Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene.

Regulators of G-protein signaling (RGS) constitute a family of GTPase-activating proteins with varying tissue-specific expression patterns and G-protein alpha subunit specificities. Here, we describe the molecular cloning of the human RGS-r/RGS16 cDNA, encoding a predicted polypeptide of 23kDa that shows 86% identity to mouse RGS-r. Northern blot analysis shows that, like the mouse Rgs-r message, hRGS-r mRNA is abundantly expressed in retina, with lower levels of expression in most other tissues examined. Characterization of the genomic organization of the hRGS-r gene shows that it consists of five exons and four introns. We have also mapped the human RGS-r /RGS16 gene to chromosome 1q25-1q31 by fluorescence in situ hybridzation. Analysis of human ESTs reveals that at least five members of the RGS gene family map to chromosome 1q, suggesting that at least part of the RGS family arose through gene duplication. The chromosomal location, retinal abundance, and presumed function of the human RGS-r protein in desensitizing photoreceptor signaling make the RGS-r/RGS16 locus a candidate for mutations responsible for retinitis pigmentosa with para-arteriolar preservation of retinal pigment epithelium (RP-PPRE or RP12), an autosomal recessive disorder previously mapped to 1q31.

Molecular cloning and expression analysis of rat Rgs12 and Rgs14.

We report the cloning of two novel rat regulators of G-protein signaling (RGS) cDNAs using a degenerate PCR strategy. The rRgs12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. We have also identified the human orthologue of rRgs12 by alignment of cosmid sequences in the database which map the human RGS12 gene to chromosome 4p16.3. Furthermore, we identified human ESTs with high homology to rRgs14 which map to human chromosome 5qter. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRgs12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. We have also identified conserved regions in RGS12 which are similar to protein domains found in mouse rhophilin and coiled-coil proteins suggesting possible interactions with ras-like G-proteins.

Cloning of human lymphocyte-specific interferon regulatory factor (hLSIRF/hIRF4) and mapping of the gene to 6p23-p25.

The interferon regulatory factor (IRF) genes encode a family of transcription factors involved in the transcriptional regulation of interferon and the interferon stimulated genes through recognition of the interferon stimulated response element. We previously reported the cloning of a murine lymphocyte-specific IRF (mLSIRF), which was rapidly induced following B- or T-cell receptor crosslinking. To study the role of LSIRF in human lymphocyte development, we have cloned the complete 5.3-kb cDNA for the human homolog (hLSIRF). hLSIRF is a protein of 450 amino acids with a predicted molecular weight of 51.6 kDa and possesses 92% identity at the amino acid level to mLSIRF, including near identity in the DNA-binding domain. In Northern blot analysis, a single transcript of approximately 5 kb was highly expressed in spleen and peripheral blood lymphocyte. hLSIRF mRNA was rapidly induced in peripheral T cells after crosslinking the T-cell receptor. Analysis of tumor cell lines showed that hLSIRF mRNA was basally expressed in most B- but not T-cell lines. Surprisingly hLSIRF mRNA was also found in the melanoma line G361 and is expressed in normal melanocytes as well. Sequence from a genomic clone for hLSIRF was compared to that from mouse and revealed an identical exon-intron structure and a conserved PU-1-binding motif in the promoter. By FISH analysis, hLSIRF was mapped to 6p23-p25.

Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene.

The megakaryocyte growth and development factor (MGDF) is a cytokine that regulates megakaryocyte development and is a ligand for the MPL receptor. In this study, we describe the genomic structure of the human MGDF gene. The MGDF gene was found to consist of seven exons and six introns spanning 8 kilobases. The protein is encoded by exons 3 through 7. The human MGDF gene has been mapped to chromosome 3q26.3. In addition to the previously described full-length cDNA, two cDNA variants were isolated from human fetal liver. Comparison of these two cDNA sequences with the genomic sequence indicates that they arise by differential splicing.

Structural and functional aspects of the multiplicity of Neu differentiation factors.

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor.

A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF) has been isolated from a human peripheral blood lymphocyte cDNA library. The expression of this gene is induced by pokeweed mitogen and superinduced by cycloheximide. It is also induced in the T-lymphoblastoid cell line HUT 78 after phorbol ester (phorbol myristate acetate) treatment. The predominant mRNA for PBEF is approximately 2.4 kb long and codes for a 52-kDa secreted protein. The 3' untranslated region of the mRNA has multiple TATT motifs, usually found in cytokine and oncogene messages. The PBEF gene is mainly transcribed in human bone marrow, liver tissue, and muscle. We have expressed PBEF in COS 7 and PA317 cells and have tested the biological activities of the conditioned medium as well as the antibody-purified protein in different in vitro assays. PBEF itself had no activity but synergized the pre-B-cell colony formation activity of stem cell factor and interleukin 7. In the presence of PBEF, the number of pre-B-cell colonies was increased by at least 70% above the amount stimulated by stem cell factor plus interleukin 7. No effect of PBEF was found with cells of myeloid or erythroid lineages. These data define PBEF as a novel cytokine which acts on early B-lineage precursor cells.

Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4.

The multicomponent enzyme, naphthalene dioxygenase, initiates the metabolism of naphthalene by Pseudomonas putida strains G7 (PpG7) and NCIB 9816-4 (Pp9816-4). The genes involved (nahAaAbAcAd) are encoded by the NAH7 and pDTG1 plasmids, respectively, and form part of the nah operon. The locations of the structural genes were determined on previously cloned fragments of DNA. The nucleotide (nt) sequences were determined for nahAaAb from Pp9816-4 and for nahAaAbAcAd from PpG7. The appropriate open reading frames were identified using N-terminal amino acid sequences determined from the purified proteins. The two nt sequences showed 93% homology, with the least homology seen upstream from the promoter region.

Effects of stem cell factor on osteoclast-like cell formation in long-term human marrow cultures.

Stem cell factor (SCF) is a newly described hematopoietic growth factor that stimulates the growth of primitive hematopoietic progenitors and mast cells. Since the osteoclast precursor is hematopoietic in origin, we tested SCF for its capacity to stimulate the formation of osteoclast-like multinucleated cells (MNC) in long-term human marrow cultures. These MNC express an osteoclast phenotype and form resorption lacunae on calcified matrices. Addition of SCF alone (0.1 pg/ml to 100 ng/ml) to long-term marrow cultures did not increase MNC formation. However, treatment of these cultures sequentially with SCF for 1 week followed by 1,25-(OH)2D3 for the second and third weeks of culture significantly enhanced MNC formation. [3H]Thymidine incorporation studies showed that SCF increased the proliferation of MNC precursors. These data suggested that SCF was acting on early MNC precursors. We then tested the capacity of SCF to stimulate the formation of colonies of committed precursors for osteoclast-like MNC. SCF (20 pg/ml to 20 ng/ml) enhanced osteoclast precursor formation in unfractionated bone marrow mononuclear cells but was unable to increase osteoclast precursor formation when a highly purified population of hematopoietic precursors was used as the target cells for SCF. These data suggest that SCF works in concert with other factors produced by nonhematopoietic marrow cells to increase the precursor pool for osteoclasts and that other factors, such as 1,25-(OH)2D3, complete the differentiation process to the mature osteoclast.

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.

Vasculitic neuropathy mimicking Guillain-Barré syndrome.

Vasculitic neuropathy classically manifests as a subacute mononeuritis multiplex or polyneuropathy. Patients with a Guillain-Barré syndrome (GBS)-like presentation are usually not considered to have an underlying vasculitis. We describe 2 patients with rapidly progressive areflexic paralysis that was initially diagnosed as GBS, in whom vasculitic neuropathy was found histologically.

Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor.

The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report, we focus on the human counterpart of the mouse Steel (Sl) gene. Alternative splicing of the human SCF pre-mRNA transcript results in secreted and membrane-bound forms of the protein. To investigate the role of these two forms of human SCF, we targeted an immortalized stromal cell line derived from fetal murine homozygous (Sl/Sl) SCF-deficient embryos for gene transfer of various human cDNAs encoding SCF. We report that stable stromal cell transfectants can differentially process the two forms of human SCF protein product. We also demonstrate that both soluble SCF and membrane-bound SCF are active in increasing the number of human progenitor cells in the context of stromal cell cultures, although in a qualitatively different manner. Hence, the membrane-bound form of SCF may play an important role in the cell-cell interactions observed between stromal and hematopoietic cells both in vitro and in vivo.

Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit.

We recently reported that a 44 kd glycoprotein secreted by transformed fibroblasts stimulates tyrosine phosphorylation of the product of the neu proto-oncogene and induces differentiation of mammary tumor cells to milk-producing, growth-arrested cells. A partial amino acid sequence of the protein, termed Neu differentiation factor (NDF), enabled cloning of the corresponding complementary DNA. The deduced structure of the precursor of NDF indicated that it is a transmembrane protein whose extracellular portion contains an EGF-like domain that probably functions as a receptor recognition site. In addition, the ectodomain contains one immunoglobulin homology unit. Despite the lack of a recognizable hydrophobic signal peptide at the N-terminus, a recombinant NDF, like the natural molecule, is released into the medium of transfected COS-7 cells in a biologically active form. Northern blot analysis indicated the existence of several NDF transcripts, the major ones being 1.8, 2.6, and 6.7 kb in size. Transformation by the ras oncogene dramatically elevated the expression of NDF in fibroblasts.

Lateralization of epileptic foci by magnetic resonance imaging in temporal lobe epilepsy.

A retrospective single-blind study was carried out to assess the reliability of magnetic resonance imaging (MRI) for determining lateralization of the electrographic focus in 45 patients with intractable temporal lobe epilepsy. With strictly defined MRI diagnostic criteria, the electroencephalographic (EEG) focus was correctly lateralized in 86% of patients. Excluding patients with structural lesions, the criteria provided for correct lateralization of the epileptogenic focus in 78% and false lateralization in 5%. Hippocampal atrophy on T1-weighted images and increased signal intensity from mesial structures on T2-weighted scans were highly reliable for lateralization. Postoperative outcome did not differ between the patients with normal and those with abnormal findings on MRI, but the group sample was inadequate to assess the issue of surgical outcome. These findings suggest that with appropriate techniques and strictly defined diagnostic criteria, MRI can provide reliable seizure lateralization in patients with intractable temporal lobe epilepsy.

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

Secretion of glycosylated human erythropoietin from yeast directed by the alpha-factor leader region.

The pre-pro alpha-factor leader region of the yeast MF alpha 1 gene was used to direct the secretion of the human glycoprotein, erythropoietin (EPO), into the culture medium. An examination of the role of expression level on secretion of biologically active EPO indicated that there are several rate-limiting steps. These include processing of the alpha-factor-EPO precursor protein by the KEX2-encoded endoproteinase and transport of the protein through the secretory pathway. The rate-limiting steps for transport were early in the secretory pathway, probably from the endoplasmic reticulum to the Golgi apparatus.

Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region.

The Saccharomyces cerevisiae secretory process was studied by evaluating secretion efficiency, processing efficiency, and the efficiency of protein folding for hybrid proteins containing the yeast prepro-alpha-factor leader region. Secretion of three proteins, beta-endorphin, calcitonin, and a consensus alpha-interferon (IFN-Con1), were compared in terms of secretion efficiency into the culture medium, beta-Endorphin and calcitonin, both small proteins, were found to be efficiently secreted from logarithmically grown cells. In contrast, the larger IFN-Con1 accumulated in the periplasmic space and cell wall. The glycosylated, unprocessed prepro-alpha-factor/IFN-Con1 fusion protein was also found to be secreted into the culture medium. The presence of (Glu-Ala) dipeptides in the alpha-factor spacer peptide increased the efficiency of cleavage at Lys-Arg in the prepro-alpha-factor/IFN-Con1 protein fusion. Purified secreted IFN-Con1 was structurally characterized to determine the effect of passage through the yeast secretory pathway on the fidelity and efficiency of protein folding. The disulfide structure of the secreted protein was found to be identical with that reported for the native human alpha-interferons.

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene.

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.