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Clinical Competence↗
Biomedical subjects
S Suggs
Publications and source records attributed to S Suggs.
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Electroencephalography↗
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.
Animals↗
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.
Amino Acid Sequence↗
Cloning and expression of the human erythropoietin gene.
The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide probes. The entire coding region of the gene is contained in a 5.4-kilobase HindIII-BamHI fragment. The gene contains four intervening sequences (1562 base pairs) and five exons (582 base pairs). It encodes a 27-amino acid signal peptide and a 166-amino acid mature protein with a calculated Mr of 18,399. The erythropoietin gene, when introduced into Chinese hamster ovary cells, produces erythropoietin that is biologically active in vitro and in vivo.
Amino Acid Sequence↗
Replication of bacteriophage M13 IX. Requirement of the Escherichia coli dnaG function for M13 duplex DNA replication.
Temperature-shift experiments with an Escherichia coli dnaG strain indicate a requirement for the dnaG function for M13 phage production only at an early stage of infection. Mutant cells infected at nonpermissive temperature form the parental RF (SS leads to RF) but do not replicate further. A shift to nonpermissive temperature after infection inhibits RF leads to RF replication but not RF leads to SS synthesis. The synthesis of both strands of the duplex RF was inhibited equally after a temperature shift during RF leads to RF replication. We infer that the dnaG protein is required for M13 production only during RF replication and that it is required for the synthesis of both strands of the RF.
Centrifugation, Density Gradient↗
Stability of cytosine-containing deoxyribonucleic acid after infection by certain T4 rII-D deletion mutants.
When T-even phage infect Escherichia coli, synthesis of host deoxyribonucleic acid (DNA) rapidly ceases. If the phage carry a mutation in a gene essential for phage DNA synthesis, then the infected bacteria should make no DNA, either host DNA or phage DNA. However, we have found that infection with certain T4 gene 56 (deoxycytidine triphosphatase)-rII double mutants leads to substantial DNA synthesis. Only rII deletion mutations which extend into the middle third of the adjacent, nonessential D region lead to the anomalous DNA synthesis, when combined with a gene 56 mutation; the requirement probably is that the deletion extend into the D2a transcriptional unit identified by Sederoff et al. Genetic evidence indicates that the observed anomalous DNA synthesis is synthesis of phage DNA. We suggest that the D2a region controls, directly or indirectly, a nuclease involved in the breakdown of cytosine-containing DNA. In the absence of the D2a product, the cytosine-containing phage DNA made by the gene 56 mutant is stabilized.
Cell Survival↗