A cyclic nucleotide-gated channel inhibits sensory axon outgrowth in larval and adult Caenorhabditis elegans: a distinct pathway for maintenance of sensory axon structure.

The tax-2 and tax-4 genes of C. elegans encode two subunits of a cyclic nucleotide-gated channel that is required for chemosensation, thermosensation and normal axon outgrowth of some sensory neurons. Here we show that, in tax-2 and tax-4 mutants, young larvae have superficially normal axons, but axon outgrowth resumes in inappropriate regions in late larval stages. Using a temperature-sensitive mutation in tax-2, we find that tax-2 activity is required during the adult stage to preserve normal axon morphology. These results indicate that tax-2 and tax-4 are required for the maintenance of correct axon structure, and reveal an unexpected plasticity that allows C. elegans axons to be remodeled long after their initial connections have been established. TAX-2 and TAX-4 have been proposed to form a transduction channel for chemosensation and thermosensation, and tax-2 activity is required in the adult stage for normal chemotaxis to NaCl and odorants. Animals mutant for the daf-11 gene have axon phenotypes that are similar to those of tax-2 and tax 4 mutants; this axon phenotype also has a late time of action. daf-11 regulates a developmental process called dauer larva formation that is controlled by sensory stimuli, and tax-2 and tax-4 can either stimulate or inhibit dauer larva formation in different contexts.

A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans.

In vertebrate visual and olfactory systems, a cyclic nucleotide-gated channel couples receptor activation to electrical activity of the sensory neurons. The Caenorhabditis elegans tax-2 gene is required for some forms of olfaction, for chemosensation of salts, and for thermosensation. We show here that tax-2 encodes a predicted subunit of a cyclic nucleotide-gated channel that is expressed in olfactory, gustatory, and thermosensory neurons, implicating this channel in multiple sensory modalities. Some sensory neurons display axon outgrowth defects in tax-2 mutants. Thus, the channel has an unexpected role in sensory neuron development in addition to its role in sensation. Consistent with this proposed dual function, a Tax-2::GFP fusion protein is present both in sensory cilia and in sensory axons.

Double targeted gene replacement for creating null mutants.

We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.

Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes.

We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.

Stable DNA transfection of a wide range of trypanosomatids.

We have shown that the Leishmania major transfection vector pR-NEO (or derivatives thereof) can be introduced and stably maintained in four species complexes of pathogenic Leishmania (L. tropica, L. mexicana, L. donovani, L. braziliensis), and the genera Endotrypanum and Crithidia; transfection of Trypanosoma cruzi or Trypanosoma brucei was not successful. Quantitative plating assays showed that the transfection efficiencies were high in L. major and Leishmania amazonensis (5x10(-5)/cell) and about 10-fold less for Leishmania panamaensis and Crithidia. Leishmania donovani transfected with pR-NEO retained the ability to infect hamsters, and amastigotes recovered after 2 months yielded G418-resistant promastigotes which retained high levels of extrachromosomal pR-NEO DNA. In promastigotes, the transfected DNA existed as extrachromosomal circles, and expressed the predicted 2.4-kb hybrid NEO/DHFR-TS mRNA bearing the trans-spliced miniexon. Large quantitative differences were observed only in Crithidia: relative to transfected Leishmania species, the copy number of pR-NEO was elevated 20-fold, while the levels of the NEO/DHRFR-TS mRNA or Escherichia coli beta-galactosidase (synthesized from the expression vector pX-beta GAL) were reduced 80 and more than 1000-fold, respectively. Thus, genetic signals derived from L. major DNA that mediate RNA expression or stability are recognized by the heterologous Leishmania species but less efficiently by Crithidia. These studies suggest that pR-NEO derived vectors may be applied to the study of genes expressed throughout the life cycle in a wide range of pathogenic trypanosomatids.

Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.

Trypanosomatid protozoan parasites cause several important tropical diseases and have been a fertile ground for the discovery of molecular paradigms such as trans-splicing and RNA editing. Transfection-based methods for the study of these organisms have recently been developed, and we have now designed an expression vector, pX, which contains only 2.3 kilobases of Leishmania DNA and can be stably transfected with high efficiency. Genes encoding Escherichia coli beta-galactosidase or a Leishmania amazonensis protective membrane glycoprotein (GP46A/M-2) were inserted into the pX expression site and transfected into Leishmania major, where they directed the synthesis of high levels of mRNAs formed by 5' and 3' processing events occurring predominantly at the sites used by the normal transcripts. Colony assays and immunoblot analysis showed that both proteins were produced; enzymatically active beta-galactosidase comprised approximately 1% of total protein. Sizes of the GP46A protein synthesized in transfected L. major or L. amazonensis were similar and differed from the predominant L. amazonensis GP46, suggesting that the GP46A gene may encode a variant GP46 family member. Because these vectors function efficiently in pathogenic species of Leishmania, pX will facilitate the genetic analyses of parasite proteins crucial for infectivity as well as the identification of cis-acting elements mediating transcription and replication.

Recurrent de novo appearance of small linear DNAs in Leishmania major and relationship to extra-chromosomal DNAs in other species.

We have detected several new chromosome-sized DNAs in lines derived from the LT252 isolate of Leishmania major. These DNAs appeared de novo in two clonal lines undergoing methotrexate (MTX) selection (clone 7-R50, clone 15-R50), in a stably MTX-resistant population reverting from MTX pressure (R1000-11-P55rev), and spontaneously during routine serial passage of the wild-type LT252 line in vitro (LT252+). No association of these new DNAs with drug resistance was detected. The new chromosomes were present in multiple copies, stably maintained, linear, and hybridized to a telomere-specific probe, and ranged in size from 180 to 220 kb. Southern blot hybridization revealed that all four new DNAs were related, and the family was designated the 715 class of small linear DNAs (SLDs). A 715-class SLD hybridization probe also identified small chromosomes described previously in Leishmania donovani (LD-1, HU-3 minichromosome) and Leishmania braziliensis (LD-1); LD-1 is known to be related to a smaller circular DNA, CD-1. A cloned probe derived from CD-1 (from K. Stuart and C. Tripp) identified two of the four L. major 715 class of SLDs in addition to the LD-1 DNAs of L. donovani and L. braziliensis, however the 715-class SLD probe did not identify CD-1 itself. L. major and L. donovani possess a homologous 1.5 Mb chromosome containing both the CD-1 and 715 sequences within a 40-kb region, whose size remained unaltered following appearance of the SLDs. These data suggest that SLDs and CD-1 may arise from an evolutionarily conserved chromosomal reservoir.

Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

The partial pressure of isoflurane or halothane does not affect their solubility in rabbit blood or brain or human brain: inhaled anesthetics obey Henry's Law.

We tested the possibility that the solubility of halothane or isoflurane in rabbit blood or human or rabbit brain does not obey Henry's Law. We measured the blood/gas and brain/gas partition coefficients for both anesthetics at approximately 1 MAC and at 0.01 MAC at 37 degrees C. The partition coefficients determined at the high vs low partial pressures did not differ. We conclude that the solution of isoflurane and halothane in blood and brain obeys Henry's Law.

Effect of sodium ascorbate concentration on the stability of samples for determination of serum folate levels.

Sodium ascorbate can be used as a preservative of patient samples for folate assay when freezing of serum is impractical. To evaluate the effect of sodium ascorbate on folate levels in human serum, it was added to pooled human sera in 1 g/l increments from 0 to 10 g/l serum. Free folate levels remained constant when the sodium ascorbate concentration was 6 g or less per liter of serum. At more than 6 g/l serum, free folate levels decreased. Bound folate levels increased when sodium ascorbate levels were 4 g/l or less, but remained stable at more than 4 g/l. A sodium ascorbate concentration of 5.0 +/- 1 g/l serum provided optimal preservation of folate in patient samples, indicated by obtaining constant values for four days when serum was kept at room temperature.