Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

Normal and Mutant Glycine Transfer RNAs

THE glycine-specific tRNAs of E. coli can be grouped into three subspecies which are separated by chromatography on benzoylated DEAE cellulose (BDC): tRNA^Gly₁ (GGG), tRNA^Gly₂ (GGA/G) and tRNA^Gly₃ (GGU/C)^1,2. The tRNA^Gly₁ and tRNA^Gly₂ are specified by the genes, glyU and glyT, respectively, which have been located at 55 and 77 minutes on the E. coli chromosome. Suppressors of tryptophan A gene (trpA) missense mutations and partial diploid strains have been used extensively to characterize the glycine tRNA structural genes (Table 1)^1–3. A common property of these suppressor mutations is that the altered tRNA^Gly is no longer aminoacylated at the normal rate by the glycyl tRNA synthetase (GRS). When ordinary loading conditions are used virtually none of the suppressor tRNA species are amino-acylated. These studies have shown that single gene copies are normally present at the glyT and glyU loci.     

<Emphasis Type="Italic">In vitro</Emphasis> Synthesis of tRNA<Superscript>Tyr</Superscript> Precursors and their Conversion to 4S RNA

Three bacterial-specific RNA messengers, transcribed in vitro from phage ?80psu₃ DNA, contain the nucleotide sequence corresponding to the tRNA^Tyr gene carried by this phage. As there is only one copy of this gene in the phage genome, there are thought to be three promoter sites on the DNA template.     

Capacity assessment of <Emphasis Type="Italic">Myzus persicae,Aphis gossypii</Emphasis> and <Emphasis Type="Italic">Aphis spiraecola</Emphasis> (Hemiptera: Aphididae) to acquire and retain PVY<Superscript>NTN</Superscript> in Tunisia

The study was carried out to investigate the ability of three aphids, Myzus persicae, Aphis gossypii and Aphis spiraecola, to acquire and retain the Potato Virus Y (PVY) isolate, PVY^NTN. Tobacco plants, Nicotiana tabacum var. Xanthi, were used as test plant for the virus inoculation and aphid acquisition. The serological test double-antibody sandwich enzyme-linked immunosorbent assay was applied for virus detection on the test plants and aphids. Furthermore, virus retention by aphids was also assessed using a monoclonal anti-PVY^N. Although a duration of 2 min was enough for the virus acquisition, the three tested aphids showed different capacities to retain PVY^NTN. The retention of PVY^NTN was 3 h for M. persicae and A. spiraecola, and 2 h for A. gossypii. This study provides basic information of the virus retention by potato-colonizing aphid species, which may increase our understanding of PVY epidemiology in Tunisia.     

Discovery of an acidic,thermostable and highly NADP<Superscript>+</Superscript> dependent formate dehydrogenase from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> NRRL B-30929

Objectives

To identify a robust NADP⁺ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

A new NADP⁺ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6?×?histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP⁺ and NAD⁺, respectively and its T_m value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K_M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP⁺), NADP⁺ and NAD⁺, respectively.

Conclusions

An NADP⁺ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.

     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> W3110 for the production of <Emphasis Type="SmallCaps">l</Emphasis>-methionine

In this study, we constructed an l-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metA ^Fbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMA^Fbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

The Effect of Mutation <Emphasis Type="Italic">dominant spotting-Yurlovo</Emphasis> (<Emphasis Type="Italic">Kit</Emphasis><Superscript><Emphasis Type="Italic">W-Y</Emphasis></Superscript>) on Spermatogenesis,Early Embryogenesis,and Fertility of C57BL/6JY Mice

The effect of mutation Kit ^W-Y found in C57BL/6 mice on fertility, spermatogenesis, and early embryogenesis of mice have been studied. If heterozygotes Kit ^W /+ are crossed with wild-type mice, fertility decreases by 20%. Homozygotes Kit ^W-Y /Kit ^W-Y and compounds Kit ^W-Y /Kit ^Ssm are nonviable. The study of spermatogenesis in Kit ^W /+ mice has demonstrated a negative effect of this mutation on spermatocytes. Histological examination of the testes of mutant males has shown local empty spaces in seminal ducts. Electron microscopic examination of synaptonemal complexes have demonstrated desynapsis disturbance in some nuclei at the diplotene stage of meiotic prophase I. However, these disturbances do not cause a decrease in the number of fertilized oocytes/ova. The decrease in fertility is accounted for disturbances of early embryogenesis. In vivo and in vitro analyses of early embryogenesis have demonstrated that cleavage divisions are asynchronous in Kit ^W-Y /+ heterozygous embryos. Some of these embryos die before implantation, and others cleave more rapidly than wild-type embryos, which give them selective advantage during the postimplantation period of embryogenesis. The pattern of Kit ^W-Y expression during spermatogenesis and embryogenesis mimics potential human pathology, which makes these mutants an interesting and valuable object for genetics and developmental biology.     

Complete mitochondrial DNA sequence and phylogenetic analysis of Zhikong scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis> (Bivalvia: Pectinidae)

The complete mitochondrial genome of Zhikong scallop Chlamys farreri is 21,695 bp in length and contains 12 protein-coding genes (the atp8 gene is absent, as in most bivalves), 2 ribosomal RNA genes, and 22 transfer RNA genes. The heavy strand has an overall A+T content of 58.7%. GC and AT skews for the mt genome of C. farreri are 0.337 and ?0.184, respectively, indicating the nucleotide bias against C and A. The mitochondrial gene order of C. farreri differs drastically from the scallops Argopecten irradians, Mimachlamys nobilis and Placopecten magellanicus, which belong to the same family Pectinidae. 6623 bp non-coding nucleotides exist intergenically in the mitogenome of C. farreri, with a large continuous sequence (4763 bp) between tRNA ^Val and tRNA ^Asn . Two repeat families are found in the large continuous sequence, which seems to be a common feature of scallops. Phylogenetic analysis based on 12 concatenated amino acid sequences of protein-coding genes supports the monophyly of Pectinidae and paraphyletic Pteriomorphia with respect to Heteroconchia.     

Loss of ncm<Superscript>5</Superscript> and mcm<Superscript>5</Superscript> wobble uridine side chains results in an altered metabolic profile

Introduction

The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm⁵) and 5-methoxycarbonylmethyl (mcm⁵) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified \( {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} \), which in wild-type strains are modified with mcm⁵s²U, partially suppress phenotypes of an elp3Δ strain.

Objectives

To identify metabolic alterations in an elp3Δ strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified \( {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} \).

Method

Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3Δ strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing \( {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} \). The temperature sensitive elp3Δ strain derivatives were cultivated at permissive (30 °C) or semi-permissive (34 °C) growth conditions.

Results

Culturing an elp3Δ strain at 30 or 34 °C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3Δ strain carrying the wild-type ELP3 gene. Overexpression of hypomodified \( {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} \) suppressed a subset of the metabolic alterations observed in the elp3Δ strain.

Conclusion

Our results suggest that the presence of ncm⁵- and mcm⁵-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.