Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli. Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a β-lyase activity that nicks DNA 3′ to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7,8-dihydroxy-8-oxo-2′-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix–hairpin–helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2

A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5′–3′ exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed λSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.

Geranyl diphosphate synthase: Cloning, expression, and characterization of this prenyltransferase as a heterodimer

Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer.

Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae

The isolation and study of Anopheles gambiae genes that are differentially expressed in development, notably in tissues associated with the maturation and transmission of the malaria parasite, is important for the elucidation of basic molecular mechanisms underlying vector–parasite interactions. We have used the differential display technique to screen for mRNAs specifically expressed in adult males, females, and midgut tissues of blood-fed and unfed females. We also screened for mRNAs specifically induced upon bacterial infection of larval stage mosquitoes. We have characterized 19 distinct cDNAs, most of which show developmentally regulated expression specificity during the mosquito life cycle. The most interesting are six new sequences that are midgut-specific in the adult, three of which are also modulated by blood-feeding. The gut-specific sequences encode a maltase, a V-ATPase subunit, a GTP binding protein, two different lectins, and a nontrypsin serine protease. The latter sequence is also induced in larvae subjected to bacterial challenge. With the exception of a mitochondrial DNA fragment, the other 18 sequences constitute expressed genomic sequence tags, 4 of which have been mapped cytogenetically.

Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria and eukarya. We report here the identification and characterization of the SSB of an archaeon, Methanococcus jannaschii. The M. jannaschii SSB (mjaSSB) has significant amino acid sequence similarity to the eukaryotic SSB, replication protein A (RPA), and contains four tandem repeats of the core single-stranded DNA (ssDNA) binding domain originally defined by structural studies of RPA. Homologous SSBs are encoded by the genomes of other archaeal species, including Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus. The purified mjaSSB binds to ssDNA with high affinity and selectivity. The apparent association constant for binding to ssDNA is similar to that of RPA under comparable experimental conditions, and the affinity for ssDNA exceeds that for double-stranded DNA by at least two orders of magnitude. The binding site size for mjaSSB is ≈20 nucleotides. Given that RPA is related to mjaSSB at the sequence level and to Escherichia coli SSB at the structural level, we conclude that the SSBs of archaea, eukarya, and bacteria share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.

Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR

It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of β-galactosidase from a diphtheria tox promoter/operator–lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of β-galactosidase expression from a diphtheria tox promoter/operator–lacZ transcriptional fusion.

Cloning and characterization of TDD5, an androgen target gene that is differentially repressed by testosterone and dihydrotestosterone

By using mRNA polymerase chain reaction differential display technique (DDPCR), we have identified one early responsive cDNA fragment, TDD5, from a 5α-reductase-deficient T cell hybridoma. The DDPCR profiles of TDD5 suggest that its expression can be repressed by testosterone (T) within 2 hr. More importantly, both DDPCR and Northern blot analysis further demonstrated that the expression of TDD5 was differentially repressed by T and dihydrotestosterone (DHT) at the mRNA level. To our knowledge, this is the first androgen target gene to show a preference in response to T over DHT in cell culture. TDD5 is expressed in several tissues with particular abundance in kidney. Full-length TDD5 cDNA (2,916 bp) encodes a protein with a calculated molecular weight of 42,000. Finally, our animal studies further confirm that TDD5 mRNA levels can be repressed to the basal level 8 hr after DHT administration. The isolation and characterization of the early-responsive androgen target gene TDD5 and the fact that TDD5 mRNA level can be differentially regulated by T and DHT may provide a useful tool to study the molecular mechanism of androgen preference on target gene regulation.

Identification and characterization of a human protein kinase related to budding yeast Cdc7p

The Cdc7p protein kinase is essential for the G1/S transition and initiation of DNA replication during the cell division cycle in Saccharomyces cerevisiae. Cdc7p appears to be an evolutionarily conserved protein, since a homolog Hsk1 has been isolated from Schizosaccharomyces pombe. Here, we report the isolation of a human cDNA, HsCdc7, whose product is closely related in sequence to Cdc7p and Hsk1. The HsCdc7 cDNA encodes a protein of 574 amino acids with predicted size of 64 kDa. HsCdc7 contains the conserved subdomains common to all protein-serine/threonine kinases and three “kinase inserts” that are characteristic of Cdc7p and Hsk1. Immune complexes of HsCdc7 from cell lysates were able to phosphorylate histone H1 in vitro. Indirect immunofluorescence staining demonstrated that HsCdc7 protein was predominantly localized in the nucleus. Although the expression levels of HsCdc7 appeared to be constant throughout the cell cycle, the protein kinase activity of HsCdc7 increased during S phase of the cell cycle at approximately the same time as that of Cdk2. These results, together with the functions of Cdc7p in yeast, suggest that HsCdc7 may phosphorylate critical substrate(s) that regulate the G1/S phase transition and/or DNA replication in mammalian cells.

Cloning and characterization of a corepressor and potential component of the nuclear hormone receptor repression complex

Nuclear hormone receptors are potent repressors of transcription in the unliganded state. We describe here the cloning of a nuclear receptor corepressor that we call SUN-CoR (Small Unique Nuclear receptor CoRepressor), which shows no homology to previously described nuclear hormone receptor corepressors, N-CoR, or SMRT. SUN-CoR is a highly basic, 16-kDa nuclear protein that is expressed at high levels in adult tissues and is induced during adipocyte and myogenic differentiation. SUN-CoR potentiates transcriptional repression by thyroid hormone receptor and RevErb in vivo, represses transcription when fused to a heterologous DNA binding domain, and interacts with RevErb as well as with thyroid hormone receptor in vitro. SUN-CoR also interacts with N-CoR and SMRT in vitro and with endogenous N-CoR in cells. We conclude that SUN-CoR is a corepressor and may function as an additional component of the complex involved in transcriptional repression by unliganded and orphan nuclear hormone receptors.

Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum

The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Immunoisolation and Characterization of a Subdomain of the Endoplasmic Reticulum That Concentrates Proteins Involved in COPII Vesicle Biogenesis

Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi. We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.

Cloning and Characterization of peter pan, a Novel Drosophila Gene Required for Larval Growth

We identified a new Drosophila gene, peter pan (ppan), in a screen for larval growth–defective mutants. ppan mutant larvae do not grow and show minimal DNA replication but can survive until well after their heterozygotic siblings have pupariated. We cloned the ppan gene by P-element plasmid rescue. ppan belongs to a highly conserved gene family that includes Saccharomyces cerevisiae SSF1 and SSF2, as well as Schizosaccharomyces pombe, Arabidopsis, Caenorhabditis elegans, mouse, and human homologues. Deletion of both SSF1 and SSF2 in yeast is lethal, and depletion of the gene products causes cell division arrest. Mosaic analysis of ppan mutant clones in Drosophila imaginal disks and ovaries demonstrates that ppan is cell autonomous and required for normal mitotic growth but is not absolutely required for general biosynthesis or DNA replication. Overexpression of the wild-type gene causes cell death and disrupts the normal development of adult structures. The ppan gene family appears to have an essential and evolutionarily conserved role in cell growth.