The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.     

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.     

Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences in Paramecium tetraurelia

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.     

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.     

Genetic Control of Resistance to the Sterol 14α-Demethylase Inhibitor Fungicide Prochloraz in the Cereal Eyespot Pathogen Tapesia yallundae

Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth by 50% {IG₅₀} for each was ≤0.03 mg/liter]) and field isolates from France and New Zealand with differing levels of resistance (PR11 [IG₅₀ = 0.5 mg/liter], PR1 [IG₅₀ = 1.0 mg/liter], and 11-3-18 [IG₅₀ = 2.4 mg/liter]) yielded progeny showing a bimodal distribution, with an even number of sensitive and resistant progeny. This indicated the segregation of a single major gene for resistance in each cross, which was confirmed by the use of backcrosses, crosses between F₁ progeny, and control crosses between sensitive parents. However, there was also evidence of additional quantitative genetic components responsible for the increased IG₅₀s of the more resistant isolates. A further cross was made between isolate PR11 and an F₁ progeny arising from isolate 11-3-18, and this also yielded progeny which were entirely prochloraz resistant. This suggested that resistance genes were allelic in these two isolates, with resistance conferred by a gene at the same locus (or closely linked loci), despite the fact that the isolates (PR11 and 11-3-18) originated from different continents.     

Mutations in Pdd1 Reveal Distinct Requirements for Its Chromodomain and Chromoshadow Domain in Directing Histone Methylation and Heterochromatin Elimination

Pdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation of Tetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.     

Apparent TRANS Control of Murine βglucuronidase Synthesis by a Temporal Genetic Element

A difference in the heat-inactivation kinetics between the β-glucuronidases of C3HeB/FeJ and C57Bl/6J mice was utilized to assess the mode of action of a temporal genetic element in controlling the expression of the β-glucuronidase structural gene Gus. The heat-inactivation kinetics of liver and kidney β-glucuronidase from F₁ C3HeB/FeJ x C57Bl/6J animals were intermediate with respect to the parental enzyme patterns, suggesting that equal concentrations of the two allelic products were present in β-glucuronidase tetramers of F₁ progeny. β-glucuronidase heteropolymers assembled in vivo under conditions where equal concentrations of the two structural alleles of the enzyme were known to be present also exhibited intermediate heat-inactivation kinetics. These observations are consistent with a trans mode of action of a genetic element that controls the rate of murine β-glucuronidase synthesis.     

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.     

ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component,Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψ_m) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential F_oF₁-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψ_m that manifests as a decreased growth phenotype, indicating that the F_oF₁-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane F_o-moiety. These Dk cells generate the Δψ_m by combining the hydrolytic activity of the matrix-facing F₁-ATPase and the electrogenic exchange of ATP⁴- for ADP³- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric F_oF₁-ATPase complexes were identified. In fact, the immunoprecipitation of a F₁-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψ_m of Dk cells. Thus, even in the absence of subunit a, a portion of the F_oF₁-ATPase is assembled in Dk cells.     

In Vivo Correlation of Glucose Metabolism,Cell Density and Microcirculatory Parameters in Patients with Head and Neck Cancer: Initial Results Using Simultaneous PET/MRI

Objective

To demonstrate the feasibility of simultaneous acquisition of ¹⁸F-FDG-PET, diffusion-weighted imaging (DWI) and T1-weighted dynamic contrast-enhanced MRI (T1w-DCE) in an integrated simultaneous PET/MRI in patients with head and neck squamous cell cancer (HNSCC) and to investigate possible correlations between these parameters.

Methods

17 patients that had given informed consent (15 male, 2 female) with biopsy-proven HNSCC underwent simultaneous ¹⁸F-FDG-PET/MRI including DWI and T1w-DCE. SUV_max, SUV_mean, ADC_mean, ADC_min and K ^trans, k _ep and v _e were measured for each tumour and correlated using Spearman’s ρ.

Results

Significant correlations were observed between SUV_mean and K ^trans (ρ = 0.43; p ≤ 0.05); SUV_mean and k _ep (ρ = 0.44; p ≤ 0.05); K ^trans and k _ep (ρ = 0.53; p ≤ 0.05); and between k _ep and v _e (ρ = -0.74; p ≤ 0.01). There was a trend towards statistical significance when correlating SUV_max and ADC_min (ρ = -0.35; p = 0.08); SUV_max and K ^trans (ρ = 0.37; p = 0.07); SUV_max and k _ep (ρ = 0.39; p = 0.06); and ADC_mean and v _e (ρ = 0.4; p = 0.06).

Conclusion

Simultaneous ¹⁸F-FDG-PET/MRI including DWI and T1w-DCE in patients with HNSCC is feasible and allows depiction of complex interactions between glucose metabolism, microcirculatory parameters and cellular density.     

Solution Structure,Determined by Nuclear Magnetic Resonance,of the b30-82 Domain of Subunit b of Escherichia coli F1Fo ATP Synthase

Subunit b, the peripheral stalk of bacterial F₁F_o ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and δ-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an α-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 Å. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both α-helices in b22-156.ATP synthesis by oxidative phosphorylation or photophosphorylation is a multistep membrane-located process that provides the bulk of cellular energy in eukaryotes and many prokaryotes. The majority of ATP synthesis is accomplished by the enzyme ATP synthase (EC 3.6.1.34), also called F₁F_o ATP synthase, which, in its simplest form, as in bacteria, is composed of eight different subunits (α₃, β₃, γ, δ, ɛ, a, b₂, and c_9-12). This multisubunit complex is divided into the F₁ headpiece, α₃:β₃, and a membrane-embedded ion-translocating part known as F_o, to which F₁ is attached by a central and a peripheral stalk (1, 5, 25). ATP is synthesized or hydrolyzed on the α₃:β₃ hexamer, and the energy provided for or released during that process is transmitted to the membrane-bound F_o sector, consisting of subunits a and c and part of subunit b (30, 31). The energy coupling between the two active domains occurs via the stalk part(s) (6). The central stalk is made of subunits γ and ɛ, and the peripheral stalk is formed by subunits δ and b. The peripheral stalk, which lies at the edge of the multisubunit assembly of the F₁F_o ATP synthase, acts as a stator to counter the tendency of the α₃:β₃ hexamer to follow the rotation of the central stalk and the attached c-ring, and to anchor the membrane-embedded a subunit (17, 36).In Escherichia coli, subunit b with its 156 residues extends with its soluble part (b_sol; b21-156) from the top of the F₁ sector down, into, and across the membrane, where it is associated with subunit a (2, 15, 32, 34). The 156-residue b subunit has been divided into four functional domains (28). They are, in order from the N to the C terminus; the membrane domain, the tether region, the dimerization domain, and the δ-binding domain. The structure of the synthesized 33-residue peptide comprising the N-terminal membrane-spanning region has been solved by ¹H NMR, showing an α-helical feature (14). The crystallographic structure of the major part of the dimerization domain, b62-122, revealed an α-helix with a length of 9.0 nm (12). Most recently, the NMR solution structure of the very C-terminal segment, b140-156, which interacts with the C terminus of subunit δ (δ91-177), has been determined by NMR spectroscopy (26). This molecule adopts a stable helix formation in solution with a flexible tail between amino acid residues 140 and 145. SAXS (26) and analytical ultracentrifuge studies have indicated that the soluble domain of subunit b (b21-156, b22-156) is dimeric in solution (12). So far, no high-resolution structure of the tether domain, including residues 25 to 52, or the N-terminal segment of the dimerization domain, which is formed by residues 53 to 122, is available (14).Here, we have turned our attention to the production and purification of residues 30 to 82 of subunit b (b30-82) from E. coli F₁F_o ATP synthase, which forms the remaining unsolved structural segment of subunit b. The structural features of this segment have been determined in solution using NMR spectroscopy. The introduction of a cysteine residue into b22-156 at four positions resulted in different intersubunit disulfide patterns, giving insight into the proximity of the residues.     

Transpiration- and growth-induced water potentials in maize

Recent evidence from leaves and stems indicates that gradients in water potential (ψ_w) necessary for water movement through growing tissues are larger than previously assumed. Because growth is sensitive to tissue ψ_w and the behavior of these gradients has not been investigated in transpiring plants, we examined the water status of all the growing and mature vegetative tissues of maize (Zea mays L.) during high and low rates of transpiration. The ψ_w measured in the mature regions of the plant responded primarily to transpiration, while the ψ_w in the growing regions was affected both by transpiration and growth. The transpiration-induced potentials of the mature tissue formed a gradient of decreasing ψ_w along the transpiration stream while the growth-induced potentials formed a gradient of decreasing ψ_w from the transpiration stream to the expanding cells in the growing tissue. The growth-induced gradient in ψ_w within the leaf remained fairly constant as the xylem ψ_w decreased during the day and was associated with a decreased osmotic potential (ψ_s) of the growing region (osmotic adjustment). The growth-induced gradient in ψ_w was not caused by excision of the tissue because intact maize stems exhibited a similar ψ_w. These observations support the concept that large gradients in ψ_w are required to maintain water flow to expanding cells within all the vegetative tissues and suggest that the maintenance of a favorable gradient in ψ_w for cell enlargement may be an important role for osmotic adjustment.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

Tetrahymena Pot2 Is a Developmentally Regulated Paralog of Pot1 That Localizes to Chromosome Breakage Sites but Not to Telomeres

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3′ overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.     

Mapping determinants of gene expression plasticity by genetical genomics in C. elegans

Recent genetical genomics studies have provided intimate views on gene regulatory networks. Gene expression variations between genetically different individuals have been mapped to the causal regulatory regions, termed expression quantitative trait loci. Whether the environment-induced plastic response of gene expression also shows heritable difference has not yet been studied. Here we show that differential expression induced by temperatures of 16 °C and 24 °C has a strong genetic component in Caenorhabditis elegans recombinant inbred strains derived from a cross between strains CB4856 (Hawaii) and N2 (Bristol). No less than 59% of 308 trans-acting genes showed a significant eQTL-by-environment interaction, here termed plasticity quantitative trait loci. In contrast, only 8% of an estimated 188 cis-acting genes showed such interaction. This indicates that heritable differences in plastic responses of gene expression are largely regulated in trans. This regulation is spread over many different regulators. However, for one group of trans-genes we found prominent evidence for a common master regulator: a transband of 66 coregulated genes appeared at 24 °C. Our results suggest widespread genetic variation of differential expression responses to environmental impacts and demonstrate the potential of genetical genomics for mapping the molecular determinants of phenotypic plasticity.     

Two-Component Anti-Staphylococcus aureus Lantibiotic Activity Produced by Staphylococcus aureus C55

Staphylococcus aureus C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C₈ and C₁₈) chromatography. The yield following C₈ chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill S. aureus or Micrococcus luteus but not S. epidermidis strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.     

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria

The PUFAs include many bioactive lipids. The microbial metabolism of C₁₈ PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C₂₀ PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C₂₀ PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C₂₀ PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C₁₈ and C₂₀ PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.     

ANALYSIS OF THE GEOTROPIC ORIENTATION OF YOUNG RATS. II

1. Equations describing the geotropic orientation of young rats as a function of the inclination of the surface on which creeping take place, under standardized conditions, are found to be of similar form but with different values of the contained constants, when several different, genetically stabilized lines or races are compared. The values of these constants are characteristic for the several races. 2. The biological "reality" of the differences between young rats of two races, as given mathematical form in terms of these parameters and coefficients, can be submitted to radical test by investigating their behavior in inheritance. A simple result favorable to the inquiry would be decisive; a complex, non-clear result would not however be definitely unfavorable to the view that "real" differences in behavior are in question. The actual result is of a kind demonstrating (a) the efficiency of the original formulations, and (b), at the same time, the definite inheritance of certain quantitative aspects of geotropic behavior. 3. On the assumption that orientation on a sloping surface is achieved when, within a threshold difference, the tension-excitations on the two sides of the body (legs) are the same, the angle of oriented progression (θ) can be taken as a direct measure of the total excitation. This is consistent with the equation, accurately obeyed by our initial races, Δ cos θ/Δ sin α = - const., where α is the slope of the surface. 4. The total excitation of tension-receptors must be regarded as involving, over a gross interval of time, (1) the total array of receptors with thresholds below a certain value, a function of the stretching force, and (2) the frequency of change of tension. The latter, largely determined (it is assumed) by the frequency of stepping, should be proportional to the speed of progression. This speed is directly proportional to log sin α. Hence Δθ/Δ log sin α. plotted against sin α, should give a picture of the distribution of effective thresholds among the available tension-receptors in terms of the exciting component of gravity. For the races investigated this distribution can be resolved in each case into three groups. 5. A "variability number" is employed which permits the demonstration that the variability of θ as measured is (1) definitely controlled by α, and is (2) a characteristic number for each of the pure races used. 6. By attaching a weight to rats of one race it is found that Δθ/Δα is modified in a manner concordant with the assumption that the three "groups of sense organs" are in fact discrete. 7. In race K these three groups (I, II, III) are large, in race A, small (i, ii, iii). F₁ rats of the cross between these two races show i, ii, III. 8. F₁ individuals back-crossed to A give in the progeny two sorts of individuals, in equal numbers: i, ii, III and i, ii, iii. 9. F₁ individuals back-crossed to K are expected to give in the progeny four types of individuals, I, II; i, II; I, ii; i, ii. In the numbers available these classes are reasonably clear, and occur with equal frequency. 10. It is pointed out that these considerations imply a mode of definition of a gene somewhat different from that commonly employed by tacit assumption; namely, a definition of the effect in inheritance as a function of some controlling, independent variable.