Cloning of cDNA encoding a novel mouse DNA topoisomerase III (Topo IIIbeta) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis

We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3beta). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The overall sequence of mTOP3beta has a 48 and 36% identity with mouse TOP3alpha at the nucleotide and amino acid level, respectively. DNA topoisomerase IIIbeta was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase IIIbeta had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37 degreesC and complete relaxation was observed at higher temperatures. mTOP3beta mRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3beta mRNA in the testis increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase.     

Immunochemical and biochemical identification of the rice seed protein encoded by cDNA clone A3-12

Previously isolated cDNA clone A3-12 that was expressed in E. coli as the fusion protein with Trp E showed immunoreactivity with the mouse antibody raised against isolated alpha-globulin from rice seed. The N-terminal amino acid sequences determined for the purified alpha-globulin and its tryptic peptides were identical with the deduced amino acid sequence reported, except for two residues at the protein N terminus. An error in the reported sequence was confirmed by re-sequencing the cDNA, the nucleotide sequence for the two N-terminal residues being shown to be CAGCTG and not CACGTG. Thus, the protein encoded by cDNA clone A3-12 was identified to be the major rice seed globulin, alpha-globulin, with an apparent molecular mass of 26kDa.     

Cloning and molecular characterization of plant aldehyde oxidase

Primary structural information of a plant aldehyde oxidase (AO), which was purified from maize coleoptiles using indole-3-acetaldehyde as a substrate, was obtained by sequencing a series of cleavage peptides, permitting the cloning of the corresponding cDNA (zmAO-1). The complete nucleotide sequence was determined; the deduced amino acid sequence encodes a protein of 1358 amino acid residues of Mr 146,681, which is consistent with the size of the AO monomeric subunit. There is a significant similarity with AO from mammals and xanthine dehydrogenases from various sources. The maize AO polypeptide contains consensus sequences for iron-sulfur centers and a putative molybdopterin cofactor-binding domain. In addition, another cDNA (zmAO-2), highly homologous to zmAO-1 at both the nucleotide and amino acid sequence levels, was cloned. zmAO-2 would encode a protein of 1349 amino acid residues of Mr 145,173 and has molecular characteristics similar to those of zmAO-1. zmAO-1 was expressed at a high level in roots, which was closely correlated with immunoblotting results using antiserum raised against the purified maize AO protein, whereas zmAO-2 was expressed at a higher level in coleoptiles than in roots. We propose each zmAO may have a unique function during plant development.     

Molecular cloning of a murine cDNA encoding a novel protein, p38-2G4, which varies with the cell cycle

Proliferating cells express genes active in cell cycle control. The modulation of control genes and factors are required to maintain critical cell cycle activities. We used a set of monoclonal antibodies prepared against DNA-binding proteins from Ehrlich ascites tumor cells in immunofluorescent microscopy to screen for proteins showing cell cycle-specific staining patterns. Here, we report cloning and characterizing of a novel mitogen-inducible gene from murine macrophages that predicts a cell cycle-specifically modulated nuclear protein of 38 kDa, designated p38-2G4. p38-2G4 displayed a speckled pattern of varying fluorescence intensity confined to the nucleus, but sparing the nucleoli. Strongly stained granules were observed between G1 and mid S phase, followed by a less abundant punctated arrangement toward the end of S phase, and negative fluorescence at the S/G2 transition. Thereafter, the nuclear staining reappeared. Additionally, p38-2G4 expression vanished in G0-arrested cells and was restored after release from growth arrest. p38-2G4 conserved in vertebrates by means of immunofluorescence data contains a number of putative phosphorylation sites, a cryptic nuclear localization signal, and an amphipathic helical domain. Our cDNA and its deduced amino acid sequence is related to a Schizosaccharomyces pombe gene encoding a 42-kDa protein that associates with curved DNA, suggesting that we have cloned the murine homologue of the S. pombe gene which defines a novel cell cycle-specifically modified and proliferation-associated nuclear protein in mammals.     

Cloning and sequencing of cDNA encoding mouse GTP cyclohydrolase I

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a mouse brain cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 994 base pairs. The coding region encoded a protein of 241 amino acid residues with a calculated molecular mass of 27,014 daltons. The deduced amino acid sequence of mouse GTP cyclohydrolase I was found to be highly homologous to rat (96%) and human type 1 (89%) enzymes.     

Molecular cloning and characterization of a novel human classic cadherin homologous with mouse muscle cadherin

We used a novel cDNA cloning method based on the cadherin-beta-catenin protein interaction and identified a new human classic-type cadherin, which we named cadherin-15, from adult brain and skeletal muscle cDNA libraries. Sequence analysis revealed that this cadherin was closely related to mouse muscle cadherin and seemed to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra 31-amino acid sequence at its C terminus that has been found neither in mouse muscle cadherin nor in any other known classic cadherin. Analysis of cadherin-15 protein expressed in L fibroblasts showed that it was cleaved proteolytically, expressed on the cell surfaces as a mature form of about 124-kDa, and functioned as a cell-cell adhesion molecule in a homophilic and specific manner, but Ca2+ did not protect it against degradation by trypsin. Our findings also suggest that cadherin-15 mediates cell-cell adhesion with a binding strength comparable to that of E-cadherin.     

Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis

The gene coding for the CAMP factor from a strain of Streptococcus uberis (ATCC 9927) was cloned in Escherichia coli. Chromosomal DNA from Streptococcus uberis was used to construct a gene library in plasmid pTZ18R and six CAMP-reaction positive clones were obtained from a total of 10,000 transformants. One clone, pJLD21, was subcloned and the CAMP factor gene was located in a 3.2 kb BamHI fragment. The nucleotide sequence of Streptococcus uberis CAMP factor gene was determined and the deduced amino acid sequence is highly homologous to the corresponding Streptococcus agalactiae protein. Immunoblot analysis revealed that the recombinant strain pJLD21 expressed a protein with a molecular weight of 28 000. Antibodies raised against purified Streptococcus uberis CAMP factor cross-reacted with Streptococcus agalactiae protein B.     

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.     

Isolation of a 65-kDa protein from white muscle of warm temperature-acclimated goldfish (Carassius auratus)

A 65-kDa protein expressed in association with warm temperature acclimation of goldfish (Carassius auratus) was purified from epaxial muscle by successive ion-exchange, gel filtration, and reversed-phase columns while monitoring immuno-reaction with a specific antibody. A total of 517 micrograms of the 65-kDa protein was obtained from 23.4 g of the muscle of 30 degrees C-acclimated fish. The purified 65-kDa protein gave one band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was determined to be 65,000 by gel filtration and SDS-PAGE, demonstrating that it consists of a single polypeptide chain; 44 amino acid residues were determined by N-terminal amino acid sequencing. The amino acid stretch was comparatively rich in histidine and phenylalanine. Homology search in the National Biomedical Research Foundation and Swiss-Prot bank did not identify any known amino acid sequence with significant homology to the 44-amino acid stretch of the 65-kDa protein, suggesting it to be a novel protein.     

Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages

We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophage protein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis.     

Cloning and characterization of a novel zinc finger protein that associates with nuclear matrix

We have recently reported that the nuclei of B16 melanoma cells are intensely stained with anti-rat vitronectin (Vn) antibody, which reacts with both mouse and rat Vn. In the present study, we characterized the protein immunoreactive with the antibody using NIH3T3 cells, whose nuclei were also stained with the antibody. Western blot analysis showed that a protein with an approximate molecular weight of 75 kDa (p75), which was distinct from Vn, existed in the nuclear fraction, and, more specifically, in the nuclear matrix fraction, of NIH3T3 cells. Screening of an NIH3T3 cDNA library resulted in the isolation of a nearly full-length cDNA clone encoding p75. A database search revealed that the cDNA represents a novel gene. The deduced amino acid sequence showed that the protein is 580 amino acids long and contains two C2H2-type zinc finger motifs and glutamic acid-rich domains in the C-terminal region. When a fusion protein of green fluorescence protein and p75 was expressed in NIH3T3 cells, fluorescence was preferentially observed in the nuclei, demonstrating that the protein has a nuclear localization signal. The p75 protein, termed ZAN75, exhibited DNA-binding activity in a zinc-dependent manner. Southern blot analysis demonstrated that the ZAN75 gene exists in a single copy in the mouse genome and that a closely related gene is also present in chicken, rat, and human. Northern blot analysis showed that the ZAN75 gene is ubiquitously expressed in adult mouse tissues. In the cell cycle of NIH3T3 cells, expression was low in the G0/G1 phase, increased during the G1 phase, and persisted during the S and G2/M phases, suggesting that ZAN75 plays a role in regulating cell growth.     

Adult T-cell leukemia/lymphoma in London: clinical experience of 21 cases

An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences. However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues. The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN. One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini. The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site. Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B. thuringiensis CryIA delta-endotoxin.     

Effect of naloxone on the habituation of novelty-induced hypoalgesia: the collateral inhibition hypothesis revisited

The prtT gene, coding for trypsinlike proteolytic activity, has been isolated from Porphyromonas gingivalis ATCC 53977. This gene is present immediately downstream from the sod gene on a 5.9-kb DNA fragment from the organism isolated in Escherichia coli. The complete nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the enzyme corresponds to a 53.9-kDa protein with an estimated pI of 11.85. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography also indicated a similar molecular size for the protease. The enzyme was purified to near homogeneity following anion-exchange and gel-filtration chromatography. The purified enzyme also exhibited a single protein species with a size of approximately 53 kDa. Enzyme activity was strongly dependent upon the presence of reducing agents (dithiothreitol, cysteine, and 2-mercaptoethanol) and was also stimulated in the presence of calcium ions. A comparison of the properties of the prtT gene product with comparable parameters of proteases previously purified from different strains of P. gingivalis suggested that the cloned protease represents a previously uncharacterized enzyme.     

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.     

Determinants of cholesterol screening and treatment patterns. Insights for decision-makers

We isolated rat UCP2 cDNA, which has been proposed to play an important role in mammalian thermogenesis and body weight regulation. The nucleotide sequence of the cDNA revealed that the rat UCP2 protein is composed of 309 amino acid residues, and is 99% and 95% identical to the mouse and human proteins, respectively. The molecular weight of rat UCP2, calculated from the predicted amino acid sequence, was 33,369, and the UCP2 protein of this size was detected when the cDNA was expressed in vitro. Northern blot analysis revealed that the corresponding mRNA is approximately 1.7 kb in size, and is expressed in a variety of rat organs, with predominant expression in the heart, lung and spleen. UCP2 mRNA levels in the heart, liver, muscle and epididymal adipose tissue of Zucker fatty (fa/fa) rats were comparable to those in the lean littermates, while ob mRNA level markedly increased in the epididymal adipose tissue of Zucker (fa/fa) rats.     

Identification and characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.     

Vascular malformations of the jaw bones. Report on nine patients

Partial complementary DNA (cDNA) for thymidine phosphorylase (dThdPase) was cloned by means of a polymerase chain reaction. There was complete sequence identity between the amino acid sequence deduced from the nucleotide sequence of a clone (288 nucleotides) and the residues of platelet-derived endothelial cell growth factor (PD-ECGF). The amino acid sequence of all four peptide fragments from purified human dThdPase could be aligned with that of PD-ECGF. Our data indicate that residues 125-244 of PD-ECGF are identical to the sequence of human dThdPase. The molecular weights of human dThdPase and recombinant PD-ECGF (rPD-ECGF) that lacks 10 amino acids at the amino terminal were 55 and 52 kDa, respectively. Anti-PD-ECGF antibody recognized dThdPase, and anti-dThdPase antibody recognized rPD-ECGF. rPD-ECGF had dThdPase activity and its specific activity was similar to that of purified human dThdPase. dThdPase activity and molecules were detected in COS cells transfected with human PD-ECGF cDNA, but not in nontransfected cells. The sizes of PD-ECGF and dThdPase in the transfected COS cells were identical. These data suggest that human dThdPase is identical to PD-ECGF.