Biochemical genetics of Es-14 (formerly Es-Si) and a new esterase variation,Es-15, of the laboratory rat (Rattus norvegicus): Biochemistry,tissue expression,and linkage to Es-1 in linkage group V

The segregation of rat esterases controlled by loci residing in linkage group V (LGV) has been studied in two backcross series, (LEW/Han × BN/Han)F₁ × LEW/Han and (LEW/Han × LE/Han)F₁ × LEW/Han. Es-14 (formerly Es-Si) was shown to be closely linked to Es-1. A new esterase locus, Es-15, was described which codes for a liver isozyme. The distribution pattern of three alleles at the Es-15 locus is presented for 52 independent inbred strains. Close linkage of Es-15 to Es-14 and to Es-1 was established, proposing the following gene order: [Es-2, Es-10]—[ES-1, ES-14, ES-15]. The esterase loci on LGV are thus separated into two gene clusters, cluster 1 and cluster 2. These conclusions are supported by the strain distribution patterns of the two RI strain series, LXB and DXE.Otto von Deimling was supported by the Deutsche Forschungsgemeinschaft (De 315/2-1, communication No. 56).     

Polymorphism of esterase 11 in Mus musculus,a further esterase locus on chromosome 8

A further esterase, esterase 11, which exhibits a polymorphism detectable by electrophoresis, has been observed in the house mouse, Mus musculus. In 15 inbred strains and two outbred strains, the ES-11A phenotype has been found, composed of two bands of enzyme activity of greater anodal electrophoretic mobility than the two bands of the ES-11B phenotype found in one inbred strain, one wild stock, and 101 wild mice. In F₁ hybrids (IS/Cam×C57 BL/Gr), the phenotype shown corresponds to a mixture of the two parental phenotypes. In backcrosses, ES-11 segregates as an autosomal gene, designated Es-11, closely linked to Es-2 and Es-5 on chromosome 8.This work was supported by the Medical Research Council.     

Antifungal compounds from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus

The cell-free culture filtrate (CCF) was prepared from a culture of an Aspergillus flavus antagonist, Bacillus subtilis B-FS06. The CCF inhibited the growth and spore germination of A. flavus at a series of concentrations (10, 25, 50%) (v/v). It still retained the activity after treatment at pH values ranging from 2 to 12 for 24 h or at 100 °C for 30 min. The antifungal activity, however, was reduced by 30% after treatment at 121 °C for 20 min. After purification by anion exchange chromatography, gel filtration chromatography and HPLC, the active compounds revealed six ion peaks: [M–H] m/z = 1006.78, 1020.71, 1034.74, 1049.54, 1056.78, and 1071.64 by using electrospray ionization mass spectrometry (ESI-MS) analysis. In the presence of the active compounds at 200 μg/g, the growth of A. flavus on peanuts was completely inhibited. Ting Zhang and Zhi-Qi Shi contributed equally to this work.     

Genetic control of enzyme polymorphisms in the California vole,Microtus californicus

Inheritance of 15 polymorphic isozymes was investigated in captive Microtus californicus. Eleven of the isozymes show patterns consistent with a Mendelian model of inheritance: glycerol-3-phosphate dehydrogenase (GPD), lactate dehydrogenases A and B (LDH-A and LDH-B), malic enzyme 2 (ME-2), isocitrate dehydrogenase 1 (ICD-1), phosphogluconate dehydrogenase (PGD), glutamate-oxaloacetate transaminase 1 (GOT-1) phosphoglucomutase 2 (PGM-2), leucine aminopeptidase (LAP), glucosephosphate isomerase (GPI), and esterase 2 (ES-2, from kidney). four of the isozymes show patterns that cannot be interpreted by a simple genetic model: esterases 1 and 4 (ES-1, ES-4, from hemolysate), esterase 3 (ES-3, from plasma), and protein 1 (PT-1). The following pairs of loci are assorting independently: LAP and PGD, LAP and PGM-2, GOT-1 and PGD, GOT-1 and GPD, LAP and GPD, GPD and PGD, GPI and PGD. Data from one test cross mating indicate that GPD and PGM-2 are loosely linked with recombination about 30%. Additional data are needed to confirm this relationship.This study was conducted while the first author was the recipient of an NIH Traineeship. The Departments of Genetics and Zoology provided financial support for the maintenance of the animal colony. This work was supported in part by an NIH-Biomed grant (3-S05-RR-07006-08S1) to W. Z. Lidicker.     

Polymorphism of esterase-10 in Mus musculus

An esterase, esterase-10, in the house mouse, Mus musculus, is specific for esters of 4-methylumbelliferone and exhibits a polymorphism detectable by electrophoresis. Fifteen inbred strains and two outbred strains have been examined for this polymorphism, and two phenotypes, ES-10A and ES-10B, have been observed. Each phenotype manifests itself as a single band of enzyme activity, but under the electrophoretic conditions used the ES-10A phenotype has less anodal electrophoretic mobility than the ES-10B phenotype. In F₁ hybrids (C3H/He/Lac×C57BL/Gr) a third phenotype was observed, ES-10AB, consisting of three bands of enzyme activity, two of which correspond to the parental forms and the third with intermediate mobility. The triple-band pattern in the F₁ hybrids indicates that esterase-10 is a dimeric enzyme protein.This work was supported by the Medical Research Council.     

Esr,a second locus in the house mouse controlling esterase-5

Electrophoretic variation characterized by the presence (ES-5B⁺) or absence (ES-5B^–) of esterase-5B in the plasma of the house mouse has been observed. It is suggested that the expression of esterase-5B is controlled by an autosomal locus, Esr, linked to Ldr-1 on chromosome 6, in addition to the presumptive structural locus Es-5, which is located on chromosome 8. A gene order of Lyt-3-Esr-Ldr-1 was determined by two crosses.Supported by the Deutsche Forschungsgemeinschaft (SFB 46).This is communication No. 33 of a research program devoted to the investigation of cellular distribution and genetics of nonspecific esterases.     

Genetically defined lysosomal acetylesterase EC 3.1.1.6 in the cauda epididymidis of mouse,rat, and man

After inhibition by bis-p-nitrophenyl phosphate and subsequent staining for esterase using naphthol AS-D acetate as the substrate, a strong lysosomal esterase was demonstrated in the cauda epididymidis of mouse, rat, and man. Owing to its behaviour towards the classifying inhibitors eserine, diisopropyl fluorophosphate, bis-p-nitrophenyl phosphate, and p-chloromercuriphenylsulphonate, this lysosomal esterase was shown to be an acetylesterase (EC 3.1.1.6). Control experiments involving isoelectric focusing revealed that this acetylesterase was identical with the genetically defined homologues ES-17, ES-6, and ES-A4 in mouse, rat, and man, respectively.     

Responses of alfalfa pollen and callus to filtrates from two isolates of Fusarium oxysporum

Summary For 25 Medicago sativa plants, measurements were made of in vitro pollen germination and growth and callus growth on mediums containing culture filtrate from two isolates of Fusarium oxysporum f. sp. medicaginis. In vivo resistance was determined by field inoculation with one isolate. There was no correlation between any in vitro measurement and in vivo resistance, and none of the in vitro measurements on control mediums were correlated. The only significant correlation for the same measurement between the two isolates was for pollen-tube length (r = 0.65). Percent of pollen germination was negatively correlated with callus growth for both isolates. Earlier work showed that callus growth in the presence of culture filtrate was linked to plant resistance, thus it appeared that percent pollen germination on culture filtrate had decreased for the more resistant plants. The haploid pollen may be used as a progeny test for identifying heterotic loci conferring resistance to Fusarium, but if the pollen of this plant species is used in direct selection by exposure to culture filtrate, the level of resistance to Fusarium may decrease.     

Esterase-30 (ES-30) of the house mouse: Biochemical characterization and genetics of a new carboxylesterase isozyme linked to cluster-2 loci on chromosome 8

A new carboxylesterase isozyme (EC 3.1.1.1), designated ES-30, is described in mouse liver. Two phenotypes were distinguished, ES-30A, a possible null type, was found in SPE/Pas and in other lines derived fromMus spretus, and ES-30B was found in BALB/cJ and other laboratory inbred strains. ES-30B is characterized by a distinct electrophoretic band when stained using 5-bromoindoxyl acetate as the substrate. After isolation and purification from other esterases by ion-exchange chromatography and molecular sieving, the molecular mass was estimated by two independent methods to be 62 and 64 kDa, respectively. The activity of ES-30B is higher in adult males than in females and can be stimulatedin vivo by testosterone. The distribution of phenotypes on the progeny of a backcross series suggests a separate locus,Es-30, with the allele a for absence andb for presence of the isozyme. LocusEs-30 is shown to be closely linked toEs-2 and toEs-7 of cluster-2 on chromosome 8. The gene orderEs-9—Got-2—(Es-2, Es-7, Es-30) is suggested. This work was supported by the Deutsche Forschungsgemeinschaft. This is communication No. 72 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Study of the antifungal activity of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> LCH001 in vitro and identification of its antifungal components

An Acinetobacter strain, given the code name LCH001 and having the potential to be an endophytic antagonist, has been isolated from healthy stems of the plant Cinnamomum camphora (L.) Presl, guided by an in vitro screening technique. The bacterium inhibited the growth of several phytopathogenic fungi such as Cryphonectria parasitica, Glomerella glycines, Phytophthora capsici, Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani. Biochemical, physiological, and 16S rDNA sequence analysis proved that it is Acinetobacter baumannii. When the filtrate from the fermentation broth of strain LCH001 was tested in vitro and in vivo, it showed strong growth inhibition against several phytopathogens including P. capsici, F. graminearum, and R. solani, indicating that suppression of the growth of the fungi was due to the presence of antifungal compounds in the culture broth. Moreover, the antifungal activity of the culture filtrate was significantly correlated with the cell growth of strain LCH001. The active metabolites in the filtrate were relatively thermally stable, but were sensitive to acidic conditions. Three antifungal compounds were isolated from the culture broth by absorption onto macropore resin, ethanol extraction, chromatography on silica gel or LH-20 columns, and crystallization. The structures of the bioactive compounds were identified by spectroscopic methods as isomers of iturin A, namely, iturin A2, iturin A3, and iturin A6. The characterization of an unusual endophytic bacterial strain LCH001 and its bioactive components may provide an alternative resource for the biocontrol of plant diseases.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Genetic variation and biochemical properties of esterase-18 (ES-18) in the laboratory rat (Rattus norvegicus): A new locus of esterase cluster 2 in linkage group V

A new liver-specific rat carboxylesterase isozyme (EC 3.1.1.1) designated esterase-18 (ES-18) is described. Genetic variation of ES-18 was examined in 93 inbred strains and substrains and a structural locusEs-18 was suggested, coding for either the presence (Es-18 ^a) or the absence (Es-18 ^b) of the isozyme. Linkage studies involving two backcross series revealed thatEs-18 resides in cluster 2 of LGV. No recombination betweenEs-18 and other cluster 2 loci was found in 19 lines of two RI strain sets or in the backcross series.R. K. was supported by the Sonderforschungsbereich 146 (Versuchstierforschung). O.D. was supported by the Deutsche Forschungsgemeinschaft (De 315/2). This is communication No. 65 of a research program devoted to the cellular distribution, regulation, and genetics of nonspecific esterases.     

Ion measurements by X-ray microanalysis in unfixed,frozen, hydrated plant cells of species differing in salt tolerance

A technique is described for X-ray microanalysis of unfixed, frozen, hydrated higher plant cells using a scanning electron microscope in conjunction with a cryostage. Freezing in liquid N₂ is the only preparative step required. Using this method, ion distribution was compared in the roots of Zea mays L. (termed a salt excluder) and Hordeum vulgare L. (which is rather more tolerant), both grown in the presence of NaCl. Distinct differences were observed between the two species in Na, K and Cl distribution. Evidence is presented to support the hypothesis that reabsorption of Na from the xylem sap in the mature regions of the root may occur in salt-sensitive glycophytes such as Z. mays.     

<Emphasis Type="Italic">AtTBP2</Emphasis> and <Emphasis Type="Italic">AtTRP2</Emphasis> in <Emphasis Type="Italic">Arabidopsis</Emphasis> encode proteins that bind plant telomeric DNA and induce DNA bending in vitro

Telomeric DNA-binding proteins (TBPs) are crucial components that regulate the structure and function of eukaryotic telomeres and are evolutionarily conserved. We have identified two homologues of AtTBP1 (for Arabidopsis thaliana telomeric DNA binding protein 1), designated as AtTBP2 and AtTRP2, which encode proteins that specifically bind to the telomeric DNA of this plant. These proteins show extensive homology with other known plant TBPs. The isolated C-terminal segments of these proteins were capable of sequence-specific binding to duplex telomeric plant DNA in vitro. DNA bending assays using the Arabidopsis TBPs revealed that AtTBP1 and AtTBP2 have DNA-bending abilities comparable to that of the human homologue hTRF1, and higher than those of AtTRP1 and AtTRP2.     

Importance of Nutrient Competition and Allelopathic Effects in Suppression of the Green Alga Scenedesmus obliquus by the Macrophytes Chara, Elodea and Myriophyllum

Possible allelopathic effects of substances released from the macrophytes Chara globularis, Elodea canadensis, Myriophyllum spicatum on the common green alga Scenedesmus obliquus were tested in the laboratory with plastic plants and untreated medium as controls. A two-phase approach was used in which first the effects of physical presence of plants was studied (phase I) followed by the effects of plant culture filtrates (phase II). In the presence of plastic plants growth was reduced only marginally, but strong growth inhibition of Scenedesmus occurred in the physical presence of all macrophytes. In contrast, filtrates from Chara had no growth inhibitory effect on Scenedesmus. Myriophyllum filtrate reduced particle-based growth rate by 7% compared to filtration controls, while Elodea culture filtrate reduced volume-based growth by 12%, chlorophyll-based growth by 28% and particle-based growth by 15%. Photosystem II-efficiency of Scenedesmus was reduced in all three macrophyte treatments in phase I, but not in filtrates from macrophyte cultures (phase II). Thus, while enzyme activity or other physiological aspects may have been affected, the current study yielded no proof for allelopathically active compounds being directed at photosynthesis. Mean particle volume (MPV) of Scenedesmus was not influenced by macrophyte exudates and cultures remained dominated by unicells. The strong growth inhibitory effects found for Scenedesmus in the physical presence of macrophytes, but not in plastic controls, and no or weaker response in nutrient-enriched filtrates, suggest nutrient competition was a more powerful driving factor than allelochemicals. However, the experimental design does not exclude disappearance of allelochemicals during the filtration process.     

AtHVA22 gene family in Arabidopsis: phylogenetic relationship,ABA and stress regulation,and tissue-specific expression

HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.). Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes. Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis. These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other. Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, bandc, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots. The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor. AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses. These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression. ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene. Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues. In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves. The expression level of these homologues in immature siliques is the lowest among all tissues analyzed. It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.     

Chemical evidence for the mechanism of the biodecoloration of Amaranth by <Emphasis Type="Italic">Trametes versicolor</Emphasis>

The white-rot fungus Trametes versicolor decolorized Amaranth. The hypothesis that the carbon structure of Amaranth was broken down in smaller mass fragments was investigated analyzing the products of decoloration. FTIR spectroscopy, ion chromatography, sulfite and ammonia analysis were used to compare the culture filtrate before dye addition, with the pure dye, the culture filtrate after dye addition, and the culture filtrate during the treatment. The hypothesis of polymerization of the decoloration products was tested by spectrophotometric analysis of dialysates of the pure dye, the culture filtrate before dye addition, and the culture filtrates after dye addition and decoloration. FTIR showed that the signals typical for the azo group disappeared after decoloration, while new peaks appeared that were characteristic of substituted naphthalenic or benzenic compounds. Ion chromatography showed that the level of sulfate in the treatment increased when compared with the level of the sulfate in control, suggesting that the sulfonic groups were being stripped from Amaranth’s structure and metabolized to sulfate. Sulfite measurements for the treatment and controls showed no significant difference, and were well below the saturation concentration for sulfite in water, confirming that the medium was aerobic. Ammonia concentration did not change with the decoloration. Absorbance scans after dialysis of decolorized samples showed no new peaks, suggesting that the decoloration products were not polymerized. These observations suggests that the decoloration mechanism starts with the azo link removal, followed by desulfonation, naphthalene ring opening, and the formation of smaller mass fragments, similar to fungal metabolites.     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

Esterase-17 (ES-17): Characterization and genetic location on chromosome 9 of a bis-p-nitrophenyl phosphate-resistant esterase of the house mouse (Mus musculus)

Genetic variation of a new codominantly inherited esterase, designated ES-17, has been discovered in the house mouse using isoelectric focusing in polyacrylamide gels. The ES-17 A phenotype (three bands; isoelectric points, betweenpH 5.55 andpH 5.90) was found in C57 BL/10Sn. LP/J possessed the Es-17B phenotype (three bands; isoelectric points,pH 5.05–5.55). ES-17 was present in all tissues examined, except for hemolysate and serum, and was most clearly expressed in the small intestine. Because of its reaction toward various substrates and inhibitors, ES-17 has tentatively been classified as acetyl esterase (EC 3.1.1.6). ES-17 was shown to be controlled by the structural locusEs-17, located on chromosome 9. From test-cross data, a gene order ofEs-17-8.7±2.5 map units-Mpi-1-10.2±2.7 map units-Mod-1 was established. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 46). This is communication No. 35 of a research program devoted to the cellular distribution and genetics of nonspecific esterases.