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PubMed2003

Comparative study of regulation of RTA-responsive genes in Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8.

Replication and transcription activator (RTA) (also referred to as ORF50), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/(human herpesvirus 8), plays a critical role in balancing the viral life cycle between latency and lytic replication. RTA has been shown to act as a strong transcription activator for several downstream genes of KSHV. Direct binding of RTA to DNA is thought to be one of the important mechanisms for transactivation of target genes, while indirect mechanisms are also implicated in RTA transactivation of certain selected genes. This study demonstrated direct binding of the DNA-binding domain of RTA (Rdbd) to a Kaposin (Kpsn) promoter sequence, which is highly homologous to the RTA-responsive element (RRE) of the PAN promoter. We undertook a comparative study of the RREs of PAN RNA, ORF57, vIL-6, and Kpsn to understand how RTA regulates gene expression during lytic replication. Comparing RNA abundance and transcription initiation rates of these RTA target genes in virus-infected cells suggested that the transcription initiation rate of the promoters is a major determinant of viral gene expression, rather than stability of the transcripts. RTA-mediated transactivation of reporters containing each RRE showed that their promoter strengths in a transient-transfection system were comparable to their transcription rates during reactivation. Moreover, our electrophoretic mobility shift assays of each RRE demonstrated that the highly purified Rdbd protein directly bound to the RREs. Based on these results, we conclude that direct binding of RTA to these target sequences contributes to their gene expression to various extents during the lytic life cycle of KSHV.

Base Sequence↗
PubMed1997

Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit.

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

Amino Acid Sequence↗
PubMed1999

Identification and initial characterization of calcyclin and phospholipase A2 in equine conceptuses.

For development to proceed normally, the appropriate genes must be expressed in the correct tissues and in the correct time frame. Knowledge of gene expression during development provides information about the changes taking place within the conceptus as well as possible reasons for pregnancy failure. However, little is known about gene expression during development in the equine conceptus. In this study, we examined differences in gene expression between day 12 and day 15 equine conceptuses by suppression subtractive hybridization. This technique was used to isolate transcripts that are more abundantly expressed in day 15 conceptuses compared to day 12 conceptuses. Between day 12 and 15 of pregnancy in horses, maternal recognition of pregnancy occurs, gastrulation is taking place, and mesoderm is beginning to form. Fifty cDNA clones were isolated, sequenced, and compared to known sequences in the GenBank database. Two cDNA clones identified that were of primary interest were calcyclin and phospholipase A2. Calcyclin is a calcium-binding protein of the S-100 protein family that has been found in mouse decidua and trophoblast. Calcyclin was found to be expressed in both day 12 and 15 equine conceptuses, with approximately a 30-fold increase in transcript abundance between days 12 and 15. Phospholipase A2 is an enzyme that cleaves phospholipids to release fatty acids and is involved in arachidonic acid release needed for prostaglandin, thromboxane, and leukotriene synthesis. Multiple forms of PLA2, that appear to be differentially regulated in day 12 and 15 conceptuses, were detected by northern blotting.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean (Glycine max L.) epicotyls.

Brassinosteroids promote elongation and regulate gene expression in soybean (Glycine max L.) stems. We constructed a cDNA library from brassinosteroid-treated soybean epicotyls and used differential hybridization to isolate a cDNA (pBRU1) corresponding to a transcript whose abundance is increased by brassinosteroid treatment. Sequence analysis of pBRU1 revealed an open reading frame of 283 amino acids with a putative signal peptide of 29 amino acids. The sequence had extensive homology (77% identity, 89% similarity) over 114 contiguous amino acids to the meri-5 gene of Arabidopsis thaliana (J.I. Medford, J.S. Elmer, H.J. Klee [1991] Plant Cell 3: 359-370), and significant homology (48% identity, 62% similarity) to a xyloglucan endotransglycosylase localized in the cell walls of nasturtium (J. de Silva, C.D. Jarman, D.A. Arrowsmith, M.S. Stronach, S. Chengappa, C. Sidebottom, J.S. Reid [1993] Plant J 3: 701-711). RNase protection studies showed that BRU1 transcript levels are not increased by 1.0 microM auxins, cytokinins, abscisic acid, or gibberellic acid and that BRU1 expression is highest in stem tissue. Findings from studies with run-on transcripts from isolated soybean nuclei most likely indicate that the regulation of BRU1 by brassinosteroids is largely posttranscriptional. The elevated levels of BRU1 transcripts in elongating tissue and the homology with a xyloglucan endotransglycosylase suggest a possible role for the BRU1 protein in brassinosteroid-stimulated elongation.

Amino Acid Sequence↗
PubMed1999

Changes in the gene expression of GABAA receptor subunit mRNAs in the septum of rats subjected to pentylenetetrazol-induced kindling.

Chemical kindling was induced in rats by long-term administration of pentylenetetrazol (PTZ) (30 mg/kg three times a week for 9 weeks). The effects of such kindling on the abundance of transcripts encoding subunits of the gamma-aminobutyric acid type A (GABAA) receptor in the brain were measured by RNase protection assay. Kindled rats were examined either 3 or 30 days after discontinuation of PTZ treatment. The amounts of gamma2L and gamma2S subunit mRNAs were significantly increased in the hippocampus and cerebral cortex of kindled rats 3 and 30 days after treatment discontinuation, compared with those observed in control rats, and these effects were prevented by the concomitant administration of the anticonvulsant abecarnil. In contrast, the amounts of alpha1 and beta2 subunit mRNAs in these two brain regions did not differ significantly between kindled and control rats. The abundance of alpha1, beta2, gamma2L and gamma2S subunit mRNAs was decreased in the septum of rats 3 or 30 days after discontinuation of treatment with PTZ either alone or in combination with abecarnil. The amounts of none of the four subunit mRNAs measured differed significantly between the striatum or frontal cortex of kindled rats and control rats 3 days after drug discontinuation. Immunohistochemical analysis with antibodies to choline acetyltransferase revealed a marked decrease in the number of cholinergic neurons in the septum of kindled rats 30 days after discontinuation of PTZ treatment; this effect was not prevented by the administration of abecarnil. These results suggest that long-term treatment with PTZ induces a loss of GABAA receptors in the septum.

Animals↗
PubMed1995

Coordinate patterns of expression of type I and III collagens during mouse development.

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.

Animals↗
PubMed2003

Interfering with different steps of protein synthesis explored by transcriptional profiling of Escherichia coli K-12.

Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level. A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents. Real-time reverse transcriptase PCRanalysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response. Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons. Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents. Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts. However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain.

Aminoglycosides↗
PubMed1996

Inactivation of a novel neuropeptide Y/peptide YY receptor gene in primate species.

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to a family of structurally related peptides which have numerous functions in both neural and endocrine signaling. By homology screening, we cloned a novel gene sharing the highest homology with the NPY Y1 receptor gene from humans, rabbits, and several other species. This novel gene of rabbit encodes a functional NPY/PYY receptor, designated Y2b, which prefers NPY13-36 rather than [Leu31,Pro34]NPY despite its higher identity with the Y1 receptor. Although, at low levels, mRNA was detected in the tissues and brain regions, including hypothalamus. Further, sequence data revealed that this gene is the orthologue of the recently cloned mouse novel NPY receptor, Y5. However, our study demonstrates that the receptor function of this gene has been inactivated in primates by a frameshift mutation occurring early in primate evolution. This novel NPY receptor represents the first neurotransmitter receptor identified that has universally lost its receptor function in primate species. Interestingly, despite its inactivation in humans, the transcripts were abundantly detected in the heart and skeletal muscle, suggesting a novel function of the human gene.

Amino Acid Sequence↗
PubMed1992

Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes.

Previous analyses of the uteroglobin gene promoter revealed a GT1 box which is also found in the SV40 enhancer. The GT1 element in the context of the uteroglobin promoter is active in Ishikawa cells, a human endometrial cell line, but not in HeLa cells. Here we report the cloning by recognition site screening of two factors (SPR-1 and SPR-2) which bind to this GT1 motif. SPR-1 and SPR-2 are homologues of the transcription factor Sp1. All three proteins are closely related members of a gene family encoding proteins with very similar structural features. Like Sp1, SPR-1 and SPR-2 contain glutamine and serine/threonine rich amino acid stretches. Most significantly, the DNA binding domains of all three proteins are highly conserved and they recognize GT as well as GC boxes identically. SPR-2 mRNA is expressed ubiquitously, whereas SPR-1 transcripts are abundant in the brain but barely detectable in other organs. The possible function of these factors for the activity of the uteroglobin promoter is discussed.

Amino Acid Sequence↗
PubMed1996

Molecular structure and genetic mapping of the mouse gastrin gene.

The mouse gastrin gene has three exons totalling 460 bp and a deduced preprogastrin of 101 amino acids. The sequence of murine gastrin-34 is 94% identical to rat gastrin-34 and 76% identical to human gastrin-34. At Arg79, mouse progastrin has a unique cleavage site that might allow species-specific synthesis of gastrin-13. Northern analysis and RT-PCR demonstrated that gastrin gene transcripts are abundant in mouse stomach and duodenum and present at low levels in brain, ovary and pancreas, similar to the pattern described for other mammals. The gastrin gene was mapped to the distal region of mouse chromosome 11.

Amino Acid Sequence↗
PubMed1988

Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

Amino Acid Sequence↗
PubMed1993

Glucocorticoid-induced increase in plasma corticosteroid-binding globulin levels in fetal sheep is associated with increased biosynthesis and alterations in glycosylation.

In fetal sheep, there is a concomitant prepartum rise in cortisol and corticosteroid-binding globulin (CBG) that maintains a low free plasma cortisol level and allows for a low negative feedback effect of cortisol on the secretion of ACTH from the fetal pituitary. However, the stimulus for the prepartum increase in CBG and the mechanism(s) of this effect are not known. It has been proposed that glucocorticoids increase CBG concentrations, and therefore, we infused fetal sheep with the synthetic glucocorticoid dexamethasone (DEX; 2 micrograms/min over 15 min every 2 h for 96 h, n = 5) or saline (n = 5). The plasma corticosteroid-binding capacity increased from 30.0 +/- 2.4 to 55.6 +/- 7.7 and 92.6 +/- 11.1 ng/ml at 48 and 96 h, respectively, of DEX infusion. To examine possible mechanisms of increasing fetal plasma CBG, we first cloned and sequenced a sheep CBG cDNA and purified the protein. This allowed us to deduce the primary structure of ovine CBG and to demonstrate that hepatic CBG mRNA abundance (single transcript of 1.8 kilobases) rose from 0.9 +/- 0.2 to 3.6 +/- 1.6 arbitrary units after 96 h of DEX treatment. Fetal DEX treatment produced a significant increase (7.1 +/- 1.2% to 13.1 +/- 1.4%) in the Concanavalin-A-binding forms of CBG that predominate in adult sheep plasma. There was negligible transfer of purified [125I]CBG from the ewe to fetal plasma, urine, or amniotic fluid. We also injected adult sheep with DEX (10 mg/day for 4 days) and demonstrated a significant decrease in plasma corticosteroid-binding capacity by 24 h, which remained suppressed for the duration of the study. After 96 h of DEX treatment, there was also a significant decrease in adult hepatic CBG mRNA abundance. We conclude that glucocorticoids increase fetal plasma CBG in part by increased hepatic biosynthesis. It may also be accentuated by a change in the glycosylation of CBG, but cannot be attributed to transplacental transfer. Furthermore, glucocorticoid treatment exerts opposite effects on CBG biosynthesis in fetal and adult sheep.

Amino Acid Sequence↗
PubMed1992

Complementary DNA cloning and expression of Pit-1/GHF-1 from the domestic turkey.

The transition from egg laying to incubation activity in birds is associated with a dramatic rise in serum prolactin levels. To further our understanding of the regulation of prolactin gene expression in birds, a cDNA clone encoding turkey Pit-1/GHF-1 was isolated. The turkey cDNA, designated tPit-1/GHF-1, was 1,123 nucleotides in length and encoded a protein of 327 amino acids, including a conserved 80-amino-acid POU-specific domain and a 60-amino-acid POU homeodomain. tPit-1/GHF-1 POU-specific domain and POU-homeodomain showed 94-95% amino acid identity with the corresponding rat Pit-1/GHF-1 domains. At its amino terminus, tPit-1/GHF-1 contained a 26-amino-acid insertion comparable to that found in the rat variant isoform, Pit-1 beta. Two other insertions of 38 and 7 amino acids were present and were not found in the mammalian protein. Levels of tPit-1/GHF-1 mRNA in pituitary tissue were examined at different phases of the turkey reproductive cycle by Northern blotting. tPit-1/GHF-1 mRNA was expressed as a 3.5-kb transcript, whose abundance remained relatively constant throughout the reproductive cycle. Thus, the dramatic rise in prolactin mRNA, observed during hyperprolactinemia in incubating turkey hens, was not associated with a concomitant increase in tPit-1/GHF-1 gene expression.

Amino Acid Sequence↗
PubMed2006

Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco.

The role of the redox state of the apoplast in hormone responses, signaling cascades, and gene expression was studied in transgenic tobacco (Nicotiana tabacum) plants with modified cell wall-localized ascorbate oxidase (AO). High AO activity specifically decreased the ascorbic acid (AA) content of the apoplast and altered plant growth responses triggered by hormones. Auxin stimulated shoot growth only when the apoplastic AA pool was reduced in wild-type or AO antisense lines. Oxidation of apoplastic AA in AO sense lines was associated with loss of the auxin response, higher mitogen-activated protein kinase activities, and susceptibility to a virulent strain of the pathogen Pseudomonas syringae. The total leaf glutathione pool, the ratio of reduced glutathione to glutathione disulfide, and glutathione reductase activities were similar in the leaves of all lines. However, AO sense leaves exhibited significantly lower dehydroascorbate reductase and ascorbate peroxidase activities than wild-type and antisense leaves. The abundance of mRNAs encoding antioxidant enzymes was similar in all lines. However, the day/night rhythms in the abundance of transcripts encoding the three catalase isoforms were changed in response to the AA content of the apoplast. Other transcripts influenced by AO included photorespiratory genes and a plasma membrane Ca(2+) channel-associated gene. We conclude that the redox state of the apoplast modulates plant growth and defense responses by regulating signal transduction cascades and gene expression patterns. Hence, AO activity, which modulates the redox state of the apoplastic AA pool, strongly influences the responses of plant cells to external and internal stimuli.

Ascorbate Oxidase↗
PubMed2002

Suppression of epithelial ion transport transcripts during pneumococcal acute otitis media in the rat.

Until recently, it was not feasible to conduct genome-wide screening for gene transcript variations that play key roles in the pathogenesis of otitis media. In this study microarray technology was used to profile differential gene expression patterns from rat middle ear mucosa at 12 and 48 h after Streptococcus pneumoniae challenge. Real-time polymerase chain reaction was performed for independent verification of the microarray results. Three ion transport mRNAs were simultaneously suppressed more than 4-fold at 12 h in bacteria-challenged ears, including Na,K-ATPase alpha I subunit (SPATPa1), sodium channel beta 2 subunit (SCNB2) and sodium-hydrogen exchange protein isoform 2 subunit (NHE2). At 48 h after infection, the mRNA levels of SCNB2 and NHE2 had decreased 7- and 10-fold, respectively, whereas the relatively abundant SPATPa1 transcript showed recovery. The downregulation of Na(+)-transporting transcripts suggests a reduced number of epithelial cells and transporting proteins and/or the dysfunction of sodium transporters secondary to the bacterial infection. These changes can disrupt the coupling of the apical Na + entry and basolateral Na + extrusion, deplete the electrochemical Na+ transmembrane gradient, disrupt the intracellular osmotic equilibrium and lead to intracellular acidification and the accumulation of excess sodium, water and other organic and inorganic molecules in the middle ear cavity. Any or all of these changes may contribute to the initiation and persistence of middle ear mucosa inflammation and effusion during an episode of bacterial acute otitis media.

Acute Disease↗
PubMed2004

Developing pineapple fruit has a small transcriptome dominated by metallothionein.

In a first step toward understanding the molecular basis of pineapple fruit development, a sequencing project was initiated to survey a range of expressed sequences from green unripe and yellow ripe fruit tissue. A highly abundant metallothionein transcript was identified during library construction, and was estimated to account for up to 50% of all EST library clones. Library clones with metallothionein subtracted were sequenced, and 408 unripe green and 1140 ripe yellow edited EST clone sequences were retrieved. Clone redundancy was high, with the combined 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of the EST clone sequences clustered within 13.5% and 9.3% of contigs from green unripe and yellow ripe libraries, respectively, indicating that a small subset of genes dominate the majority of the transcriptome. Furthermore, sequence cluster analysis, northern analysis, and functional classification revealed major differences between genes expressed in the unripe green and ripe yellow fruit tissues. Abundant genes identified from the green fruit include a fruit bromelain and a bromelain inhibitor. Abundant genes identified in the yellow fruit library include a MADS box gene, and several genes normally associated with protein synthesis, including homologues of ribosomal L10 and the translation factors SUI1 and eIF5A. Both the green unripe and yellow ripe libraries contained high proportions of clones associated with oxidative stress responses and the detoxification of free radicals.

Ananas↗
PubMed1997

Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea.

A new member of the aquaporin (AQP) family has been identified from rat testis. This gene, referred as aquaporin 7 (AQP7), encodes a 269-amino acid protein that contained the conserved NPA motifs of MIP family proteins. AQP7 has the amino acid sequence homology with other aquaporins ( approximately 30%), and it is highest with AQP3 (48%), suggesting that both AQP3 and AQP7 belong to a subfamily in the MIP family. Injection of AQP7-cRNA into Xenopus oocytes expressed a 26-kDa protein detected by immunoblotting. The expression of AQP7 in oocytes stimulated the osmotic water permeability by 10-fold which was not inhibited by 0.3 mM mercury chloride. The Arrhenius activation energy for the stimulated water permeability was low (2.1 kcal/mol). AQP7 also facilitated glycerol and urea transport by 5- and 9-fold, respectively. The activation energy for glycerol was also low (5.3 kcal/mol after the correction of the endogenous glycerol permeability of oocytes). Northern blot analysis revealed a 1.5-kilobase pair transcript expressed abundantly in testis. In situ hybridization of testis revealed the expression of AQP7 at late spermatids in seminiferous tubules. The immunohistochemistry of testis localized the AQP7 expression at late spermatids and at maturing sperms. AQP7 may play an important role in sperm function.

Amino Acid Sequence↗
PubMed2004

Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae.

Laccase (EC 1.10.3.2) is an enzyme with p-diphenol oxidase activity that is a member of a group of proteins collectively known as multicopper, or blue copper, oxidases. Laccase is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the 10 histidines and one cysteine that form the copper-binding centers, as well as a methionine in the T1 copper center. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino-terminal sequence characterized by a unique domain consisting of several conserved cysteine, aromatic, and charged residues. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that AgLac1 was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malpighian tubules, and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. MsLac2 showed strong expression in the pharate pupal and reduced expression in the early pupal epidermis, consistent with the laccases' presumed role in cuticle sclerotization.

Amino Acid Sequence↗