Variation in mutation rates caused by RB69pol fidelity mutants can be rationalized on the basis of their kinetic behavior and crystal structures

We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3′-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations.     

Fatty Acid accumulation in maturing flaxseeds as influenced by environment

The effects of temperature and light on boll and shoot maturity and on the accumulation of fatty acids in developing seeds of flax (Linum usitatissimum L.) were determined in controlled environments. Palmitic and linoleic acids decreased but linolenic increased in percent as seed formation progressed. In the same period, oleic acid increased in percent in 1 variety and decreased in another. Increased temperatures hastened these changes and resulted in decreased iodine value of the oil at maturity.     

Bathy Phytochromes in Rhizobial Soil Bacteria

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.Phytochromes are biological photoreceptors that were discovered in plants, where they control development throughout the life cycle in manifold ways (21, 33). Today, a large number of homologs are known also from cyanobacteria, other bacteria, and fungi, which are termed cyanobacterial phytochromes (Cphs), bacteriophytochromes (BphPs), and fungal phytochromes (Fphs), respectively (20, 24). The chromophore is autocatalytically assembled within the N-terminal part of the protein, the photosensory core module (PCM), which contains the PAS, GAF, and PHY domains (30). Typically, phytochromes are converted by light between two spectrally different forms, the red-absorbing Pr and the far-red-absorbing Pfr forms. Photoconversion is initiated by an isomerization of the covalently bound bilin chromophore (32).Plant and cyanobacterial phytochromes incorporate phytochromobilin (PΦB) and phycocyanobilin (PCB) as natural chromophores, respectively, which are covalently bound to Cys residues in the GAF domains. All characterized phytochromes that belong to these groups have a Pr ground state. Plant phytochromes can undergo dark conversion of Pfr to Pr (5), whereas the Pfr form of typical cyanobacterial phytochromes is stable in darkness (26).Bacteriophytochromes utilize biliverdin (BV) instead as a natural chromophore (1), which is covalently attached to a Cys residue in the N terminus of the PAS domain (26). Since the conjugated system of BV is longer than that of PΦB or PCB, the absorption maxima of bacteriophytochromes are found at higher wavelengths than those of cyanobacterial or plant homologs.With the discovery of a bacterial phytochrome from Bradyrhizobium sp. strain ORS278, termed BrBphP1, the first phytochrome with a Pfr ground state and dark conversion from Pr to Pfr was found (10). Thereafter, five more phytochromes with dark conversion of Pr to Pfr were described: Rhodopseudomonas palustris BphP1 (RpBphP1) from strain CEA001, RpBphP5, and RpBphP6 from strain CGA009 (11); Agrobacterium tumefaciens Agp2 (or AtBphP2) from strain C58 (18); and Pseudomonas aeruginosa BphP1 (PaBphP1) (40). These phytochromes are now termed bathy phytochromes because the absorption maxima of their ground states are bathochromically (to longer wavelengths) shifted compared to those of all other phytochromes.Moreover, some other bacterial phytochromes with unusual properties have been described. In the Ppr from Rhodospirillum centenum, a photoactive yellow protein (PYP) domain is fused to the N terminus of a phytochrome homolog. The phytochrome part of Ppr assembles with BV to form a Pr adduct. However, irradiation does not result in the formation of Pfr but in a bleaching of the Pr spectrum (23). The BV adduct of RpBphP3 from R. palustris, which has a Pr ground state, photoconverts to the so-called Pnr form with a blue-shifted absorption maximum (12). RpBphP4 from R. palustris strains Ha2 and BisB5 and Bradyrhizobium BphP3 (BrBphP3) from Bradyrhizobium BTAi1, both with a Pr ground state, photoconvert into a long-lived MetaR form (8, 42). MetaRa and MetaRc are intermediates in the photoconversion from Pr to Pfr of prototypical phytochromes (3). BphP3 from the Bradyrhizobium strain ORS 278 is an exception among bacteriophytochromes as it binds PCB as a natural chromophore. This phytochrome adopts a so-called Po (P-orange) ground state with an absorbance maximum in the orange range (11, 15). Upon irradiation, this phytochrome converts into the Pr form. RpBphP4 from R. palustris CGA009 lacks the biliverdin binding cysteine and does not bind a chromophore (42).With the rapidly growing number of bacterial genome sequences, many new bacterial phytochromes are being discovered. Thus, a large and increasing number of newly identified phytochromes remain spectroscopically uncharacterized. We established an in vivo photometry approach which allowed the rapid acquisition of spectral information about phytochromes from intact bacterial cells. In the beginning period of plant phytochrome research, in vivo photometry was extensively applied (4, 6, 29, 34). This method, in fact, allowed the identification of phytochromes for the first time in plant tissues (6), which led to the purification of phytochromes from plant extracts (37). Here, we apply in vivo photometry for the first time to organisms outside the plant kingdom. This method is especially useful for studying species with single phytochrome genes. The approach is also helpful for comparing properties of native phytochromes in vivo and of their recombinant proteins in vitro.In the present study, we concentrate on nonphotosynthetic species of the order Rhizobiales which belongs to the Alphaproteobacteria. The family Rhizobiaceae comprises plant-interacting soil bacteria. A. tumefaciens and Agrobacterium vitis can transfer genes into plants to induce plant tumors, whereas many other Rhizobiaceae can live as plant symbionts in nodules of stems or roots in which they assimilate molecular nitrogen to produce NH₄⁺, which is used by the plant for synthesis of amino acids and other nitrogen-containing molecules. A. tumefaciens C58 contains two phytochromes, termed Agp1 (or AtBphP1) and Agp2 (or AtBphP2), that have been characterized as recombinant proteins (14, 18, 26, 35) and whose spectral activities have been measured in extracts of wild-type and knockout mutants (31). A large number of phytochromes from photosynthetic Bradyrhizobium and Rhodopseudomonas species, which also belong to the order Rhizobiales, have been characterized as recombinant proteins (11), some of which have already been noted above.It turned out that most of our analyzed phytochromes undergo dark conversion of Pr to Pfr and thus belong to the group of bathy phytochromes. Such phytochromes, which absorb at around 750 nm, clearly dominate among Rhizobiales. We propose that this specific property reflects an adaptation to the light regime in the soil. Our studies also suggest that bacterial phytochromes with a C-terminal response regulator have evolved from a bathy phytochrome progenitor.     

Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia

A new needle nematode, Longidorus carniolensissp. n., recovered from the soil around the roots of grapevine Vitis vinifera L. from Slovenia, is described and illustrated. Longidorus carniolensisis an amphimictic species, characterised by females with a moderately long (L=5.6-8.2 mm) and plump (a=51-72.4, ave. 66.3) body, assuming a spiral to C-shape when heat relaxed. Head region continuous, anteriorly almost flat, lip region 23-25 μm wide; guiding ring situated posteriorly (42-47 μm, 43-50 μm in males), odontostyle long (ave. 146.6 (136-157) μm); pharyngeal glands with normal location, their nuclei of approximately equal size; tail bluntly conoidal to almost hemispherical. Males abundant, spicules slender and long (122-145 μm), ventromedian supplements 13-17, irregularly spaced, preceded by an adanal pair. Four juvenile stages present, the first stage juvenile with bluntly conoidal tail. Codes for identifying the new species when using the key by Chen et al. (1997) are: A 56, B 4, C 4, D 1, E 4, F 35, G 1, H 1, I 2. The new species is morphologically the most similar to Longidorus poessneckensis Altherr, 1974, Longidorus macrosoma Hooper, 1961, Longidorus caespiticola Hooper, 1961, Longidorus helveticus Lamberti et al., 2001, Longidorus macroteromucronatus Altherr, 1974, Longidorus pius Barsi & Lamberti, 2001, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram et al. 2008, Longidorus silvae Roca, 1993, Longidorus iuglandis Roca et al., 1985, Longidorus vinearum Bravo & Roca, 1995 and Longidorus major Roca & d'Erico, 1987, but differs from these species either by the body and odontostyle length, position of guide ring, head region and tail shape or the shape of the first stage juvenile tail. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other speciesof the genus Longidorus with known sequences. Relationships of Longidorus carniolensissp. n. with other Longidorus species based on analysis of this DNA fragment and morphology are discussed.     

Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.     

Biological roles of APP in the epidermis

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimer's disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.     

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

Extension of a de novo TIM barrel with a rationally designed secondary structure element

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four‐fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub‐problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high‐resolution X‐ray structure for one variant and compare it to our design model. The successful extension of this robust TIM‐barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.     

Potential of promotion of alleles by genome editing to improve quantitative traits in livestock breeding programs

Background

Genome editing (GE) is a method that enables specific nucleotides in the genome of an individual to be changed. To date, use of GE in livestock has focussed on simple traits that are controlled by a few quantitative trait nucleotides (QTN) with large effects. The aim of this study was to evaluate the potential of GE to improve quantitative traits that are controlled by many QTN, referred to here as promotion of alleles by genome editing (PAGE).

Methods

Multiple scenarios were simulated to test alternative PAGE strategies for a quantitative trait. They differed in (i) the number of edits per sire (0 to 100), (ii) the number of edits per generation (0 to 500), and (iii) the extent of use of PAGE (i.e. editing all sires or only a proportion of them). The base line scenario involved selecting individuals on true breeding values (i.e., genomic selection only (GS only)-genomic selection with perfect accuracy) for several generations. Alternative scenarios complemented this base line scenario with PAGE (GS + PAGE). The effect of different PAGE strategies was quantified by comparing response to selection, changes in allele frequencies, the number of distinct QTN edited, the sum of absolute effects of the edited QTN per generation, and inbreeding.

Results

Response to selection after 20 generations was between 1.08 and 4.12 times higher with GS + PAGE than with GS only. Increases in response to selection were larger with more edits per sire and more sires edited. When the total resources for PAGE were limited, editing a few sires for many QTN resulted in greater response to selection and inbreeding compared to editing many sires for a few QTN. Between the scenarios GS only and GS + PAGE, there was little difference in the average change in QTN allele frequencies, but there was a major difference for the QTN with the largest effects. The sum of the effects of the edited QTN decreased across generations.

Conclusions

This study showed that PAGE has great potential for application in livestock breeding programs, but inbreeding needs to be managed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0135-3) contains supplementary material, which is available to authorized users.