Deletion mutagenesis of stem cell factor defines the C-terminal sequences essential for its biological activity.

We constructed a series of murine stem cell factor (mSCF) cDNAs which were sequentially truncated at the 3' termini. The resultant six mutant cDNA encode N-terminal 183, 179, 162, 149, 142 and 133 amino acid residues of the mature mSCF protein fused to the heterogeneous C-terminal peptides derived from the linker sequences. Each mutant cDNA was transiently expressed in COS cells, and the cultured supernatant was assayed for its ability to support the growth of a human factor-dependent cell line, TF-1 and to enhance colony formation by murine hematopoietic progenitor cells. The results showed that as few as N-terminal 142 but not 133 amino acid residues of mSCF remained biologically active in vitro, suggesting that the region of 9 amino acids from Asp134 to Ser142 containing a Cys138-mediated disulfide bond may contribute to the C-terminal end of the active subdomain of mSCF.     

Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase delta.

The cDNA of human DNA polymerase delta was cloned. The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa. Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb. Monoclonal and polyclonal antibodies to the C-terminal 20 residues specifically immunoblotted the human pol delta catalytic polypeptide. A multiple sequence alignment was constructed. This showed that human pol delta is closely related to yeast pol delta and the herpes virus DNA polymerases. The levels of pol delta message were found to be induced concomitantly with DNA pol delta activity and DNA synthesis in serum restimulated proliferating IMR90 cultured cells. The human pol delta gene was localized to chromosome 19 by Southern blotting of EcoRI digested DNA from a panel of rodent/human cell hybrids.     

cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.     

Molecular cloning of the cDNA of human X chromosomal gene (CCG1) which complements the temperature-sensitive G1 mutants, tsBN462 and ts13, of the BHK cell line.

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13 has a ts defect in G1 progression and belongs to the same complementation group as the ts13 cell line. We cloned human cDNA which can complement both tsBN462 and ts13 mutations, from the cDNA library of the secondary ts+ transformant (K-1-1) of tsBN462 cells using, as a probe, the isolated human X chromosomal genomic DNA. The cloned DNA is 5.3 kb long and has an open reading frame of 4662 bp, encoding a protein of 178,768 daltons. The putative protein is hydrophilic with a tandem repeat of 120 amino acids in the C-terminal region. An amino acid sequence (PPKKKRRV), similar to the consensus sequence for the nuclear translocation signal, is located immediately before the tandem repeat of amino acids.     

Sequencing of full-length cDNA encoding the alpha and beta subunits of human casein kinase II from human platelets and megakaryocytic cells. Expression of the casein kinase IIalpha intronless gene in a megakaryocytic cell line

Casein kinase II (CKII) is a ubiquitous protein kinase composed of two subunits, alpha and beta, that can use both ATP and GTP as phosphoryl donors. Two genes located on two separate chromosomes were identified for CKIIalpha: one on chromosome 20 band 13 with an approximate size of 20 kb and a second on chromosome 11 band 15.5-p15.4 that is the same size as the cDNA of locus 20 kb (1.2 kb) and does not contain any introns. The two genes differ in four amino acids. Recently, it has been demonstrated that a membrane-associated platelet-derived CKII phosphorylates coagulation factor Va. The mRNA encoding the platelet CKII was isolated from fresh human platelets, and the corresponding cDNAs encoding the alpha and beta subunits of human platelet CKII were produced and sequenced. The cDNA for platelet CKIIalpha was found to be 99.7% homologous to the CKIIalpha intronless gene, having the same characteristic amino acid residues at positions 128, 256, 287, and 351. However, the cDNA of platelet CKIIalpha has a different amino acid at position 236 (Arg --> His), which is not found in the intronless gene. The cDNA of the CKIIbeta subunit was completely identical with the sequence of the CKIIbeta subunit isolated from other tissues. Since platelets arise from megakaryocytes, mRNA was isolated from the megakaryocytic cell line MEG-01 and the cDNA for CKIIalpha was cloned and sequenced. The cDNA was found to be identical to the intronless gene found in platelets. We have also investigated the expression of the intronless gene in several other cell lines. Expression of the intronless gene was only found in cell line MEG-01. Our data demonstrate expression of the CKIIalpha intronless gene in megakaryocytes and platelets.     

Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA.

Human beta 2-glycoprotein (beta 2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature beta 2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the beta 2gpI cDNA. Upon isoelectric focusing, recombinant beta 2gpI obtained from expression of beta 2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as beta 2gpI isolated from plasma, and at least 5 polypeptides were visible.     

Structure-function studies on human macrophage colony-stimulating factor (M-CSF)

M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells. An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro. Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo. The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used. Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro. The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core. Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants. A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering. Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization. Mol Reprod Dev 46:31–38, 1997. © 1997 Wiley-Liss, Inc.     

Cloning and characterization of a single-stranded DNA binding protein that specifically recognizes deoxycytidine stretch.

We previously identified a G-rich silencer element involved in negative regulation of catalase gene expression in some hepatoma cells (Mol. Cell. Biol., (1992), 12, 2525-2533). To study a nuclear binding protein for this element, we screened cDNA libraries from a rat ascites hepatoma cell line by binding with a synthetic oligonucleotide probe and obtained several clones. One of them, designated SW, was studied in detail. A clone (SW2) of this series contained a near full length cDNA encoding a putative peptide with 463 amino acid residues. We isolated this peptide as a fusion protein. It was found that the protein strongly bound to the C-stretch of the DNA sequence in a single strand specific fashion, but absolutely did not to G-rich sequence. The protein bound weakly to the corresponding double-stranded DNA as well as to C-rich RNA sequence. This protein, though not the expected one, was found to be a novel protein whose DNA binding domain was located on the region containing at least 75 amino acid residues of the carboxyl terminus. A proline rich region was also observed in the middle part of the protein. Northern blot profiles indicated extensive and slight expression of both 2.0 kb and 2.7 kb mRNA species in some hepatoma cell lines and in the rat liver, respectively.     

C-terminal amino acid determination of the transmembrane subunits of the human platelet fibrinogen receptor, the GPIIb/IIIa complex

Glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the Ca2(+)-dependent GPIIb/IIIa complex, which acts as the fibrinogen receptor on activated platelets. GPIIb and GPIIIa are synthesized as single peptide chains. The GPIIb precursor is processed proteolytically to yield two disulphide-bonded chains, GPIIb alpha and GPIIb beta. The GPIIb/IIIa complex has two membrane attachment sites located at the C-termini of GPIIb beta and GPIIIa. The short cytoplasmic tails of GPIIb beta and/or GPIIIa become most likely associated to the cytoskeleton of activated platelets. In the present work the C-terminal amino acid residues of platelet GPIIb beta and GPIIIa have been analyzed by protein-chemical methods and compared with those predicted from cDNA analysis. We were able to confirm the positions of the C-termini in both glycoproteins and the identity of the C-terminus predicted for GPIIIa, i.e. threonine. However, glutamine, not glutamic acid as predicted for GPIIb beta from the human erythroleukemic cell line and megakaryocyte cells, was found to be the C-terminal amino acid of GPIIb beta. This indicates that the glutamic acid in the GPIIb precursor is posttranslationally modified to glutamine.     

Multiple roles of M-CSF in human osteoclastogenesis

Although the critical role of M-CSF in osteoclastogenesis is well documented, there has been no detailed analysis of how it regulates human osteoclast formation and function in vitro. We used a human osteoclastogenesis model employing CFU-GM osteoclast precursors cultured for 14 days on dentine with RANKL, with varying exposure to exogenous human M-CSF. Short-term treatment of precursors with M-CSF (10-100 ng/mL) resulted in increased proliferation with or without RANKL. Treatment with M-CSF (1-100 ng/mL) for 14 days caused a biphasic concentration-dependent stimulation of formation, fusion, and resorption peaking at 10-50 ng/mL and almost complete abolition of resorption at 100 ng/mL. Time-course studies using M-CSF (25 ng/mL) showed that osteoclast size, nuclei/cell, and resorption increased with longer duration of M-CSF treatment. When treatment was restricted to the first 4 days, M-CSF (25-100 ng/mL) stimulated formation of normal numbers of osteoclasts that resorbed less. Blockade of endogenous M-CSF signaling with neutralizing M-CSF antibody during the first week of culture extensively inhibited osteoclastogenesis, whereas blockade during the second week produced only a small reduction in resorption. Treatment with M-CSF during the second week of culture caused a small increase in osteoclast number and a concentration-dependent increase in cytoplasmic spreading with inhibition of resorption. We have shown that M-CSF modulates multiple steps of human osteoclastogenesis, including proliferation, differentiation and fusion of precursors. In the later stages of osteoclastogenesis, M-CSF modulates osteoclast-resorbing activity, but is not required for survival. Modulation of M-CSF signaling is a potential therapeutic target for conditions associated with excess bone resorption.     

Isolation of human cDNA clones of myb-related genes, A-myb and B-myb.

cDNA clones of the myb-related genes A-myb and B-myb were obtained by screening human cDNA libraries. The predicted open reading frame of B-myb could encode a protein of 700 amino acid residues. Although the C-terminal end has not been cloned yet, an almost entire coding region of A-myb, which is 745 amino acid long, was determined. The A-myb and B-myb proteins are highly homologous with the myb protein in three regions. Domain I, which is 161 amino acid long, is well conserved in the myb gene family. The homology between human-myb and A-myb in domain I is 90% at the amino acid level. Domain II, which is about 85 amino acid long, is less well conserved. Although it is a short stretch, domain III is found in the C-terminal region. The mRNAs of A-myb and B-myb were 5.0 and 2.6 kb, respectively. The mRNA expression pattern of the myb gene family in various tumors is presented.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells.

Three cDNA clones of rat renal dipeptidase (rrDP) were isolated from rat renal and pulmonary cDNA libraries using a DNA fragment of human renal DP cDNA clone, MDP4, as a probe. The complete amino acid sequence deduced from the cDNA contains 410 amino acid residues, beginning with a signal peptide of 16 amino acid residues. RNA blot hybridization analysis showed that 1.6 and 2.2 kb mRNAs were expressed in lung and kidney, however, only 1.6 kb mRNA was detected in small intestine. COS-1 cells transfected with the cDNA expressed enzymatically active rrDP.     

Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.     

Structure of human milk bile salt activated lipase

The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda gt11 and lambda gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.