Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells.

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis.

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.

Shipboard testing of the efficacy of SeaKleen as a ballast water treatment to eliminate non-indigenous species aboard a working tanker in Pacific waters.

Trials were conducted aboard the tanker Seabulk Mariner to test a natural product, SeaKleen, as a biocide controlling non-indigenous populations of plankton and bacteria in ballast water. SeaKleen was dosed into matched ballast tanks at two different concentrations, 0.8 mg L(-1) active ingredient (a.i.) and 1.6 mg L(-1) a.i. during ballasting off the Oregon coast during a three-day passage to Prince William Sound, Alaska. Live organism counts from treated ballast water were compared with those from untreated (control tank) water collected from the same source location. Shipboard chemical analyses were made to verify dose and quantify chemical degradation and residuals following dilution. Results indicated that both SeaKleen doses resulted in complete zooplankton and phytoplankton mortality and that the higher dose (1.6 mg L(-1) a.i.) caused a two-log removal of culturable bacteria over a 92 h grow-out period. Spectrophotometry confirmed initial dosing to within 5% of nominal values. Shipboard bioassays were conducted using larval fish (Cyprinodon variegatus), brine shrimp (Artemia salina) and the bioluminescent dinoflagellate Pyrocystis lunula. Exposure of the test organisms to water drawn from treated ballast tanks 48 h after SeaKleen was added to the tanks resulted in 100% mortalities in Cyprinodon and Pyrocystis at both doses. Corresponding mortalities for Artemia larvae were 100% and 60% for high and low SeaKleen doses, respectively. Toxicity testing of treated water, subjected to varying dilutions, indicated that residual toxicity to even the most sensitive organisms would be eliminated once the discharge had dispersed beyond 100 feet from the vessel.

Repair of UV-induced lesions in Xenopus laevis oocytes.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.

Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene.

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene.

We have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression (Z. Chen, M. L. Harless, D. A. Wright, and R. E. Kellems, Mol. Cell. Biol. 10:4555-4564, 1990). Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase III promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. We identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, we have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of our findings, we hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene.

We have previously shown that a transcription arrest site near the 5' end of the murine adenosine deaminase (ADA) gene is significantly involved in the regulation of ADA gene expression. To facilitate the analysis of this transcription arrest site, we have analyzed the transcription products from cloned ADA gene fragments injected into Xenopus laevis oocytes. When genomic fragments spanning the 5' end of the ADA gene were injected into oocytes, a 96-nucleotide (nt) ADA RNA was the major transcription product. The 5' end of this RNA mapped to the transcription initiation site for the ADA gene, and its 3' terminus mapped 7 nt downstream of the translation initiation codon within exon 1. A 300-base-pair fragment of genomic DNA spanning the 5' end of the ADA gene was sufficient to generate the 96-nt transcript which accounted for approximately one-half of the transcription products from injected templates. Deletion of a segment of approximately 65 base pairs, located immediately downstream of the 3' terminus of the 96-nt transcript, resulted in a substantial reduction in the synthesis of the 96-nt transcript and a corresponding increase in the production of larger transcripts. These studies show that the transcriptional apparatus of X. laevis oocytes responds to the transcription arrest site associated with exon 1 of the murine ADA gene and that oocyte injections provide a convenient functional assay for additional mechanistic studies.

Naphthoquinones as broad spectrum biocides for treatment of ship's ballast water: toxicity to phytoplankton and bacteria.

Current UN International Maritime Organization legislation mandates the phased introduction of ballast water treatment technologies capable of complying with rigorous standards related to removal of waterborne organisms. Doubts concerning mechanical treatments at very high ballasting rates have renewed interest in chemical treatment for very large vessels. High removal rates for biota require broad spectrum biocides that are safe to transport and handle and pose no corrosion problems for ships' structure. The current study focuses on the naphthoquinone group of compounds and extends a previously reported set of screening bioassays with an investigation of the toxicity of four naphthoquinones to select protists and prokaryotes, representative of typical ballast water organisms. Vegetative dinoflagellate cysts exposed to 2.0 mg/L of the naphthoquinones juglone, plumbagin, menadione and naphthazarin showed varying degrees of chloroplast destruction, with menadione demonstrating the most potency. Laboratory and mesocosm exposures of various phytoplankton genera to menadione showed toxicity at 1.0 mg/L. Juglone demonstrated the most bactericidal activity as judged by a Deltatox assay (Vibrio fischeri) and by acridine orange counts of natural bacterial populations.

The influence of water quality on the toxicity and degradation of juglone (5-hydroxy 1,4-naphthoquinone).

This study was part of a broader investigation of low molecular weight quinones under consideration as biocides for the control of aquatic nuisance species (ANS). Preliminary investigations identified the 2-ring naphthoquinones as broad spectrum biocides controlling a wide range of aquatic organisms. All biocides were relatively short-lived in saline waters, with half-lives between 5 and 30h. Juglone (5-hydroxy 1,4-naphthoquinone) and plumbagin (5-hydroxy-2-methyl-1,4- naphthoquinone) showed the greatest toxicity against most aquatic organisms. These qualities formed the basis for a patent focusing on these two compounds as biocides for ANS control, with juglone identified as the more cost-effective of the two. Although juglone has been extensively studied as a plant toxin and reducing agent, remarkably little information exists on its use as an aquatic biocide. We describe the toxicity of juglone over the range of water quality parameters likely to be encountered in ballast water, a major vector for ANS. Tests indicated that its molecular stability was enhanced in freshwater and particularly under neutral to acid conditions. This was supported by results of bioassays on the freshwater cladoceran Daphnia magna that indicated enhanced juglone toxicity at pHs of < or =6.7. A low octanol:water partition coefficient for juglone indicated little capacity for these compounds to be adsorbed by suspended particulates and for bioaccumulation. These properties together with their relatively rapid degradation (t1/2 < or =30h), particularly in the marine environment, indicated a low the risk of residual toxicity associated with the release of juglone-treated water.

Screening of natural product biocides for control of non-indigenous species.

Several benzo-, naphtho- and anthraquinones were tested for their efficacy as biocides in controlling aquatic nuisance species in ships' ballast water. A requirement of this application was broad spectrum aquatic toxicity, coupled with a relatively rapid rate of degradation, in order to comply with coastal discharge requirements. Compounds were screened using a suite of toxicity bioassays designed to establish their relative toxicity to an array of planktonic organisms including larval bivalves Dreissena and Crassostrea, various developmental stages of the estuarine copepod Eurytemora affinis, brine shrimp larvae (Artemia salina), the freshwater invasive water flea Bythotrephes, larval sheepshead minnows CCyprinodon variegates) and two unicellular algal genera Isochrysis and Neochloris.. The majority of the data were recorded as the lowest concentration of the test compound resulting in complete mortality or inactivation of test organisms (LC ,m). The naphthoquinones juglone, plumbagin, menadione and naphthazarin showed the highest toxicity to the broadest range of organisms, often at levels much less than 1 mg l(-1), and most of the attention was focused on this group. While plumbagin and juglone appeared overall to be the most toxic compounds, it was concluded that menadione was probably the most cost-effective candidate compound for shipboard use for controlling invasive species in ballast water, particularly in view of the large volumes of water that would require treatment.

Two sex-linked loci in the leopard frog, Rana pipiens.

Crosses involving one heterozygous parent were performed to test the inheritance of enzymes in the leopard frog, Rana pipiens. After metamorphosis, offspring were sexed and tissue extracts from them were analyzed by gel electrophoresis. Enzyme genotype and sex showed independent assortment for 10 of 12 enzymes heterozygous in the male parent. However, among the offspring of males heterozygous for peptidase C (Pep-C) or superoxide dismutase 1 (SOD-1), male progeny tend to inherit one allele, whereas female progeny tend to inherit the other. Data from several different crosses yield recombination frequencies of 8.6% between sex and SOD-1, 6.9% between SOD-1 and Pep-C and 12.1% between sex and Pep-C. When the female parent is heterozygous for these enzymes no significant difference is seen, in the offspring, between male and female homozygotes and heterozygotes. These results confirm that males are the heterogametic sex in R. pipiens and suggest that sex is determined by a small number of genes on otherwise identical X and Y chromosomes.

Potential retroviruses in plants: Tat1 is related to a group of Arabidopsis thaliana Ty3/gypsy retrotransposons that encode envelope-like proteins.

Tat1 was originally identified as an insertion near the Arabidopsis thaliana SAM1 gene. We provide evidence that Tat1 is a retrotransposon and that previously described insertions are solo long terminal repeats (LTRs) left behind after the deletion of coding regions of full-length elements. Three Tat1 insertions were characterized that have retrotransposon features, including a primer binding site complementary to an A. thaliana asparagine tRNA and an open reading frame (ORF) with approximately 44% amino acid sequence similarity to the gag protein of the Zea mays retrotransposon Zeon-1. Tat1 elements have large, polymorphic 3' noncoding regions that may contain transduced DNA sequences; a 477-base insertion in the 3' noncoding region of the Tat1-3 element contains part of a related retrotransposon and sequences similar to the nontranslated leader sequence of AT-P5C1, a gene for pyrroline-5-carboxylate reductase. Analysis of DNA sequences generated by the A. thaliana genome project identified 10 families of Ty3/gypsy retrotransposons, which share up to 51 and 62% amino-acid similarity to the ORFs of Tat1 and the A. thaliana Athila element, respectively. Phylogenetic analyses resolved the plant Ty3/gypsy elements into two lineages, one of which includes homologs of Tat1 and Athila. Four families of A. thaliana elements within the Tat/Athila lineage encode a conserved ORF after integrase at a position occupied by the envelope gene in retroviruses and in some insect Ty3/gypsy retrotransposons. Like retroviral envelope genes, this ORF encodes a transmembrane domain and, in some insertions, a putative secretory signal sequence. This suggests that Tat/Athila retrotransposons may produce enveloped virions and may be infectious.

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Meiotic origin of triploidy in the frog detected by genetic analysis of enzyme polymorphisms.

A female frog heterozygous at two unlinked loci, specifying electrophoretic forms of mannosephosphate isomerase (MPI) and malate dehydrogenase (MDH) was crossed to male frogs homozygous for different alleles at each locus. In the offspring approximately ten percent proved to be triploid according to nucleolar and chromosome counts of tail tip cells. Most of these triploids had both maternal alleles at the MDH and MPI loci suggesting that the first meiotic division was repressed. Others seemed to represent a repressed second meiotic division and one animal, a pentaploid, could only have resulted from inhibition of both meiotic divisions of the egg. Densitometer tracings of starch gels stained for 6 phosphogluconate and isocitrate dehydrogenases, expected to be heterozygous in a particular cross, demonstrated that the triploids had twice as much maternal as paternal gene product for each locus, similar to patterns found in triploids produced by nuclear transplantation.

Multiple non-LTR retrotransposons in the genome of Arabidopsis thaliana.

DNA sequence analysis near the Arabidopsis thaliana ABI3 gene revealed the presence of a non-LTR retrotransposon insertion that we have designated Ta11-1. This insertion is 6.2 kb in length and encodes two overlapping reading frames with similarity to non-LTR retrotransposon proteins, including reverse transcriptase. A polymerase chain reaction assay was developed based on conserved amino acid sequences shared between the Ta11-1 reverse transcriptase and those of non-LTR retrotransposons from other species. Seventeen additional A. thaliana reverse transcriptases were identified that range in nucleotide similarity from 48-88% (Ta12-Ta28). Phylogenetic analyses indicated that the A. thaliana sequences are more closely related to each other than to elements from other organisms, consistent with the vertical evolution of these sequences over most of their evolutionary history. One sequence, Ta17, is located in the mitochondrial genome. The remaining are nuclear and of low copy number among 17 diverse A. thaliana ecotypes tested, suggesting that they are not highly active in transposition. The paucity of retrotransposons and the small genome size of A. thaliana support the hypothesis that most repetitive sequences have been lost from the genome and that mechanisms may exist to prevent amplification of extant element families.

Autosomal modifiers of the bobbed phenotype are a major component of the rDNA magnification paradox in Drosophila melanogaster.

rDNA magnification in Drosophila melanogaster is defined experimentally as the ability of bb/Ybb- males to produce exceptional progeny that are wild type with respect to rDNA associated phenotypes. Here, we show that some of these bobbed-plus progeny result not from genetic reversion at the bb locus but rather from variants at two or more autosomal loci that ameliorate the bobbed phenotype of rDNA deficient males in Drosophila. In doing so we resolve several aspects of a long-standing paradox concerning the phenomenon of rDNA magnification. This problem arose from the use of two genetic assays, which were presumed to be identical, but paradoxically, produced conflicting data on both the kinetics of reversion and the stability of magnified bb+ chromosomes. We resolve this problem by demonstrating that in one assay bobbed-plus progeny arise primarily by genetic reversion at the bobbed locus, whereas in the other assay bobbed-plus progeny arise both by reversion and by an epistatic effect of autosomal modifiers on the bobbed phenotype. We further show that such modifiers can facilitate the appearance of phenotypically bobbed-plus progeny even under conditions where genetic reversion is blocked by magnification defective mutants. Finally, we present a speculative model relating the action of these modifiers to the large increases in rDNA content observed in males undergoing magnification.

The mouse genome contains two expressed intronless retroposed pseudogenes for the sentrin/sumo-1/PIC1 conjugating enzyme Ubc9.

The ubiquitin conjugating (ubc) E2 enzyme ubc-9 conjugates the ubiquitin-like peptide sentrin/SUMO-1/PIC1 to target proteins which include the Fas antigen. We show that the mouse genome contains four copies of the ubc-9 gene. These include a structural ubc-9 gene consisting of seven exons which encode a protein identical to human ubc-9, and three intronless processed pseudogenes. The open reading frames (ORF) of two of the pseudogenes, ubc9-psi1 and ubc9-psi2, correspond to the cDNA of ubc-9 and encode for proteins which differ from ubc9 by three and one amino acid substitutions respectively. The third pseudogene, ubc9-psi3, contains many mutations and stop codons. ubc9-psi1 and ubc9-psi2 are flanked by 5'- and 3'-untranslated (UT) regions homologous to those of the structural ubc-9 gene. Both genes contain a polyA tail and direct repeats at both ends suggesting that they arose by mRNA retroposition. Both ubc9-psi1 and ubc9-psi2 are transcribed into mRNA in murine cells. In contrast to ubc9, the protein products of ubc9-psil and ubc9-psi2 fail to bind Fas and to complement an yeast conditional ubc9 mutant. These results suggest that ubc9-psi1 and ubc9-psi2 encode for proteins that may interact with targets that differ from those recognized by ubc-9.

MPM-12: a monoclonal antibody that predominantly stains mitotic cells and recognizes a protein kinase.

The monoclonal antibody MPM-12, raised by using partially purified extract of mitotic HeLa cells as the immunogen, preferentially stains the cytoplasm of mitotic cells by indirect immunofluorescence without exhibiting any species specificity. On immunoblots, MPM-12 recognizes three bands, of 155, 88, and 68 kDa, in mitotic HeLa cell extract but only the 68-kDa band in interphase cell extract. The 68-kDa band seems to be associated with chromatin while the other two are not. All three MPM-12 reactive peptides are phosphorylated, and the phosphorylation seems to be required for MPM-12 reactivity. The MPM-12 immunocomplexes exhibit autophosphorylating and histone H1 kinase activity.

Chromosome condensing ability of mitotic proteins diminished by the substitution of phenylalanine with parafluorophenylalanine.

One of the objectives of this study was to develop a method for the reversible arrest of HeLa cells in G2 phase by using p-fluorophenylalanine (FPA), an analog of phenylalanine. Addition of 0.5 mM of FPA to synchronized HeLa cells during the middle of S phase, i.e., 4 h after reversal of the double thymidine block, arrested 93% of the cells in G2 phase. After the drug was removed from the medium, these G2 cells proceeded into mitosis synchronously. The results of the study also indicate that incorporation of FPA into mitotic proteins results in the inhibition of chromosome condensation as assayed by their ability to induce premature chromosome condensation in G1 HeLa cells and meiotic maturation in amphibian oocytes.

Carbohydrate feedings 1 h before exercise improves cycling performance.

The effects of consuming two different amounts of liquid carbohydrate 1 h before exercise on the metabolic responses during exercise and on exercise performance were determined. Subjects consumed either 1.1 g (LC) or 2.2 g (HC) carbohydrate/kg body mass or a placebo (P). Subjects cycled at 70% of maximal oxygen consumption (VO2max) for 90 min and then underwent a performance trial. Blood glucose and insulin responses during exercise were different among the three trials. Total carbohydrate oxidation was greater for the carbohydrate trials compared with P. Time-trial performance was significantly improved by LC and HC. Despite elevated insulin concentrations at the start of and during exercise, and despite an initial drop in blood glucose, consumption of between 1.1 and 2.2 g liquid carbohydrate/kg body mass 60 min before moderately intense prolonged exercise can improve performance, presumably via enhanced carbohydrate oxidation.