Dose Titration of Walleye Dermal Sarcoma (WDS) Tumor Filtrate.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 10(10) viral RNA copies in 100 µL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 10(10) inoculum, or approximately 10(7) viral RNA copies.

Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.

Dose Titration of Walleye Dermal Sarcoma (WDS) Tumor Filtrate.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 1010 viral RNA copies in 100 µL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 1010 inoculum, or approximately 107 viral RNA copies.

High-level expression of a synthetic red-shifted GFP coding region incorporated into transgenic chloroplasts.

We describe here a synthetic red-shifted variant of GFP that can be introduced into tobacco plastid genomes and is highly expressed in regenerated plants that appear normal and fertile. The variant contains the S65G and S72A mutations which shift the absorption maximum from the 395 nm of wild-type GFP closer to 488 nm, a wavelength emitted by a laser commonly used in confocal microscopy. In addition to enhanced fluorescence, the removal of significant absorption below 450 nm will potentially facilitate double-labelling experiments. The variant GFP encoded by the synthetic gene can be expressed at a high level, forming approximately 5% of total leaf protein.

New reports and a redescription of Porrocaecum heteropterum (Diesing, 1851) (Ascarididae), a rare nematode parasitic in South American.threskiornithid birds1.

Porrocaecum heteropterum (Diesing, 1851) (Nematoda, Ascarididae) is reported parasitising the white-faced ibis Plegadis chihi and the black-faced ibis Theristicus melanopis melanopis (Ciconiiformes, Threskiornithidae) from the Provinces of Buenos Aires and Neuquén, Argentina. This nematode has been reported very few times in the literature, mainly from Brazilian threskiornithids, and there have been no new reports following a redescription of the species given in 1957. This paper provides new host and locality records for this rather rare species, as well as some additional morphological data, mainly based on SEM studies, which complement the previous descriptions. The scarce and sporadic records of this species seem to indicate not only a defined host-specificity towards threskiornithid birds but also that the acquisition of this parasite is possible only when certain ecological barriers, including food availability, feeding habits and environmental conditions, are surmounted.

Tapironema coronatum n. gen., n. sp. (Trichostrongyloidea-Cooperiidae-Obeliscoidinae), a parasite of Holochilus brasiliensis and Tapirus terrestris.

In this paper we provide a description of Tapironema coronatum n. gen. n. sp. (Trichostrongyloidea, Obeliscoidinae) from the cricetid Holochilus brasiliensis or "water rat" in Argentina (Type material) and from Tapirus terrestris in French Guyana (voucher material in poor condition). The new genus is characterized by a corona radiata, an oesophageal tooth, a bilaterally synlophe with about 73 (male), 122 (female) cuticular ridges, a caudal bursa pattern 2-1-2 with rays 5 and 6 parallel and close together and rays 5 longer than rays 3. The most closely related genus is the monospecific Teporingonema Harris, 1985, from a Mexican lagomorph, Romerolagus. The cephalic extremity of this parasite is redescribed after the type-material. The systematic position of Teporingonema amongst the Obeliscoidinae is defined and the hypotheses concerning the origin of this sub family are provided.

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco.

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.

A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited.

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Sequencing, processing, and localization of the petunia CMS-associated mitochondrial protein.

The petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. Pcf transcripts are modified by editing at 11 sites. Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region. Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989). N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa. Through pulse-chase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence. Antibodies to synthetic peptides encoded by the atp9 and coxII portions of pcf recognized petunia ATP9 or COXII but no other mitochondrial proteins on immunoblots. Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes. Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria.

Editing of rps3/rpl16 transcripts creates a premature truncation of the rpl16 open reading frame.

Overlapping open reading frames corresponding to maize mitochondrial genes rps3 and rpl16 have been found in Petunia mitochondrial DNA. The DNA region associated with these two genes is part of the Petunia mitochondrial recombination repeat and is iterated three times. Analysis of transcripts from these genes shows that there is RNA editing of the coding regions and that one of the editing sites detected in the open reading frame overlap creates a premature stop codon in the rpl16 sequence. No transcripts were detected that were unedited at this site. Thus, in Petunia editing of rpl16 appears to render this gene nonfunctional.

Editing of pre-mRNAs can occur before cis- and trans-splicing in Petunia mitochondria.

Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription.

Characterization of coccidial proteins by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Two dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D SDS-PAGE) has been used to produce 'fingerprint' maps of the proteins from each of the 7 species of Eimeria which infect the chicken. All 7 species could be identified from their array of polypeptides but few differences were detected between strains of the same species. Alterations to the polypeptide array associated with the stage of sporulation of the oocysts were observed. Iodination of sporozoites, 2D SDS-PAGE, autoradiography and immunoblotting techniques were combined to identify polypeptides with a surface moiety and those which were antigenic.

Determinants of heat shock-induced chromosome puffing.

Modified Drosophila heat shock genes were introduced into the germ line by P element transformation. The genes were altered such that several factors could be tested for their influence upon chromosome puffing. Deletion of promoter sequences upstream of position -73 of an hsp70-IacZ hybrid gene was sufficient to abolish puffing. Analysis of progressive 5' deletions defines a 16 bp interval that contains sequences required for both heat-induced puffing and gene expression. An internal deletion of the hsp70-IacZ gene that reduces the transcript size from 9 kb to 0.8 kb results in a dramatic reduction in puff size. The chromosomal insertion sites of 26 variant hsp70 or hsp26 genes fail to influence puffing greatly with one marked exception. This transformant possesses an insert that fails to puff and exhibits a tissue-restricted pattern of expression. These results indicate that variation in either promoter strength or transcript length have profound effects on puffing.

Localization and expression of transformed DNA sequences within heat shock puffs of Drosophila melanogaster.

In situ hybridization at high resolution with biotin-labeled DNA was used to locate specific transcriptional units within the chromosomal puffs of normal heat shock loci and of new heat shock loci generated by transformation. This method resolves copies of the hsp70 gene that are separated by 40 kb within the 87C heat shock locus. In the case of two new puff loci generated by transformation, the heat-activated transcript is encoded by sequences that reside entirely within the puff domain and the activated promoter is positioned asymmetrically within the puff. However, an adjacent promoter and its transcriptional unit which do not appear to be induced by heat shock are also within the puff. These results support the idea that a chromosomal puff is a structure that facilitates or results from vigorous transcription, but that the structural change alone is insufficient to induce transcription.

New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene.

A hybrid gene that consists of the Drosophila heat shock gene, hsp70, fused to the E. coli beta-galactosidase gene has been introduced into the Drosophila germline by the P element microinjection method. This hybrid includes 194 bp of sequence upstream of the start of the hsp70 transcript. Three strains of transformed flies were isolated and characterized by DNA blotting experiments and by in situ hybridization to polytene chromosomes. Strain Bg61 has a single insert of the hybrid gene at the tip of chromosome 3L, site 61A, and the insert consists of a structure that is consistent with P-element-mediated transposition. Strain Bg9,61 has inserts at both 61A and 9E, while Bg64 has a single insert at 64D. Heat shock induces the formation of a large chromosomal puff at all three sites. These puffs appear and regress with kinetics indistinguishable from the puffing of the heat shock locus, 87C, from which the hsp70 gene, used in these studies, was isolated. The beta-galactosidase activity in the transformants is inducible by heat shock and shows a widespread distribution throughout the tissues of larvae and adults.

Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phosphatidylinositol turnover in GH pituitary cells: a possible second step of TRH action.

TRH was found to rapidly influence 32PO4 incorporation into phospholipids of PRL-secreting GH pituitary cells. Analogs of TRH were found to exert similar effects, with potencies related to receptor-binding affinity. Additional PRL-releasing agents were also tested. Bombesin exerted a similar effect, whereas vasoactive intestinal polypeptide, 8-bromo cAMP, phosphodiesterase inhibitors, 50 mM K+, and scorpion venom toxin had no influence. Cationophore A23187 stimulated phospholipid labeling in a manner distinguishable from that of TRH. Chromatographic analysis showed the action of TRH to be restricted to the labeling of phosphatidylinositol and phosphatidic acid. Kinetic studies indicated a rapid influence of TRH on phosphatidylinositol breakdown, with subsequent accelerated 32PO4 incorporation into phosphatidylinositol and phosphatidic acid. These studied identified a rapid, receptor-mediated, cAMP-independent action of TRH on phospholipid metabolism. Similar effects of other hormones are believed to be involved in promoting cellular Ca2+ translocation. The rapid onset of the response reported here suggests that this event may play a role in mediating the PRL-releasing effects of TRH and bombesin in GH cells.

Thyrotropin-releasing hormone and cyclic AMP activate distinctive pathways of protein phosphorylation in GH pituitary cells.

The studies reported here were undertaken to clarify the cellular mechanism of the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), in clonal, hormone-responsive GH pituitary cells and to assess the possibility of a role for cyclic AMP as a mediator of TRH action. We investigated patterns of protein phosphorylation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of high speed supernatant and pellet fractions from untreated and treated GH cells. Brief treatment of cells with agents which elevate or mimic cellular cyclic AMP (8-bromo cyclic AMP, dibutyryl cyclic AMP, vasoactive intestinal polypeptide or cholera toxin) stimulated the phosphorylation of five supernatant peptides (41, 45, 47, 72, and 82 kilodaltons) and one pellet peptide (135 kilodaltons) and decreased the phosphorylation of one supernatant peptide (55 kilodaltons). In contrast, TRH promoted the phosphorylation of four different supernatant peptides (two 59, 65, and 80 kilodaltons). In addition, TRH also stimulated the phosphorylation of cyclic AMP-responsive 41-, 45-, and 82-kilodalton supernatant peptides and 135-kilodalton pellet protein and decreased the phosphorylation of 55-kilodalton supernatant peptide. Altered labeling of 47- and 72-kilodalton supernatant peptides, however, was not observed with TRH. Time course studies, as well as the overlapping biological action of TRH and vasoactive intestinal polypeptide, lead us to conclude that these peptide hormones utilize distinct, parallel pathways which converge at some late step. Furthermore, the results indicate that effects of TRH are mediated by a cyclic AMP-independent pathway.

Ribosome biosynthesis in Tetrahymena thermophila. IV. Regulation of ribosomal RNA synthesis in growing and growth arrested cells.

Three parameters involved in the production of new ribosomal RNA (rRNA) were measured in Tetrahymena thermophilia: (i) the rate of synthesis of the rRNA precursor, (ii) the rate of processing of the RNA precursor and rRNA intermediates and (iii) the efficiency of utilization of the rRNA precursor in producing mature ribosomal RNA. These parameters were measured in cells in exponential growth and in cells starved in a dilute salt solution. Growing cells synthesize rRNA 20 times faster and process rRNA precursors and intermediates 10 to 15 times more rapidly than do starved cells. Both utilize their rRNA precursors with an efficiency of one in converting them to mature rRNA.

Ribosome biosynthesis in Tetrahymena thermophila. III. Regulation of ribosomal RNA degradation in growing and growth arrested cells.

We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient-deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one-half hours lasting for six hours is followed by a slower exponential degradation with a half-life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.