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PubMed2001

Subunit heterogeneity of cytoplasmic dynein: Differential expression of 14 kDa dynein light chains in rat hippocampus.

Cytoplasmic dynein is a multi-subunit protein complex in which each subunit is encoded by a few genes. How these subunit isoforms are assembled and regulated to mediate the diverse functions of cytoplasmic dynein is unknown. We previously have shown that two highly conserved 14 kDa dynein light chains, Tctex-1 and RP3, have different cargo-binding abilities. In this report, coimmunoprecipitation revealed that Tctex-1 and RP3 were present in mutually exclusive dynein complexes of brain. Two specific antibodies were used to examine the localization of these two dynein light chains in adult rat hippocampal formation and cerebral cortex. By light microscopy, Tctex-1 and RP3 immunoreactivities exhibited distinct and almost complementary distribution patterns in both brain regions. In hippocampal formation, Tctex-1 immunoreactivity was most enriched in somata of newly generated granule cells and scant in the mature granule and pyramidal cell somata. In contrast, RP3 immunoreactivity was abundant in pyramidal and granule cell somata. Ultrastructural analysis of the dentate gyrus revealed both dynein light chains were associated with various membranous organelles that often were affiliated with microtubules. In addition, Tctex-1 and RP3 immunoreactivities were preferentially and highly enriched on membranous organelles and/or vesicles of axon terminals and dendritic spines, respectively. These results suggest that dynein complexes with different subunit composition, and possibly function, are expressed differentially in a spatially and temporally regulated manner. Furthermore, Tctex-1 and RP3 may play important roles in synaptic functions.

Animals↗
PubMed1999

Hunting for differentially expressed genes.

Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Several methods have been used for this purpose, including differential hybridization, cDNA subtraction, differential display and, more recently, DNA chips. Subtractive hybridization has significantly improved after the polymerase chain reaction was incorporated into the original method and many new protocols have been established. Recently, the availability of the wellknown coding sequences for some organisms has greatly facilitated gene expression analysis using high-density microarrays. Here, we describe some of these modifications and discuss the benefits and drawbacks of the various methods corresponding to the main advances in this field.

Gene Expression↗
PubMed1996

The sensory innervation of the mouse spinal cord may be patterned by differential expression of and differential responsiveness to semaphorins.

To better understand the regulatory processes underlying axonal pathfinding we analyzed the embryonic expression of seven murine semaphorin genes by in situ hybridization In the spinal cord, transcripts of all seven semaphorin genes were detected from Embryonic Day 11.5 (E11.5) onward and restricted to distinct regions at E15.5. Interestingly, semE, F, and G mRNAs were in addition differentially expressed in the ventricular zone of the telencephalon. In order to correlate these expression patterns to the behavior of different types of sensory afferents, we tested their response to recombinant semaphorin proteins. Specific subpopulations of dorsal root ganglion sensory neurons displayed a developmentally regulated differential response to Sem D. Whereas extension of both NGF- and NT-3-dependent neurites was inhibited by Sem D at E12.5, only neurites formed in the presence of NGF responded at E14.5. This suggests that Sem D may be involved in preventing an early penetration of the spinal cord by sensory afferents and subsequently shaping their lamina-specific termination.

Animals↗
PubMed2003

The novel murine Ca2+-binding protein, Scarf, is differentially expressed during epidermal differentiation.

Calcium (Ca2+) signaling-dependent systems, such as the epidermal differentiation process, must effectively respond to variations in Ca2+ concentration. Members of the Ca2+-binding proteins play a central function in the transduction of Ca2+ signals, exerting their roles through a Ca2+-dependent interaction with their target proteins, spatially and temporally. By performing a suppression subtractive hybridization screen we identified a novel mouse gene, Scarf (skin calmodulin-related factor), which has homology to calmodulin (CaM)-like Ca2+-binding protein genes and is exclusively expressed in differentiating keratinocytes in the epidermis. The Scarf open reading frame encodes a 148-amino acid protein that contains four conserved EF-hand motifs (predicted to be Ca2+-binding domains) and has homology to mouse CaM, human CaM-like protein, hClp, and human CaM-like skin protein, hClsp. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca2+-binding method. By Southern blot and computational genome sequence analysis, a highly related gene, Scarf2, was found 15 kb downstream of Scarf on mouse chromosome 13. The functional Scarf Ca2+-binding domains suggest a role in the regulation of epidermal differentiation through the control of Ca2+-mediated signaling.

Amino Acid Motifs↗
PubMed2005

Microarray profiling of genes differentially expressed during erythroid differentiation of murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are widely used to study erythroid differentiation thanks to their ability to terminally differentiate in vitro in response to chemical induction. At the molecular level, not much is known of their terminal differentiation apart from activation of adult-type globin gene expression. We examined changes in gene expression during the terminal differentiation of these cells using microarray-based technology. We identified 180 genes whose expression changed significantly during differentiation. The microarray data were analyzed by hierarchical and k-means clustering and confirmed by semi-quantitative RT-PCR. We identified several genes including H1f0, Bnip3, Mgl2, ST7L, and Cbll1 that could be useful markers for erythropoiesis. These genetic markers should be a valuable resource both as potential regulators in functional studies of erythroid differentiation, and as straightforward cell type markers.

Acetamides↗
PubMed1993

Arbitrary primed PCR fingerprinting of RNA applied to mapping differentially expressed genes.

Differential gene expression between various tissues and developmental stages or between cells in vitro under different growth conditions can be rapidly and efficiently compared using the RNA arbitrarily primed polymerase chain reaction (RAP) fingerprinting method (Welsh et al., 1992b; Liang and Pardee, 1992). In RAP, a primer of arbitrary sequence primes both first and second strand cDNA synthesis. The mixture of products is then PCR amplified and resolved electrophoretically, yielding highly reproducible fingerprints that are tissue-specific or growth condition-specific. Differences between fingerprints arise from differentially expressed genes, as verified by Northern blot analysis. RAP can be performed on the RNA samples using various DNA primers. Each two day experiment yields a sample of approximately twenty cDNA products per lane making the identification of differentially or developmentally regulated genes no longer rate limiting. Those PCR products representing genes that are regulated can be cloned from the gel and sequenced. Sequences can be compared to the DNA and protein sequence databases to identify homologs, motifs and members of gene families. The clones can be placed on the genetic map as Expression Tagged Sites (ETS, Adams et al., 1991a).

Animals↗
PubMed2000

Identification of differentially expressed genes in the visual structures of brain using high-density cDNA grids.

The hybridization patterns of 18,371 high-density-grid-arrayed non-redundant complementary DNA (cDNA) clones were examined using three different sources of cDNA probes. The first set of probes was synthesized from mRNA isolated from visual brain areas MT and V4 of Vervet monkey. The second set of probes was derived from cDNA libraries constructed from two micro dissected sets of layers of the monkey Lateral Geniculate Nucleus layers within the visual pathway, namely the magnocellular and parvocellular layers. The third set of cDNA probes was synthesized from the subtracted fractions of the cDNAs enriched for either the magnocellular or the parvocellular layers of the Lateral Geniculate Nucleus. Software, linked directly to the Genbank database, was developed to aid in the rapid identification of both expressed and differentially expressed genes. Our results indicate that both the cDNA probes synthesized from mRNA and cDNA libraries can identify similar fractions of expressed genes. However, the subtracted cDNA probes improve the efficiency of detection for those genes that are expressed at much lower abundance. Analyses of these results for the differential expression patterns of these genes were validated by semi-quantitative PCR on the DNA derived from the whole tissue cDNA libraries. A list of some known genes that are statistically differentially expressed within the magnocellular layers of the LGN and area MT in the primate visual areas is derived.

Animals↗
PubMed1995

The melanoma differentiation-associated gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21, is differentially expressed during growth, differentiation and progression in human melanoma cells.

The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) induces terminal differentiation with an irreversible loss of proliferative capacity in human melanoma cells. Using subtraction hybridization, cDNAs were identified that display enhanced expression in terminally differentiated and growth arrested human melanoma cells (Jiang and Fisher, 1993; Jiang et al., 1994a). A specific melanoma differentiation-associated (mda) cDNA, mda-6, is described whose expression inversely correlates with melanoma progression and growth. mda-6 is identical to WAF1/CIP1/SDI1 that encodes the M(r) 21,000 protein (p21) that is an inhibitor of cyclin-dependent kinases. Actively growing normal melanocyte, SV40-immortalized human melanocyte and dysplastic nevus cell lines synthesize elevated levels of mda-6 mRNA; whereas, actively proliferating radial and early vertical growth phase primary melanomas as well as metastatic human melanoma cells produce reduced levels of mda-6 mRNA. Treatment of primary and metastatic human melanoma cells with IFN-beta + MEZ results in growth inhibition and an increase in mda-6 expression. mda-6 expression also increases when human melanoma cells are grown to high saturation densities or when grown in serum-free medium. Using anti-p53 and anti-p21 antibodies, an inverse correlation is found between p53 and p21 protein levels during growth arrest and differentiation. Induction of growth arrest and terminal differentiation in H0-1 human melanoma cells by IFN-beta + MEZ results in a temporal decrease in wild-type p53 protein levels with a corresponding increase in p21 levels. In the Matrigel-assisted melanoma progression model, mda-6 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. In metastatic human melanoma cells displaying a loss of metastatic potential resulting from introduction of a normal human chromosome 6, mda-6 mRNA levels increase. Taken together, these studies indicate that mda-6 (p21) may function as a negative regulator of melanoma growth, progression and metastasis.

Amino Acid Sequence↗
PubMed1993

Differential expression of leukocyte differentiation antigens in small round blue cell sarcomas.

BACKGROUND: Small round blue cell sarcomas (SRBCS) comprise a group of cytomorphologically poorly differentiated neoplasms that are characterized by different histogenesis and biologic behavior. METHODS: Twenty-seven well-characterized SRBCS were examined immunohistochemically to detect the expression of a panel of leukocyte differentiation (CD) antigens and class I (HLA-A,B,C) and class II (HLA-DR) major histocompatibility complex antigen. RESULTS: Although various cell surface antigens were detectable in SRBCS, the pan-leukocytic-histiocytic CD53 antigen was absent in the neoplastic population of all tumors studied; this finding allowed the authors to discriminate these lesions from lymphomas and leukemias. Some antigens had a differential pattern of expression in the SRBCS group, in particular in the undifferentiated tumor cell populations. In most instances, neuroblastomas (NB) and ganglioneuroblastomas (GNB) were CD9+/CD24+/CD56+ but CD40-/HLA-A,B,C-. Rhabdomyosarcomas (RMS) were CD56+ in all specimens and CD9+ in many samples; generally, they showed CD24-/CD40-/HLA-A,B,C-. Ewing sarcomas (ES) and peripheral primitive neuroectodermal tumors (pPNET) were HLA-A,B,C+/CD40+ but CD9-/CD24-/CD56- in most instances. Thus, with few exceptions, the expression of CD9 and CD56 and the simultaneous absence of HLA-A,B,C and CD40 differentiated GNB, NB, and RMS from ES and pPNET. GNB, NB, and RMS differed in regard to their CD24 expression. CONCLUSIONS: These data show that various types of SRBCS have different patterns of cell surface antigens. Therefore, these antigens are considered to be helpful in the immunophenotypic subclassification of SRBCS: The immunophenotypic similarities between ES and pPNET, however, might be an additional argument for a close relationship between these two lesions.

Antigens, CD↗
PubMed1990

Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells.

In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded to TSH by regaining a characteristic epithelial shape and high Tg mRNA content. 32 h after the replacement of EGF by TSH, cells in mitosis presented the same distribution of the Tg mRNA content as the rest of the cell population. This implies that cell cycling (at least 27 h, as previously shown) did not affect the induction of the Tg gene which is clearly detectable after a time lag of at least 24 h. The data unequivocally show that the reexpression of differentiation and proliferative activity are separate but fully compatible processes when induced by cAMP in thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Representation of differential expression: a new approach to study differential gene expression in trypanosomatids.

During the past five years, several methods have been described that allow the isolation and cloning of stage-specific or cell-specific genes. The characterization of genes expressed at different stages of parasite development is of the utmost importance for the understanding of the mechanisms involved in the regulation of gene expression. Here, Samuel Goldenberg and Marco Aurelio Krieger describe a method for the amplification and cloning of Trypanosoma cruzi genes expressed specifically at different times of the metacyclogenesis process. This method, representation of differential expression (RDE), should be useful for the isolation and cloning of any trypanosomatid gene transcribing differentially expressed messenger RNA.

Journal Article↗
PubMed2005

Differentially expressed genes in endothelial differentiation.

By screening differentially expressed genes in mouse embryonic stem (ES) cells by subtractive hybridization, we identified three conserved but uncharacterized genes encoding bromodomain containing 3 (BRD3), protein lysine methyltransferase (PLM), and kelch domain containing 2 (KLHDC2), which were downregulated during endothelial differentiation. An RNA blot study showed that these genes were markedly expressed in undifferentiated ES cells, whereas the expression was reduced upon endothelial differentiation; a study of mouse endothelium showed a significant reduction in the expression of BRD3. A study of human BRD3, located on chromosome 9 at q34, a region susceptible to genomic rearrangement, showed an altered expression in 4 of 12 patients with bladder cancer, compared with adjacent noncancerous tissues. Taken together with the result of siRNA inhibition showing the positive regulation of cell proliferation by BRD3, it is suggested that this molecule plays a role in allowing cells to enter the proliferative phase of the angiogenic process.

Animals↗
PubMed1997

Identification of differentially expressed mRNAs during rat C6 glial cell differentiation by mRNA fingerprinting using arbitrarily primed PCR (RAP).

Differentiation of glial cells is controlled by a complex program of differential expressions of many genes. To identify differentially expressed genes that are involved in rat C6 glial cell differentiation induced by dibutyryl cyclic AMP and theophylline, mRNA fingerprinting using arbitrarily primed PCR (RAP) was used. Four cDNA fragments, that were differentially expressed during differentiation, were isolated. Sequence analysis revealed that one of them, abundantly expressed during differentiation, was homologous to a hamster calcium-dependent serine protease. Another one was highly similar to rabbit dystrobrevin and the other two clones were identical to rat triose phosphate isomerase and calnexin. The results obtained suggest that the expressions of particular genes were changed and that RAP is a useful method to identify genes which are differentially expressed during glial cell differentiation.

Animals↗
PubMed2007

Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart.

In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.

Animals↗
PubMed2002

Identification of differentially expressed genes in normal and malignant prostate by electronic profiling of expressed sequence tags.

Differentially expressed genes between corresponding normal and cancertissue can advance our understanding of the molecular basis of malignancy and potentially serve as biomarkers or prognostic markers of malignancy. To identify differentially expressed genes in prostate cancer, we used a procedure combining electronic expression profiling of the prostate expressed sequence tag (EST) database and molecular biology techniques. A novel electronic expression-profiling algorithm was developed to search publicly available EST sequences for genes that show significant differential expression in prostate cancer compared with normal prostate tissue. Approximately 600 genes expressed in prostate were identified through adequate EST counts of ESTs for electronic profiling. Of these 600 genes, 9 showed statistically significant differences in their EST counts between cancer and normal prostate and were further analyzed. The predictions associated with electronic profiling were experimentally verified for two genes, cysteine-rich secretory protein 3 (CRISP-3) and deadenylating nuclease (DAN), using real-time reverse transcription-PCR with total RNA extracted from cells isolated by laser capture microdissection. In five of five Gleason score 6 cancer cases, CRISP-3 expression was increased >50 fold, whereas the expression of DAN was reduced by >80%.

Algorithms↗
PubMed2005

Characterization of pollen tube development in Pinus strobus (Eastern white pine) through proteomic analysis of differentially expressed proteins.

The differentially expressed proteins in pollen tubes indicate their specific roles in this stage of male gametophyte development. To isolate these proteins, 2-DE was done using ungerminated pollen and 2-day-old pollen tubes of Pinus strobus. Results show that 645 and 647 protein spots were clearly resolved from pollen grains and pollen tubes, respectively. Thirty-eight protein spots were expressed only in pollen tubes, while 19 increased in intensity. MALDI-TOF MS was used to generate tryptic peptide masses that were submitted to Mascot for identification. Of the differentially expressed proteins, 12% matched with hypothetical proteins, 33% did not hit any protein, and for the 55%, a putative function was assigned based on similarity of sequences with previously characterized proteins. Therefore, pollen tube development can be characterized by the cellular activities that involve metabolism, stress/defense response, gene regulation, signal transduction, and cell wall formation. This study expands our understanding of the changes in protein expression associated with pollen tube development and provides insights into the molecular programs that separate the development of the pollen tubes from pollen grains. This is the first report that describes a global analysis of differentially expressed proteins from the pollen tube of any seed plant.

Cell Wall↗
PubMed2002

Differential expression of the PTEN tumor suppressor protein in fetal and adult neuroendocrine tissues and tumors: progressive loss of PTEN expression in poorly differentiated neuroendocrine neoplasms.

Genetic alteration and loss of expression of tumor suppressor gene PTEN has been found in carcinomas of the breast, prostate, and endometrium, as well as in gliomas. PTEN expression in neural crest/neuroendocrine (NC/NE) tissues and in neoplasms has not been reported. This study examines PTEN expression in embryonal, fetal, and adult tissues by immunohistochemistry. The authors found high PTEN expression in embryonal, fetal, and adult NC/NE tissues. The authors also study the PTEN expression in NC/NE neoplasms (N = 37), including 5 melanocytic nevi, 2 melanomas, 9 carcinoids, 2 moderately differentiated neuroendocrine carcinomas, 13 poorly differentiated neuroendocrine carcinomas, 2 paragangliomas, 2 pheochromocytomas, 2 medullary thyroid carcinomas, and 1 neuroblastoma. All carcinoid tumors and melanocytic nevi showed moderate or strong immunostaining for PTEN. In contrast, the majority of poorly differentiated neuroendocrine carcinomas (7 of 13) were negative for PTEN (54%); the remainder showed diminished reactivity. The two melanomas studied were also negative for PTEN immunostaining. The paragangliomas, pheochromocytomas, medullary thyroid carcinomas, and neuroblastoma all showed a strong PTEN stain. The authors postulate that PTEN is a differentiation marker for NC/NE tissue and tumors and that loss of PTEN expression may represent an important step in the progression of NE tumors.

Adult↗
PubMed2001

Identification of differentially expressed genes in mouse development using differential display and in situ hybridization.

A common problem in developmental biology is the isolation of genes that are expressed differentially between two closely matched tissue populations, for example, between wild-type and mutant mouse embryos generated by targeted gene disruption. Typically, the applicable experimental methodologies are limited by the amount of mRNA that can be obtained for cDNA library construction and/or expression analysis. Differential display is a polymerase chain reaction (PCR)-based RNA fingerprinting technique that is ideally suited for the identification of differentially expressed transcripts when only limiting amounts of tissue are available, as is frequently encountered in studies of vertebrate development. Here, I describe protocols for differential display using arbitrarily primed reverse transcription PCR and for the subsequent verification of differential gene expression that are adapted for molecular genetic studies of mouse embryogenesis. The overall strategy involves two steps: First, RNA samples isolated from two nearly identical populations of embryos or microdissected embryonic tissues are compared by differential display, and candidate differentially expressed transcripts are identified. Second, these candidate transcripts are analyzed for differential expression in vivo using nonradioactive whole-mount or section in situ hybridization. In principle, this strategy permits the efficient isolation of genes that are differentially expressed during early mouse embryogenesis.

Animals↗