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PubMed2001

foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent on the C-terminal domain.

Xenopus foxD5a, the full-length fork head gene previously described as a PCR fragment (XFLIP), is first detectable at stage II of oogenesis. Low-abundance maternal transcripts are localized to the animal hemisphere of the cleavage embryo, and protein can be translocated to the nucleus prior to the onset of zygotic transcription. Zygotic expression is strongest in the presumptive neural ectoderm at gastrula and neural plate stages, but there is minor paraxial mesodermal expression during primary gastrulation that becomes significant in the tail bud during secondary gastrulation. Expression of foxD5a in animal cap explants induces elongation and expression of mesodermal, neural-inducing, and early neural-specifying genes, indicating a role in dorsal axis formation. Zygotic foxD5a expression is induced strongly by siamois, moderately by cerberus, weakly by Wnt8 and noggin, and not by chordin in animal cap explants. Expression of foxD5a in whole embryos has differential dorsal and ventral effects. Ventral mRNA injection induces partial secondary axes composed of expanded mesodermal and epidermal tissues, but does not induce ectopic neural tissues. Dorsal mRNA injection causes hypertrophy of the neural plate and expansion of early neural genes (sox3 and otx2), but this is not the result of increased proliferation or expanded neural-inducing mesoderm. The neural plate appears to be maintained in an immature state because otx2 expression is expanded and expression of en2, Krox20, proneural genes (Xnrgn1, neuroD) and a neural differentiation gene (n-tubulin) is repressed in foxD5a-expressing cells. These results indicate that foxD5a maintains an undifferentiated neural ectoderm after neural induction. Expression of foxD5a constructs fused with the engrailed repressor domain or with the VP16 activation domain demonstrates that FoxD5a acts as a transcriptional repressor in axis formation and neural plate expansion. Deletion constructs indicate that this activity requires the C-terminal domain of the protein.

Amino Acid Sequence↗

PubMed1998

Photoregulation of cot-1, a kinase-encoding gene involved in hyphal growth in Neurospora crassa.

Blue light plays a key role as an environmental signal in the regulation of growth and development of fungi and plants. Here we demonstrate that in Neurospora crassa hyphae branch more frequently in cultures grown in light. Previous studies had identified cot-1 as a gene that controls apical hyphal cell elongation. In the cot-1 mutant, cessation of elongation is accompanied by hyperbranching. Here we demonstrate that the cot-1 gene encodes two transcript species of about 2100 nt (cot-1 (s)) and about 2400 nt (cot-1 (l)) in length and that the ratio of both transcript species abundance is photoregulated. The origin of the difference between cot-1 (l) and cot-1 (s) was localized to the 5' end of the cot-1 transcripts, suggesting that two COT1 isoforms with different activities may be formed. Both light effects, on branching and on cot-1 expression, were dependent on functional wc-1 and wc-2 gene products. In addition to light, L-sorbose comprises another environmental cue that controls hyphal branching in N. crassa. In the presence of L-sorbose, photoregulation of cot-1 was blocked, suggesting the involvement of alternative and potentially interdependent signaling pathways for the regulation of hyphal elongation/branching.

Base Sequence↗

PubMed1989

Transcriptional control of adrenal catecholamine and opiate peptide transmitter genes.

In the rat, decreasing transsynaptic activity through adrenal denervation, nicotinic receptor blockade, or explanation is associated with an increase in preproenkephalin mRNA, enkephalin prohormone and peptide. In contrast, catecholamine pathways remain unchanged under similar conditions. Since it is not known whether changes in messenger RNA result from stabilization or increased synthesis, we exploited transcription 'run-on' assays to measure the rate of transmitter gene read out. Tyrosine hydroxylase message (TH-mRNA) was found to be the most abundantly produced transcript in the unmanipulated control rat adrenal medulla. TH-mRNA was produced in excess of twice the rate of transcription of the structural gene beta-actin. In contrast, preproenkephalin transcription occurred at a much lower rate (60% of the actin gene and only 25% of tyrosine hydroxylase gene transcription). All transcripts were inhibited by the polymerase II inhibitor, alpha-amanitin. After two days in explant culture, the rate of enkephalin transcription increased approximately 2-fold (to the same level as actin transcription); while tyrosine hydroxylase transcriptional activity fell to 30% of actin level. To analyze cellular mechanisms, explants were depolarized with potassium chloride. Enkephalin gene transcription was observed to be 2.5-fold less when grown under depolarizing conditions (50 mM KCl) than in control explants. On the other hand, tyrosine hydroxylase gene read-out was unchanged, similar to results obtained when TH catalytic activity was measured. These data indicate that membrane depolarization can selectively regulate expression of a transmitter gene product and are consistent with a proposed transsynaptic regulatory mechanism controlling biosynthesis of adrenal opiate peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗

PubMed1998

Photoregulation of cot-1, a Kinase-Encoding Gene Involved in Hyphal Growth in Neurospora crassa.

Blue light plays a key role as an environmental signal in the regulation of growth and development of fungi and plants. Here we demonstrate that in Neurospora crassa hyphae branch more frequently in cultures grown in light. Previous studies had identified cot-1 as a gene that controls apical hyphal cell elongation. In the cot-1 mutant, cessation of elongation is accompanied by hyperbranching. Here we demonstrate that the cot-1 gene encodes two transcript species of about 2100 nt (cot-1 (s)) and about 2400 nt (cot-1 (l)) in length and that the ratio of both transcript species abundance is photoregulated. The origin of the difference between cot-1 (l) and cot-1 (s) was localized to the 5' end of the cot-1 transcripts, suggesting that two COT1 isoforms with different activities may be formed. Both light effects, on branching and on cot-1 expression, were dependent on functional wc-1 and wc-2 gene products. In addition to light, l-sorbose comprises another environmental cue that controls hyphal branching in N. crassa. In the presence of l-sorbose, photoregulation of cot-1 was blocked, suggesting the involvement of alternative and potentially interdependent signaling pathways for the regulation of hyphal elongation/branching. Copyright 1998 Academic Press.

Journal Article↗

PubMed1996

The growth hormone-dependent decrease in hepatic fatty acid synthase mRNA is the result of a decrease in gene transcription.

The present study was conducted to determine the chronic effects of porcine growth hormone administration on fatty acid synthase (FAS) mRNA abundance and gene transcription in growing rats. Growth hormone treatment increased growth rate approximately 27% (P<0.01). Porcine growth hormone decreased FAS mRNA levels by 55%. The reduction in FAS mRNA was due to a marked decrease in transcription of the FAS gene (decreased by 80%). In contrast, porcine growth hormone did not affect mRNA abundance or transcription rate of another insulin-regulated gene, phosphoenolpyruvate carboxykinase. In summary, our results have established that chronic treatment with growth hormone decreases FAS mRNA by decreasing the transcription rate of the gene. Furthermore, they suggest that the effects of growth hormone are specific and are not mediated by general changes in insulin-responsive gene expression in liver.

Animals↗

PubMed2004

The cryptochrome (cry) gene and a mating isolation mechanism in tephritid fruit flies.

Two sibling species of tephritid fruit fly, Bactrocera tryoni and Bactrocera neohumeralis, are differentiated by their time of mating, which is genetically determined and requires interactions between the endogenous circadian clock and light intensity. The cryptochrome (cry) gene, a light-sensitive component of the circadian clock, was isolated in the two Bactrocera species. The putative amino acid sequence is identical in the two species. In the brain, in situ hybridization showed that cry is expressed in the lateral and dorsal regions of the central brain where PER immunostaining was also observed and in a peripheral cell cluster of the antennal lobes. Levels of cry mRNA were analyzed in whole head, brain, and antennae. In whole head, cry is abundantly and constantly expressed. However, in brain and antennae the transcript cycles in abundance, with higher levels during the day than at night, and cry transcripts are more abundant in the brain and antennae of B. neohumeralis than in that of B. tryoni. Strikingly, these results are duplicated in hybrid lines, generated by rare mating between B. tryoni and B. neohumeralis and then selected on the basis of mating time, suggesting a role for the cry gene in the mating isolation mechanism that differentiates the species.

Amino Acid Sequence↗

PubMed1997

Stage-independent splicing of transcripts two heterogeneous neighboring genes in Leishmania amazonensis.

Gene expression in trypanosomatid protozoa is largely regulated posttranscriptionally, e.g., 5' splice leader addition and 3' polyadenylation of mRNAs. We examined these events in Leishmania by mapping the splice sites of the transcripts from two different, but closely linked single-copy genes 2.3 kb apart. The coding regions of the approx. 1 kb upstream gene (P36) and the approx. 1.4 kb downstream gene (NAGT) produce approx. 2 and 3 kb mRNAs, respectively. Both genes were overexpressed in cells that were transfected with this bicistronic unit (> or = 7.5 kb), taking advantage of the NAGT as a selectable marker for tunicamycin-resistance. The transcripts from both genes were spliced constitutively at both ends, irrespective of their episomal or chromosomal expression in both leishmanial stages. Primer extension of the 5' UTRs and S1 nuclease protection of the 3' UTRs initially identified the major splice sites, corresponding to the genomic sequence at -205 bp and + approx. 900 bp of P36, and -1012 bp and + approx. 600 bp of NAGT. These splice sites, consistent with the size of the major transcripts, are among those mapped precisely by sequencing RT-PCR amplified 5' and 3' UTRs. The additional sites mapped by the latter are minor alternatives, especially abundant for transcripts of the downstream NAGT. All these minor splice sites are closer than the major splice sites to the coding region, indicating that the most distant splice sites are preferentially used. This preference creates a 387 bp 'gap' with polypyrimidine tracts in the intergenic region consistent with the model coupling splice leader addition with polyadenylation in pre-mRNA processing. The stage-independence of these events suggests that the 7.5 kb dicistronic unit is suitable for constructing Leishmania-specific constitutive expression vectors.

Animals↗

PubMed2006

Glycoprotein 340 in normal human ocular surface tissues and tear film.

The tear film is a complex mixture of secreted fluid, ions, proteins, glycoproteins, and lipids that lubricates and protects the ocular surface. Recently, several antimicrobial peptides have been described in the tear fluid. In this study, we describe the presence of the large secreted glycoprotein gp340 in the tear film. Western blot analysis showed that gp340 is abundant in secreted tears and in the lacrimal glands. Lesser amounts of gp340 were detected in the cornea and conjunctiva. Consistent with Western blot data, reverse transcription-PCR and real-time quantitative PCR showed that gp340 transcripts were abundant in lacrimal gland tissue and were also present in the cornea and conjunctiva. Immunohistochemistry localized gp340 to the acinar cells of the lacrimal gland and the deeper layers of the conjunctival epithelium. gp340 was not detected in conjunctival goblet cells. In the cornea, gp340 was present only in a peripheral band of basal epithelial cells, suggesting that gp340 may play a role in the cycle of corneal epithelial renewal. To determine if tear film gp340 may function as a bacterial agglutinin as it does in saliva, tears were incubated with streptococcal cells and the formation of bacterial aggregates was monitored. Addition of tears to late-exponential-phase Streptococcus mutans cells resulted in time- and dose-dependent aggregation of the bacteria. Furthermore, Western blot analysis confirmed the presence of cell-associated gp340 in isolated bacterial aggregates. The ocular pathogen Staphylococcus aureus, but not Pseudomonas aeruginosa, also aggregated when incubated with tears. These results suggest that gp340 is a normal component of the tear film and that the glycoprotein may function as a bacterial agglutinin.

Adult↗

PubMed1994

An alternatively spliced form of Pit-1 represses prolactin gene expression.

The pituitary-specific transcription factor Pit-1 is required for expression of the PRL gene. Transcription of the PRL gene in the anterior pituitary is both activated and repressed in response to neuroendocrine signals. The molecular events that mediate repression are unknown. Transplantation of GH3 pituitary tumor cells from culture to female Wistar-Furth rats resulted in repression of PRL gene expression. When the transplanted cells were returned to culture, PRL gene expression was rapidly activated. We used this model to study potential mechanisms by which PRL gene expression was silenced. In addition to the appropriate size Pit-1 proteins of 33 and 31 kilodaltons, smaller forms of the transcription factor, migrating at approximately 27 and 24 kilodaltons, were found in transplanted cells in which PRL gene expression was repressed. These smaller forms of Pit-1 protein were observed to disappear when transplanted cells were returned to culture, coincident with the activation of PRL gene expression. A transcript approximately 170 base pairs shorter than expected for that encoding full-length Pit-1 was detected in transplanted GH3 cell RNA by polymerase chain reaction. The shorter Pit-1 transcript was abundant only in GH3 cells after in vivo passage and was not readily detected in transplanted cells after as little as 12 h in culture. This shorter transcript was found to result from excision of sequence corresponding to exon IV and encodes a Pit-1 protein lacking 54 amino acids of the POU-specific domain. Gene transfer studies demonstrated this alternative form of Pit-1 inhibited PRL promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1987

Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos.

Considerable evidence indicates that the 2-cell stage is a critical period of mouse embryo development when a transition from maternal to zygotic genomic control takes place. The overall changes in the structure of the mRNA population as a result of this transition were explored using a random cDNA library of 69 clones derived from late 2-cell embryos. The prevalence of the cloned sequences was analysed by dot hybridization of the cDNA clones with labelled cDNA probes synthesized to poly(A)+ RNA from different stages of development from 1-cell through blastocyst. The number of copies of individual transcripts was quantitatively estimated by comparison to standard clones of known prevalence. About one half of the transcripts that gave a measurable reaction at the 2-cell and later stages were not represented detectably in egg RNA, suggesting that a large set of zygote-specific genes not included in the maternal gene set becomes transcriptionally active in the 2-cell embryo. Six of the cDNA clones represented B1 and B2 repeat sequences. As measured by hybridization with labelled cDNA, B1 and B2 transcripts were abundantly expressed throughout cleavage, being represented by about 10(5) to 10(6) copies per embryo. However, the developmental pattern of prevalence was different for the two transcripts suggesting that their expression is regulated independently. The results of this study corroborate previous evidence derived from protein synthetic patterns and in vitro translation experiments that a major qualitative shift in the mRNA population occurs in the 2-cell embryo.

Animals↗

PubMed2004

Effects of morphine on gene expression in the rat amygdala.

Influence of morphine self-administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self-administer morphine, each having two 'yoked' control animals, receiving passive injections of either morphine or saline. After 40 sessions of self-administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real-time PCR showed significant changes in abundance of five transcripts: gamma protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self-administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, gamma1 subunit of GABAA receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC gamma, gamma1 subunit of GABAA and hsp70 genes.

Amygdala↗

PubMed1985

The Gv-1 locus coordinately regulates the expression of multiple endogenous murine retroviruses.

The analysis of the normal expression of endogenous retroviral sequences in congenic 129 GIX+ and GIX- mouse strains suggests the action of a regulatory element, encoded by Gv-1, functioning in trans to control the expression of these sequences. At steady state, the abundance of polyadenylated retroviral transcripts of different length cosegregates with the Gv-1 genotype. The presence of low but detectable quantities of these transcripts in antigen-negative mice indicates however, that Gv-1 may be distinct from the structural genes encoding retroviral proteins. We report here that retroviral transcripts, derived from distinct proviruses, are expressed in a tissue-specific manner and are coordinately regulated by the Gv-1 locus. The structure of several of the abundant transcripts demonstrates further that they are transcribed from independent defective endogenous proviral genomes exhibiting extensive deletions of env coding regions as well as modified U3 regions distinct from those found in exogenous retroviral transcripts.

Animals↗

PubMed1993

Isolation of cDNAs for two closely related members of the axolotl Wnt family, Awnt-5A and Awnt-5B, and analysis of their expression during development.

To characterize molecular interactions between cells in the early amphibian embryo, we have isolated cDNAs for two members of the axolotl (Ambystoma mexicanum) Wnt family, Awnt-5A and Awnt-5B. The encoded proteins share 83% amino acid identity. Using a reverse transcription-polymerase chain reaction (RT-PCR) assay, we find that Awnt-5A transcripts are abundant in the blastula until gastrulation, barely detectable during gastrulation, and increase again during neurulation. They are detected throughout the remaining development and in hatched larvae. In contrast, transcripts for Awnt-5B are undetectable in the blastula. They appear with gastrulation, are present throughout neurulation and organogenesis, and decrease to barely detectable levels in hatched larvae. PCR reactions performed using cDNA library-phage DNA templates derived from whole neurulae versus embryos with the neuroectoderm removed suggest that, in the neurula, Awnt-5A transcripts are present in neuroectodermal as well as non-neuroectodermal tissues while Awnt-5B mRNAs are predominantly localized in the neuroectoderm. To localize Awnt-5A expression in embryos before gastrulation, early gastrulae were dissected by cutting along the animal-vegetal and future dorso-ventral axes and analyzed by RT-PCR. At this early stage, Awnt-5A transcripts appear to be predominantly localized in the dorso-vegetal region of the embryo. These results suggest that the two closely related Awnt-5 genes participate in different morphogenetic processes during early axolotl development.

Ambystoma mexicanum↗

PubMed2005

A highly conserved 6S RNA structure is required for regulation of transcription.

6S RNA, a highly abundant noncoding RNA, regulates transcription through interaction with RNA polymerase in Escherichia coli. Computer searches identified 6S RNAs widely among gamma-proteobacteria. Biochemical approaches were required to identify more divergent 6S RNAs. Two Bacillus subtilis RNAs were found to interact with the housekeeping form of RNA polymerase, thereby establishing them as 6S RNAs. A third B. subtilis RNA was discovered with distinct RNA polymerase-binding activity. Phylogenetic comparison and analysis of mutant RNAs revealed that a conserved secondary structure containing a single-stranded central bulge within a highly double-stranded molecule was essential for 6S RNA function in vivo and in vitro. Reconstitution experiments established the marked specificity of 6S RNA interactions for sigma(70)-RNA polymerase, as well as the ability of 6S RNA to directly inhibit transcription. These data highlight the critical importance of structural characteristics for 6S RNA activity.

Bacillus subtilis↗

PubMed1987

Regulatory elements that modulate expression of human c-myc.

Regulation of transcription from the proto-oncogene c-myc apparently plays an important part in cellular proliferation and the genesis of diverse tumors. Here, we report that the abundance of transcripts from the two principal promoters for human c-myc (P1 and P2) is governed by a composite of positive and negative regulators, located within a 2.3-kb domain upstream of the gene. In actively proliferating cells, the action of the positive elements is apparently dominant over that of the single negative regulator that we have identified. Nuclear proteins bind specifically to nucleotide sequences within the negative regulator and at least one of the positive regulators. The cooperative and counteracting actions of the regulatory elements described here presumably contribute to the plasticity of transcription from c-myc and may be affected by the tumorigenic damage that sometimes afflicts c-myc.

Animals↗

PubMed1997

Expression of cell cycle genes during Sesbania rostrata stem nodule development.

Upon infection of Sesbania rostrata with Azorhizobium caulinodans, nodules are formed on roots and stems. Stem nodules develop from abundantly distributed dormant root primordia. To acquire more insight into the meristem organization during stem nodule development, the expression patterns of a mitotic B1-type cyclin gene (Sesro; CycB1;1), a cyclin-dependent kinase gene (Cdc-2-1Sr), and a histone H4 gene (H4-1Sr) of S. rostrata were followed by in situ hybridization. Cdc2-1Sr transcripts were found in all cells of uninfected and infected root primordia. In uninfected root primordia, Sesro;CycB1;1 transcripts were detected in a few cells of the apical root meristem whereas H4-1Sr transcripts were abundant in this region. Interestingly, after inoculation with A. caulinodans, H4-1Sr transcripts disappeared in the root meristem and a patchy pattern of Sesro;CycB1;1 and H4-1Sr expression appeared in the cortex of the root primordium, reflecting the formation of globular nodule primordia. When bacterial invasion started, a distal nodule meristem was delimited wherein Sesro;CycB1;1 and H4-1Sr expression was concentrated. Approximately 1 week after inoculation, meristem activity ceased, indicated by the loss of Sesro;CycB1;1 and H4-1Sr expression.

Amino Acid Sequence↗

PubMed1999

Expansion of interferon-gamma-producing lung lymphocytes in mouse silicosis.

Silicosis is characterized by mononuclear cell inflammation with macrophage activation, accumulation of lymphocytes, and fibrosis. Interferon-gamma (IFN-gamma) is a lymphocyte cytokine with broad effects, particularly macrophage activation. Mice exposed to an aerosol of cristobalite silica (70 mg/m3, 12 d, 5 h/d) developed diffuse pulmonary pathologic changes with macrophage, lymphocyte, and neutrophil recruitment, and increased lung collagen. IFN-gamma messenger RNA (mRNA) was more abundant by semiquantitative reverse transcription-polymerase chain reaction in the lungs of silica-exposed mice than in control animals. IFN-gamma mRNA transcripts were detected by in situ hybridization with digoxigenin-labeled complementary DNA probes in normal mouse lung tissue within bronchial-associated lymphoid tissues (BALT). In silica- exposed mice, mononuclear cells with IFN-gamma mRNA were more numerous in the silicotic lesions and enlarged BALT structures. Lung-cell suspensions were prepared by enzyme digestion, stained with fluorescent-labeled antibodies against intracellular cytokines, and enumerated by flow cytometry. The percentage of cells producing IFN-gamma was increased in silicotic mice (19% versus 11%). Interleukin (IL)-4 mRNA transcripts were less abundant in the lung tissue from silica-exposed mice than in control mice. Cells staining for IL-4 mRNA were found rarely in either the air-sham or the silica-exposed mouse lungs, and almost all appeared to be within BALT structures. Approximately 3% of cells stained for IL-4 in the digested lungs from both groups. Similar cytokine patterns were observed in mediastinal lymph node/thymus and spleen tissues. The augmented IFN-gamma response, with IL-4 unchanged or decreased, in the lung lesions and lymphoid tissue of mice with silicosis suggests a Th-1-like lymphocyte-mediated immune-inflammatory response.

Aerosols↗

PubMed1999

Expression analysis of new Ly49 genes: most transcripts of Ly49j lack the transmembrane domain.

Five new Ly49 genes, named Ly49j-n, have recently been identified in C57BL/6 mice. This study examined the expression of three of these new genes, Ly49j, k, and n. To determine whether the Ly49j, k, and n genes were transcribed, gene-specific primers were used to amplify cDNA clones for each gene from C57BL/6 interleukin-2-activated natural killer (NK) cell cDNA. A full-length cDNA for Ly49j was detected which encodes a 267 amino acid protein and shares approximately 96% nucleotide identity with Ly49c and i. COS cells transfected with the Ly49j cDNA were shown to react with the monoclonal antibody 8H7, suggesting that the gene likely encodes a functional protein. Many different sized Ly49k and n transcripts were observed, although it is likely that they do not encode functional proteins due to missing exons or severe truncations in the open reading frames. Interestingly, the most abundant Ly49j transcript detected was shown to lack exon 3, which encodes the transmembrane domain. Similar studies performed on the same source of NK cell cDNA using Ly49c- and i-specific primers revealed the presence of transmembrane-less Ly49i transcripts, although at a much lower frequency than observed for Ly49j. We also detected Ly49g and h transcripts lacking the transmembrane domain. Despite the absence of the transmembrane region, the resulting Ly49 transcripts maintain their open reading frames, and therefore could potentially encode cytoplasmic proteins with a role in NK cell function.

Alternative Splicing↗