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D S Gross

Publications and source records attributed to D S Gross.

At least 37 records · Page 2Linked to original sources
PubMed1990

Genomic footprinting of the yeast HSP82 promoter reveals marked distortion of the DNA helix and constitutive occupancy of heat shock and TATA elements.

We describe here for the first time successful application of the hydroxyl radical technique for genomic footprinting. In combination with two complementary techniques, DNase I footprinting and dimethyl sulfate methylation protection, we have obtained a high-resolution map of the promoter region of the yeast HSP82 heat shock gene, which resides within a constitutive nuclease hypersensitive site. We find that irrespective of transcriptional state, basal or induced, only one of three putative heat shock elements, HSE1, and the TATA box are tightly bound by proteins, presumably heat shock factor (HSF) and TFIID, respectively. Whereas the HSE1-associated factor binds tightly within the major groove of DNA, as discerned by protection of guanine residues from methylation by dimethyl sulfate in intact cells, the TATA factor appears to bind principally to the sugar-phosphate backbone, as revealed by strong protection from hydroxyl radical cleavage in whole-cell lysates. In addition, while HSE1 is strongly footprinted by DNase I in lysates, the TATA box is only weakly footprinted. Strikingly, both elements are associated with marked distortion of the DNA double helix in chromatin. Protein binding to HSE1 appears to cause a non-B-conformation, on the basis of a local 12 base-pair periodicity of hydroxyl radical protection and the presence of multiple DNase I hyperreactive cleavages flanking HSE1, whose pattern changes following heat shock. Similarly, helix distortion is evident in the vicinity of the TATA box, since hydroxyl radical detects a lower strand-specific hypersensitive site at the dyad center of an adjacent polypurine tract. Finally, the absence of discernable modulation in the DNase I cleavage pattern argues against the presence of a specifically positioned nucleosome within the IISP82 promoter region.

Base Sequence↗
PubMed1990

Promoter function and in situ protein/DNA interactions upstream of the yeast HSP90 heat shock genes.

We have mapped in vivo protein/DNA interactions within the upstream regulatory regions of the two yeast HSP90 genes, and have begun mutagenizing footprinted sequences in an effort to identify the cis-acting determinants of heat shock transcription. Genomic footprinting of the HSP82 promotor using chemical and enzymatic nucleases reveals that irrespective of transcriptional state, the most proximal of three heat shock elements, HSE1, is occupied along both sugar-phosphate backbones as well as within its major groove, while the TATA box is bound along both sugar-phosphate backbones. Distorted DNA structure is associated with each constitutively bound factor: protein binding to HSE1 appears to induce a local A-form-like helical conformation, whereas occupancy of the TATA box is associated with strand-specific nuclease hypersensitivity of an adjacent polypurine tract. In situ mutagenesis experiments indicate that HSE1 is absolutely required for both basal and induced expression, and that basal transcription can be preferentially abolished by point mutations within this sequence. In contrast, point mutations within the TATA element have the reverse effect, as induced transcription is more significantly affected. Similar to HSE1 point mutants, we have found that basal transcription is preferentially repressed by an HMRE silencer element when it is transplaced approximately 1 kb upstream of the HSP82 start site. Finally, a complementary footprinting analysis of the upstream region of the constitutively expressed HSC82 gene reveals the presence of three discrete protein complexes. These map to the TATA box, the promotor-distal heat shock element, C.HSE1, and a novel sequence upstream of C. HSE1, suggesting that the 10-fold higher basal transcription of HSC82 stems, at least in part, from a non-HSE-binding factor.

Base Sequence↗
PubMed1989

Basal-level expression of the yeast HSP82 gene requires a heat shock regulatory element.

Previous studies have shown that heat shock factor is constitutively bound to heat shock elements in Saccharomyces cerevisiae. We demonstrate that mutation of the heat shock element closest to the TATA box of the yeast HSP82 promoter abolishes basal-level transcription without markedly affecting inducibility. The mutated heat shock element no longer bound putative heat shock factor, either in vitro or in vivo, but still resided within a nuclease-hypersensitive site in the chromatin. Thus, constitutive binding of heat shock factor to heat shock elements in S. cerevisiae appears to functionally direct basal-level transcription.

Base Sequence↗
PubMed1988

Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes.

We describe a vacuum blotting procedure for transferring DNA fragments from conventional polyacrylamide sequencing gels to nylon membranes. The method employs a combination of vacuum-assisted diffusion (effected by a standard gel drier) and an osmotic gradient (effected by over- and underlying filters presoaked in ammonium acetate). Fragments up to 310 nucleotides in length transfer at 40-60% efficiency within 90 min. When combined with indirect end-labelling, the method allows genomic sequencing of a single-copy gene of Saccharomyces cerevisiae employing as little as 5 micrograms DNA per lane.

Autoradiography↗
PubMed1986

The ubiquitous potential Z-forming sequence of eucaryotes, (dT-dG)n . (dC-dA)n, is not detectable in the genomes of eubacteria, archaebacteria, or mitochondria.

The potential Z-forming sequence (dT-dG)n . (dC-dA)n is an abundant, interspersed repeat element that is ubiquitous in eucaryotic nuclear genomes. We report that in contrast to eucaryotic nuclear DNA, the genomes of eubacteria, archaebacteria, and mitochondria lack this sequence, since even a single tract of greater than or equal to 14 base pairs in length is not detectable through either hybridization or sequence analysis. Interestingly, the phylogenetic distribution of the (dT-dG)n . (dC-dA)n repeat exhibits a striking parallel to that of (dT-dC)n . (dG-dA)n, but not to other homocopolymeric sequences such as (dC-dG)n . (dC-dG)n or (dT-dA)n . (dT-dA)n.

Archaea↗
PubMed1985

Sexual responsiveness and its relationship to vaginal stimulation-produced analgesia in the rat.

Increasing amounts of pressure applied to the cervix produce a dose-response-like elevation of pain threshold in rats. This vaginal stimulation-produced analgesia (VSPA) is facilitated in animals given estrogen (E) doses sufficient to induce high levels of sexual receptivity. It has been proposed that enhancement of VSPA may serve to decrease any noxious input associated with multiple intromissions by the male. In this study, the anti-nociceptive effect of VSPA was compared in animals given E doses insufficient to increase receptivity with animals made receptive using subthreshold E levels + progesterone (P) in an attempt to determine if enhancement of VSPA is associated with the receptive state of the animal or the dose of E used. Tail flick latencies and tail shock vocalization thresholds were measured in groups of E, E + P and oil-treated rats during application of 0, 100 and 200 g of force on the cervix. Within oil, E and E + P-treated animals, significant increases in tail flick latencies were observed at 100 and 200 g with respect to baseline (0 g). Moreover, at 100 g of force E treated animals displayed a significant increase in tail flick latency over oil and E + P treated rats. In contrast, tail shock vocalization was increased at 100 and 200 g levels of probing in oil and E + P groups but was not facilitated by E. In the present study, as in previous work, VSPA was potentiated by E; however, this potentiation was not correlated with steroid-induced receptivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesia↗
PubMed1985

Gonadotropin-releasing hormone within the organum vasculosum lamina terminalis in the ovariectomized, estrogen/progesterone-treated rat: a quantitative immunocytochemical study using image analysis.

No specific function has been ascribed to the high gonadotropin-releasing hormone (GnRH) content of the organum vasculosum lamina terminalis (OVLT). The objective of this study was to determine whether levels of GnRH within the OVLT are altered during cyclic gonadotropin secretion. GnRH levels were determined at various times during an estrogen/progesterone (E/P)-induced LH surge. Groups of E/P and sesame oil-treated animals were decapitated at 12.00 h, 14.00 h, 16.00 h, 18.00 h, and 22.00 h following the P or oil treatment. Morphological localization, as well as quantitation of immunoreactive GnRH within discrete regions of the brain was achieved by combining unlabeled antibody immunocytochemistry with computerized image analysis. Analysis of GnRH levels in the OVLT revealed that at any of the 5 times examined, there was: (1) no significant difference among controls, (2) no significant difference among E/P-treated animals, and (3) no significant difference between E/P-treated versus control animals. In contrast, ME GnRH levels in E/P-treated rats showed the expected decrease prior to the onset of the LH surge. These findings suggest that the level of GnRH detected in axon terminals within the OVLT cannot be related directly to the serum LH status of ovariectomized, E/P-treated rats. It is therefore possible that GnRH within the OVLT might function in a neuromodulatory role, rather than as a direct regulator of cyclic gonadotropin secretion.

Animals↗
PubMed1985

Chromatin structure of the potential Z-forming sequence (dT-dG)n X (dC-dA)n. Evidence for an "alternating-B" conformation.

The sequence (dT-dG)n X (dC-dA)n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG)n X (dC-dA)n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG)n X (dC-dA)n elements instead adopt an "alternating-B" conformation on the nucleosomal surface.

Animals↗
PubMed1985

Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system.

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.

3,3'-Diaminobenzidine↗
PubMed1984

The mammalian hypophysial pars tuberalis: a comparative immunocytochemical study.

Despite its occurrence in most vertebrate species, the function of the hypophysial pars tuberalis (PT) remains obscure. Recent immunocytochemical studies have demonstrated the presence of hormone-containing cells in the few species studied. In the present study the secretory cell composition of the PT was characterized in a variety of mammals using immunocytochemistry. Species studies were the mouse, rat, guinea pig, rabbit, sheep, rhesus monkey, baboon, and human. Antisera were chosen on the basis of their ability to identify a distinct cell population in the pars distalis. A total of 21 antisera were used to identify GH, PRL, ACTH, beta-endorphin, LH, FSH, and TSH. Gonadotropes were identified in the PT of all eight species and were the predominant immunoreactive cell type in the human, baboon, rhesus monkey, sheep, guinea pig, rabbit, and mouse. Thyrotropes were detected in all species except the sheep. They were the predominant cell type in the rat but were less common than gonadotropes in other species. No other secretory cell types were found, with the exception of occasional somatotropes and mammotropes in some human specimens, and small clusters of opiocorticotropes in the guinea pig. Thus the general pattern in the mammalian PT is the presence of gonadotropes and thyrotropes and the absence of other pituitary cell types. In the human, baboon, and rat, all PT parenchymal cells can be identified immunocytochemically. However, in the rhesus monkey, sheep, guinea pig, rabbit, and mouse, the majority of PT cells do not react with any antisera, and thus their function is unknown. Follicles are common in the PT of most mammalian species, however, the luminal contents do not react with antisera to adenohypophysial hormones.

Animals↗
PubMed1984

The ovine pars tuberalis: a naturally occurring source of partially purified gonadotropes which secrete luteinizing hormone in vitro.

The pars tuberalis and pars distalis are constant subdivisions of the vertebrate adenohypophysis. Unlike the pars distalis, however, direct evidence of an endocrine function for the pars tuberalis is lacking. The present immunocytochemical study shows the ovine pars tuberalis to be unique in that 1) its only immunoreactive hormone-containing cell type is the gonadotrope; and 2) the vast majority of pars tuberalis cells contain no identifiable pituitary hormones. In vitro superfusion studies demonstrate that the ovine pars tuberalis is capable of baseline and GnRH-stimulated gonadotropin secretion which, on a per mg basis, is qualitatively and quantitatively similar to the secretory capacity of the pars distalis.

Animals↗
PubMed1983

Distribution of immunoreactive somatostatin in the primate hypothalamus.

Immunocytochemical methods were used to compare the localization of somatostatin (SRIF) in the human and rhesus monkey hypothalamus. The distribution of SRIF-containing cell bodies and fibers is similar in the two species. Perikarya are located predominantly in the periventricular region and to a lesser extent in the ventromedial nucleus. Fibers occur in dense clusters within the periventricular region, ventromedial nucleus, arcuate nucleus, median eminence, and pericommissural area of both species. Analysis of serial sections suggests that fibers originate from cells in the periventricular region, extend ventrally through the ventromedial and arcuate nuclei to terminate around the portal vessels of the infundibular stalk, and thereby participate in the regulation of anterior pituitary function. Somatostatinergic fibers are also found surrounding non-immunoreactive perikarya in the ventromedial nucleus and periventricular region of both primates. This arrangement may support somatostatin's postulated role as a neurotransmitter or neuromodulator. The strong similarity between the localization of hypothalamic SRIF in the human and rhesus monkey supports the use of the rhesus monkey as a model for the study of somatostatin as a neuroendocrine regulatory in the human.

Adult↗
PubMed1983

5-methylcytosine is localized in nucleosomes that contain histone H1.

We have developed a procedure for purifying highly specific polyclonal antibodies against 5-methylcytosine. These antibodies were used to probe the distribution of 5-methylcytosine among fractionated nucleosomes of mouse cell chromatin. Our results demonstrate that at least 80% of the 5-methylcytosine is localized in nucleosomes that contain histone H1. Native nucleosomes that lack histone H1 or possess high mobility group proteins package DNA that is 1.6- to 2.3-fold undermethylated. We suggest that the preferential association of methylated sequences with histone H1 has functional significance because DNA methylation has been linked to gene inactivation and histone H1 is known to promote chromatin condensation.

5-Methylcytosine↗
PubMed1983

Hormone production in the hypophysial pars tuberalis of intact and hypophysectomized rats.

The endocrine function of the hypophysial pars tuberalis is currently unknown. Recent immunocytochemical and electron microscopic studies have shown the presence of gonadotropin-producing cells in this tissue in intact rats. Because the pars tuberalis is not destroyed by hypophysectomy, the objective of the present study was to determine whether this gland is activated to produce pituitary hormones following hypophysectomy. Hormone-producing cells were identified by the unlabeled antibody peroxidase-antiperoxidase method of immunocytochemistry using primary antisera generated against the following hormones: LH, FSH, TSH, GH, PRL, ACTH, and beta-endorphin. In intact control rats, the only cell types detected in the pars tuberalis were gonadotropes and thyrotropes. Most gonadotropes contained both LH and FSH. The TSH cells were a separate cell population, and constituted the majority of pars tuberalis parenchymal cells. As early as 1 week after hypophysectomy, a hyperplastic and hypertrophic response was noted in the gonadotropes, and both cell types showed cytological features characteristic of enhanced hormonal synthesis and secretion. These responses increased with time post hypophysectomy. Secretory cell types not present in the pars tuberalis before hypophysectomy, i.e. somatotropes, mammotropes, opiocorticotrophes, were not activated or induced to differentiate after hypophysectomy. Based on these immunocytochemical observations, the hypophysectomized rat cannot be viewed as an animal totally devoid of anterior pituitary hormones. It has the capacity, or at least the potential, for secretion of LH, FSH, and TSH.

Animals↗
PubMed1982

Prolactin status of hereditary dwarf mice.

Snell (dw/dw) and Ames (df/df) dwarf mice of both sexes were evaluated for immunoassayable PRL in plasma and for the presence of PRL-containing cells in the hypophysis. Regardless of the method of blood collection (decapitation or cardiac or orbital puncture), minimal concentrations of PRL were detected in the plasma of hereditary dwarf mice. PRL secretion was not augmented in Snell or Ames female dwarfs after treatment with perphenazine or estradiol benzoate, stimuli which greatly increased PRL release in normal female littermates. Comparison of PRL levels in dwarf animals (dw/dw or df/df) using two different homologous RIAs substantiated the observation that male and female dwarfs are PRL deficient. Mammotropes, readily detectable in the pituitary glands of all normal siblings of Snell and Ames mice, were absent from the dwarf mouse hypophysis, which was markedly reduced in size. The lack of PRL-containing cells in the dwarf mouse pituitary may explain why peripheral PRL levels in this animal are below those measured in hypophysectomized mice.

Animals↗
PubMed1981

Influence of inorganic cations and histone proteins on the terbium(III)-nucleic acid interaction.

We have studied the interaction of the fluorescent lanthanide, terbium(III) (Tb3+), with polynucleotides and linear and superhelical DNA, through employment of mono- and multivalent cations as competitive inhibitors. Increasingly effective competitive inhibiton of the Tb3+-nucleic acid interaction was achieved, for the most part, in the cation order monovalent less than divalent less than tetravalent. The divalent cation Cu2+ proved to be an exception to this trend, and was the strongest competitive inhibitor or all cations tested, exhibiting an affinity for Tb3+ binding sites over twice that of Tb3+ itself. Unexpectedly, a narrow range of low sodium ion concentration (8--20 mM) was found to be effective in inducing localized unwinding or unstacking of linear and supercoiled DNA double helices, a phenomenon detectable through the use of both Tb3+ fluorescence enhancement and ultraviolet spectroscopy. Within a similar range of low sodium ion concentration, moreover, histone H1 was substantially more effective in displacing terbium ion from DNA than either histones H2B or H4, but at higher ionic strength, this difference was absent. These results further confirm the sensitivity and specificity of Tb3+ as a conformational probe of nucleic acids.

Animals↗