Cells from the human leukemia cell line HL-60 undergo terminal differentiation when exposed to inducing agents. Differentiation of these cells is always accompanied by withdrawal from the cell cycle. Here we describe the isolation of a cDNA encoding a novel serine protease that is present in HL-60 cells and is down-regulated during induced differentiation of these cells. We have named this protease myeloblastin. Down-regulation of myeloblastin mRNA occurs with both monocytic and granulocytic inducers. Myeloblastin mRNA is undetectable in fully differentiated HL-60 cells as well as in human peripheral blood monocytes. We found that regulation of myeloblastin mRNA in HL-60 cells is serum dependent. Inhibition of myeloblastin expression by an antisense oligodeoxynucleotide inhibits proliferation and induces differentiation of promyelocyte-like leukemia cells.

The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5' of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic beta-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.

SummaryTwo glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3,4-isomerase.

Cells from the human leukemia cell line HL-60 undergo terminal monocyte-like differentiation after exposure to either the active circulating form of vitamin D3, 1, is not confined to the target tissues of intestine and bone, suggesting that the role of this hormone extends beyond its classical role in mineral metabolism (1)(2)(3). A number of human leukemia cells, such as HL-60, a promyelocyte-like cell line (4-6), and U937, a histiocytic monoblast-like lymphoma cell line (7-9), can be induced to differentiate into cells with monocyte-macrophage characteristics when treated with 1,25-(OH)2D3. Murao et al. (4) have suggested that 1,25-(OH)2D3 is an important native compound in inducing monocyte-macrophage differentiation. Little is known about the genes that are regulated by 1,25-(OH)2D3 during monocytic-macrophagic differentiation. We have recently derived and analyzed a clonal variant of HL-60, iOlO, that permits us to study several intermediate steps in monocytic differentiation (10). In the iOlO model system, cells were made partially resistant to differentiation by phorbol 12-myristate 13-acetate (PMA) or 1,25-(OH)2D3 such that when used singly they are arrested at discrete steps in differentiation.Complete maturation of iOlO cells is attained when PMA and 1,25-(OH)2D3 are used simultaneously. Furthermore, when the two inducers are added sequentially, cells must be treated first with 1,25-(OH)2D3 to fully differentiate (10).To identify genes that are induced by 1,25-(OH)2D3 at a discrete step in HL-60 differentiation, cDNA subtraction was applied to our iOlO system. We present here the molecular cloning and characterization of a cDNA, pD3-137, corresponding to a gene that is preferentially induced (13,14). The double-stranded cDNA was digested with S1 nuclease (Boehringer Mannheim), then 3' dC-tailed and annealed with a Pst I-cut 3' dG-tailed pUC9 vector previously purified by using an oligo(dC)-cellulose column to remove nontailed molecules (13,14). Fifteen microliters of annealing mixture (5 ng vector) was taken for transforming 100 ,ul of competent RRI bacteria Abbreviations: PMA, phorbol 12-myristate 13-acetate; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PBM, peripheral blood monocytes; FBPase, fructose 1,6-bisphosphatase. tPresent address: Columbia University College of Physicians and Surgeons,

The molecular mechanism by which interleukin 6 (IL-6) induces terminal differentiation of B cells was investigated in a subpopulation of the clonal human Blymphoblastoid cell line CESS selected for high density of cell surface IgGi. Induction of CESS cells with IL-6 resulted in a 15-fold preferential accumulation of secreted-specific yV (YVs) mRNA but not of the alternatively processed membranespecific Vi (ylm) mRNA. Similarly, p, mRNA but not the Sum mRNA of the nonproductively rearranged I% heavy-chain allele was also increased. Accompanying the differential accumulation of yVs mRNA was a 4.5-fold increase in A light-chain mRNA, leading to secretion of IgGi. Analyses of transcription in isolated nuclei demonstrated that transcriptional activation was the primary mechanism for quantitative increase of immunoglobulin mRNAs (5.5-fold for yV and jA and at least 2-fold for A). Since polymerase loading is diminished by 75% before reaching the downstream ylm polyadenylylation site in CESS cells, irrespective of IL-6 induction, transcriptional pausing/ termination appears intrinsic and contributes to the selection of Vys and ylm polyadenylylation sites in activated B cells.