Biomedical subjects

Y M Bae

Publications and source records attributed to Y M Bae.

24 records · Page 2Linked to original sources

PubMed1991

A monoclonal antibody to human leukocyte common antigen, SHL-1, and its use for formalin-fixed, paraffin-embedded tissues.

The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.

Antibodies, Monoclonal↗

PubMed1991

Genetic analysis of the attenuator of the Rhizobium meliloti trpE(G) gene.

It was previously reported that transcription of the Rhizobium meliloti trpE(G) gene starts at the adenine residue of the AUG codon of the leader peptide coding sequence (trpL), suggesting that translation of the trpL sequence starts without the Shine-Dalgarno sequence. We constructed mutations replacing the AUG codon of the trpL sequence with AAG or ACG. These mutations reduced the expression of a trpL'-'lacZ fusion gene to 0.1 and 0.2% of the wild-type level, respectively, indicating that the AUG codon is the translation initiation codon for the trpL coding sequence. In addition, these mutations, as well as a mutation converting the eighth codon (UCG) of the trpL sequence to UGA, abolished regulation by attenuation when introduced upstream of the tandem tryptophan codons in a trpE'-'lacZ fusion. Mutations affecting the stability of the probable antiterminator and terminator secondary structures in trpL mRNA were also constructed. Studies using these mutations indicate that the attenuator of R. meliloti functions in a way analogous to that of the Escherichia coli trp attenuator.

Base Sequence↗

PubMed1991

Mutations that affect activity of the Rhizobium meliloti trpE(G) promoter in Rhizobium meliloti and Escherichia coli.

The cloned Rhizobium meliloti trpE(G) gene is not expressed in Escherichia coli. Oligonucleotide-directed mutagenesis was used to introduce base substitution mutations in the promoter region of this gene. Three separate mutations that increased homology of the putative -10 region of this promoter with the E. coli -10 promoter consensus sequence by 1 bp converted this promoter to an active promoter in E. coli. A deletion extending to position -43 from the 5' side had a minor effect on transcription in R. meliloti. However, transcription was nearly eliminated when a deletion extended to position -33, indicating that the crucial domain of the R. meliloti trpE(G) promoter begins in the region downstream of position -43. The R. meliloti trpE(G) promoter has two regions that show homology with the E. coli -35 and -10 promoter consensus sequences. Mutations in these putative -35 and -10 regions, but not in the spacer region, affected promoter strength in R. meliloti. By comparing four known R. meliloti promoter sequences, we identified a highly conserved trimer near position -35 (5'-TTG-3') but no noticeably conserved sequence near position -10.

Bacterial Proteins↗

PubMed1991

Requirements for in vitro growth of human thymocytes.

Since it is difficult to study human thymocyte maturation in vitro, we have developed an in vitro thymocyte culture system which has allowed us to select the optimal growth conditions for thymocyte subpopulations. Three thymocyte subpopulations (CD3-CD1-, CD1+CD3-, and CD3+CD1-) were isolated by a single step percoll density gradient centrifugation and indirect panning procedure using anti-CD1 and anti-CD3 monoclonal antibodies, and their purity was checked by flow cytometry. The combination of concanavalin A (Con A), tetradecanoylphorbol acetate (TPA), and IL-2 was shown to be the most reliable stimulus for the proliferation of CD3-CD1- thymocytes for up to 15 days in a culture system in vitro. Flow cytometric analysis for the phenotypic change of CD3-CD1- thymocytes revealed a steady increase of CD3 antigen after a 3-day cultivation, whereas there was no change in CD1 antigen intensity. A combination of Con A and IL-2 was both sufficient and necessary to induce growth of CD3+CD1- thymocytes. The major population of immature cortical thymocytes (CD3-CD1+ or CD3+CD1+), which are considered to be the most unresponsive dead-end cells, could not be maintained or stimulated with any combination used in this experiment, even in the presence of thymic accessory cells.

Antigens, CD↗

PubMed1990

The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition.

In Rhizobium meliloti, the genes involved in biosynthesis of the amino acid tryptophan are found at three separate chromosomal locations. Of the three gene clusters, trpE(G), trpDC, and trpFBA, only the trpE(G) gene is regulated by the end product of the pathway, tryptophan. We found that trpE(G) mRNA contains a leader transcript that terminates at a stem-loop structure in a putative transcription attenuator. The level of this leader transcript was constant regardless of the amount of tryptophan in the growth medium. However, the level of full-length trpE(G) mRNA decreased as the amount of tryptophan increased. The beta-galactosidase activity of an R. meliloti strain carrying a trpL'-'lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE(G)'-'lacZ fusion decreased as the tryptophan concentration increased. These data indicate that transcription of the R. meliloti trpE(G) gene is regulated only by attenuation. We also found that the product of the trpE(G) gene, anthranilate synthase, is feedback inhibited by tryptophan.

Anthranilate Synthase↗

PubMed1989

Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli.

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site.

Amino Acid Sequence↗