Isolation and characterization of a cDNA that encodes the peptide core of the secretory granule proteoglycan of human promyelocytic leukemia HL-60 cells

A cDNA that encodes the peptide core of the secretory granule proteoglycan of the human promyelocytic leukemic cell line, HL-60, has been isolated and analyzed. When human genomic DNA was digested and probed under conditions of low stringency with a rat cDNA that encodes a Mr = 18,600 serine/glycine-rich proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1321-1325) and basophilic leukemia-1 cells (Avraham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chem. 263, 7292-7296), a number of DNA fragments were identified. A HL-60 cell-derived cDNA library was therefore screened under conditions of low stringency with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. Analysis of the resulting human cDNA clones indicated that the proteoglycan peptide core that is expressed in HL-60 cells is Mr = 17,600 and contains an 18-amino acid glycosaminoglycan attachment region that consists primarily of alternating serin and glycine. Northern blot analysis of total RNA probed with the human cDNA revealed that the major message for this proteoglycan peptide core in HL-60 cells is approximately 1.3 kilobase pairs in size. When a Southern blot of digested human genomic DNA was probed with the human cDNA, three bands of approximately 6, 9, and 12 kilobase pairs were detected. However, when the Southern blot was probed with the XmnI----3' fragment of this human cDNA, one prominent band was detected, indicating that a single gene encodes this protein in the human. Analysis of the DNA from human/mouse and human/hamster somatic cell hybrids probed with the human cDNA demonstrated that the gene that encodes this molecule resides on human chromosome 10. Because the proteoglycans that are present in the secretory granules of different types of rat and mouse mast cells possess small peptide cores that are rich in serine and glycine, we propose that this HL-60 cell-3 derived cDNA encodes the peptide core of the proteoglycan that is expressed in the secretory granules of this human promyelocytic cell.     

Characterization of two distinct positive cis-acting elements in the mouse alpha 1 (III) collagen promoter

We have identified two distinct sequence elements in the mouse alpha 1(III) collagen promoter which are protected from DNase I digestion by the binding of factors present in crude nuclear extracts of NIH 3T3 fibroblasts. Small substitution mutations were introduced into these promoter elements and shown by the gel retardation (gel mobility shift) and DNase I protection assays to decrease or eliminate factor binding to the mutated element but not to the remaining wild-type element, indicating that two distinct factors recognize these separate promoter regions. Region A appears to bind a factor related to the Jun/AP-1 protein, whereas the factor binding to region B remains as yet unidentified. Mutagenesis of either region decreased the activity of the alpha 1(III) collagen promoter in DNA transfection assays by about 3-fold for the A region (located between - 122 and - 106) and about 5-fold for the B region (located between -83 and -61). These results indicate that regions A and B in the mouse alpha 1(III) collagen promoter are positive cis-regulatory elements, independently binding two distinct trans-activating factors.     

Isolation of a cDNA that encodes the peptide core of the secretory granule proteoglycan of rat basophilic leukemia-1 cells and assessment of its homology to the human analogue

It has been previously shown that a single gene is used to encode the peptide core of the extracellular proteoglycan of rat L2 yolk sac tumor cells and the intracellular proteoglycan of rat basophilic leukemia (RBL)-1 cells. In order to determine if the predicted amino acid sequences of these proteoglycans are identical as well as to isolate a full length cDNA encoding a rat secretory granule proteoglycan, a cDNA library was prepared from RBL-1 cells and screened with the 165-base pair 5'----XmnI fragment of pPG-1, a partial cDNA which encodes the rat L2 cell proteoglycan peptide core. Based on the consensus nucleotide sequence of two full length RBL-1 cell-derived cDNAs, the 5' untranslated region of the mRNA that is expressed in RBL-1 cells is shorter than that expressed in the rat L2 cells although the coding regions of the mRNAs from the two cell types are identical. These findings indicate that the targeting of proteoglycans to an intracellular or extracellular compartment is a cell-specific event which is independent of the translated peptide core. Since the RBL-1 cell and the rat L2 cell proteoglycans have different types of glycosaminoglycans bound to them, it can also be concluded that the selection of the type of glycosaminoglycan that will be synthesized onto a peptide core is a cell-specific event which is not exclusively dependent on the translated peptide core. When the predicted amino acid sequence of the RBL-1 cell proteoglycan peptide core was compared to the predicted sequence of the homologous human molecule from HL-60 cells, 48% of the amino acids were identical. The N terminus was the most highly conserved area of the molecule. This region of the peptide core, which precedes the serine-glycine repeat region, is likely to be of critical importance for the biosynthesis and/or function of these proteoglycans. Analysis of 10 different mouse/hamster somatic cell hybrid lines with a SspI----3' fragment of the rat L2 cell cDNA revealed that, as in the human, the gene that encodes the mouse analogue of this peptide core resides on chromosome 10.     

Analysis of cis-acting elements present in the CD20/B1 antigen promoter.

A genomic clone spanning a large portion of the 5' untranscribed region of the CD20 gene was isolated. Deletion analysis of subcloned fragments identified several regulatory elements. A major positive cis-acting element was localized between base pairs -290/-186. A second positive regulatory element was localized between -454/-280 and negative regulatory elements were present in the region between bp -828/-454. The sequence -280/-186 conferred B cell-specific expression on a heterologous, TATA box containing c-fos promoter. Electrophoretic mobility shift assays with overlapping oligonucleotide probes spanning -280/-186 revealed that a 25-bp probe (-225/-201) bound a nuclear protein present in B cell lines expressing the CD20/B1 antigen but not in Jurkat (T cell), U937 (promonocytic), U251 (glioma), or HeLa cells. To confirm the functional significance of this sequence, a trimer of this region was subcloned into the c-fos promoter containing CAT plasmid. Expression was observed only in BJA-B and HS-Sultan cells but not in CD20/B1- cell lines. This sequence element is also important in phorbol ester-induced CD20 expression in the pre-B cell line BP-697. These results partially characterize several regulatory elements present in the CD20 promoter that are likely important in the B cell-specific expression of the CD20 gene.     

Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S locus glycoprotein gene promoter.

The promoter of the S Locus Glycoprotein (SLG) gene of Brassica is a tightly regulated promoter that is active specifically in reproductive organs. In transgenic tobacco, this promoter is active exclusively in cells of the pistil and in pollen. We transformed tobacco with truncated versions of the SLG13 promoter fused to the beta-glucuronidase reporter gene. We show that the promoter has a modular organization and consists of separable DNA elements that independently specify pistil- and pollen-specific expression. A 196-bp region (-339 to -143) is sufficient to confer stigma and style specificity to the marker gene. Two distinct, but functionally redundant, domains (-415 to -291 and -117 to -8) allow specific expression of the gene in pollen. The functional domains identified within the SLG13 promoter contain sequence elements that are highly conserved in different alleles of the SLG gene and in the S Locus Related SLR1 gene.