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Biomedical subjects

G An

Publications and source records attributed to G An.

At least 91 records · Page 5Linked to original sources
PubMed1994

Developmental and environmental regulation of two ribosomal protein genes in tobacco.

Two cDNA clones, TSC29 and TSC40, were isolated from a cDNA library prepared from three-day-old tobacco cell suspension grown to early exponential stage. DNA sequence analyses and database searches revealed that the TSC29 transcript encodes a protein which is highly homologous to eukaryotic 60S ribosomal (r)-protein L25 and that the TSC40 product is homologous to rat 60S r-protein L34. Southern blot analysis showed that the putative r-protein genes are members of multigene families. Transcript levels of both genes were most abundant in three-day-old cell suspension and declined in older cultures. Transcripts were also present in plant vegetative and reproductive organs. However, for TSC40 in particular, the mRNA levels were lower in plant organs than in three-day-old cell suspension. Stems and roots exhibited higher expression than leaves and flowers, indicating that these clones are differentially regulated in various cell types. Both genes were expressed at low levels in mature seeds but transcript levels significantly increased after one day of germination, remained at a high level until day 4, and declined after day 5. In situ localization experiments with germinating seedlings revealed that the TSC29 transcript was preferentially localized in root tips, epidermis, and endosperm. Wounding increased the steady-state mRNA amounts of these r-protein genes, and 2,4-dichlorophenoxyacetic acid and benzyladenine further increased the transcript level.

Amino Acid Sequence↗
PubMed1994

A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity.

The nopaline synthase (nos) promoter is expressed in a wide range of plant cell types and regulated by various developmental and environmental factors. The nos upstream control region essential for this regulation was studied by means of synthetic oligomers using transient and stable transformation systems. Insertion of a 20 nucleotide sequence containing two hexamer motifs and a spacer region into deletion mutants lacking the upstream control region was essential for promoter activity. Mutation of one or more nucleotides of either hexamer sequence significantly altered the strength of expression of the nos promoter. Point mutations within the spacer region also strongly influenced promoter strength. Insertion of multiple copies of the 20 nucleotide sequence into the nonfunctional deletion mutants proportionally increased the promoter activity. These results suggest that this twenty nucleotide sequence is essential for the nos promoter to function. Substitution of the nos element with the ocs or 35S as-1 which contain similar hexamer motifs restored not only promoter activity but also responses to wounding, auxin, methyl jasmonate, and salicylic acid.

Amino Acid Oxidoreductases↗
PubMed1994

Expression of MUC2 gene is down-regulated by vitamin A at the transcriptional level in vitro in tracheobronchial epithelial cells.

The functional role of airway mucin in the respiratory system is well recognized. The isolation of mucin cDNA clones, MUC genes, introduces new information regarding the structure of the mucin core protein; however, the nature of the authentic core protein of airway mucin is still unresolved. In this communication, the effects of vitamin A on the regulation of MUC2 gene expression in primary tracheobronchial epithelial (TBE) cells of human and nonhuman primates were examined. Vitamin A has been recognized as one of the most important nutrients in the regulation of airway mucous cell differentiation. The expression of the MUC2 gene has been demonstrated in both rat and human tracheal tissues. The monkey cDNA clone MT80 was isolated from a cDNA library derived from vitamin A-depleted cultures of monkey TBE cells using a synthetic oligonucleotide probe corresponding to the 69 nucleotides of a tandemly repeated sequence in human MUC2 cDNA. DNA sequencing revealed a similar tandemly repeated sequence, except that 72 oligonucleotide repeats were observed in the monkey cDNA clone. Using the MT80 cDNA as a probe, the expression of the MUC2 gene was studied in vitro. The corresponding MUC2 message level in primary cultures of monkey TBE cells was down-regulated by vitamin A. This result was consistently demonstrated in primary human and hamster TBE cultures. The down-regulation was both time- and dosage-dependent on vitamin A. A nuclear run-on assay demonstrated a decrease in the transcriptional rate of the MUC2 gene in nuclei isolated from vitamin A-treated cultures. These results suggest that MUC2 gene expression in TBE cells is transcriptionally down-regulated by vitamin A.

Amino Acid Sequence↗
PubMed1994

A PCR differential screening method for rapid isolation of clones from a cDNA library.

We have developed a new two-step differential screening method that allows the rapid isolation of induced or suppressed pure gene clones from a lambda phage cDNA library. This method involves a primary differential screening step and a PCR differential screening step. From the primary screening step, impure pools of positive cDNA clones are obtained. Each pool of clones is then amplified directly by PCR using two primers flanking the cloning site in the vector. The PCR products are run on two duplicate agarose gels and blotted onto two filter strips. These two filters are then subjected to differential Southern hybridization with different cDNA probes. Each pure positive cDNA band on the gel is identified and selected for further subcloning. This method has three advantages over the traditional differential screening method. First, several rounds of plaque rescreening are replaced by a single PCR screening. Second, plaque hybridization is replaced by a more reliable and accurate PCR DNA Southern hybridization. Third, the time-consuming and tedious phage DNA isolation step is eliminated, and subcloning is facilitated by direct cloning of the pure PCR product to a plasmid vector. We have successfully used this method to isolate ozone-responsive genes from a cDNA library of monkey respiratory airways.

Animals↗
PubMed1993

Isolation and characterization of the human spr1 gene and its regulation of expression by phorbol ester and cyclic AMP.

The small proline-rich protein gene (spr1) is a marker whose expression is frequently associated with squamous cell differentiation. We observed that the expression of the spr1 gene is strongly induced by phorbol 12-myristate 13-acetate (PMA). Both the time course result and the nuclear run-on transcriptional assay suggested that the regulation of spr1 expression by PMA is controlled at the transcriptional level. To understand the nature of this regulation, human genomic clones of the spr1 gene were isolated. DNA sequence analysis revealed that the human spr1 gene contains two exons and a single intron located within the 5'-untranslated region. An AP-1 binding site (TGAGTCA) is found at -142, and a putative cyclic AMP-responsive element (TGAGGTCA) at -597 base pairs upstream of the transcription start site. A chimeric construct containing the 5'-flanking region of the spr1 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect HeLa cells or monkey primary TBE cells. The CAT activity in transfected cells is stimulated 7.5-11-fold by PMA, and the stimulation is inhibited by a protein kinase C inhibitor or by pretreating cells with PMA to down-regulate the protein kinase C activity. The CAT activity is also stimulated 3.5-fold by dibutyryl cyclic AMP, a protein kinase A activator. The stimulations by PMA and cAMP are additive. These results suggest that protein kinase C and probably protein kinase A play important roles in regulating the transcription of the spr1 gene.

Amino Acid Sequence↗
PubMed1993

cDNA cloning of a hnRNP A1 isoform and its regulation by retinol in monkey tracheobronchial epithelial cells.

An isoform of the hnRNP A1 was cloned from a cDNA library of monkey tracheobronchial epithelial (TBE) cells by differential hybridization. The cDNA clone MT77 has an insert of 1756 base pairs and the DNA sequence shares high homology to both human A1 alpha-type and beta-type isoforms with the exception of several differences in the coding and noncoding regions. Like the other two isoforms, MT77 has two polyadenylation sites. A probe prepared from the MT77 clone hybridizes to two message bands at 1.4 and 1.8 kb. Both messages were found in a polysomal preparation, suggesting that both messages are used in A1 protein synthesis. The expression of the A1 gene in monkey TBE cells is stimulated by vitamin A (retinol). The results of nuclear run-on transcriptional assays suggest that this stimulation occurs at the transcriptional level. Furthermore, this effect is not prevented, but superinduced, by cycloheximide. These results suggest that vitamin A may be directly involved in regulating A1 transcription through a mechanism similar to the interactions between the retinoic acid responsive elements and the nuclear receptors of retinoic acid.

Amino Acid Sequence↗
PubMed1993

Expression of c-fos and c-jun family genes after focal cerebral ischemia.

The expression of the protooncogenes, c-fos, jun B, c-jun, and jun D was investigated in a rat focal cerebral ischemia model by Northern analysis and in situ hybridization. Severe ischemia (reduction of regional blood flow by 88-92%) in this model is confined to cerebral cortex irrigated by the right middle cerebral artery. Ischemia for 30 minutes, which caused only slight cortical damage (infarct size, < 10 mm3), induced both jun B and c-fos mRNAs exclusively in the right cerebral cortex. Ischemia for 90 minutes, which led to large cortical infarction (infarct size, > 140 mm3), also induced the expression of these two genes in the right cerebral cortex as well as the ipsilateral hippocampus. The latter sustained very mild ischemia (reduction of regional blood flow by 10-20%). The coinduction of jun B and c-fos expression occurred immediately after reperfusion and peaked at 60 minutes after reperfusion. The expression of c-jun was enhanced in a similar pattern, but at a much lower magnitude. In contrast, no change in jun D expression was observed. Nuclear run-on assays indicated that the increase in c-fos, jun B, and c-jun mRNA levels was due to the increase of transcription rate in these genes. Mobility shift assays showed a basal DNA binding activity of transcription factor AP-1 in the right cerebral cortex. Ischemia for 30 or 90 minutes followed by reperfusion for 4 hours resulted in a four- to sixfold increase of AP-1 binding activity. The enhanced DNA binding activity persisted for as long as 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression of a squamous cell marker, the spr1 gene, is posttranscriptionally down-regulated by retinol in airway epithelium.

Vitamin A (retinol) is required for the normal mucociliary differentiation of respiratory epithelium. A depletion of vitamin A promotes squamous cell metaplasia. To understand how vitamin A suppresses squamous cell differentiation, the expression of a squamous cell differentiation marker, the small proline-rich protein gene (spr1), was studied in cultured monkey tracheobronchial epithelial (TBE) cells. The expression of the spr1 gene was inhibited about 40 fold by retinol. The mRNA levels of the spr1 gene started to decline within 6 h of retinol treatment and reached a minimum level after 7 days. The inhibition by retinol was concentration dependent and did not require concurrent protein synthesis. The inhibition of the spr1 mRNA by retinol was not due to a decrease in the transcription rate of its gene but due to a decrease in its stability, as determined by nuclear run-on assays and mRNA half-life measurement, respectively. This result was further supported by a DNA transfection study using a chimeric construct containing the spr1 promoter region and the chloramphenicol acetyltransferase (CAT) reporter gene. The CAT activity in transfected cells was not inhibited by retinol. These results suggest that spr1 gene expression is posttranscriptionally down-regulated by retinol.

Animals↗
PubMed1993

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor.

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization experiments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.

Actin Depolymerizing Factors↗
PubMed1993

Identification of methyl jasmonate and salicylic acid response elements from the nopaline synthase (nos) promoter.

Transgenic tobacco plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (cat) were studied for their inducibility by salicylic acid (SA) or methyl jasmonate (MJ) treatments. Either chemical significantly increased CAT activity to a level much higher than that achieved by wounding. Northern blot analysis showed a corresponding increase in mRNA levels. After 20 h of induction of flowering plants, the response to MJ treatment was weaker in old leaves compared with young leaves, whereas the SA response was stronger in old leaves. Kinetic experiments showed that the SA response was much faster than the MJ response, suggesting that the induction mechanism of the nos promoter by these chemicals may differ. Deletion analysis showed that both SA and MJ responses require the DNA sequence between -119 and -112 from the transcription initiation site. This region contains the hexamer sequence (TGACGT) that has been found to be an important regulatory element for several promoters. The MJ response was also reduced by deletions of the CAAT box region or the sequence between -112 and -101, whereas the SA response was not significantly affected by these deletions. This suggests that the nos upstream region containing the hexamer motif is essential for the SA or MJ response and that the CAAT box region and the sequence immediately downstream from the hexamer motif are required for maximum induction by MJ.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1993

A small proline-rich protein regulated by vitamin A in tracheal epithelial cells is induced in lung tumors.

In cell-free translations of RNA from primary cultures of pig trachea surface epithelial cells, we observed that a 20 kD proline-rich protein (sPRP) is induced during culturing (Biochem. Biophys. Res. Commun. 1990; 172:1304-1309). Subsequently, a cDNA encoding sPRP has been cloned from pig tracheal cell mRNA and sequenced. This cDNA shows a high similarity to cDNAs cloned from monkey tracheal cells cultured in vitamin A-free medium and from UV-irradiated human epidermal keratinocytes. Amino acid sequences from these cDNAs are exceptionally rich in proline, glutamine, cysteine, and lysine but contain no aromatic amino acids. Two repeats of 12 amino acids on the N-terminus are followed by multiple 8 amino acid repeats. When compared with monkey trachea and human keratinocyte cDNAs, the sPRP cDNA from pig trachea has an additional 24 bp nucleotide repeat. Antiserum raised to a synthetic peptide (23 amino acids) on the C-terminus of sPRP (C23-antiserum) reacted with the 20 kD sPRP in immunoprecipitations from cell-free translations. On Northern blot analysis, sPRP cDNA hybridized to RNAs of similar sizes in tracheal cells from cat, rabbit, and lamb. sPRP was not detected in tracheal cells that were cultured with 10(-9) M arotinoid. Since sPRP is considered a putative squamous cell differentiation marker, experiments using lung tumors were performed. sPRP mRNA levels were dramatically increased in squamous lung tumors that were induced by injecting hamsters with 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone, a tobacco-specific nitrosamine. In situ hybridization with tissue sections prepared from these lung tumors revealed that cells around the keratin pearls contained high levels of sPRP mRNA.

Amino Acid Sequence↗
PubMed1993

Expression of immediate early gene and growth factor mRNAs in a focal cerebral ischemia model in the rat.

BACKGROUND AND PURPOSE: Increased release of excitatory neurotransmitters leading to activation of signal transduction has been noted after cerebral ischemia. One of the biochemical events in the signaling processes is an alteration of gene expression. A focal cerebral ischemia-reperfusion model in the rat was used to study the postischemic alteration of selected proto-oncogene and neurotrophin gene expression. METHODS: Focal ischemia in the cerebral cortex irrigated by the right middle cerebral artery was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries for 30 or 90 minutes. Expression of selected proto-oncogenes and neurotrophin genes was studied with Northern blot analysis and in situ hybridization. RESULTS: Northern blot analysis showed that an increase in the expression of selected proto-oncogenes including c-fos, c-jun, jun B, zif/268, and nur 77 were noted 30 to 90 minutes into postischemic reperfusion. In situ hybridization revealed different regional distribution of c-fos mRNA signal under different experimental conditions. Nuclear run-on experiments showed that the increase in c-fos and jun B mRNA signals was due to an increase in the transcription rate. An increase in AP-1 binding activity in the ischemic cortex was also noted. Increased expression of brain-derived neurotrophic factor and nerve growth factor mRNAs were noted subsequent to the expression of immediate early genes in the ischemic cortex and adjacent areas in a biphasic pattern. CONCLUSIONS: Results are in agreement with the contention that increased c-fos and jun B expression leads to increased AP-1 binding activity, which in turn has been linked to the enhanced expression of neurotrophin genes.

Animals↗
PubMed1992

Induction of Krox-20 expression after focal cerebral ischemia.

Krox-20 is one of the transcription factors of "zinc finger" family. The expression of Krox-20 was investigated in a rat focal cerebral ischemia model by Northern blot analysis and in situ hybridization. Northern blot analysis showed that ischemia for 30 min which caused little cortical infarction induced a 29-fold increase in Krox-20 mRNA signal exclusively in the ischemic cortex. Ischemia for 90 min which led to large cortical infarction induced Krox-20 mRNA not only in the ischemic cortex but also in the ipsilateral hippocampus. The induction of Krox-20 mRNA was rapid, transient and appeared to be controlled at the transcriptional level, as indicated by nuclear run-on assays. The regional induction of Krox-20 mRNA was further confirmed by in situ hybridization. These results suggest that the expression of transcription factor genes may play a role in the post-ischemic changes of the injured brain.

Brain Ischemia↗
PubMed1992

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Amino Acid Sequence↗
PubMed1992

Thioredoxin gene expression is transcriptionally up-regulated by retinol in monkey conducting airway epithelial cells.

Using the differential hybridization technique, a cDNA clone, MT78, was isolated from the cDNA library of retinol-treated monkey tracheobronchial (TBE) epithelial cells. MT78 has a high sequence homology to human thioredoxin. The cDNA insert contains 506 nucleotides which encodes a peptide of 105 amino acids. The deduced peptide contains the highly conserved sequence Cys-Gly-Pro-Cys, found at the active site of all thioredoxins. The expression of the thioredoxin gene is stimulated 8-10 fold by vitamin A (retinol) in monkey TBE cells. The expression is significantly enhanced within 4 h after the vitamin A treatment and concurrent protein synthesis is not required for this enhancement. These results, in conjunction with the nuclear run-on transcriptional assay, support the conclusion that thioredoxin gene is transcriptionally up-regulated by retinol and/or its metabolites.

Amino Acid Sequence↗