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Biomedical subjects

R Wu

Publications and source records attributed to R Wu.

At least 271 records · Page 15Linked to original sources
PubMed1994

Identification of internal variation in the pseudoautosomal VNTR DXYS17, with nonrandom distribution of the alleles on the X and the Y chromosomes.

The PCR technique was used to analyze the DXYS17 locus in the pseudoautosomal region of the X and the Y chromosomes. Analysis on an automated DNA sequencer allowed for sensitive and highly accurate typing of 16 different alleles with a size between 480 and 1,100 bp. Two DXYS17 alleles migrated with the same size on agarose or denaturing polyacrylamide gels but with different mobilities on nondenaturing polyacrylamide gels. Sequence analysis showed that, while an identical number of repeats were present in both alleles, differences in the composition of the units were observed. The origin of these differences was found in the 28- and 33-bp units, which only had a specific repeat pattern at the 5' and 3' ends of the region. The genotype distribution for DXYS17 in a Caucasian population did not deviate from the values expected under Hardy-Weinberg equilibrium. However, the frequency of one allele and one genotype was significantly different between males and females. Segregation analysis showed that this difference was the result of a nonrandom distribution of certain alleles on the sex chromosomes in males.

Alleles↗
PubMed1993

Preparation and characterization of a bifunctionally spin-labeled mutant of murine epidermal growth factor for saturation-transfer electron paramagnetic resonance studies of the growth factor/receptor complex.

In this report we describe the production of a [Lys3,Tyr22] murine epidermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N-succinimidyl)-[15N,2H16]doxyl-2-spiro-4'-pimelate ([15N,2H16]BSSDP) in order to study the rotational dynamics of the EGF/EGF receptor complex by saturation-transfer electron paramagnetic resonance (ST-EPR). Previous results [Faulkner-O'Brien et al. (1991) Biochemistry 30,8976-8985] indicated that the reaction of [15N,2H16]BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of the EGF/EGF receptor complex because the major product, in which [15N,2H16]BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the more tightly coupled bidentate product was unstable. Using oligonucleotide-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for [15N,2H16]BSSDP labeling. The [Lys3,Tyr22] mEGF was expressed in Escherichia coli HB101 transformed with a pIN-III-ompA3-[Lys3,Tyr22] mEGF plasmid and was purified from the bacterial periplasm using a simple two step purification method. The [15N,2H16]BSSDP reacted with [Lys3,Tyr22]mEGF in high yield, and EPR analysis of the major product revealed tight motional coupling between the spin probe and the protein. Biological activity, as assessed by stimulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label. The [15N,2H16]BSSDP-modified [Lys3,Tyr22] mEGF was shown to be equipotent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF binding to the EGF receptor in A431 membrane vesicles. Using the [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR.

Amino Acid Sequence↗
PubMed1993

Inhibition of epidermal growth factor-like growth factor secretion in tracheobronchial epithelial cells by vitamin A.

Vitamin A deficiency of respiratory tract epithelium results in the phenomenon of squamous cell metaplasia. The mechanisms by which vitamin A regulates airway epithelial cell growth and differentiation are not completely understood. In this study, we focused on the effects of vitamin A (retinol) on growth of human and non-human primate tracheobronchial epithelial (TBE) cells in culture. Retinol and its derivatives have little growth-stimulatory effect on TBE cells that are maintained in primary culture in a serum-free medium supplemented with 6 hormonal supplements: insulin, transferrin, epidermal growth factor (EGF), hydrocortisone, cholera toxin, and bovine hypothalamus extract. However, it was observed that retinol exhibited dose-dependent inhibition of TBE cell growth when EGF was removed from this serum-free culture condition. This inhibition can be reversed if EGF or the conditioned medium of primary TBE cells that are maintained in vitamin A-deficient condition is added. This type of EGF-retinol interacting phenomenon was not observed with the 5 remaining hormonal supplements. Analysis of 125I-labeled EGF binding shows a down-regulation of the high affinity binding sites (Kd = 0.09 nM) on TBE cells grown in the absence of vitamin A. These results suggest that TBE cells are capable of secreting an EGF-like growth factor in the absence of vitamin A. The possibility that transforming growth factor-alpha (TGF-alpha) is involved in this phenomenon is further examined by antibodies specific to TGF-alpha and its binding to an EGF-receptor. Using the TGF-alpha antibody, the presence of a TGF-alpha-specific antigen was found to be 3-fold higher in the conditioned medium obtained from the vitamin A-deficient cultures than that derived from retinol-treated cultures. Furthermore, the antibody neutralizing the TGF-alpha binding to an EGF receptor was able to reduce the DNA synthesis associated with the vitamin A deficiency. These results suggest that vitamin A plays an important regulatory role in the paracrine/autocrine secretion of EGF/TGF-alpha-like mitogen in TBE cell cultures.

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

A new method for specific cleavage of megabase-size chromosomal DNA by lambda-terminase.

The development of methods for cleavage of DNA at specific site(s) that are widely spaced would facilitate physical mapping of large genomes. Several methods for rare and specific cleavage of chromosomal DNAs require a nearly complete methylation of a given type of restriction site except the one that is specifically protected. It is expected that as the target DNA increases in length, it will become less likely to achieve nearly complete methylation. The intron-encoded endonucleases may also provide a capability to cleave megabase-sized DNA segments due to their very large recognition sequences. However, there are endogenous cleavage sites in the chromosomes of most organisms. We present here a new method to specifically cleave intact chromosomal DNA using lambda-terminase. A plasmid containing two specific cleavage sites (cohesive-end sites) for lambda-terminase was specifically introduced into the E.coli genome and into chromosome V of S.cerevisiae. Chromosomal DNA was prepared from the resulting strains, and then cleaved with lambda-terminase. The results showed that the 4.7-megabase pair (Mb) circular E.coli chromosome and the 0.58-Mb linear yeast chromosome V were specifically cleaved at the desired sites with very high efficiencies. The approach of using the lambda-terminase cleavage reaction is a simple one-step procedure with a high specificity which is particularly suitable for mapping very large genomes of eucaryotes.

Bacteriophage lambda↗
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 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

Co-amplification of the cystic fibrosis delta F508 mutation with the HLA DQA1 sequence in single cell PCR: implications for improved assessment of polar bodies and blastomeres in preimplantation diagnosis.

We have developed a heminested PCR (polymerase chain reaction) method, performed on single cells, for the analysis of the most common cystic fibrosis (CF) mutation (delta F508). As a quality control, the polymorphic exon 2 of the HLA DQA1 locus was co-amplified from the same cell. With a non-radioactive reverse dot-blot assay, the genotype of these two loci could be determined. Experiments on 98 single fibroblasts, heterozygous for the CFTR and the DQA1 locus, showed that amplification of either locus could be obtained in 97 per cent of the cases, but only 90 per cent showed heterozygosity for CF, 75 per cent showed heterozygosity for DQA1, and 74 per cent showed heterozygosity for both CF and DQA1. Contaminations detected only after DQA1 typing occurred in 3 per cent of our samples. Error rate calculations based on our experimental PCR data indicate that single blastomere diagnosis would lead to unacceptable errors, i.e., an affected fetus, in less than 1 per cent of the cases. The risk of undetected crossing-over or the dubious results that crossing-over could generate, would make isolated polar body diagnosis at the present time very difficult. The combined approach of PCR on polar bodies followed by confirmation of the diagnosis on blastomeres, however, should give a solid base for preimplantation diagnosis of monogenic disorders.

Base Sequence↗