Sex-hormones in Neurospora crassa further studies on its biological properties

Sexual hormones, isolated from the single strains (EmA, Ema) and cross (EmA × Ema) cultures ofNeurospora crassa have been found to restore the fertility of a completely sterile strain obtained from the selfed progeny of EmA. In an attempt to see the effects of different concentrations (dilutions) of the cross-extract on EmA and also on the cross EmA×Ema, different threshold values were indicated.Part of the work reported here is from the Ph. D. dissertation of the author and the remaining portion is from his graduate work, previously not reported elsewhere.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.     

Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima

Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles – wall-forming bodies – found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52–70%), with some α-helix (28–43%) but a relatively low percentage of β-sheet (1–11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42 kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein’s propensity to form dityrosine bonds.     

Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

The promoter-elements of some abiotic stress-inducible genes from cereals interact with a nuclear protein from tobacco

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.     

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em 1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.     

Wolbachia‐induced feminisation newly found in Eurema hecabe,a sibling species of Eurema mandarina (Lepidoptera: Pieridae)

1. Complete feminisation of genetic males into functional females, a unique case among insects, is known in Eurema mandarina (former Eurema hecabe Y type) that are infected with two strains of Wolbachia, wCI^Em and wFem^Em. 2. Here, we newly found that a proportion of wild‐caught E. hecabe (former E. hecabe B type) produced only female offspring. Cytogenetic observations indicated that individuals of E. hecabe displaying the all‐female trait were genetically male (i.e. feminisation). 3. Multilocus sequence typing analyses demonstrated that the feminised individuals of E. hecabe were infected with two Wolbachia strains, wCI^Eh and wFem^Eh, that were indistinguishable from wCI^Em and wFem^Em, respectively. 4. Even identical strains of Wolbachia can be regulated differently depending on the host genetic background. Therefore, we compared the infection densities and vertical transmission efficiencies of Wolbachia between feminised E. mandarina and E. hecabe, but detected no significant differences in these traits. 5. The possible routes by which the two Wolbachia strains have transferred between E. mandarina and E. hecabe are discussed.     

Identification of the theta-type minimal replicon of theLactococcus lactis subsp.lactis CNRZ270 lactose protease plasmid pUCL22

The replication region of pUCL22, the lactose-protease plasmid ofLactococcus lactis subsp.lactis (Lc. lactis) CNRZ270, was isolated on an 18-kbBamHI fragment, by cloning into pUCB300, anEscherichia coli vector encoding Em^r, and selecting for Em^r inLc. lactis MG1614. Subcloning and deletion analysis localized the replication region, namedRep22, on a 2.3-kb fragment. Replicons based on this region followed a theta-type mechanism of replication inLc. lactis. An internal 1251-bpDraI fragment ofRep22 used as a probe hybridized with numerous plasmids in bacteria from the generaLactococcus, Lactobacillus, andLeuconostoc. In some strains, two or three coresident plasmids hybridized with the probe in stringent conditions. It appears, therefore, that this family of theta-type replicons is widely distributed in lactic acid bacteria and contains several incompatibility groups.     

Gene note. Characterization of an Em-like gene of Brassica napus

Em-like proteins are present in dry seeds of several species playing a role in the desiccation tolerance of the embryo. A novel Em gene (BnEm6) of Brassica napus was identified using a PCR-walking method. BnEm6 shares high amino acid sequence identity with other known Em proteins and a similar pattern of expression.     

Light Sensitivity of the Antagonism of Cladosporium Species to Races of Melampsora larici-populina Kleb. on Cultivars of Populus×euramericana (Dode) Guinier

Antagonism of five species of Cladosporium to production of uredinia by three races of Melampsora larici-populina on leaf disks of poplar (Populas × euramericana) was generally higher with races A and E but lower with race D, at high light intensity (400 μ Em^?2s^?1) than at low light intensity (100 μ Em^?2s^?1). Colonization by all species of Cladosporium, of the uredinia induced by the races on the cultivars, was higher at the low than at the high light intensity. The extent of reduction in colonization with increasing light intensity depended on the race, cultivar and species of Cladosporium involved. While the species of Cladosporium was the most important major factor affecting both uredinial production and colonization, race, cultivar, light intensity of incubation and species of Cladosporium interacted significantly to determine disease level (uredinial number) on the leaf disks and the percentage of these uredinia subsequently colonized by the Cladosporium spp.     

Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos

Substantial quantities of mRNA encoding the abundant Em polypeptide accumulate, in planta, in developing embryos of maize (Zea mays L.). By contrast, accumulation of Em mRNA is only barely detectable in embryos with the vp-5/vp-5 genotype [an abscisic acid (ABA)-deficient viviparous phenotype]. Em mRNA is not detectable within viviparous embryos of the vp-1/vp-1 genotype that are non-responsive to ABA. Culture of immature wild-type and vp-5/vp-5 embryos in the presence of exogenous ABA or of an osmotically active agent prevents precocious germination and results in expression of the Em genes. When vp-1/vp-1 embryos are cultured under similar conditions, only the application of osmotic stress prevents precocious germination. However, Em mRNA does not accumulate either in ABA-treated or stressed, arrested embryos, indicating a requirement for ABA perception through a VP-1-mediated mechanism for Em gene expression. Nevertheless, vp-1/vp-1 embryos do show both ABA and stress responses at the molecular level. Treatment with ABA causes the accumulation of mRNA encoding a polypeptide of approx. 30 kDa, whilst osmotic stress induces the accumulation both of a 30-kDa polypeptide and a set of approx. 20-kDa polypeptides. This indicates the existence of discrete, parallel ABA and stress response pathways in developing maize embryos.Abbreviations ABA abscisic acid - cDNA copy-DNA - DAP days after pollination - kDa kilodaltons - MS Murashige and Skoog medium - LEA late embryogenesis abundant - NEpHGE non-equilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Molecular characterization of the erythromycin resistance plasmid pPV142 from Staphylococcus simulans

The 2.5-kb erythromycin resistance (Em^R) plasmid pPV142 of Staphylococcus simulans 13044 was isolated and characterized. Sequence analysis identified ORF1 and ORF2 encoding a 158-residue replication protein (Rep142) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV142 shared homology with the Em^R plasmid pPV141 (2.4 kb) of S. chromogenes 3688 and other Em^R plasmids known to exist in staphylococci and bacilli. Based on the presence of a 61-bp repeat upstream of the ermM gene, pPV142 is apparently a unique member of the pSN2 family of Em^R plasmids able to express erythromycin resistance constitutively.     

Photosensitized Preparation of Fluorescent Luminarosine and Analogues*

Abstract

Optimized conditions for photosensitized preparation of green-yellow fluorescent (λ_Em = 530 nm) 7-β-D-ribofuranosylamino)pyrido[2,1 -h]pteridin-11-ium-5-olate (luminarosine) and its 2′-deoxy- and 2′-O-methyl- analogues, the key compounds in our studies on the synthesis of fluorescence-labeled oligonucleotides, have been developed.

     

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta,Canada

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.     

Cloning of a tyrosinase gene in Streptococcus thermophilus

Summary The streptococcal cloning vector pIL253 (4.96-kbp, Em^r) was used to introduce the Streptomyces antibioticus tyrosinase (mel) gene (1.56-kbp) into S. thermophilus, an important microbe in dairy fermentations. Electrotransformants of S. thermophilus ST128 contained 6.51-kbp recombinant plasmids which probed positively in Southern hybridizations with the biotin-labeled mel fragment. Western blots of cell extracts resolved by SDS-PAGE showed the presence of a ca. 31-kDa band thus confirming the synthesis of tyrosinase protein by genetic transformants.     

Abscisic acid-insensitive mutations provide evidence for stage-specific signal pathways regulating expression of an Arabidopsis late embryo genesis-abundant (lea) gene

An Arabidopsis homolog of the abscisic acid (ABA)-inducible cotton D19 and wheat Em genes was cloned and its expression assayed at two developmental stages in wild-type, ABA-deficient (aba) and three ABA-insensitive (abi) lines of Arabidopsis thaliana. Expression of this gene was reduced slightly in seeds of aba mutants and approximately ten-fold in abi3 mutants, but seed expression was not decreased in either abi1 or abi2 monogenic mutants. In contrast, the abi1 and abi2 mutants showed a very slight reduction of ABA inducibility in 8-day-old plants, while the responses of aba and abi3 mutants were comparable to that of wild type. Although previous studies have shown that none of the abi mutations show completely stage-specific effects, the results reported here indicate that the importance of each of the ABI loci in regulating this single gene is stage-dependent. Furthermore, the fact that none of the abi mutations show more than minor effects on exogenous ABA inducibility of the Arabidopsis D19/Em homolog in young plants suggests that an additional ABA signalling pathway may be operating during vegetative growth.     

Measurement of the oxidation reduction potential of the EPR detectable active centre of the molybdenum iron protein of Chromatium nitrogenase

The midpoint oxidation-reduction potential of the EPR detectable centre of the molybdenum iron protein of Chromatium nitrogenase has been measured. Two centres with identical EPR spectra but different midpoint potentials were detected. The measured midpoint potentials are (1) Em_7.5 = ?60 mV and (2) Em_7.5 = ?260 mV. The midpoint potentials were not affected by other components of the nitrogen fixing system.     

The primary quinone acceptor and the membrane-bound c-type cytochromes of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum: A spectroscopic and thermodynamic study

The mid-point potential (Em_7.0) of the primary quinone acceptor (Qa) and the biochemical features (Em_7.0 and apparent molecular mass, MM) of the membrane bound c-type cytochromes (cyt) involved in photosynthetic electron transfer of the halophilic phototrophic bacterium Rhodospirillum (Rs.) salinarum were determined. A tetrahemic RC bound cytochrome was found (MM of 39.8 kDa) with Em_7.0 of the hemes equal to +304, +98, +21, –134 (± 8) mV as determined by dark equilibrium redox titrations in the isolated purified form. The highest potential heme (Em_7.0 = +304 mV, band at 556 nm) was able to reduce the photo-oxidized reaction center (P⁺) in a sub-millisecond ( 20 s) time scale reaction, acting most likely as the direct electron donor to P⁺). The midpoint potential of the primary electron donor (Em_7.0 = + 455 mV) was found to be close to that reported for the primary donor of the non-halophilic Rhodospirillum species Rs. rubrum, whereas the quinone primary electron acceptor (Qa) was different showing the spectral features of a menaquinone molecule with Em_7.0 at –128 (± 5) mV. A membrane bound c-type heme with Em_7.0 of 259 (± 1) and MM of 40 kDa was also isolated and referred to an orthodox cytochrome c₁). The present data on the photosynthetic apparatus, along with the previous results on the respiratory system [Moschettini et al. (1997) Arch Microbiol 168: 302-309], suggest that Rs. salinarum is biochemically distinct from Rs. rubrum, the most representative specie of the genus.