Isolation and characterization of the gene encoding the heavy chain of Drosophila kinesin.

An antiserum that recognizes the heavy chain of Drosophila kinesin was used to isolate Drosophila cDNA clones. Immunoblot analysis of the proteolytic fragments of the protein produced by one of the cDNA clones has demonstrated that the cDNA clones encode the heavy chain of Drosophila kinesin. The in vitro-synthesized product of the largest cDNA comigrates with Drosophila kinesin heavy chain on NaDodSO4/polyacrylamide gels and binds to taxol-stabilized microtubules in the presence of the nonhydrolyzable analogue of ATP, 5'-adenylyl imidodiphosphate, but not in the presence of ATP or 0.1 M KCl. Analysis of the cDNA clones suggests that there is a single gene encoding kinesin heavy chain in Drosophila located at polytene chromosome position 53A. However, Southern hybridization analyses suggest the presence of related sequences in the Drosophila genome.     

Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II.

Rabbit antibodies specific to yeast DNA topoisomerase I were used in immunological screening of a Saccharomyces cerevisiae genomic DNA library in Escherichia coli. One of the clones identified by its expression of antigenic determinants of the yeast enzyme is shown to contain the coding sequence of the enzyme: no active DNA topoisomerase I is detectable in cell extracts when insertion or deletion mutations are introduced into a 2-kilobase-pair (kb) region of the sequence in a haploid yeast genome. Blot hybridizations show that there is a single copy of the cloned sequence per haploid and that the sequence is transcribed to give a 2.7-kb poly(A)+ message. Mutants in which 1.7 kb of the sequence is deleted are viable. Temperature-shift experiments using synchronously grown cells of a delta top1 top2 temperature-sensitive (ts) double mutant and its isogenic top2 ts strain show that, whereas mitotic blocks can prevent killing of the top2 ts mutant at a nonpermissive temperature, the same treatments are ineffective in preventing cell death of the delta top1 top2 ts double mutant. These experiments suggest that in yeast DNA topoisomerase I serves a role auxiliary to DNA topoisomerase II.     

Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments.     

Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.     

Identification of a vaccinia virus gene encoding a type I DNA topoisomerase.

Vaccinia virus encapsidates a type I DNA topoisomerase (EC 5.99.1.2). The enzyme was purified from virus cores to apparent homogeneity, yielding a protein of Mr 32,000. The amino-terminal sequence of the isolated Mr 32,000 polypeptide was determined and used to map the putative structural gene for the vaccinia topoisomerase to the H7r open reading frame of the vaccinia genome. This gene encodes a 314-amino acid polypeptide containing a region homologous to a region of the type I topoisomerase from the yeast Saccharomyces cerevisiae.     

A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase.

A protein kinase activity has been identified that is tightly associated with the purified Drosophila type II DNA topoisomerase. The kinase and topoisomerase activities are not separated when the enzyme is subjected to analytical chromatography (phosphocellulose, single-strand DNA agarose, and Sephacryl S-300) and analytical glycerol gradient sedimentation. These two activities are also inactivated to the same extent by either heat or N-ethylmaleimide treatment. The evidence, however, does not rule out the possibility that the kinase activity resides in a polypeptide other than the topoisomerase polypeptide. The topoisomerase-associated protein kinase activity is not stimulated by Ca2+ or cyclic nucleotides. It shows a broad substrate range, including the DNA topoisomerase itself, casein, phosvitin, and histones. Phosphoamino acid analysis identified phosphoserine and phosphothreonine in polypeptides modified by the topoisomerase-associated protein kinase. No similar activity has been identified previously in Drosophila melanogaster.     

Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020.     

Isolation and characterization of the gene encoding 2,3-oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae.

The ERG7 gene encoding oxidosqualene-lanosterol cyclase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] from Saccharomyces cerevisiae has been cloned by genetic complementation of a cyclase-deficient erg7 strain. The DNA sequence of this gene has been determined and found to contain an open reading frame of 2196 nt (including stop codon) that encodes a predicted protein of 731 amino acids. The predicted molecular mass of the S. cerevisiae cyclase, 83.4 kDa, is similar to the predicted molecular masses of the oxidosqualene-lanosterol cyclase from Candida albicans and the oxidosqualene-cycloartenol cyclase from Arabidopsis thaliana, as well as to the molecular masses assigned to vertebrate oxidosqualene-lanosterol cyclases; however, it is substantially larger than the molecular mass assigned to purified S. cerevisiae cyclase. At the level of DNA and predicted amino acid sequences, the S. cerevisiae and C. albicans cyclases share 56% and 63% identity, respectively. Tryptophan and tyrosine residues are unusually abundant in the predicted amino acid sequences of (oxido)-squalene cyclases, leading to a hypothesis that electron-rich aromatic side chains from these residues are essential features of cyclase active sites.     

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.     

Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.

A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.     

Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

Several DNA topoisomerase II (Topo II; EC 5.99.1.3) partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.     

Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades.     

In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction.

DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complex-lamina fraction. Indirect immunofluorescence analyses of larval cryosections have confirmed the nuclear localization of topoisomerase II in situ. Although apparently excluded from the nucleolus, the topoisomerase protein is otherwise distributed throughout the interior of interphase nuclei. Similar immunocytochemical studies performed with permeabilized whole giant cells from third-instar larval salivary glands have shown topoisomerase II to be largely restricted to the polytene chromosomes. Upon nuclear disassembly during mitosis, the topoisomerase polypeptide appears to redistribute diffusely throughout the cell. Faint immunofluorescent staining of mitotic chromosomes is also observed.     

Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro.

The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA.