Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes.

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.     

Characterization of cDNA encoding the mouse DNA topoisomerase II that can complement the budding yeast top2 mutation.

Several cDNA clones encoding mouse DNA topoisomerase II were obtained from a mouse spermatocyte cDNA library and the entire coding sequence of the gene was determined. The mouse DNA topoisomerase II consists of 1528 amino acids with a molecular weight of 173 kDa. It shares significant homologies with the other eucaryotic enzymes, although species-specific sequences are observed in their highly charged C-terminal regions. The complete mouse TOP2 cDNA was put under yeast GAL1 promoter and examined for complementation of top2ts mutation in S.cerevisiae. We found that the cloned mouse gene could rescue the temperature-sensitive top2ts mutation, depending on its induction by galactose. The functional expression of the mouse DNA topoisomerase II in yeast was further confirmed by enzymatic assays and by immunological methods with antibodies specific for the mouse enzyme.     

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.     

Homology between mammalian DNA polymerase beta and terminal deoxynucleotidyltransferase

Nucleotide sequence analysis of the cDNA and the genomic clones for rat DNA polymerase beta revealed the existence of a 1,005-base pair open reading frame capable of encoding a Mr = 38,269 polypeptide of 335 amino acid residues. The region of 174 amino acid residues between the 42nd and 215th residues of the DNA polymerase beta polypeptide has extensive amino acid sequence homology with the region between the 195th and 366th residues of human terminal deoxynucleotidyltransferase. The two enzymes share extensive homology not only in primary structures but also in the computer-derived higher structures in these particular regions. The genes for DNA polymerase beta and terminal deoxynucleotidyltransferase are proposed to be derived from a common ancestral DNA polymerase gene.     

Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24.

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.     

Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene

We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.     

Molecular cloning and sequence analysis of cDNA encoding human kidney D-amino acid oxidase

cDNA clones encoding D-amino acid oxidase were isolated from a human kidney cDNA library by hybridization with cDNA for the pig enzyme. The cDNA insert of 2.0 kilobase pairs long provided coding information for a protein consisting of 347 amino acids. The molecular mass of the enzyme was calculated to be 39,410 Da. The amino acid sequence similarity between the pig and human enzymes is 84.4%, and among the active site residues proposed from chemical modification studies, methionine-110 of the pig enzyme was replaced by threonine. Northern blot analysis confirmed the expression of an mRNA of 2.0 kilobases encoding the D-amino acid oxidase in human kidney.     

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.     

cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine-rich region. Furthermore, unlike human furin, Dfurin1 possessed an extended amino-terminal region in which a potential transmembrane anchor was present.     

Molecular cloning and expression of four actin isoforms during Artemia development.

Four cDNA clones coding for different Artemia actin isoforms have been isolated. Three of the clones contain the complete coding sequences while the fourth one lacks 145 bases, coding for the 49 amino terminal amino acids of the protein. The amino acid sequences predicted for the four actin isoforms identified are highly homologous to insect actins as well as to vertebrate cytoplasmic actins. The four identified cDNA clones code for mRNAs of 5.2, 1.9, 1.6 and 1.8 kb, respectively, whose expression is regulated during development. Three of the actin mRNAs are present in cryptobiotic embryos while the other is not. The steady-state levels of all four mRNAs increase during development to reach maximal levels by 10-15 hours of development and decrease thereafter. The total number of actin genes encoded in the Artemia genome has been estimated as 8 to 10 by Southern analysis of total DNA.     

Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells.

Three cDNA clones of rat renal dipeptidase (rrDP) were isolated from rat renal and pulmonary cDNA libraries using a DNA fragment of human renal DP cDNA clone, MDP4, as a probe. The complete amino acid sequence deduced from the cDNA contains 410 amino acid residues, beginning with a signal peptide of 16 amino acid residues. RNA blot hybridization analysis showed that 1.6 and 2.2 kb mRNAs were expressed in lung and kidney, however, only 1.6 kb mRNA was detected in small intestine. COS-1 cells transfected with the cDNA expressed enzymatically active rrDP.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.     

Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II

Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other. Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta. Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model.     

Drosophila RNA polymerase II elongation factor DmS-II has homology to mouse S-II and sequence similarity to yeast PPR2.

DmSII is a Drosophila RNA polymerase II elongation factor which suppresses pausing by RNA polymerase II at specific sites on double stranded templates. Using antibodies produced against the purified protein, a Drosophila cDNA expression library was screened and a cDNA was isolated which encoded a portion of DmSII. When this cDNA was used to probe Kc cell mRNA the predominant species was found to be 1.4 kb in length. The original cDNA was used to screen a Drosophila Kc cell cDNA library resulting in the isolation of a 1.4 kb cDNA which was then sequenced. The deduced protein sequence for DmSII exhibited high similarity to mouse SII protein sequence. In addition, significant sequence similarity was found with the protein encoded by the yeast gene PPR2, which is involved in regulation of URA4 gene expression. The comparison of amino acid sequences suggests that DmSII is comprised of two domains homologous to mouse SII separated by a flexible, serine rich region of low homology. The shorter yeast protein has sequence similarity only to the carboxy terminal domain.