Microbial genetic diversity and ciliate community structure along an environmental gradient in coastal soil

We investigated the microbial genetic diversity and ciliate community in coastal soil from five sites with an environmental gradient using denaturing gradient gel electrophoresis (DGGE), gene sequencing and the Ludox–QPS method. The analyses of both the 16S ribosomal RNA (rRNA) gene and 18S rRNA gene DGGE resulted in equal or even a higher number of bands found in the samples taken from the high-salinity sites IV and V than in those taken from the low-to-moderate-salinity sites I–III. Cluster analysis of both DGGE profiles classified the five sites into three main groups (sites I, II and III and IV and V), which corresponded well to the analysis of environmental factors. There were 13 species observed at site I, three species at site II and nine species at site III, while no active ciliates were observed at the high-salinity sites IV and V. By contrast, the ciliate-specific DGGE revealed a higher number of bands in the samples taken from the high-salinity soil. Furthermore, gene sequencing suggested that the ciliates in the high-salinity soil comprised forms originating not only from soil but also from marine environments. The data indicate that saline soil may maintain a high diversity of ciliates and soil salinity might be the most influential factor regulating the community structure of ciliates.     

The Effect of the Quantitative Protargol Stain and Lugol's and Bouin's Fixatives On Cell Size: A More Accurate Estimate of Ciliate Species Biomass

ABSTRACT. The quantitative protargol stain (QPS) is used to estimate ciliate biomass and species composition from mixed field samples. Length, width, breadth and volume of live Euplotes sp., Eutintinnus sp., Strobilidium spiralis, Strombidium acutum , and Gymnodinium sanguineum were compared with 0.6% acid Lugol's fixed, 5% Bouin's fixed, and QPS cells. Cells shrank due to treatments (ANOVA and Tukey's test, α= 0.05). Protistan post-fixation cell volume (as a percentage of live volume) was 55%-80% for acid Lugol's fixed, 40%-70% for Bouin's fixed, and 30%-65% for QPS. Each species shrank to a different extent; cytostructural elements apparently alter the effect of fixation. Egestion is likely not the main cause of shrinkage since the autotroph, G. sanguineum , shrank to the same extent as the heterotrophs when stained by QPS. If field studies do not consider fixation effects on cell size, biomass may be underestimated. We recommend, for studies on planktonic ciliates, either acid Lugol's and QPS be used concurrently or QPS be used alone and biovolume values divided by 0.4 to correct for shrinkage. We stress that this is a rough estimate as this value ranges from 0.3 to 0.45 for planktonic protists.     

A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94–100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9–97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.     

Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Ciliophora: Peritrichia) inferred from mitochondrial, nuclear, and morphological markers

Protist diversity is currently a much debated issue in eukaryotic microbiology. Recent evidence suggests that morphological and genetic diversity might be decoupled in some groups of protists, including ciliates, and that these organisms might be much more diverse than their morphology implies. We sought to assess the genetic and morphological diversity of Carchesium polypinum, a widely distributed peritrich ciliate. The mitochondrial marker cytochrome c oxidase subunit I and the nuclear small subunit ribosomal RNA were used to examine genetic diversity. For the morphological assessment, live microscopy and Protargol staining were used. The mitochondrial marker revealed six robust, deeply diverging, and strongly supported clades, while the nuclear gene was congruent for three of these clades. There were no major differences among individuals from the different clades in any of the morphological features examined. Thus, the underlying genetic diversity in C. polypinum is greater than what its morphology suggests, indicating that morphology and genetics are not congruent in this organism. Furthermore, because the clades identified by the mitochondrial marker are so genetically diverse and are confirmed by a conserved nuclear marker in at least three cases, we propose that C. polypinum be designated as a "cryptic species complex." Our results provide another example where species diversity can be underestimated in microbial eukaryotes when using only morphological criteria to estimate species richness.     

Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles

The assembly pathway of small nuclear ribonucleoprotein (snRNP) particles in the cytoplasm of L929 mouse fibroblasts was analyzed by observing the nuclear accumulation of snRNP proteins. Immunoprecipitations of nuclear and cytoplasmic fractions after a pulse label and chase indicate that the snRNP D, E, F, and G proteins assemble first, followed by the small nuclear RNA (snRNA), then the snRNP B protein and, in the case of the U1 snRNP, the A and C proteins. The snRNP B' protein is not detected in the L929 cells. The U1-specific A and C proteins can enter the nucleus in the absence of snRNP assembly, suggesting that these proteins exchange on the mature nuclear snRNP particles. Two-dimensional electrophoresis using nonequilibrium pH gradient electrophoresis identifies the A, B, B", C, D, E, F, and G proteins in a distribution similar to that reported previously by immunoprecipitation (Sauterer, R. A., and Zieve, G. W. (1989) J. Biol. Chem., submitted for publication). The D protein appears in multiple isoelectric variants in the cytoplasm and shifts toward more basic variants during maturation. Kinetic experiments analyzed by two-dimensional electrophoresis indicate a quantitative maturation of the cytoplasmic B protein into nuclear particles. Quantitative densitometry of immunoprecipitated stable nuclear snRNPs labeled with [35S] methionine corrected for the published methionine content of the A, B, C, D, and E proteins indicates that the mature nuclear U1 snRNP probably contains four copies of D, two copies each of B, C, and A, and one copy of E.     

An Optimized Protocol of Protargol Staining for Ciliated Protozoa

Protargol staining is a crucial method to reveal the infraciliature of ciliates, which is the most important morphological character for species identification. In the present study, Wilbert's protocol of protargol staining was emended mainly toward the highly happened improper bleaching. Through reciprocal treatments, both insufficient and excessive bleachings were much eliminated from the protargol protocol and the tests performed with four different species of ciliates established that the stainings were considerably improved and more reliable with optimized bleaching. Compared to the original protocol, the optimized method was proved to be more suitable for the groups difficult to stain, and it is also friendlier for the beginners and researchers in related fields.     

Staining Flagellate Protozoa by Various Silver-Protein Compounds

Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites. The samples consisted of one of Protargol (Winthrop's prewar German), one of Protargol S (Winthrop-Stearns, Inc., present manufacture, Commission Certified), and five of the experimental batches (4Z, 20, 20B, 22, and 36) by H. A. Davenport and collaborators (1952). The pre-war Protargol is rated best and batch 22 second best for staining of all flagellates. Protargol S gave uniform, but only fair results, with all organisms, while batch 20B, better than Protargol S in several instances, was poorer in a few. Thus Protargol S and batch 20B are rated third, as about equal. Batch 4Z is rated fourth; and batch 20, which stained some species, fifth. Batch 36 stained no protozoa. The tests show that while the present Protargol S is usable for protozoa, it is still inferior to the old German variety. Since some of the experimental batches, none of which was made by exactly the same process, gave promising results, further study of the relation between manufacturing process and subsequent staining reaction should be fruitful.     

Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol

Protargol (silver proteinate) impregnation is a common method used to identify and characterize ciliated protozoa. Unfortunately, chemical companies have stopped producing the ‘strong’ protargol powder used in this method. Based on an in-house protocol for its synthesis published in 2013, more than 10 batches of protargol powder were produced and subsequently applied in taxonomic studies. During these studies, the protocol for protargol powder synthesis was slightly modified and employed a peptone not originally listed in the 2013 protocol. This modification improved the results of the impregnation protocol. Protargol preparations of hypotrichs were optimized by adjusting the pH during staining rather than during the synthesis. The pH was adjusted to 7.5–7.6, and an acetone developer was used. While the conditions used in this study are not completely comparable to those using the commercially produced protargol, access to this information could help researchers investigate the diversity of ciliates, particularly hypotrichs.     

Staining Flagellate Protozoa by Various Silver-Protein Compounds

Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites. The samples consisted of one of Protargol (Winthrop's prewar German), one of Protargol S (Winthrop-Stearns, Inc., present manufacture, Commission Certified), and five of the experimental batches (4Z, 20, 20B, 22, and 36) by H. A. Davenport and collaborators (1952). The pre-war Protargol is rated best and batch 22 second best for staining of all flagellates. Protargol S gave uniform, but only fair results, with all organisms, while batch 20B, better than Protargol S in several instances, was poorer in a few. Thus Protargol S and batch 20B are rated third, as about equal. Batch 4Z is rated fourth; and batch 20, which stained some species, fifth. Batch 36 stained no protozoa. The tests show that while the present Protargol S is usable for protozoa, it is still inferior to the old German variety. Since some of the experimental batches, none of which was made by exactly the same process, gave promising results, further study of the relation between manufacturing process and subsequent staining reaction should be fruitful.     

Evidence against the incorporation into protein of amino acids directly from the membrane transport system in rat heart

The possibility suggested recently [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433; and Adamson, L.F., Herington, A.C. and Bornstein, J. (1972) Biochim. Biophys. Acta 282, 352-365] that protein synthesis takes place using amino acids directly from the membrane transport system and not from an intracellular pool has been investigated in rat heart. The tissue was perfused first for 30 min with either [14C]glycine or [14C]leucine and then for a further 30 min with identical medium containing [3H]glycine or [3H]leucine, respectively. After an initial lag, [14C]glycine was incorporated into protein at a linear rate up to 60 min. The [3H]glycine was accumulated into tissue water and incorporated just as readily as the [14C]glycine had been. The rate of total protein synthesis agrees with literature values only if intracellular and not extracellular specific activity values are used in the calculation. Some glycine was converted to serine or threonine. Leucine influx and efflux were very rapid in contrast to the relatively slow exchange reported for incubated tissues [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433]. The results are consistent with the existence of an intracellular precursor pool for glycine. Some possible reasons for the discrepancies between this and the other studies are discussed.     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation

We have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well‐characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized β–carotene ketolase gene optimized for maize codon usage, and a wild‐type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports (Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O'Neill, J. and Li, L. ( 2008 ) J. Exp. Bot. 59, 213–223; Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Küpper, H., Earle, E.D., Cao, J. and Li, L. ( 2006 ) Plant Cell 18, 3594–3605), we found that the wild‐type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm‐specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.     

The phylogenetic problem of Huia (Amphibia: Ranidae)

A taxonomic consensus for the diverse and pan-global frog family Ranidae is lacking. A recently proposed classification of living amphibians [Frost, D.R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006. The amphibian tree of life. B. Am. Mus. Nat. Hist. 297, 1-370] included expansion of the Southeast Asian ranid frog genus Huia from seven to 47 species, but without having studied the type species of Huia. This study tested the monophyly of this concept of Huia by sampling the type species and putative members of Huia. Molecular phylogenetic analyses consistently recovered the type species H. cavitympanum as the sister taxon to other Bornean-endemic species in the genus Meristogenys, rendering all previously published concepts of Huia as polyphyletic. Members of Huia sensu [Frost, D.R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006. The amphibian tree of life. B. Am. Mus. Nat. Hist. 297, 1-370.] appear in four places within the family Ranidae. A clade containing the type species of Odorrana is phylogenetically unrelated to the type species of Huia, and Odorrana is removed from synonymy with Huia. These findings underscore the need to include relevant type species in phylogenetic studies before proposing sweeping taxonomic changes. The molecular phylogenetic analyses revealed a high degree of homoplasy in larval and adult morphology of Asian ranid frogs. Detailed studies are needed to identify morphological synapomorphies that unite members in these major clades of ranid frogs.     

Enzymatic cleavage of peptide-linked radiolabels from immunoconjugates.

We have incorporated peptides selected by combinatorial library [Peterson, J. J., and Meares, C. F. (1998) Bioconjugate Chem. 9, 618-626) into peptide-linked radiolabeled immunoconjugates of the form DOTA-peptide-antibody. Decapeptide linkers -GFQGVQFAGF- and -GFGSVQFAGF-, selected for cleavage by human liver cathepsin B, were rapidly digested in vitro when compared to the simple model tetrapeptide motif of the prototype -GGGF- [Li, M., and Meares, C. F. (1993) Bioconjugate Chem. 4, 275-283]. Cleavage properties of these library-selected substrates for cathepsin B compared favorably with decapeptide linkers -GLVGGAGAGF- and -GGFLGLGAGF-, which incorporate two of the most labile extended cathepsin B substrates from the literature. The decapeptide linker -GFGSTFFAGF-, selected from the library for cleavage by human liver cathepsin D, was rapidly digested by cathepsin D while the others were not.     

An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli

The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.     

Ubiquitination of free cyclin D1 is independent of phosphorylation on threonine 286

Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.     

Characterization of the regulatory thioredoxin site of phosphoribulokinase

Phosphoribulokinase is light-regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/disulfide groups. To identify the cysteinyl residues that are involved in regulation, the S-carboxymethyl labeling patterns of the fully reduced (active) and oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced, [14C]carboxymethylated enzyme contained four labeled peptides, all of which were purified and sequenced by Edman degradation. If the enzyme was oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid) prior to carboxymethylation and tryptic digestion, only two labeled peptides were observed, thereby revealing the identity of the regulatory cysteines as Cys-16 and Cys-55. The former was previously implicated as part of the nucleotide-binding domain of the active site (Porter, M.A., and Hartman, F.C. (1986) Biochemistry 25, 7314-7318), a conclusion reinforced by the present observation that the sequence around the Cys-16 is similar to a consensus sequence of ATP-binding sites from a number of proteins of diverse phylogenetic origin (Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermondson, M. (1986) Nature 323, 448-450). The regulatory disulfide of phosphoribulokinase was found to be intrasubunit based on the stoichiometry of the oxidation and the failure to resolve oxidized and reduced enzyme by gel filtration under dissociation conditions.     

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).     

Interactions in hypoxic and hypercapnic breathing are genetically linked to mouse chromosomes 1 and 5.

The genetic basis for differences in the regulation of breathing is certainly multigenic. The present paper builds on a well-established genetic model of differences in breathing using inbred mouse strains. We tested the interactive effects of hypoxia and hypercapnia in two strains of mice known for variation in hypercapnic ventilatory sensitivity (HCVS); i.e., high gain in C57BL/6J (B6) and low gain in C3H/HeJ (C3) mice. Strain differences in the magnitude and pattern of breathing were measured during normoxia [inspired O(2) fraction (Fi(O(2))) = 0.21] and hypoxia (Fi(O(2)) = 0.10) with mild or severe hypercapnia (inspired CO(2) fraction = 0.03 or 0.08) using whole body plethysmography. At each level of Fi(O(2)), the change in minute ventilation (Ve) from 3 to 8% CO(2) was computed, and the strain differences between B6 and C3 mice in HCVS were maintained. Inheritance patterns showed potentiation effects of hypoxia on HCVS (i.e., CO(2) potentiation) unique to the B6C3F1/J offspring of B6 and C3 progenitors; i.e., the change in Ve from 3 to 8% CO(2) was significantly greater (P < 0.01) with hypoxia relative to normoxia in F1 mice. Linkage analysis using intercross progeny (F2; n = 52) of B6 and C3 progenitors revealed two significant quantitative trait loci associated with variable HCVS phenotypes. After normalization for body weight, variation in Ve responses during 8% CO(2) in hypoxia was linked to mouse chromosome 1 (logarithm of the odds ratio = 4.4) in an interval between 68 and 89 cM (i.e., between D1Mit14 and D1Mit291). The second quantitative trait loci linked differences in CO(2) potentiation to mouse chromosome 5 (logarithm of the odds ratio = 3.7) in a region between 7 and 29 cM (i.e., centered at D5Mit66). In conclusion, these results support the hypothesis that a minimum of two significant genes modulate the interactive effects of hypoxia and hypercapnia in this genetic model.