, their inhibition correlated with an overactivation of NF-kappa B, particularly for glutathione peroxidase. Inversely, we tested the response of glutathione peroxidase-transfected cells on NF-kappa B activation, which was lower as compared with the parental cells. Furthermore, interleukin-6 production also correlated perfectly with the reduced level of NF-kappa B activation is these experiments. The results clearly show that NF-kappa B activation is, strongly dependent on the antioxidant potential of the cells, especially on the activity of reduced glutathione-dependent enzymes such as glutathione peroxidase. The results support the hypothesis that the level of the oxidised glutathione:reduced glutathione ratio and the activity of intracellular antioxidant enzymes play a major role in NF-kappa B tine tuning.

Transgenic glutathione peroxidase mouse models for neuroprotection studies.

Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.

Protein synthesis in hyperoxic endothelial cells: evidence for translational defect.

To study the effects of hyperoxia on protein synthesis in primary cultures of porcine aortic endothelial cells, we exposed confluent cells to different O2 concentrations for various durations. Exposure to 95% O2 for 5 days resulted in a 71% inhibition of [3H]phenylalanine incorporation into total proteins. When compared with control cells, we observed no changes in 1) the pool size of free cytoplasmic phenylalanine and of phenylalanine attached to transfer RNA (tRNA), 2) the rate of protein degradation, and 3) the rate of charging of tRNA with phenylalanine. We found that under hyperoxic conditions 1) the incorporation of [3H]-uridine into total and polyadenylated RNA was increased, 2) the efficiency of extracted messenger RNA to direct protein synthesis in a reticulocyte lysate was maintained, 3) the proportion of polymeric to monomeric ribosomes was slightly increased, and 4) the rate of elongation, as measured by the ribosomal transit time, was decreased. Thus the reduction in protein synthesis in hyperoxic cells appears to result primarily from defects at the translational level in polypeptide chain elongation.

Neurobehavioral changes in mice chronically exposed to methylmercury during fetal and early postnatal development.

Pregnant C57BL/6 mice were chronically treated with 0, 4, 6, or 8 ppm of methylmercury chloride (MeHg) in drinking water during fetal and early postnatal development. Four behavioral functions were analyzed in female and male offspring between the age of 6 and 12 weeks: motor coordination learning on the rotarod; training to spatial alternation in the standard T maze followed by a working memory test with delays; spontaneous locomotion and rearings in the open field; reference and working memory assessment in the modified T maze [Behav. Neurosci. 102 (1988) 635]. Chronic perinatal treatment with MeHg resulted in moderate brain levels of mercury near birth which rapidly decreased during nursing. MeHg exerted an effect on the performance of females, but not of males, on two of the four measurements. All treated females exhibited less locomotion than control mice when the open field was new, but not in the following four sessions when the environment was becoming increasingly familiar. Working memory was impaired in females treated with 6 and 8 ppm of MeHg in the modified T maze, but not on the test with delays in the standard T maze. Taken together, these results show that chronic exposure to MeHg during fetal and postnatal development had sex-dependent effects on horizontal exploration and on working memory in the modified T maze, and no effects on motor coordination learning and reference memory.

Neurobehavioral changes in mice treated with methylmercury at two different stages of fetal development.

Pregnant C57BL/6 mice were orally given daily doses of 4 or 6 mg/kg of methylmercury chloride (MeHg) or vehicle during either gestational days 7-9 (GD7-9) or days 12-14 (GD12-14). Their female offspring were tested between 6 and 16 weeks of age on a variety of behavioral tasks. Motor coordination on the rotarod and visual discrimination learning in the Y maze were not affected by administration of MeHg either at GD7-9 or at GD12-14. In the open field, the total number of square crossings was lower in mice treated with 4 and 6 mg/kg of MeHg at GD12-14 than in control mice whether the environment was new or familiar, but prenatal administration of MeHg at GD7-9 had no effect on this measure. Administration of MeHg either at GD7-9 or at GD12-14 had no effect on the percentage of central square crossings or on the frequency of rearings in the open field. On spatial alternation training in the T maze, both treated groups in Condition GD7-9 and the group treated with 6 mg/kg at GD12-14 required more sessions to reach the learning criterion than their respective vehicle groups. When spatial alternation was tested with delays, treated groups did not differ from their respective control groups. In the radial arm maze, the performance of mice treated at GD7-9 was normal, but reference memory and working memory were impaired by administration of MeHg at GD12-14. In mice treated with 4 mg/kg of MeHg, reference memory was impaired only on the first block of trials, whereas in mice treated with 6 mg/kg, the deficit persisted on all blocks of trials. Overall, these results indicate that prenatal administration of MeHg at GD12-14 had more detrimental effects on behavioral performance than administration at GD7-9. It reduced locomotor activity and impaired reference memory for egocentric and allocentric spatial information as well as working memory for places.

Emerging roles of thioredoxin cycle enzymes in the central nervous system.

The thioredoxins (Trxs) constitute a family of enzymes which catalyze the reduction of protein disulfide bonds. Recent animal studies have revealed the importance of the Trx superfamily in various experimental systems. For example, the homozygous disruption of the genes encoding cytoplasmic (TRX1) or mitochondrial Trx (TRX2) in mice generates lethal embryonic phenotypes. In contrast, transgenic mice overexpressing TRX1 show an extended life span and are relatively resistant to ischemia- mediated brain damage. In addition to their capacity to detoxify peroxides in concert with peroxiredoxins and Trx reductases, Trx isozymes perform multiple redox signaling functions mediated by their specific interaction with various proteins, including redox-regulated kinases and transcription factors. Recent studies indicate that specific isoforms of Trx cycle enzymes, targeted to different cell compartments, are key regulators of fundamental processes, such as gene expression, cell growth and apoptosis. The present review is primarily focused on the emerging neuroprotective role of these proteins in the central nervous system.

Structure and expression of a human gene coding for a 71 kd heat shock 'cognate' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.

Increased resistance of GPx-1 transgenic mice to tumor promoter-induced loss of glutathione peroxidase activity in skin.

Reactive oxygen species formation is strongly suspected to play a role in multistep carcinogenesis, notably in tumor promotion. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces peroxide production, oxidative damage to DNA and inflammation in mouse skin. TPA is also known to cause a decrease in the activity of several antioxidant enzymes including glutathione peroxidases (GPx). The observation that several anti-oxidants can inhibit TPA-mediated tumor promotion suggests that a decline in GPx activity could contribute to tumor promotion. We report here the effects of TPA on GPx activity in the skin of transgenic GPx mice that contain human GPx-1 transgenes under the regulation of a metallothionein IIA promoter. As expected, no significant difference in basal level of skin GPx activity was detected in the 3 lines of tg-MT-GPx mice investigated compared with non-transgenic controls. A single topical application of TPA induced gradually, over 20 hr, a small but detectable increase in GPx mRNA and protein levels in skin of non-transgenic mice and a contrasting decrease in both selenium-dependent and selenium-independent GPx activity. The extent of GPx induction was more pronounced in transgenic mice, and in contrast with non-transgenic mice, no significant loss of GPx activity was observed in the TPA-treated skin of these mice. Transgenic mice may, therefore, offer a novel model suitable to assess the role of GPx-1 in skin carcinogenesis, without the potential disadvantage of abnormally high levels of GPx activity produced constitutively in other transgenic models.

Distinct oxidoresistance phenotype of human T47D cells transfected by rat glutathione S-transferase Yc expression vectors.

We have investigated the role of a glutathione S-transferase (GST) with inherent peroxidase activity in the cellular defense against lipid peroxidation and free radical-mediated oxidative damage. Stable transfectants of human T47D cells were generated which express recombinant rat GST-Yc from a human cytomegalovirus promoter-based expression vector. Among several GST-Yc transfectants characterized, two of them contained, respectively, 2- and 3-fold higher GST activity than parental cells or control transfectants and, respectively, 4-5- and 8-10-fold higher selenium-independent glutathione peroxidase activity. Cellular growth kinetics and rates of [3H]thymidine incorporation showed that both transfectants were more resistant to oxidative shocks mediated by cumene hydroperoxide or singlet oxygen generated by photosensitized rose bengal than were T47D cells and control transfectants. In contrast, a T47D transfectant, which expressed high levels of recombinant selenoglutathione peroxidase and showed enhanced resistance to cumene hydroperoxide (Mirault, M.-E., Tremblay, A., Beaudoin, N., and Tremblay, M. J. (1991) J. Biol. Chem. 266, 20752-20760), was as sensitive as parental cells to singlet oxygen. No difference was found in growth sensitivity to 1-h shock treatments with the quinonoid drug daunomycin, irrespective of GST-Yc or selenoglutathione peroxidase overexpression in these cells.

Overexpression of seleno-glutathione peroxidase by gene transfer enhances the resistance of T47D human breast cells to clastogenic oxidants.

The role of seleno-glutathione peroxidase (GSHPx; EC 1.11.1.9) in the cellular defense against oxidative stress was selectively investigated in novel cell models. Expression vectors designed to overexpress human GSHPx efficiently in a broad range of mammalian cells were used to transfect T47D human breast cells which contain very low levels of endogenous GSHPx. Several stable transfectants expressing GSHPx to various extents, up to 10-100 times more than parental cells, were isolated and characterized. Growth inhibition kinetics following transient exposure to increasing concentrations of H2O2, cumene hydroperoxide or menadione (an intracellular source of free radicals and reactive oxygen intermediates) showed that transfectants overexpressing GSHPx were considerably more resistant than control T47D cell derivatives to each of these oxidants. A sensitive DNA end-labeling procedure was used as a novel approach to compare relative extents of DNA strand breakage in these cells. In contrast to the extensive DNA damage induced in control transfectants by 1-h exposure to cytotoxic concentrations of menadione, the extent of DNA breakage detected in GSHPx-rich transfectants was remarkably reduced (6- to 9-fold, p less than 0.005).

Differential expression of hsp70 stress proteins in human endothelial cells exposed to heat shock and hydrogen peroxide.

The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45 degrees C), exposure to hydrogen peroxide (H2O2) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (O2- plus H2O2), as well as exposure to 95% O2, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45 degrees C; (2) treatment with a 5-mM H2O2 bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95% O2. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and H2O2-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to H2O2 treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.

Preferential localization of DNA damage induced by depurination and bleomycin in a plasmid containing a scaffold-associated region.

Recent evidence suggests that DNA damage of various origins is not randomly distributed in the genome but appears to be clustered in unidentified hypersensitive regions of the chromatin. A model was proposed that stipulates that unpaired DNA stretches, such as those found in scaffold- (or matrix)-associated regions (SARs) under torsional strain, are candidate regions of hypersensitivity to DNA damage in vivo. In this study, we assessed in vitro the relative susceptibility of supercoiled plasmids containing a SAR or chromatin loop DNA segment to DNA damage induced by acid-catalyzed depurination or FeIII-bleomycin. Single-strand specific S1 nuclease was used in combination with 3'-end-labeling to detect single-strand breaks or gaps, after cleavage of abasic sites or removal of 3'-phosphoglycolates by Escherichia coli endonuclease IV. The optimal conditions of DNA cleavage specificity by S1 nuclease were determined. Using these conditions, the DNA cleavage patterns obtained showed (i) a preferential localization of S1 hypersensitive sites in the SAR DNA as compared with plasmid or chromatin loop DNA and (ii) a strikingly similar localization of DNA damage with the two clastogenic treatments.

Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels.

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.

Regulation of heat-shock genes: a DNA sequence upstream of Drosophila hsp70 genes is essential for their induction in monkey cells.

Heat-shock genes coding for heat-shock protein 70 (HSP70) in Drosophila melanogaster were subcloned into an SV40/plasmid recombinant capable of replication in permissive monkey COS cells. Following transfection of COS cells, no significant amount of Drosophila hsp70 RNA was detected at 37 degrees C. In contrast, a heat-shock at 43 degrees C or arsenite poisoning at 37 degrees C induced the massive production of Drosophila hsp70 RNA of correct size and faithful 5' ends. After heat-shock, the efficiency of hsp70 transcription in COS cells containing 2-4 X 10(4) gene copies was found to be 15-30% of that measured in Drosophila, on a per gene basis. By testing a series of 5' deletion mutants in this inducible transcription assay it was found that a sequence less than 70 bp long, directly upstream of the hsp70 gene, was essential for the heat or arsenite induction of transcription.

Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold.

Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells. We find one loop for each of the tandemly repeated histone gene clusters. The attachment site is localized in the A + T rich H1-H3 spacer on a 657 bp fragment. In the cluster of the hsp70 heat-shock genes, in both control and heat-shocked cells, we find two attachment sites in close proximity upstream of regulatory elements. The transcribed sequences are not associated with the nuclear scaffold in control or in heat-shocked cells. A family of attachment sites related by hybridization to those of the hsp70 genes was discovered.

Genetic and molecular analysis of the 87A7 and 87C1 heat-inducible loci of D. melanogaster.

Two different types of heat-inducible sequences are found at the cytogenetic loci 87A7 and 87C1 of D. melanogaster. One of these codes for the 70,000 dalton heat shock protein (hsp 70) and is found at both loci. The other type of sequence (alpha beta) codes for an RNA of unknown function and is found only at 87C1. We have completed a study of the organization of the two loci, using deficiencies that delete one or other locus, and have estimated the number of the hsp 70 genes at each locus. Thus in at least three strains of files there are a total of five coding sequences, three at 87C1 and two at 87A7. Restriction mapping of the coding regions at the two loci reveals that each of the two cytogenetic loci has its own characteristic coding sequence. The overall organization of the two loci appears to differ considerably. The alpha beta and hsp 70 heat-induced sequences at 87C1 are closely linked and are contained within two Eco RI restriction fragments.