A regulated RNA binding protein also possesses aconitase activity.

A clone for the iron-responsive element (IRE)-binding protein (IRE-BP) has been transfected and expressed in mouse fibroblasts. The IRE-BP gene product binds IREs with high affinity and specificity. Amino acid alignments reveal that the IRE-BP is 30% identical to mitochondrial aconitase. The 18 active site residues of mitochondrial aconitase are identical to those in the IRE-BP, suggesting that the IRE-BP may possess aconitase activity. After purification of native IRE-BP and immunoaffinity purification of transfected and expressed IRE-BP, we demonstrate that the purified IRE-BP has aconitase activity.     

Isolation of the gene encoding the Hin recombinational enhancer binding protein.

In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer. We have cloned and determined the primary sequence of the gene encoding Fis. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus. The gene encoding Fis maps at 72 min on the Escherichia coli chromosome. The construction of mutant strains of E. coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo.     

A fission-yeast gene encoding a protein with features of protein-tyrosine-phosphatases.

Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosine-phosphatases (PTPases) were used to amplify DNA fragments from a Schizosaccharomyces pombe cDNA library by polymerase chain reaction (PCR) methods. A cloned PCR product predicted peptide sequences similar to those found in PTPases but not identical to any published sequences. A S. pombe gene, designated pyp1+, was identified in a cDNA library with this PCR probe, cloned, and sequenced. The sequence of the gene predicted a 550-amino acid protein with Mr 61,586, which includes amino acid sequences that are highly conserved in mammalian PTPases. Disruption of the pyp1+ gene resulted in viable cells. Overexpression of the pyp1+ gene in S. pombe permitted detection of a protein of apparent Mr 63,000.     

Purification, properties, and immunocytochemical localization of human liver peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.

A molecular understanding of genetic disease in which peroxisomal functions are impaired depends on analysis of the structure of normal and mutant enzymes of peroxisomes. We report experiments describing the isolation, characterization, and immunocytochemical localization of enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE) of the peroxisomal fatty acid beta-oxidation system from normal human liver and compared it with that of rat liver enzyme. The human enzyme, purified approximately equal to 2300-fold by ion-exchange chromatography, is homogeneous as judged by NaDodSO4/PAGE. This PBE is localized exclusively in the matrix of peroxisomes in liver cells by the protein A/gold immunocytochemical method. The human PBE is similar to rat enzyme in size (Mr, approximately equal to 79,000), isoelectric point (pI, 9.8), pH optima, molecular structure as observed by rotary shadowing, and peptide pattern on NaDodSO4/PAGE after proteolytic digestion with Staphylococcus aureus V8 protease. The human and rat enzymes differed in their immunological properties by having partial identity with each other; this is reflected in their slightly dissimilar composition of the amino acids aspartic acid, threonine, glutamic acid, tyrosine, and glycine. COOH-terminal amino acid were similar for both the enzymes: -Gly-Ser-Leu-Ile-COOH. These results suggest that the human and rat liver PBE may be different in their amino acid sequences at their antigenic sites.     

Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1.

A yeast gene encoding SSB-1, a single-stranded nucleic acid binding protein, has been isolated by screening a lambda gt11 genomic DNA library. The gene is located on a 1.84-kilobase chromosomal Bgl II-BamHI fragment. Yeast strains carrying the high-copy-number vector YEp24 with an SSB1 gene insert overproduce SSB-1 3-fold and SSB-1 mRNA 10-fold. A typical haploid cell contains about 20,000 molecules of SSB-1; thus, the cells can tolerate up to 60,000 copies. Yeast SSB-1 was expressed in Escherichia coli cells by using a phage T7 expression system. Spores containing the gene disrupted at a point within the coding sequence germinate and grow normally; thus, the gene is not essential. Protein blots show that no SSB-1 or novel immunologically related species that might retain SSB-1 activity are present in cells containing the disrupted SSB1 genes. Southern analysis and protein blots suggest the presence in yeast of a second, related, but nonidentical gene and two immunologically related proteins of 55 kDa and 75 kDa.     

Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.     

Molecular cloning of a gene encoding an ARS binding factor from the yeast Saccharomyces cerevisiae.

We report the isolation of the gene for origin binding factor 1 (OBF1) from the yeast Saccharomyces cerevisiae by screening a yeast genomic DNA library in lambda gt11 with an ARS-specific oligonucleotide probe. One recombinant encoded a fusion protein of approximately 180 kDa that bound ARS-specific oligonucleotide probes in vitro. The restriction map of this gene was determined after isolation of the complete gene by screening a yeast genomic DNA library in YEp24. Characterization of the gene for OBF1 by pulsed-field gel electrophoresis and Northern and Southern blot analyses demonstrated that (i) the gene is located in chromosome IV, (ii) the gene is a single-copy gene, (iii) the mRNA is approximately 3.8 kilobases, which could code for an approximately 130-kDa polypeptide, consistent with the reported size of OBF1. An antibody, affinity-purified using the lysogen-encoded fusion protein, specifically detected an approximately 130-kDa polypeptide in yeast extract. The isolation of the gene for OBF1 should allow further analysis of the mechanism of action of this protein in vitro and in vivo.     

Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein.

Iron-responsive elements (IREs) are stemloop structures found in the mRNAs encoding ferritin and the transferrin receptor. These elements participate in the iron-induced regulation of the translation of ferritin and the stability of the transferrin receptor mRNA. Regulation in both instances is mediated by binding of a cytosolic protein to the IREs. High-affinity binding is seen when cells are starved of iron and results in repression of ferritin translation and inhibition of transferrin receptor mRNA degradation. The IRE-binding protein (IRE-BP) has been identified as an approximately 90-kDa protein that has been purified by both affinity and conventional chromatography. In this report we use RNA affinity chromatography and two-dimensional gel electrophoresis to isolate the IRE-BP for protein sequencing. A degenerate oligonucleotide probe derived from a single peptide sequence was used to isolate a cDNA clone that encodes a protein containing 13 other sequenced peptides obtained from the IRE-BP. Consistent with previous characterization of the IRE-BP, the cDNA encodes a protein of 87 kDa with a slightly acidic pI, and the corresponding mRNA of approximately 3.6 kilobases is found in a variety of cell types. The encoded protein contains a nucleotide-binding consensus sequence and regions of cysteine and histidine clusters. This mRNA is encoded by a single gene on human chromosome 9, a finding consistent with previous localization by functional mapping. The protein contains no previously defined consensus motifs for either RNA or DNA binding. The simultaneous cloning of a different, but highly homologous, cDNA suggests that the IRE-BP is a member of a distinct gene family.     

Cloning of a cDNA encoding rat intestinal fatty acid binding protein.

Intestinal fatty acid binding protein mRNA is one of the most abundant mRNA species in the rat small intestinal epithelium. RNA transfer blot analyses disclosed that the mRNA encoding intestinal fatty acid binding protein is approximately equal to 900 nucleotides long and not represented in liver RNA. We have identified 564 nucleotides of this mRNA, including 12 nucleotides of the 5' nontranslated region, the coding portion, and 155 nucleotides of the 3' nontranslated domain. The primary translation product encoded by this mRNA contains 132 amino acids and has a Mr of 15,062. The derived protein sequence was verified by automated sequential Edman degradation of the intact polypeptide isolated from a wheat germ cell-free system. The in vitro product is NH2-terminally acetylated, a finding that is consistent with its ultimate cytoplasmic destination. Comparison of the amino acid sequence of this protein with liver fatty acid binding protein, a polypeptide specified by the most abundant small intestinal epithelial mRNA, revealed significant homology and similarity in the predicted secondary structures of their NH2-terminal domains.     

A human gene (AHNAK) encoding an unusually large protein with a 1.2-microns polyionic rod structure.

We report here the identification and partial characterization of a human gene (designated AHNAK) that encodes an unusually large protein (approximately 700 kDa). AHNAK is expressed by means of a 17.5-kilobase mRNA in diverse cellular lineages but is typically repressed in cell lines derived from human neuroblastomas and in several other types of tumors. Unique-sequence domains at the two ends of the protein flank a large internal domain (approximately 4300 amino acids) composed of highly conserved repeated elements, most of which are 128 amino acids in length. The repeated elements in turn display a redundant motif, marked by the recurrence of proline at every seventh residue. Within these sequences, hydrophobic and hydrophilic residues alternate in a manner that is incompatible with a helical coiled-coil structure. Instead, we propose a structure resembling a beta-strand but with a periodicity of 2.33. The structure would engender a polyionic rod approximately 1.2 microns long. Preliminary evidence indicates that the protein resides predominantly within the nucleus, but no function has yet been discerned.     

Identification of calreticulin as a rubella virus RNA binding protein.

Previously, we observed that sequences at the 3' end of rubella virus (RV) genomic RNA that form a stable stem-loop structure are necessary for initiation of RNA replication. A cytosolic protein found in Vero 76 cells (simian origin) specifically bound to the 3' (+)-stem-loop sequence. In the present study, we have purified the RNA binding protein and identified it as a simian homologue of human calreticulin. The purified calreticulin binds to the RV RNA with specificity similar to the protein present in cytosolic extracts. Human calreticulin antibodies recognize several forms of simian calreticulin, one of which is phosphorylated in vivo. A 2-fold increase in phosphorylation of this form of calreticulin is observed in RV-infected cells. Recombinant human calreticulin can bind RV 3' (+)-stem-loop RNA only after undergoing in vitro phosphorylation. This binding activity is abrogated by pretreatment of phosphorylated recombinant human calreticulin with alkaline phosphatase. The RV RNA was also immunoprecipitated from RV-infected UV-crosslinked Vero 76 cells by using calreticulin antibodies. Our results show that phosphorylated calreticulin is an RNA binding protein and phosphorylation is necessary for this activity. Specific binding of calreticulin to the cis-acting element of RV RNA in vivo suggests a possible role for this interaction in viral replication.     

Isolation of cDNA of an auxin-regulated gene encoding a G protein beta subunit-like protein from tobacco BY-2 cells.

The addition of 2,4-dichlorophenoxyacetic acid to tobacco BY-2 cells that had been cultured in modified Linsmaier and Skoog medium deprived of auxin for 3 days induced cell division, whereas without 2,4-dichlorophenoxy-acetic acid application, no such induction of cell division was seen. When differential cDNA screening for auxin was done at 4 hr after the addition of 2,4-dichlorophenoxyacetic acid, the cDNA of an auxin-responsive gene designated arcA was isolated. The predicted gene product of arcA is a polypeptide with a M(r) of 35,825. arcA, thus, is a plant hormone-regulated gene that encodes a protein structurally related to the beta subunit of a guanine nucleotide-binding regulatory protein, which is composed of seven repetitive segments of Trp-Asp 40-aa repeats. The possibility that arcA gene products induce cell division is discussed.     

B lymphocyte-specific protein binding near an immunoglobulin kappa-chain gene J segment.

Nuclear extracts from pre-B and B cell lines contain a nuclear DNA binding protein (kappa locus protein, KLP) that specifically recognizes a DNA sequence in the immunoglobulin kappa light chain joining (J) segment gene region. KLP is not observed in mature B cells, T cells, or nonlymphoid cell types. Two tandem binding sites for KLP designated KI and KII have been identified by methylation interference analysis to be immediately proximal to the J kappa 1 nonamer-heptamer recognition sequences and separated by 38 base pairs from each other. Fragments of DNA containing KI and KII sites compete for binding to KLP, and both protein-DNA complexes have the same electrophoretic mobility. Other flanking sequences of immunoglobulin gene fragments do not bind to KLP. The position of KLP-DNA binding and its tissue-specific expression suggest that it may be involved in the regulation of lymphoid gene DNA rearrangements by targeting recombinase to the kappa-chain gene region.     

Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor.

Gene 28 of Bacillus subtilis bacteriophage SPO1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis. Here we report the cloning of gene 28 and its complete nucleotide sequence. We infer that sigma gp28 is a 25,707-dalton protein of 220 amino acids. Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of sigma gp28 exhibits extensive homology to the gene or protein sequence of Escherichia coli sigma factor.