Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli

Summary The Escherichia coli enterotoxin STII gene is carried by Tn4521. The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2. In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition. The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition. Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition. This protein may be the transposase of Tn4521. A pair of 11 bp repeat sequences flanking the ORF was also found to be important. The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region. The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.     

Specific substrates for isolation and differentiation of Azotobacter vinelandii

Summary Resorcinol, ethylene glycol and glutaric acid have been found to be specific substrates for the enrichment of Azotobacter vinelandii in the presence of other Azotobacter species. The three compounds may also be used for species differentiation in the genus Azotobacter. In contrast to other reports strains of A. chroococcum and A. beijerinckii have been isolated which are able to use L-rhamnose for growth.Conditions for the production of a yellow pigment and green fluorescence by A. vinelandii from non-aromatic substrates on agar-plates have been tested.The production of water soluble yellow pigments in benzoate containing media cannot be used as a criterium for the presence of A. vinelandii. The pigment seems to be -hydroxy-muconic semialdehyde, the splitting product resulting from metacleavage of catechol. It may be formed by all Azotobacter species that can metabolize aromatic substrates.     

The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin

(1)Where grinding or maceration of plant tissue is impractical, immersion in methanol should be used. (2)The adaption to aqueous methanol of existing techniqus for distinguishing chlorophyll and phaeophytin in aqueous acetone was studied in detail. In methanol the spectra of both phaeophytin a and b were found to be pH sensitive. A method developed involving changes in absorbance at 665 nm is less precise than similar methods using acetone. (3)The spectra of phaeophytin b in methanol at different pH levels are anomalous. Changes in absorbance between 440 and 410 nm cannot be used for the estimation of phaeophytin. (4)Plant pigments can be transferred from methanol to aqueous acetone without degradation of chlorophyll. Modified standard techniques may then be used to measure chlorophyll a and phaeophytin a content.     

A cytological study of Pellia epiphylla (L.) Corda in Britain with reference to the status of Pellia borealis Lorbeer

Abstract

The history of Pellia borealis is outlined and its morphology compared with that of P. epiphylla. The techniques for preparing material for chromosome counts are described. Several gatherings of monoecious Pellia with large epidermal cells (P. borealis) were found to be haploid, n = 9. The size of the epidermal cells is shown to be valueless as a character for distinguishing the two species. It is concluded that only P. epiphylla should be retained as a species.

We wish to record our thanks to Dr S. Arnell for examining British material and for helpful correspondence; to Dr A. J. E. Smith for his valuable advice and criticism, and for the use of cytological facilities; and to all those who have contributed material of possible P. borealis.     

Inheritance and mapping of isoenzymes in pea (Pisum sativum L.)

Summary Three isoenzyme systems (amylase, esterase and glutamate oxaloacetate transaminase) were examined in seeds of pea (Pisum sativum L.) and shown to give clear variation in their band patterns on gel electrophoresis between different lines. The inheritance of these isoenzyme systems, and the location of their genes on the pea genome was investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase was found to be on chromosome 2, linked to the loci k and wb (wb ... 9 ... k ... 25 ... Amy). The gene for esterase was found to be linked with the gene Br (chromosome 4) but the exact location is uncertain because of the lack of the morphological markers involved in the cross. The gene for glutamate oxaloacetate transaminase was found to be on chromosome 1 and linked with the loci a and d (a... 24... Got... 41 ... d).     

Construction of Rhodococcus expression vectors and expression of the aminoalcohol dehydrogenase gene in Rhodococcus erythropolis

NADP⁺-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 produces double chiral aminoalcohols, which are used as pharmaceuticals. However, the genetic manipulation of Rhodococcus strains to increase their production of such industrially important enzymes is not well studied. Therefore, I aimed to construct Rhodococcus expression vectors, derived from the Rhodococcus–Escherichia coli shuttle vector pRET1102, to express aadh. The plasmid pRET1102 could be transformed into many actinomycete strains, including R. erythropolis. The transformation ef?ciency for a species closely related to R. erythropolis was higher than that for other actinomycete strains. Promoters of various strengths, hsp, 1200rep, and TRR, were obtained from Gram-positive bacteria. The activity of TRR was stronger than that of hsp and 1200rep. The aadh-expressing plasmid pRET1172 with TRR could be transformed into many actinomycete strains to increase their AADH production. The Rhodococcus expression vector, pRET11100, constructed by removing aadh from the pRET1172 plasmid may be useful for bioconversion.     

Electrophoretic karyotypes of some related Mucor species

Contour clamped homogeneous electric field (CHEF) gel electrophoresis was used to obtain electrophoretic karyotypes from nine Mucorstrains representing five different species (M. bainieri, M. circinelloides, M. mucedo, M. plumbeus and M. racemosus). The chromosomal banding patterns revealed high variability among the isolates. The sizes of the DNA in the Mucor chromosomes were estimated to be between 2.5 and 8.7 Mb. The total genome sizes were calculated to be between 30.0 and 44.7 Mb. The applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation is considered.     

Mineral salt requirements of Bacillus globisporus subsp. marinus strains

The mineral salt requirements of four isolates of Bacillus globisporus subsp. marinus and of two terrestrial strains of B. globisporus were investigated. In contrast to the terrestrial reference strains the marine isolates showed an obligate requirement for sodium and potassium. The sodium ion could not be replaced by potassium or by osmotically equivalent concentrations of mannitol. None of the strains proved to be dependent upon Mg²⁺ or Ca²⁺. The B. globisporus subsp. marinus strains are considered true marine bacteria. The criteria used to distinguish between marine and terrestrial Gram-negative bacteria can also be applied for Gram-positive bacteria.     

Mapping of the ras1 gene of Schizosaccharomyces pombe

Summary The ras1 gene, an oncogene homologue, is known to be essential for recognition of the mating pheromone and hence for conjugation but not for vegetative growth in Schizosaccharomyces pombe. To facilitate further characterization and genetic manipulation of this gene, we have mapped it by using S. pombe strains which carry the Saccharomyces cerevisiae LEU2 gene inserted next to ras1 on the chromosome. Crosses with tester strains revealed that ras1 is tightly linked to pro2 on chromosome I. Furthermore, we have shown that ras1 is allelic with ste5, one of the sterility genes described by O. Girgsdies. The map position previously reported for ste5 eventually turned out to be false.     

Genetics of the peroxidase isoenzymes inPetunia

Summary The structural geneprxD inPetunia codes for a slow moving anodic peroxidase whose activity is sensitive to high concentrations of hydrogen peroxide. The PRXd enzyme could be found in mature and old leaf and stem tissue of full-grown flowering plants. PRXd was found to be absent in tissues from flower corolla and root. The geneprxD is the fourth gene that codes for peroxidases in leaf and stem. Two mobility variants of the PRXd enzyme have been found among our inbred lines using starch gel system II electrophoresis. The geneprxD could be located on chromosome III by a four-point-cross involving the genesprxA, prxD, Mf1 andHt1. The order of the genes established is:Ht1 — Mf1 — prxD — prxA.     

Essential Oils of Micromeria dalmatica Benth., a Balkan Endemic Species of Section Pseudomelissa

The essential oils of 13 Greek populations of Micromeria dalmatica, a Balkan endemic species and member of the section Pseudomelissa, were examined for the first time. Among the studied populations, two main oil types could be distinguished. Type I was found to be rich in β‐pinene, limonene, and germacrene D (accounting for 55.6–70.2% of the total oil), and Type II was characterized by the preponderance of p‐menthane compounds (accounting for 64.2–89.9% of the oil). The latter oil type could be further divided into two subtypes, one comprising oils with predominance of piperitenone and piperitenone oxide and another composed of oils containing high proportions of pulegone, menthone, and isomenthone. The abundance of p‐menthane compounds is a common feature of the oils of all members of the section Pseudomelissa studied to date. However, the existence of oils of Type I has not been previously reported for M. dalmatica, neither for other members of the section Pseudomelissa.     

The linkage relations of thecochleata mutant inPisum

Thecochleata mutant inPisum is a monogenic recessive characteristic. The relations between the geneCoch and 15 well known other genes were determined.Coch is linked toR and toTl and this bringsCoch intoLamprecht's linkage-group VII.Coch was also found to be linked toGp and this is not in harmony with the above conclusion, sinceGp belongs to linkage-group V. The only possibility for an explanation at hand seems to be a translocation. This possibility could not be tested in the present material.Publication 234.     

The taxonomic history of the family Lecanicephalidae Braun, 1900, a little known group of marine cestodes

Parataenia Linton cannot exist as a cestode genus as it is preoccupied. Discobothrium van Beneden is not a junior synonym of Echeneibothrium van Beneden, and Hornellobothrium Shipley & Hornell and Eniochobothrium Shipley & Hornell are very likely not junior synonyms of Discobothrium. Hexacanalis Perrenoud should be considered a junior synonym of Cephalobothrium Shipley & Hornell. Yogeshwaria Chincholikar & Shinde, Spinocephalum Deshmukh and Flapocephalus Deshmukh are considered unrecognisable. Staurobothrium Shipley & Hornell should be returned to the Phyllobothriidae. Balanobothrium Hornell should remain in the Onchobothriidae Braun. The order Lecanicephalidea was not established by Baylis. There are no grounds for recognising the superfamily Lecanicephaloidea Southwell which contains only the Lecanicephalidae Braun. The elevation of the lecanicephalids to an order, the Lecanicephala, does not appear to be valid. To avoid further confusion the scheme set out by Braun (1900) should be used as a basis for classification of the lecanicephalids.     

Astragalus trifoliastrum (Fabaceae), a revived species for the flora of Turkey

As the result of surveying the relevant type specimens, together with macro‐ and micro‐morphological studies, chromosome counting and ITS sequencing, Astragalus trifoliastrum was found to be a species independent of A. laguriformis (with which it has peviously been synonymized). In contrast, A. wanensis, assumed to be a synonym of A. trifoliastrum, indeed appears to be identical with A. trifoliastrum. The diploid chromosome number of 2n = 16 is reported for the first time for A. trifoliastrum.     

Inhibition of Chlorella growth by the lipids of cyanobacterium Microcystis aeruginosa

The total lipids of the cyanobacterium Microcystis aeruginosa have been isolated and fractionated into its components. Of these lipid components, only the fatty acid-containing fraction inhibited the growth of the green alga Chlorella pyrenoidosa. The inhibitory activity appears to be due to linoleic and linolenic acids, which are both present in significants quantities. These acids may be the substances responsible for the reported toxicity of Microcystis aeruginosa to Chlorella.     

Evaluation of agrobacterium-mediated transformation of agricus bisporus using a range of promoters linked to hygromycin resistance

There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trip2 promoters. The Coprinus cinereus β-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp 1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with ALG-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.     

Genotype dependent radiosensitivity of autotetraploids in Trigonella foenum-graecum L.

Summary Primary trisomics were used to locate the structural loci coding for particular forms of the dimeric enzymes phosphoglucoisomerase and glutamate oxaloacetate transaminase in Lolium perenne. The polymorphy of these loci enabled triallelic trisomics to be produced. Each locus could thus be directly assigned to a particular chromosome without the need to examine segregant progeny. The loci for GOT/3 and PGI/2 were found to be located on chromosomes 2 and 6 respectively.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

Physiological and Optical Responses of the Harmful Dinoflagellate Heterocapsa circularisquama to a Range of Salinity

The responses of division rate, cell volume, cellular carbon (C) and nitrogen (N), cellular pigments and optical characteristics to changes in salinity were examined in the dinoflagellate Heterocapsa circularisquama. The physiological and optical characteristics of H. circularisquama were significantly affected by changes in salinity. When cells were exposed to different salinities, they exhibited physiological acclimation by adjusting their cellular properties associated with growth. This could be a beneficial tactic for ensuring proliferation and limiting damages induced by adverse environmental factors. The results of this study could be essential for assisting in the development of growth models for H. circularisquama.     

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.