Depression in gene expression for poly(ADP-ribose) synthetase during the interferon-gamma-induced activation process of murine macrophage tumor cells

A 2.7-kb cDNA clone coding for bovine poly(ADP-ribose) synthetase was isolated from a lambda gt11 expression library by direct immunological screening with an antiserum to the enzyme. The cDNA hybridizes to an approximately 3.8-kb bovine thymus polyadenylated RNA, which translates an immunoprecipitable 120-kDa protein with the antibody to the enzyme. The partial DNA sequence of the cDNA was determined and portions of the predicted amino acid sequence matched the sequence of 26 amino acids at the N terminal of the 41-kDa alpha-chymotryptic fragment and two cyanogen-bromide-cleaved peptides of the enzyme. A subcloned fragment from the coding region of the cDNA was used as a probe to estimate the level of mRNA for the enzyme during the interferon-gamma-induced activation process of the murine macrophage tumor P388D1 cell line. The amount of mRNA for the enzyme decreased nearly completely within 24 h after incubation in a medium containing interferon-gamma, while mRNA of the Ia antigen, one of the major histocompatibility gene products, was increased in the macrophage tumor cells by interferon-gamma as confirmed by the I-A beta cDNA as a probe. These results suggest that the gene expression for poly(ADP-ribose) synthetase is depressed during the interferon-gamma-induced activation process of macrophage tumor cells.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.     

Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.     

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.     

Differential expression of the human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes

A cDNA clone encoding human thymosin-beta 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-beta 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. Nine of 132 nucleotides were different in the coding sequences (93% homology), but the deduced amino acid sequences were identical. No signal peptide was found in the deduced protein sequence. Human thymosin-beta 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-beta 4 mRNA. Expression of the human thymosin-beta 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-beta 4 mRNA varied as a function of differentiation stage. Thymosin-beta 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-beta 4 and the immune response. Thymosin-beta 4 mRNA was more highly expressed in mature granulocytes than in immature blastic cells. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-lambda reduced the levels of thymosin-beta 4 mRNA. Its level decreased after differentiation of THP-1 cells into Ia+ macrophages, but increased after differentiation of HL-60 cells into Ia- macrophages. The pattern of thymosin-beta 4 gene expression suggests that it may play a fundamental role in the host defense mechanism.     

Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues.

Using the differential hybridization screening method between osteoblastic and fibroblastic cells, a cDNA clone coding for an osteoblast specific protein, named OSF-1, consisting of 168 amino acid residues including a possible 32 amino acid long leader sequence, was isolated from murine osteoblastic cell line MC3T3-E1. The OSF-1 gene was shown by Northern blotting analysis to be expressed in mouse calvarial osteoblast-enriched cells and in mouse brain tissues, but not in thymus, spleen, kidney, liver, lung, testis or heart. The human counterpart was also found in cDNA libraries from human osteosarcoma cell line MG63 and normal brain tissues. DNA sequence analysis revealed four amino acid sequence differences between the mouse and human, of which only one is located in the mature protein. This extremely high sequence conservation suggests that OSF-1 plays a fundamental role in bone and brain functions.     

Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Filaggrin, an intermediate filament-associated protein: structural and functional implications from the sequence of a cDNA from rat.

Filaggrin is an intermediate filament-associated protein that is involved in aggregation of keratin filaments in fully cornified cells of the mammalian epidermis, and is an important marker for epidermal differentiation. In this report, the sequence of a rat cDNA clone coding for a portion of the polymeric precursor, profilaggrin, is presented. The cDNA is 2,314 bp long with 1,875 bp of coding region ending with an A-T-rich 3' noncoding region. Genomic analysis indicates that the profilaggrin gene consists of 20 +/- 2 repeats of 1,218 bp of sequence coding for 406 amino acids, making the mRNA at least 25-27 kb in length. Each repeat consists of a filaggrin domain and a linker sequence with an estimated size of 380 and 26 amino acids, respectively. High levels of profilaggrin mRNA are found only in keratinizing epithelia. Comparison of the rat filaggrin sequence with that of mouse and human filaggrin and with the sequence of phosphorylated peptides from mouse profilaggrin indicates that the proteins share extensive amino acid sequence similarities, especially in the two phosphorylated regions. Proteolytic processing sites are also quite similar in rat and mouse. The three species show blocks of sequence that are similar in length and composition which alternate with sequences that are variable in length. This analysis suggests that the evolution of the present-day filaggrins has been constrained by maintenance of phosphorylation sites and overall amino acid composition. The cDNAs for the profilaggrins are similar in structure, reflecting genes that have simple repeating structures and lack introns within their coding regions. Mouse and rat profilaggrin terminate with a nonpolar sequence atypical of the rest of the coding region, and have similar 3' noncoding regions. To explain these observations, a novel evolutionary model is proposed.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.     

Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.