A dynamic programming approach to de novo peptide sequencing via tandem mass spectrometry.

Tandem mass spectrometry fragments a large number of molecules of the same peptide sequence into charged molecules of prefix and suffix peptide subsequences and then measures mass/charge ratios of these ions. The de novo peptide sequencing problem is to reconstruct the peptide sequence from a given tandem mass spectral data of k ions. By implicitly transforming the spectral data into an NC-spectrum graph G (V, E) where /V/ = 2k + 2, we can solve this problem in O(/V//E/) time and O(/V/2) space using dynamic programming. For an ideal noise-free spectrum with only b- and y-ions, we improve the algorithm to O(/V/ + /E/) time and O(/V/) space. Our approach can be further used to discover a modified amino acid in O(/V//E/) time. The algorithms have been implemented and tested on experimental data.

Protein factors in thyrotropic tumor nuclear extracts bind to a region of the mouse thyrotropin beta-subunit promoter essential for expression in thyrotropes.

The beta-subunit gene of TSH is specifically expressed in thyrotrope cells of the anterior pituitary gland. To define the particular TSH beta-subunit gene sequences responsible for tissue-specific expression, TSH beta promoter fragments were assessed for promoter activity by gene transfer into TSH-expressing thyrotropic tumor cells (TtT-97). Previous studies have shown that the murine TSH beta gene promoter was more efficiently used in TtT-97 cells compared to other pituitary-derived cells or nonpituitary fibroblasts and that a 191-basepair DNA sequence of the 5' flanking region between -271 and -80 was sufficient for maximal promoter activity in thyrotropes. Further deletional analysis within this region has localized the area responsible for expression in thyrotropes to a 37-basepair region between -117 and -80 up-stream of the major transcriptional initiation site. DNase-I protection assays demonstrated that this functionally defined 5' flanking area, in addition to the adjacent sequences immediately up-stream and down-stream, interacts with protein factors present in nuclear extracts from TtT-97 tumor cells. When fused to a heterologous promoter, fragments derived from the region between -271 and -80 exhibited cell-specific activity, although this was not conferred solely by the TSH beta promoter fragment from -117 to -80. Heterologous promoter activity was further stimulated when fragments containing the areas from -271 or -201 to -77 were used, suggesting combinatorial cis interactions between these regions of the TSH beta promoter. DNase-I protection studies suggest that there are multiple protein-binding domains in the mouse TSH beta 5' flanking sequence. Only the more proximal domains, which encompass important promoter elements, appear to be required for efficient expression in thyrotropes, whereas other more up-stream sites of protein interaction may be involved in regulatory aspects of TSH beta gene expression.

Thyrotrope expression and thyroid hormone inhibition map to different regions of the mouse glycoprotein hormone alpha-subunit gene promoter.

The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element.

TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally.

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.

Thyroid hormone regulates the mouse thyrotropin beta-subunit gene promoter in transfected primary thyrotropes.

In TtT 97 cells, a thyrotropin-producing mouse pituitary tumor, thyroid hormone rapidly inhibits the transcription rate of both the thyrotropin alpha- and beta-subunit (TSH beta) genes, and this closely parallels the increase in nuclear thyroid hormone receptor occupancy. In this study, we have identified regions of the mouse TSH beta gene which are involved in mediating tissue-specific and thyroid hormone-regulated expression. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the firefly luciferase gene. Following transfection by electroporation, efficient expression of TSH beta 5'-flanking luciferase constructs occurred only in cells derived from TtT 97 tumors which express the endogenous TSH beta gene. Deletion analysis demonstrated that the region of the 5'-flanking DNA between positions -271 and -80 relative to the major transcriptional start site is important for TSH beta promoter activity in thyrotropes. No expression was measurable in mouse L cells, a fibroblast line, whereas a low level of expression was seen in MGH 101A cells derived from a thyrotropic tumor which no longer expresses the TSH beta gene. Reduced expression of TSH beta constructs was also found in GH3 and GH4 pituitary tumor lines. Addition of thyroid hormone effectively inhibited the level of transient TSH beta promoter activity in TtT 97 cells in a dose-dependent manner. The inhibitory effect was more pronounced and more accurately reflected the transcription rate data when transfected cells were derived from tumors treated with thyroid hormone for 5 days prior to transfection. Deletion of all but 46 base pairs of TSH beta gene 5'-flanking DNA and 3 base pairs of the first exon had no effect on thyroid hormone inhibition. This indicates that signals sufficient for transcriptional regulation of the TSH beta gene by thyroid hormone reside in the vicinity of the proximal promoter and may act by interfering with basal transcriptional factors.

Identification of thyrotroph-specific factors and cis-acting sequences of the murine thyrotropin beta subunit gene.

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prednisolone and dexamethasone in vivo and in vitro: studies of insulin binding, deoxyglucose uptake and glucose oxidation in rat adipocytes.

We have studied the effects of dexamethasone and prednisolone in vitro and in vivo on insulin binding, deoxyglucose uptake and glucose oxidation in rat adipocytes. In the studies in vivo, rats were treated for 22 h with dexamethasone (30 micrograms/kg) or prednisolone (200 micrograms/kg). Following sacrifice, adipocytes were prepared and the results demonstrated that cells from prednisolone treated rats showed a 17% increase in insulin binding and increased rates of basal and insulin stimulated deoxyglucose uptake and glucose oxidation. Conversely, dexamethasone administration resulted in a 22% decrease in insulin binding, and decreased rates of deoxyglucose uptake and glucose oxidation by the cells. Thus, prednisolone and dexamethasone had opposite effects in vivo. In contrast to the opposite effects of the two glucocorticoids in vivo, dexamethasone and prednisolone (each at a concentration of 1 mumol/l) had similar effects on adipocytes in vitro. Incubation of adipocytes with the steroids did not alter insulin binding, while both agents led to a comparable decrease in the rates of basal and insulin stimulated deoxyglucose uptake and glucose oxidation. Thus, dexamethasone and prednisolone have opposite effects on adipocyte glucose metabolism in vivo but have similar effects in vitro.

Effects of incident light levels on photosynthetic membrane polypeptide composition and assembly in Rhodopseudomonas sphaeroides.

Cells of Rhodopseudomonas sphaeroides were grown anaerobically with incident light levels ranging between 4,500 and 400 footcandles (ca. 48,420 and 4,304 lux). Cells grown with the higher light levels had lower contents of total bacteriochlorophyll and incorporated L-[U-14C]leucine into membrane protein at higher rates than cells grown with lower light levels. The former cells also contained relatively lower amounts of light-harvesting membrane polypeptides as compared with the latter cells. In contrast, the relative amounts of reaction center membrane polypeptides were approximately the same with varying incident light levels. The relative amounts of these membrane polypeptides were correlated with differences in rates of synthesis and assembly of the polypeptides into membrane by measuring the rates of incorporation of L-[U-14C]leucine into the membrane-bound polypeptides. No significant differences in rates of turnover of these polypeptides were detected under the varying incident light levels as measured in pulse-chase radioactive labeling experiments.