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W L Kuo

Publications and source records attributed to W L Kuo.

At least 19 recordsLinked to original sources
PubMed1995

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis.

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously.

Base Sequence↗
PubMed1995

Chromosomal localization of the gamma-glutamyl carboxylase gene at 2p12.

We have used two complementary approaches to analyze the chromosomal location of the gamma-glutamyl carboxylase gene. The amplification of a carboxylase-specific genomic fragment by polymerase chain reaction (PCR) in a human-rodent hybrid cell mapping panel localized the gene to chromosome 2. Mapping by fluorescence in situ hybridization assigned the gene to p12 of chromosome 2. Our results indicate that the gamma-glutamyl carboxylase gene has a single locus in the human genome.

Animals↗
PubMed1995

Semiautomated DNA probe mapping using digital imaging microscopy: I. System development.

Algorithms have been developed to help automate the mapping of DNA sequences along metaphase chromosomes using fluorescence in situ hybridization (FISH). Custom algorithms computationally define chromosome boundaries and compute chromosomal medial axes. A dynamic regional thresholding (DRT) algorithm is described that allows reliable detection of hybridization domains, even when they differ substantially in size and intensity. Chromosomal locations are calculated by determining the fractional location of each hybridization probe along the medial axis of a metaphase chromosome relative to the short arm (FLpter). These algorithms were tested on simulated data and by analysis of the location of probes that had been previously mapped by other techniques. These algorithms allow probes to be mapped rapidly along human chromosomes with a precision of 2-3 Mb.

Algorithms↗
PubMed1995

Semiautomated DNA probe mapping using digital imaging microscopy: II. System performance.

This paper describes an evaluation of a semiautomated, multicolor image-analysis system to map cloned probes along metaphase chromosomes. Mapping with this system consists of fluorescence in situ hybridization (FISH) for probe localization, automatic acquisition of multicolor images showing total chromosomal DNA and probe location(s), and automatic determination of the fractional locations of the probes along the chromosomes relative to the short arm telomere (FLpter). The system was evaluated by mapping ten phage and ten cosmid probes previously mapped to chromosome 3 with other procedures. The standard deviations of FLpter measurements averaged 3.4 Mb and 2.6 Mb for phage and cosmid probes, respectively. With this variation, the order of two probes mapped in separate hybridizations could be determined with 95% confidence when their separation was greater than 2.5 Mb. In all cases, the probe locations and order were consistent with previous mapping data. FLpter values were converted to band locations using measurements of the band locations made using digital imaging microscopy. This proved superior to conversions made using ISCN ideograms.

Bacteriophages↗
PubMed1995

Id gene expression during development and molecular cloning of the human Id-1 gene.

Id genes encode helix-loop-helix proteins that inhibit transcription by forming inactive heterodimers with basic helix-loop-helix (bHLH) proteins. bHLH proteins normally form either homodimers or heterodimers with other bHLH proteins and bind to a DNA sequence element activating transcription. Id-containing heterodimers are inactive because Id proteins lack the basic amino acid region necessary to form a DNA-binding domain. We have examined the relative levels of Id-1 and Id-2 mRNA during normal development and in malignant tissues. In the course of these experiments we cloned and sequenced the human Id-1 cDNA. Two related cDNA molecules encoding human Id-1 mRNAs were identified. Id-1a is a cDNA of 958 nucleotides and can encode a protein of 135 amino acids. Id-1b cDNA is 1145 nucleotides, can encode a protein of 149 amino acids, and appears to be a splice variant of Id-1a. The amino acid sequence of human Id-1 is greater than 90% homologous to that of mouse Id-1. The patterns of Id-1 and Id-2 expression during mouse development vary widely, and we detected Id-1 expression in human fetal and adult tissues from lung, liver, and brain. High Id-1 mRNA expression was found in many human tumor cell lines, including those isolated from nervous system tumors. We mapped Id-2 to human chromosome 2p25.

Amino Acid Sequence↗
PubMed1994

Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line.

Insulin degrading enzyme (IDE) is an evolutionarily conserved, nonlysosomal metalloprotease that has been implicated in the cellular degradation and processing of insulin. However, the site and the mode of the action of this enzyme are unclear. We have addressed these questions by establishing several Ltk- cell lines that can overexpress human insulin-degrading enzyme (hIDE) upon glucocorticoid induction. The level of overexpression of hIDE protein and transcripts in these lines correlates well with an increase in insulin degradation in both cell lysates and intact cells. Comparison of the deduced amino acid sequences of mammalian and Drosophila IDEs reveals a conserved carboxyl-terminal peroxisomal targeting sequence (A/S-K-L), suggesting that IDE may be localized in peroxisomes. To test this possibility, we determined the cellular location of the stably transfected hIDE by both immunofluorescence and immunocryoelectron microscopy. The overexpressed hIDE predominantly colocalized with catalase in peroxisomes, although IDE was also found in the cytosol at a much lower concentration. These results demonstrate that stably transfected IDE catalyzes a rate-limiting step in cellular insulin degradation and is localized predominantly in peroxisomes.

Amino Acid Sequence↗
PubMed1994

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

Breast Neoplasms↗
PubMed1994

ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.

Protein tyrosine kinases (PTKs) play an integral role in T cell activation and differentiation. Defects in the Src-family PTKs in mice and in T cell lines have resulted in variable defects in thymic development and in T cell antigen receptor (TCR) signal transduction. Here, three siblings are described with an autosomal recessive form of severe combined immunodeficiency disease (SCID) in which ZAP-70, a non-Src PTK, is absent as a result of mutations in the ZAP-70 gene. This absence is associated with defects in TCR signal transduction, suggesting an important functional role for ZAP-70.

Amino Acid Sequence↗
PubMed1994

A candidate tumor suppressor gene in human breast cancers.

We have isolated a candidate gene (designated Brush-1) located at 13q12-q13, proximal to the retinoblastoma gene (RB1). Brush-1 codes for a 4.7-kilobase mRNA expressed at high levels in normal breast epithelium but drastically reduced in 6 of 13 breast cancer cell lines. RB1 mRNA expression is at normal levels for 5 of these 6 lines suggesting a greater importance of Brush-1 for breast cancer. Four primary breast tumors which showed no loss of heterozygosity in the 13q13-q14 region demonstrated normal levels of mRNA for both Brush-1 and RB1. However, four additional primary tumors which displayed loss of heterozygosity for this region had markedly decreased levels of Brush-1 mRNA while maintaining the normal levels for RB1. This differential loss of Brush-1 mRNA expression for both primary tumors and breast cancer cell lines is the expected pattern for a breast tumor suppressor gene.

Base Sequence↗
PubMed1993

Functional analysis of conserved residues in the active site of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.

Amino Acid Sequence↗
PubMed1993

Mutations in a zinc-binding domain of human insulin-degrading enzyme eliminate catalytic activity but not insulin binding.

Insulin-degrading enzyme is a nonlysosomal metalloprotease that initiates degradation of internalized insulin in some cells. We previously identified a potential catalytic site containing an inversion of the Zn(2+)-binding domain of the thermolysin family (Kuo, W.-L., Gehm, B. D., and Rosner, M. R. (1991) Mol. Endocrinol. 4, 1580-1591). The role of this site in catalysis was examined by mutating one of the presumptive Zn(2+)-coordinating histidines (His108) in human insulin-degrading enzyme to leucine or glutamine, which were predicted to reduce or eliminate Zn2+ binding without substantially altering secondary structure. cDNAs for the mutant and wild-type enzymes were incorporated into an expression vector and transfected into COS cells. Expression of the transfected genes was confirmed by Northern and Western blots. In contrast to the wild-type gene, which increased insulin degradation by cell extracts and intact cells several-fold, the mutated genes had no effect on insulin degradation, indicating a loss of catalytic activity. However, the mutants' ability to bind substrate was unimpaired, as affinity labeling with 125I-insulin was increased compared to the wild type. These results suggest that an intact Zn(2+)-binding domain in human insulin-degrading enzyme is required for catalytic activity and can affect, but is not required for, substrate binding.

Amino Acid Sequence↗
PubMed1993

Human gastric mucin. Identification of a unique species by expression cloning.

Gastric mucin is a large glycoprotein which is thought to play a major role in the protection of the gastrointestinal tract from acid, proteases, pathogenic microorganisms, and mechanical trauma. In this paper we describe the isolation by expression cloning and characterization of cDNAs which code for human gastric mucin. The cDNA sequence is characterized by a tandem repeat region whose individual repeat unit is 507 base pairs (169 amino acids) long. The translated sequence is rich in threonine, serine, and proline (31, 18, and 15%, respectively) and contains a relatively large amount of histidine (7.1%) and alanine (5.6%). RNA blot analysis shows a polydisperse pattern which is characteristic of mucins. Expression of this gene is highest in the stomach and gall bladder, with weaker expression in the terminal ileum and right colon. This expression pattern is different from other human mucins and indicates that this gene codes for a unique mucin. Fluorescence in situ hybridization techniques have localized this gene to chromosome 11p15.4-11p15.5. This is the third mucin to be localized to the 11p15 region and suggests a clustering of secretory mucin genes. We propose that this gene for human gastric mucin be called MUC6.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of EagI YACs from human chromosome 21.

Yeast artificial chromosomes (YACs) were made from a total EagI digest of DNA from a mouse-human chromosome 21 hybrid cell line. Approximately 3750 YACs, corresponding to 75-125 human YACs, with an average size of approximately 100 kb were recovered. Southern hybridization indicates that the chimera frequency in this library may be less than 3%. Thirty-four of the human EagI YACs were regionally assigned by a number of methods. Some YACs were regionally assigned to one of six chromosome regions by hybridization of Alu-PCR products from the YAC against Alu-PCR-amplified DNA from a panel of hybrid cell lines that contain various parts of chromosome 21. Additional YACs were regionally assigned by fluorescence in situ hybridization using either biotinylated Alu-PCR products or yeast genomic DNA from the YAC-containing strains as probes. The regionally assigned EagI YACs are located preferentially in two regions of the chromosome: near the q telomere and in the p-arm ribosomal gene region.

Animals↗
PubMed1993

Chromosomal reassignment: YACs containing both YES1 and thymidylate synthase map to the short arm of chromosome 18.

The YES1 proto-oncogene was mapped previously to human chromosome band 18q21.3 by using isotopic in situ hybridization. Using yeast artificial chromosomes (YACs) as probes and fluorescence in situ hybridization, a strong signal was detected in the region corresponding to 18p11.3. Restriction digests confirmed that the YACs contained the YES1 gene and not other cross-hybridizing, protein-tyrosine kinases. In addition, these YACs were found to contain another 18p11.32 gene, thymidylate synthase. These genes were less than 50 kb apart. Collectively, these data suggest that YES1 maps to 18p11.32 rather than to 18q21.3.

Base Sequence↗
PubMed1993

An immortalized cystic fibrosis tracheal epithelial cell line homozygous for the delta F508 CFTR mutation.

The development of transformed human airway epithelial cell lines has been important in advancing the understanding of the biochemical and genetic mechanisms underlying the cystic fibrosis (CF) defect. Since the most common mutation associated with CF is a phenylalanine deletion at position 508 (delta F508) in the CF transmembrane conductance regulator (CFTR) gene, a transformed airway epithelial cell line homozygous for this mutation will be important for determining the biologic significance of this mutation in the airways. We report the genotypic and phenotypic characterization of a delta F508 homozygote cell line derived from luminal epithelium in the trachea. The cells were transformed with a plasmid containing an origin of replication defective SV40 genome and have progressed through crisis. Immunocytochemical characterization of the cells shows that they express keratin, indicating epithelial cell origin, and that a calcium-dependent cell adhesion molecule, cellCAM 120/80, is present at plasma membrane junctions between cells. Electrophysiologically, the cells show no cAMP-dependent Cl transport. However, after treatment with the calcium ionophore, ionomycin, cells secrete Cl, albeit at a lower level than that observed in normal cells. Genetically, the cells express CFTR mRNA as determined by polymerase chain reaction amplification and CFTR protein as determined by Western hybridization analysis. Karyotypic analysis shows that 70% of the cells contain two copies of chromosome 7.

Adult↗
PubMed1993

Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues.

Insulin-degrading enzyme (IDE), a cytosolic metalloendoprotease, can degrade insulin, insulin-like growth factor-II, insulin-like growth factor-I, and transforming growth factor-alpha. While IDE has been implicated in the cellular degradation of insulin, other physiological functions of this enzyme are not known. To assess the possible role of IDE in cellular growth and development, we determined the tissue and developmental distribution of the enzyme. Rat IDE cDNA fragments and antibodies directed against human IDE were used to probe IDE transcripts and proteins in rat tissues. The results demonstrate that IDE transcripts are ubiquitous in rat tissues. The level of rIDE transcripts is high in adult rat testis, tongue, and brain; moderate in kidney, prostate, heart, muscle, liver, intestine, and skin; and low in spleen, lung, thymus, and uterus. The sizes of the major transcripts of rIDE are 3.4 and 6.3 kilobases in all tissues analyzed, except testis. Surprisingly, the highest level of rIDE mRNA in the adult rat was in the testis, and the major transcripts of rIDE in this tissue were shifted in size to 3.8 and 6.7 kilobases. Immunocytochemical analysis localized the rIDE mainly in the epithelium of prostate gland and kidney, and the cytosol of liver hepatocytes. During rat development from 6-7 days of age to adulthood, rIDE mRNA levels increased in brain, testis, and tongue; decreased in muscle and skin; and did not significantly change in other tissues examined. These studies reveal regulation of IDE or IDE-related genes in rat tissues and during rat development, suggesting that this enzyme may have multiple functions relating to cellular growth and development.

Aging↗
PubMed1993

Growth of human melanocytes from suction blister of localized vitiligo patient and from foreskins of newborns and adults by modified culture medium.

We report on the culturing of melanocytes from suction blisters from the uninvolved skin of localized (focal and segmental) vitiligo patients and from the foreskins of newborns and adults, over a long period of time using a modified culture medium composed of F-12 medium supplemented with insulin (5 mg/mL), cholera toxin (40 ng/mL), transferrin (5 mg/mL), hydrocortisone (1 mM), epithelial growth factor (20 ng/mL), endothelial cell growth supplement (ECGS) (15 mg/mL), retinol (1 x 10(-7) M), 12-o-tetradecanoyl-phorbol-13 acetates (TPA) (85 nM), and 1% fetal calf serum (FCS). This method may be used in in vitro studies on normal human melanocytes and for study of the difference between melanocytes of normal individuals and of those with vitiligo. The ability to culture melanocytes from localized vitiligo, and the inability to grow those of generalized vitiligo with this system reconfirms the difference in the pathogenesis of various types of vitiligo. Thus, culturing may be used for differentiating localized vitiligo from generalized vitiligo and may be used to guide the mode of treatment when vitiligo has just started to develop and only a few depigmented patches have appeared. The results of studies on seeding density effect and serial Dopa reaction on the melanocytes of normal individuals reveal that the use of tyrosinase activity as the assessment parameter requires the use of melanocytes cultured for less than five months at a seeding density of less than 4 x 10(4) cells/cm2. Enlarged and heavily pigmented cells, senescent cells, were observed after very long-term culture (one year and four months).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗