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PubMed1993

Human beta 1-integrin gene expression is regulated by two promoter regions.

We report the cloning of two full-length cDNAs coding for the human beta 1-integrin which diverge from each other for their 5'-untranslated sequences. Characterization of a genomic clone containing these two sequences showed that they are contiguous, spaced by 261 nucleotides, and both followed by donor splice sites. Analysis by primer extension and transient transfection in a human osteogenic sarcoma cell line (MG-63) demonstrated the existence of two independent promoters for transcription initiation. The two promoter regions are very G+C-rich, and lack both a TATA box and a CAAT box. Northern blot analysis showed that transcripts starting from the distal promoter (with respect to the first coding exon) are at least 20-fold more abundant than transcripts originating from the proximal one. The levels of both transcripts increase after transforming growth factor-beta 1 induction, however, mRNAs originating from the proximal promoter increase at an higher extent. Reverse transcriptase/polymerase chain reaction analysis performed on different human tissues and cell lines revealed that, while the distal promoter is ubiquitously active, the proximal promoter is not. These findings suggest a possible complex pattern for regulation of the human beta 1-integrin gene expression.

Base Sequence↗
PubMed1985

Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei.

Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei.

Animals↗
PubMed2006

Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering.

Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol) for the treatment of many cancers. To develop an optimal bioprocess for paclitaxel supply, taxane biosynthetic pathway regulation must be better understood. Here we examine the expression profile of paclitaxel biosynthetic pathway genes by RNA gel blot analysis and RT-PCR in the Taxus cuspidata cell line P991 and compare with taxane metabolite levels. Upon methyl jasmonate (MJ) elicitation (100 microM), paclitaxel accumulates to 3.3 mg/L and cephalomannine to 2.2 mg/L 7 days after elicitation but neither are observed before this time. 10-deacetylbaccatin III accumulates to 3.3 mg/L and baccatin III to 1.2 mg/L by day 7 after elicitation. The early pathway enzyme genes GGPPS, TASY, and T5alphaH are up-regulated by MJ elicitation within 6 h and continue through 24 h before their abundances decrease. This study reveals the preference for one side of the biosynthetic pathway branch in early taxane synthesis, where transcripts coding for TalphaH are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch (TDAT and T10betaH) are not highly expressed following elicitation. Transcripts encoding the enzymes DBBT and DBAT are up-regulated upon MJ elicitation. Their products, 10-deacetylbaccatin III and baccatin III, respectively, accumulate within 6 h of the initial increase in transcript abundance. Importantly, the steady-state levels of the two terminal enzyme transcripts (BAPT and DBTNBT) are much lower than transcripts of early pathway steps. These are potential steps in the pathway for targeted metabolic engineering to increase accumulation of paclitaxel in suspension cell culture.

Cells, Cultured↗
PubMed2000

LiSa-2, a novel human liposarcoma cell line with a high capacity for terminal adipose differentiation.

LiSa-2 is a stable cell line derived from a poorly differentiated, pleomorphic liposarcoma. In serum-containing medium, LiSa-2 cells are fibroblastoid and rapidly dividing. In a serum-free, chemically defined culture medium containing physiological concentrations of insulin, triiodothyronine and cortisol, LiSa-2 cells divide slower and, extensively storing fat, acquire adipocyte morphology. In contrast to fibroblastoid LiSa-2 cells, these adipocyte-like LiSa-2 cells highly express transcripts for peroxisome proliferator-activated receptor-gamma, lipoprotein lipase, fatty acid synthetase, hormone-sensitive lipase, adipocyte most abundant gene transcript-1, glycerol-3-phosphate-dehydrogenase and the insulin-sensitive glucose transporter-4, all of which are specific for differentiated adipocytes. However, leptin mRNA expression was demonstrated only after preventing DNA methylation by incorporation of 5-aza-deoxycytidine into cellular DNA. Functionally, adipocyte-like LiSa-2 cells show increased insulin-dependent glucose uptake and lipid synthesis and are sensitive to lipolytic agents. This cell line may serve as an in vitro model for studying the regulation of human liposarcoma differentiation and for screening drugs for induction of differentiation-associated growth arrest in liposarcomas.

Adipocytes↗
PubMed2000

Pea chloroplast FtsZ can form multimers and correct the thermosensitive defect of an Escherichia coli ftsZ mutant.

This paper reports the isolation and characterization of a cDNA encoding the FtsZ protein of pea. The protein is synthesised as a precursor molecule of 423 amino acids with a molecular mass of 44 kDa. When translated in vitro, the protein is translocated efficiently into isolated, intact pea chloroplasts, demonstrating that the protein is localised in the chloroplast. Pea FtsZ synthesised in vitro formed multimers in a calcium-dependent manner. The pea cDNA complemented the thermosensitive defect of an E. coli ftsZ mutant in vivo and converted the filamentous phenotype of the E. coli mutant into the normal wild-type morphology at 42 degrees C. However, pea FtsZ mutants that were defective in multimerisation in vitro failed to correct the phenotype of the E. coli ftsZ mutant in vivo. The pea ftsZ transcripts were abundantly present in the young leaves, but barely detectable in roots and stems and undetectable in older leaves. Light stimulated transcription of the gene significantly in young and dark-grown leaves. This study strongly suggests that the division mechanisms used by chloroplasts and bacteria show considerable similarity.

Bacterial Proteins↗
PubMed2004

Identification of transcripts expressed preferentially in hemocytes of Ciona intestinalis that can be used as molecular markers.

The immunity provided by ascidian hemocytes represents one prototype of innate immune function in vertebrates. However, there are currently no molecular markers of ascidian hemocytes. We accumulated a large number of ESTs of cDNAs derived from hemocytes of Ciona intestinalis, a cosmopolitan species of ascidian. By comparing these ESTs with those derived from other tissues and developmental stages of Ciona, we were able to extract 81 transcripts expressed abundantly and preferentially in hemocytes. Among them, the von Willebrand factor type A (vWA)-like and complement 6 (C6)-like transcripts were found to be expressed almost exclusively in hemocytes, based on RT-PCR analysis and whole mount in situ hybridization. We propose that vWA-like and C6-like can be used as molecular markers for Ciona hemocytes.

Animals↗
PubMed2001

Brassica rapa has three genes that encode proteins associated with different neutral lipids in plastids of specific tissues.

Plastid lipid-associated protein (PAP), a predominant structural protein associated with carotenoids and other non-green neutral lipids in plastids, was shown to be encoded by a single nuclear gene in several species. Here we report three PAP genes in the diploid Brassica rapa; the three PAPs are associated with different lipids in specific tissues. Pap1 and Pap2 are more similar to each other (84% amino acid sequence identity) than to Pap3 (46% and 44%, respectively) in the encoded mature proteins. Pap1 transcript was most abundant in the maturing anthers (tapetum) and in lesser amounts in leaves, fruit coats, seeds, and sepals; Pap2 transcript was abundant only in the petals; and Pap3 transcript had a wide distribution, but at minimal levels in numerous organs. Immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most organs had several nanograms of PAP1 or PAP2 per milligram of total protein, the highest amounts being in the anthers (10.9 microg x mg(-1) PAP1) and petals (6.6 microg x mg(-1) PAP2), and that they had much less PAP3 (<0.02 microg x mg(-1)). In these organs PAP was localized in isolated plastid fractions. Plants were subjected to abiotic stresses; drought and ozone reduced the levels of the three Pap transcripts, whereas mechanical wounding and altering the light intensity enhanced their levels. We conclude that the PAP gene family consists of several members whose proteins are associated with different lipids and whose expressions are controlled by distinct mechanisms. Earlier reports of the expression of one Pap gene in various organs in a species need to be re-examined.

Amino Acid Sequence↗
PubMed1992

Two promoters for the whiB sporulation gene of Streptomyces coelicolor A3(2) and their activities in relation to development.

Two transcripts corresponding to the whiB sporulation gene of Streptomyces coelicolor A3(2) that differed in length at their 5' ends by 164 nucleotides were identified by S1 mapping. Their presumptive promoters differed from each other; the more downstream, P2, resembled typical prokaryotic promoters (i.e., those recognized by the major form of RNA polymerase) at five of six positions in its -10 and -35 regions; and the more upstream, P1, was comparably similar, instead, to the previously described hrdDp1 promoter (M. J. Buttner, K. F. Chater, and M. J. Bibb, J. Bacteriol. 172:3367-3378, 1990) around -40, -10, and +1. In surface cultures of the wild-type strain, the abundance of transcripts from the weak P1 promoter showed no obvious correlation with the developmental stage, whereas transcripts from P2 were barely detectable until aerial mycelium was present and then became relatively abundant, consistent with the developmental role of whiB. Both types of transcript were detected during and, to a lesser extent, after rapid growth in liquid culture. In addition, both promoters were utilized in vitro by RNA polymerase purified from a liquid culture of S. coelicolor. Transcription from P1 and P2 was observed during surface culture in strains carrying mutations blocking aerial mycelium formation (bldA and bldB) or the formation of spores in aerial mycelium (whiA, whiB, whiG, and whiH). Thus, whiB transcription is not severely dependent on any of these developmental genes, among which whiG is the determinant of a putative sigma factor specific for, and crucial to, sporulation.

Base Sequence↗
PubMed1994

IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.

We describe the cloning from a rat islet somatostatin-producing cell line of a 1.4 kb cDNA encoding a new homeoprotein, IDX-1 (islet/duodenum homeobox-1), with close sequence similarity to the Drosophila melanogaster homeobox protein Antennapedia (Antp) and the Xenopus laevis endoderm-specific homeoprotein XlHbox8. Analyses of IDX-1 mRNA and protein in rat tissues show that IDX-1 is expressed in pancreatic islets and ducts and in the duodenum. In electrophoretic mobility shift assays IDX-1 binds to three sites in the 5' flanking region of the rat somatostatin gene. In co-transfection experiments IDX-1 transactivates reporter constructs containing somatostatin promoter sequences, and mutation of the IDX-1 binding sites attenuates transactivation. Reverse transcription-polymerase chain reaction of islet RNA using degenerate amplimers for mRNAs encoding homeoproteins indicates that IDX-1 is the most abundant of 12 different Antp-like homeodomain mRNAs expressed in adult rat islets. The pattern of expression, relative abundance and transcriptional regulatory activity suggests that IDX-1 may be involved in the regulation of islet hormone genes and in cellular differentiation in the endocrine pancreas and the duodenum.

Amino Acid Sequence↗
PubMed2005

Sinup, a novel Siaz-interacting nuclear protein, modulates neural plate formation in the zebrafish embryos.

Siah, the vertebrate homologue of the Drosophila seven in absentia (sina) gene, is well conserved from Drosophila to mammal and involved in ubiquitination and proteasome-dependent degradation of various target proteins. To identify cellular proteins interacting with Siah, we screened a zebrafish cDNA library with zebrafish Siah (Siaz) as bait in a yeast two-hybrid assay. We identified a cDNA encoding a novel protein composed of 145 amino acids and termed it as Sinup (Siaz-interacting-nuclear-protein). Sinup is a novel nuclear protein that binds to the highly conserved C-terminal protein-interacting domain of Siaz both in vivo and in vitro. During development, sinup transcripts are abundant from the one-cell stage to the early blastula and then markedly diminished, suggesting sinup largely exists as maternal transcripts. sinup overexpression induced lateral expansion of the neural plate and in consequence caused ectopic expression of otx-2 and hoxb1b during the late gastrula stage. In addition, the lateral/paraxial expression of wnt8 at the onset of gastrulation is suppressed by the forced expression of sinup while the expression levels of various dorso-ventral markers are unaffected. In contrast, interfering with sinup functions using sinup morpholino oligonucleotides gradually diminished the anterior neuroectoderm from the posterior region, and resulted in compete loss of hindbrain at the 3-somites stage. Our report suggests that sinup expression should be tightly regulated during early embryonic development for the proper neural plate formation.

Amino Acid Sequence↗
PubMed2003

Logjam encodes a predicted EMP24/GP25 protein that is required for Drosophila oviposition behavior.

A newly characterized Drosophila melanogaster gene, logjam (loj), functions in female reproduction by modulating oviposition behavior. The locus encodes at least six overlapping transcripts with unique 5' ends. P-element mutants that express very low levels of loj transcripts are unable to oviposit mature eggs. This phenotype can be rescued by the introduction of a transgene expressing the most abundant loj transcript. As for many genes that specify behavioral outputs, loj is present in the adult central nervous system (CNS). Interestingly, it is also observed in vitellogenic egg chambers, suggesting that there may be multiple functions for this gene in egg-laying behavior. loj encodes a predicted protein with homology to the EMP24/GP25 transmembrane components of cytoplasmic vesicles and likely functions in intracellular trafficking.

Alternative Splicing↗
PubMed1990

A putative transmembrane protein with histidine-rich charge clusters encoded in the H-2K/tw5 region of mice.

The H-2 complex of mice contains many genes in addition to the gene families involved in immune reactions. Some of them are believed to function in mouse development, as suggested by the findings that several embryonic lethal mutations map within or near the H-2 complex. We have analyzed the H-2K/tw5 region in an attempt to study non-H-2 genes encoded in this region. Overlapping cosmid clones spanning about 170 kilobase pairs of DNA, including the H-2K/tw5 region of the mouse, have been screened for genes expressed in embryonic carcinoma cells. A transcript of 2.8 kilobase pairs (K. Abe. J.-F. Wei, F.-S. Wei, Y.-C. Hsu, H. Uehara, K. Artzt, and D. Bennett, EMBO J. 7:3441-3449, 1988) encoded by the KE 4 gene flanking H-2K distally was identified. The transcript was abundantly expressed in embryonic carcinoma cells but was present at low levels in other tissues in adults. A cDNA for this transcript was isolated from the F9 embryonic carcinoma cell line and sequenced. It potentially encodes a protein of 436 amino acids with several interesting features. First, it contains two regions made of well-conserved repeats unusually rich in histidine residues. In the repeats, histidine alternates with other amino acids, notably glycine or serine. Second, the two histidine-rich regions are separated by three putative membrane-spanning domains. Third, the N-terminal part of the sequence shows characteristics of a signal peptide. The results indicate that the protein coded by the gene may be a transmembrane protein with histidine-rich charge clusters. A similar sequence motif found in other known genes allows speculation on the possible functional of this gene.

Amino Acid Sequence↗
PubMed1997

Induction of a ribosome-inactivating protein upon environmental stress.

Transcripts of altered abundance in RNA from unstressed and 500 mm salt-shocked Mesembryanthemum crystallinum (common ice plant) were detected by reverse-transcription differential display (RT-DD). One transcript, Rip1, was of very low abundance in unstressed plants and was strongly induced by stress. RNA blot hybridizations showed strong induction and a diurnal rhythm of transcript abundance with a maximum each day around the middle of the light phase. Rip1 encodes a reading frame of 289 amino acids (molecular mass 32,652), RIP1, with homology to single-chain ribosome inactivating proteins (rRNA N-glycosidases). The deduced amino acid sequence is 31.7% identical to pokeweed antiviral protein RIP-C (overall similarity 66.5%) with highest identity in domains of documented functional importance. RT-DD also detected mRNA for pyruvate, orthophosphate dikinase (PPDK) which has already been shown to be stress-induced in the ice plant [16]. RIP1, expressed in Escherichia coli, showed rRNA N-glycosidase activity against ice plant and rabbit reticulocyte ribosomes. The induction of Rip1 coincides with the transition period during which global changes in translation lead to adaptation of the ice plant to salt stress.

Amino Acid Sequence↗
PubMed1996

A novel MADS-box gene of potato (Solanum tuberosum L.) expressed during the early stages of tuberization.

A potato MADS-box gene cDNA (POTM1-1) from an early tuber cDNA library has been isolated and characterized. The deduced amino acid sequence of POTM1-1 cDNA encodes 250 amino acids of a putative transcription factor containing a MADS-box domain and a K-box domain. These conserved domains share high homologies to those of flower-specific homeotic proteins, TM4 of tomato and AP1 of Arabidopsis, indicating that POTM1-1 gene is a homologue of the AP1 gene family. The levels of POTM1-1 transcripts were high in axillary buds, underground stolen tips, and newly formed tubers, but relatively low in mature tubers. During axillary bud development in a model petiole-leaf cutting system, the levels of POTM1-1 transcripts were abundant in actively growing shoots and during the early stages of microtuber development. It is possible that POTM1-1 functions as a transcription factor that regulates plant developmental processes in a number of tissue types.

Base Sequence↗
PubMed1998

The aberrant expression of Lewis a antigen in intestinal metaplastic cells of gastric mucosa is caused by augmentation of Lewis enzyme expression.

Immunohistochemical staining showed an aberrant expression of Le(a) antigen in the intestinal metaplastic glands of the gastric mucosa of secretors, as reported by others. In this study, we have demonstrated for the first time that the Lewis enzyme is well colocalized with Le(a) antigen, indicating that the Lewis enzyme is responsible for Le(a) antigen synthesis in the gastric mucosa. The staining intensity of the Lewis enzyme was much stronger in the cells with intestinal metaplasia than the cells without metaplasia, regardless of the secretor status. The amount of transcript of the Lewis gene was related to the degree of metaplasia; i.e., the more severe the metaplastic change was, the more abundantly the transcripts of the Lewis gene were expressed. This augmentation of the Lewis enzyme in metaplastic tissues was also confirmed by Western blotting analysis using a specific antibody against the Lewis enzyme. We conclude that intestinal metaplastic change of gastric mucosa is usually accompanied by a marked augmentation of the Lewis enzyme expression, which results in the enhanced expression of Le(a) antigens, particularly in secretors.

Adult↗
PubMed2006

Escherichia coli transcriptome dynamics during the transition from anaerobic to aerobic conditions.

Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Several previous transcript-profiling experiments have compared separate anaerobic and aerobic cultures. Here the process of adaptation was investigated by determining changes in transcript profiles when anaerobic steady-state cultures were perturbed by the introduction of air. Within 5 min of culture aeration the abundances of transcripts associated with anaerobic metabolism were decreased, whereas transcripts associated with aerobic metabolism were increased. In addition to the rapid switch to aerobic central metabolism, transcript profiling, supported by experiments with relevant mutants, revealed transient changes suggesting that the peroxide stress response, methionine biosynthesis, and degradation of putrescine play important roles during the adaptation to aerobic conditions.

Adaptation, Physiological↗
PubMed2002

Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR.

Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.

Animals↗
PubMed1992

Molecular cloning and enzymatic analysis of the rat homolog of "PhK-gamma T," an isoform of phosphorylase kinase catalytic subunit.

Messenger RNA encoding a protein kinase closely related to the catalytic subunit of skeletal muscle phosphorylase kinase has previously been isolated from a human HeLa cell cDNA library, and cross-species Northern hybridization analysis has shown that the rat homolog of this transcript is abundant in the adult testis (Hanks, S.K. (1989) Mol. Endocrinol. 3, 110-116). We now propose that the protein encoded by this transcript be designated as "PhK-gamma T." In this article, the primary structure of the rat homolog of PhK-gamma T is described, as deduced from nucleotide sequences of cDNA and genomic clones. RNase protection analysis reveals that PhK-gamma T transcripts are actually present in a wide variety of adult rat tissues, but at levels 20-100-fold less than what is observed in the testis. In the testis, transcription of the PhK-gamma T gene is initiated at multiple sites as shown by RNase protection and primer extension. Enzymatic activity of PhK-gamma T was demonstrated using renatured bacterially expressed protein. In the presence of Ca2+/calmodulin, PhK-gamma T is able to efficiently phosphorylate glycogen phosphorylase and convert it from an inactive to an active form. We conclude that PhK-gamma T represents a true isoform of phosphorylase kinase catalytic subunit.

Amino Acid Sequence↗