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S Lee

Publications and source records attributed to S Lee.

At least 919 records · Page 51Linked to original sources
PubMed1995

Model for binding of transcription factor TFIIB to the TBP-DNA complex.

Transcription factor TFIIB is essential for the formation of RNA polymerase II initiation complexes where it binds to the TATA-binding protein (TBP) complex with DNA and recruits RNA polymerase II. TFIIB is probably a target for various activators. Several models have been proposed for the position of TFIIB in the TFIIB-TBP-DNA complex. Here we examine the structure of this complex using gel mobility-shift assays and hydroxyl-radical footprinting. TFIIB requires at least seven base pairs of DNA on either side of the TATA box to form a stable TFIIB-TBP-DNA complex. The sugar residues protected from hydroxyl-radical cleavage by the TFIIB-TBP complex were mapped on the crystal-structure model of the TBP-DNA complex. This analysis suggests that TFIIB binds beneath the concave surface of TBP, contacting DNA both upstream and downstream of the TATA box. Our model predicts that TFIIB binds close to the C-terminal stirrup of TBP and provides one explanation for why TBP needs to bend DNA.

Carbohydrate Metabolism↗
PubMed1995

Transcriptional promoter of the human alpha 1(V) collagen gene (COL5A1).

We have characterized the 5' region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of 'housekeeping' and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5' sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained within the 212 bp immediately upstream of the major transcription start site contained no consensus sequences for the binding of known transcription factors, but gel mobility shift assays showed this region to bind nuclear factors, including Sp1, at a number of sites. The major transcription start site is flanked by an upstream 34-bp oligopurine/oligopyrimidine stretch, or 'GAGA' box, and a downstream 56-bp GAGA box which contains a 10-bp mirror repeat and is sensitive to cleavage with S1 nuclease.

Amino Acid Sequence↗
PubMed1995

Burkitt translocation (8;22)(q24;q11) in a patient with multiple myeloma.

The prevalence of chromosomal abnormalities in multiple myeloma (MM) has been difficult to detect by karyotyping primarily because of the low proliferative rate of malignant plasma cells. The reported incidences of abnormal karyotypes range from 24% to 63% in bone marrows obtained from MM patients, with the higher rates being seen in aggressive disease [1-8]. Detection of abnormal karyotypes in MM has been associated with a poor prognosis. We report a MM patient with an 8;22 Burkitt translocation, the first such reported case.

Bone Marrow↗
PubMed1995

Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations.

The human VHL tumor suppressor gene has been implicated in the inherited disorder von Hippel-Lindau disease and in sporadic renal carcinoma. The homologous rat gene encodes a 185-amino acid protein that is 88% sequence identical to the aligned 213-amino acid human VHL gene product. When expressed in COS-7 cells, both the human and the rat VHL proteins showed predominant nuclear, nuclear and cytosolic, or predominant cytosolic VHL staining by immunofluorescence. A complicated pattern of cellular proteins was seen that could be specifically coimmunoprecipitated with the introduced VHL protein. A complex containing VHL and proteins of apparent molecular masses 16 and 9 kDa was the most consistently observed. Certain naturally occurring VHL missense mutations demonstrated either complete or partial loss of the p16-p9 complex. Thus, the VHL tumor suppressor gene product is a nuclear protein, perhaps capable of specifically translocating between the nucleus and the cytosol. It is likely that VHL executes its functions via formation of specific multiprotein complexes. Identification of these VHL-associated proteins will likely clarify the physiology of this tumor suppressor gene.

Amino Acid Sequence↗
PubMed1995

p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches.

Insertion/deletion (IDL) mismatches in DNA are lesions consisting of extra bases on one strand. Here, the binding of p53 and its 14 kDa C-terminal domain to DNAs containing one or three 3-cytosine IDL mismatches was examined. Electron microscopy showed that both p53 forms bound predominantly as tetramers at the lesions while single-stranded binding proteins did not bind. Gel retardation assays showed that p53 formed highly stable complexes when the DNA contained the IDL mismatches, but only unstable complexes when the DNA lacked lesions (but did contain free ends). The highly stable complexes had a half-life of > 2 hr, suggesting that upon encountering lesions, p53 may recruit other proteins to the site, providing a signal for DNA damage.

Base Composition↗
PubMed1995

Correction of radiation sensitivity in ataxia telangiectasia cells by a truncated I kappa B-alpha.

Cells from patients with ataxia telangiectasia (AT) are hypersensitive to ionizing radiation and are defective in the regulation of DNA synthesis. A complementary DNA that corrects the radiation sensitivity and DNA synthesis defects in fibroblasts from an AT group D patient was isolated by expression cloning and shown to encode a truncated form of I kappa B-alpha, an inhibitor of the nuclear factor kappa B (NF-kappa B) transcriptional activator. The parental AT fibroblasts expressed large amounts of the I kappa B-alpha transcript and showed constitutive activation of NF-kappa B. The AT fibroblasts transfected with the truncated I kappa B-alpha expressed normal amounts of the I kappa B-alpha transcript and showed regulated activation of NF-kappa B. These results suggest that aberrant regulation of NF-kappa B and I kappa B-alpha contribute to the cellular defect in AT.

Amino Acid Sequence↗
PubMed1995

Refolding of alpha 1-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation.

Recombinant alpha 1-antitrypsin (alpha 1AT) produced as inclusion bodies in Escherichia coli was purified via several steps including solubilization of the inclusion bodies in 8 M urea and refolding by direct dilution of denaturant, followed by ion-exchange chromatography. The purified recombinant alpha 1AT has an activity comparable to human plasma alpha 1AT. During refolding, prolonged incubation of the alpha 1AT polypeptides at intermediate urea concentration favored production of inactive but soluble aggregates, which could regain activity after denaturation and renaturation. Nondenaturing polyacrylamide gel electrophoresis of the aggregates revealed the existence of dimers and higher oligomers. Immunological approach to characterize conformation showed that the oligomers were distinct from the native, the cleaved, or the denatured form, but was similar to the polymers induced from the native structure in mild denaturing condition. These results suggest that the oligomers are formed through specific interactions between aggregation-competent species which are stabilized at intermediate denaturant concentration.

Escherichia coli↗
PubMed1995

Organization of the gene encoding the human Kell blood group protein.

Kell is one of the major blood group systems in human erythrocytes. It is a complex system containing a large number of different antigens. Previously we cloned the Kell cDNA, which was predicted to encode an integral membrane protein with 731 amino acids. Now we have isolated overlapping genomic clones and determined the exon-intron structure of the KEL gene; it spans approximately 21.5 kb with its coding sequence being organized in 19 exons that range in size from 63 bp to 288 bp. The size of introns ranges from 93 bp to approximately 6 kb. The donor and acceptor splice sites all conform to the consensus splicing sequences. Exon 1 encodes only the initiation amino acid, methionine, and contains a consensus Sp1 binding site. The single membrane spanning region of Kell protein is encoded in exon 3 and the putative zinc endopeptidase active site is in exon 16. The amino acids encoded by the 19 exons are identical to those of a person with a common Kell phenotype, as determined by RNA polymerase chain reaction of peripheral blood. Amplification of cDNA 5' ends, derived from human fetal liver, indicated three transcription initiation sites located 30, 81, and 120 bp upstream of the initiation codon. The 5' flanking region of KEL from -176 does not contain a TATA sequence, but has possible GATA-1 binding sites and has significant promoter activity when determined by chloramphenicol acetyltransferase activity in K562 cells.

Amino Acid Sequence↗
PubMed1995

The Drosophila yolkless gene encodes a vitellogenin receptor belonging to the low density lipoprotein receptor superfamily.

Sequence comparisons of vitellogenins from a wide range of organisms have identified regions of similarity not only to each other but also to vertebrate apolipoproteins (e.g. apoB-100 and apoE). Furthermore, the chicken vitellogenin receptor, which also binds apolipoproteins receptor (LDLR) superfamily [Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T. & Schneider, W. J. (1994) EMBO J. 13, 5165-5175]. The yolk proteins of higher dipterans are exceptional, however, and instead show similarity to lipoprotein lipases. The molecular characterization of the putative Drosophila melanogaster vitellogenin receptor gene, yolkless (yl), described in this report reveals that the protein it encodes (Yl), is also a member of the LDLR superfamily. The ovary-specific 6.5-kb yl RNA codes for a protein of approximately 210 kDa which contains all three motifs common to the LDLR class of proteins. Within this superfamily, Yl may be related more to the LDLR-related proteins (LRPs), which bind both apolipoproteins and lipoprotein lipases. The similarity of Yl to the other LDLR proteins is restricted to the putative extracellular domain. Most noticeably, the cytoplasmic domain of Yl lacks the typical NPXY sequence which is involved in receptor internalization.

Amino Acid Sequence↗
PubMed1995

Molecular basis of the Kell (K1) phenotype.

K1 (K, Kell) is a strong immunogen; its antibodies can cause severe reactions if incompatible blood is transfused and may cause hemolytic disease of the newborn in sensitized mothers. K1 is a member of the Kell blood group system, which is complex, containing over 20 different antigens. Some of the antigens are organized in allelic pairs of high and low prevalence whereas others are independently expressed. K1, which is present in 9% of the population, is antithetical to the high-prevalence K2 (k) antigen. We have determined the molecular basis of the K1/K2 polymorphism by sequencing the 19 exons of the Kell gene (KEL) of a K1/K1 person. Polymerase chain reaction was performed on genomic DNA isolated from peripheral blood and the amplified products were either directly sequenced or subcloned and sequenced. Comparisons of K1/K1 and K2/K2 DNA showed a C to T base substitution in exon 6 that predicts a threonine to methionine change at amino acid residue 193. This amino acid substitution occurs at a consensus N-glycosylation site (Asn. X. Thr) and probably prevents N-glycosylation, leading to a change in phenotype. The C to T substitution creates a Bsm I restriction enzyme site, which was tested in 42 different samples to confirm that this base change identifies the K1/K1 genotype. This test differentiates genotypes, K1/K1, K2/K2, and the K1/K2 heterozygote and should prove useful in the prenatal diagnosis of K1-related hemolytic disease of the newborn.

Amino Acid Sequence↗
PubMed1995

Imaging with intermolecular multiple-quantum coherences in solution nuclear magnetic resonance.

A magnetic resonance imaging technique based on intermolecular multiple-quantum coherences in solution (the correlated spectroscopy revamped by asymmetric z gradient echo detection or CRAZED experiment) is described here. Correlations between spins in different molecules were detected by magnetic-field gradient pulses. In order for a correlation to yield an observable signal, the separation between the two spins must be within a narrow band that depends on the area of the gradient pulses. The separation can be tuned from less than 10 micrometers to more than 1 millimeter, a convenient range for many applications.

Acetone↗
PubMed1995

Characterization of the murine mitochondrial glycerol-3-phosphate acyltransferase promoter.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate, a committed step in triacylglycerol and phospholipid biosynthesis. We have previously reported the cDNA cloning and transcriptional regulation of the murine mitochondrial GPAT (mGPAT). We now report the cloning of the 5'-flanking region of the murine mitochondrial GPAT. The transcription start site was identified by primer extension and RNase protection assays. A TATA box-like motif (TTATTAT) was located between -34 and -29 and a reverse CCAAT box (ATTGG) was located between -78 and -74, relative to the transcription start site. To begin studying mechanisms underlying transcriptional regulation of the mGPAT gene, chimeric luciferase (LUC) plasmids containing serial deletions, from -1447 to -38, of the 5'-flanking region of the murine mGPAT gene were prepared and transfected into 3T3-L1 cells. The fusion construct -1447 GPAT.LUC showed high promoter activity and deletions to -1353, -747, -322, and -86 did not markedly change the promoter activity. With all constructs, luciferase activity was 2-fold higher when plasmids were transfected into 3T3-L1 adipocytes. However, deletion of sequences between -86 and -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes. Deletion of sequences between -55 and -38 did not alter promoter activity. DNase I footprint analysis revealed a protected region between -95 and -65 which included the putative CTF/NF1 binding site. Electrophoretic mobility shift assays demonstrated a single protein-DNA complex formation. Oligonucleotides synthesized according to the CTF/NF1 consensus sequence or the adenovirus NF-1 site showed a different and more complex pattern of protein-DNA interaction and were not able to compete away the mGPAT promoter-protein complex, indicating that a distinct protein was bound to -86/-55, a region important for the basal promoter activity in 3T3-L1 cells. Luciferase activity was increased 2.8- and 8-fold when adipocytes stably transfected with -322 GPAT.LUC were treated with 5 and 25 mM glucose, respectively, in the presence of 10 nM insulin. These results indicate that carbohydrate-responsive sequences are located within -322 base pairs of the mGPAT promoter.

3T3 Cells↗