The β-tubulin gene from rat and human isolates of Pneumocystis carinii

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long-term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short-term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the beta-tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single beta-tubulin gene from a rat P. carinii isolate. There was 89-91% identity at the amino acid level to beta-tubulins from filamentous fungi, but only 79-82% identity to yeast and protozoal beta-tubulins. Also, eight introns were distributed throughout the P. carinii beta-tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. carinii beta-tubulin. The polymerase chain reaction was used to amplify a segment of P. carinii beta-tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the amino-acid level.     

Isolation of α-tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of α-tubulin

As a step towards identifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire alpha-tubulin gene (designated alpha-tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other alpha-tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the alpha-tubulin I gene itself was found, although a second gene designated alpha-II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The alpha-I and beta-tubulin genes were found to reside on different chromosomes.     

Isolation and developmental expression of tyrosinase family genes in Xenopus laevis

The tyrosinase family of genes in vertebrates consists of three related members encoding melanogenic enzymes, tyrosinase (Tyr), tyrosinase-related protein-1 (TRP-1, Tyrp1) and tyrosinase-related protein-2 (Dct, TRP-2, Tyrp2). These proteins catalyze melanin production in pigment cells and play important roles in determining vertebrate coloration. This is the first report examining melanogenic gene expression in pigment cells during embryonic development of amphibians. Xenopus provides a useful experimental system for analyzing molecular mechanisms of pigment cells. However, in this animal little information is available not only about the developmental expression but also about the isolation of pigmentation genes. In this study, we isolated homologues of Tyr, Tyrp1 and Dct in Xenopus laevis (XlTyr, XlTyrp1, and XlDct). We studied their expression during development using in situ hybridization and found that all of them are expressed in neural crest-derived melanophores, most of which migrate through the medial pathway, and in the developing diencephalon-derived retinal pigment epithelium (RPE). Further, XlDct was expressed earlier than XlTyr and XlTyrp1, which suggests that XlDct is the most suitable marker gene for melanin-producing cells among them. XlDct expression was detected in migratory melanoblasts and in the unpigmented RPE. In addition, the expression of XlDct was detected in the pineal organ. The sum of these studies suggests that expression of the tyrosinase family of genes is conserved in pigment cells of amphibians and that using XlDct as a marker gene for pigment cells will allow further study of the developmental mechanisms of pigment cell differentiation using Xenopus.     

Analysis of β-tubulin Gene Fragments from Benzimidazole-sensitive and -tolerant Cercospora beticola

Cercospora leaf spot of sugar beet, caused by the fungus Cercospora beticola, is a major foliar pathogen on sugar beet. Fungicide sprays have been used extensively to manage Cercospora leaf spot, including the benzimidazole fungicides. Resistance to benzimidazoles has been observed in isolates of C. beticola. The precise genetics of this resistance is not known in this fungus. We tested benzimidazole‐tolerant and ‐sensitive isolates and found a single mutation in the β‐tubulin gene of benzimidazole‐tolerant isolates that corresponds to a mutation known to confer benzimidazole tolerance in other ascomycetes. This mutation is predicted to cause a change from glutamic acid to alanine in the protein product. Isolates containing this mutation further show an increased sensitivity to an N‐phenylcarbamate, as would be predicted based on the mutant phenotype found in other filamentous fungi. Only a single mutation was found in isolates from different regions of the United States, isolated in different growing seasons.     

Cloning and sequence of a β-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy

This work describes the isolation and characterization of a full-length cDNA clone encoding beta-tubulin from the pathogen Pneumocystis carinii. P. carinii contains a single gene encoding beta-tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the beta-tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. carinii beta-tubulin and those of fungal beta-tubulins resistant to benomyl, a beta-tubulin-binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.     

Analysis of a maize α-tubulin gene promoter by transient expression and in transgenic tobacco plants

The pattern of expression directed by the promoter of the maize Tub α 1 gene was investigated by analysis of chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) activities in transient expression experiments of maize and tobacco protoplasts. The same promoter was also investigated by histochemical GUS analysis in transgenic tobacco plants containing promoter gene fusions. As determined by histochemical tests, the Tub α 1 promoter gene preferentially directs GUS expression in regenerating root tip meristems and pollen. This pattern corresponds to the distinctive features of natural expression of the gene in maize as determined by Northern analysis. However, no expression is observed in other meristematic tissues of the transgenic tobacco plants, as in shoot apex or in coleoptiles, which is weakly detected in maize. Analysis of the regulatory properties of 5' promoter deletions showed that the proximal region of the promoter, from positions −1410 or −449 to 15 bp upstream of the ATG, is sufficient to establish the qualitative pattern of expression in transgenic tobacco plants. Deletions to positions −352 or −117 abolished the expression in roots, but not in pollen, suggesting that upstream of these positions there are elements responsible for the pattern in root. Further deletions abolished all the promoter activity, suggesting that this promoter region contains the elements essential for expression in pollen. The different patterns and levels of transient and stable expression are discussed.     

α3, β2, and β4 Form Heterotrimeric Neuronal Nicotinic Acetylcholine Receptors in Xenopus Oocytes

Abstract: One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different β subunits assemble with an α subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for α3, β2, and β4 subunits. β2 and β4, in pairwise combination with α3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). α3β4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through α3β2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for α3, β2, and β4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both β2 and β4 subunits in functional receptors.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

Differential expression of β-conglycinin genes in nodulated and non-nodulated isolines of soybean

Nodulated (T202) and non-nodulated (T201) isolines of soybean (Glycine max [L.] Merr.) were cultivated in a rotated paddy field in Niigata, Japan. The pods, and seeds were harvested at 7-day intervals until maturity, and the subunit compositions of seed storage proteins were analyzed by SDS-PAGE. The β-subunit of β-conglycinin could scarcely be detected in the non-nodulated isoline, T201, at any period throughout seed development, although it was a major component in T202. The accumulation of α′- and α-subunits of β-conglycinin, together with the acidic and basic subunits of glycinin, appeared about one week later in seeds of T201 than in those of T202, perhaps due to a shortage of nitrogen and growth retardation. Northern hybridization could not detect the β-subunit mRNA in immature T201 seeds, while it was pronounced in T202. These results indicate that the suppression of the β-subunit in the non-nodulating isoline T201 is regulated at the level of mRNA accumulation. The α′(α)-subunit mRNAs were actively expressed in both isolines. Total nitrogen concentration was consistently lower in T201 than T202. No significant difference was observed in either the free amino acid or ureide concentrations in seeds, although the concentration of sucrose was considerably lower in T201 seeds and pods compared with T202. This result indicates the possibility that β-subunit accunmlation was regulated not only directly by total nitrogen concentration but also by carbohydrate concentrations. Nitrogen regulation of storage protein subunit levels of soybean seed were evaluated using T201 and T202. Greenhouse-grown plants were subjected to different levels and timing of nitrate treatments. The culture solution (2, 5 or 10 mM NO₃–was supplied from flowering, 42 days after planting (DAP), until maturation (137 DAP), or switched from 2 to 10 mM, or from 10 to 2 mM at 61 DAP. With a continuous 2 mM NO₃–treatment, seed dry weight and N concentration of the T201 plants were significantly lower than those in the T202 plants due to the lack of N₂ fixation by the non nodulated T201 plants. However, when adequate NO₃ was supplied, N concentration and dry weight were similar in T201 and T202 seeds. When 5 mM NO₃ was supplied, the subunit proportion of the seed storage protein was similar in non-nodulating and nodulating isolines. On the other hand, when plants received a low level of NO₃ (2 mM), the β-conglycinin proportion was lower in T201 than in T2O2. Furthermore, in the nodulating T202 plants treated with 10 mM NO₃–the proportion of β-conglycinin increased markedly. The results indicate that non-nodulated T201 has a normal, non-defective, β-subunit gene and that limited N availability decreases accumulation of β-conglycinin, whereas high N availability increases the proportion of β-conglycinin in soybean seeds, irrespective of whether N was derived from N₂ fixation or from NO₃ absorption.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Differential expression of two β-amylase genes of rye during seed development

The expression of two β-amylase loci was analysed in the developing seeds of two inbred lines of rye (Secale cereale L.), one of which was a β-amylase deficient mutant. Enzymatic activity and the contents of enzymatic protein and mRNA specific for each of an endosperm-characteristic and ubiquitous β-amylase were determined throughout the course of caryopsis development. Both loci were expressed in the developing normal line caryopses according to different temporal and quantitative patterns. The ubiquitous enzyme-specific locus β-Amy 2 was expressed earlier; both mRNA and enzymatic protein accumulated to a maximum extent at 10 to 15 days after pollination. In contrast, the highest content of mRNA for endosperm β-amylase (encoded by the β-Amy I locus) was found 20 days after pollination, and the corresponding enzymatic protein accumulated throughout seed development. The expression of the β-Amy I locus was 30- to 40-fold higher than that of the β-Amy 2 locus in terms of maximum specific mRNA accumulation. The expression product of only the β-Amy 2 locus was found in the developing mutant line caryopses. The expression pattern of this locus was similar in the developing normal and mutant line seeds in terms of the temporal accumulation of mRNA and enzymatic protein. However, an approximately 4-fold higher level of ubiquitous β-amylase-specific mRNA was found in the mutant than in the normal line caryopses, and the content of ubiquitous β-amylase protein decreased to near zero at seed maturity in the mutant line, but not in the normal line, caryopses. The enzymatic activities of both β-amylases appeared to be regulated at the level of accumulated enzymatic protein.     

Isolation of genes encoding β-D-xylanase, β-D-xylosidase and α-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B14

Abstract Prevotella ruminicola B₁4 is a strictly anaerobic, Gram-negative, polysaccharide-degrading rumen bacterium. Xylanase activity in this strain was found to be inducible, the specific activity of cells grown on xylan being increased at least 20-fold by comparison with cells grown on glucose. Ten bacteriophage clones expressing xylanase activity were isolated from a A EMBL3 genomic DNA library of P. ruminicola B₁4. These clones were shown to represent four distinct chromosomal regions, based on restriction enzyme analysis and DNA hybridisation. Three groups of clones encoded activity against oat spelt xylan but not carboxymethylcellulose (CMC). In one of these groups, represented by clone 5, activities against pNP-arabinofuranoside and pNP-xyloside were found to be encoded separately from endoxylanase activity. The fourth region encoded activity against CM cellulose and lichenan, in addition to xylan, and contains an endoglucanase/xylanase gene isolated previously.