Unlocking the secondary gene‐pool of barley with next‐generation sequencing

Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker‐assisted backcrossing the size of introgressed segments. We applied next‐generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture‐based (re)‐sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two‐enzyme‐based genotyping‐by‐sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.     

Shuttling of SOX proteins

The control of access of SOX proteins to their nuclear target genes is a powerful strategy to activate or repress complex genetic programs. The sub-cellular targeting sequences of SOX proteins are concentrated within the DNA binding motif, the HMG (for high mobility group) domain. Each SOX protein displays two different nuclear localization signals located at the N-terminal and C-terminal part of their highly conserved DNA binding domain. The N-terminal nuclear localization signal binds calmodulin and is potentially regulated by intracellular calcium signalling, while the C-terminal nuclear localization signal, which binds importin-β, responds to other signalling pathways such as cyclic AMP/protein kinase A. Mutations inducing developmental disorders like sex reversal have been reported in both NLSs of SRY, interfering with its nuclear localization and suggesting that both functional nuclear localization signal are required for its nuclear activity. A nuclear export signal is also present in the HMG box of SOX proteins. Group E SOX proteins harbour a perfect consensus nuclear export signal sequence in contrast to all other SOX proteins, which display only imperfect ones. However, observations made during mouse embryonic development suggest that non-group E SOX proteins could also be regulated by a nuclear export mechanism. The presence of nuclear localization and nuclear export signal sequences confers nucleocytoplasmic shuttling properties to SOX proteins, and suggests that cellular events regulated by SOX proteins are highly dynamic.     

Reaction of wild-type and Glu243Asp variant yeast cytochrome c oxidase with O2

We have studied internal electron transfer during the reaction of Saccharomyces cerevisiae mitochondrial cytochrome c oxidase with dioxygen. Similar absorbance changes were observed with this yeast oxidase as with the previously studied Rhodobacter sphaeroides and bovine mitochondrial oxidases, which suggests that the reaction proceeds along the same trajectory. However, notable differences were observed in rates and electron-transfer equilibrium constants of specific reaction steps, for example the ferryl (F) to oxidized (O) reaction was faster with the yeast (0.4 ms) than with the bovine oxidase (~ 1 ms) and a larger fraction Cu_A was oxidized with the yeast than with the bovine oxidase in the peroxy (P_R) to F reaction. Furthermore, upon replacement of Glu243, located at the end of the so-called D proton pathway, by Asp the P_R → F and F → O reactions were slowed by factors of ~ 3 and ~ 10, respectively, and electron transfer from Cu_A to heme a during the P_R → F reaction was not observed. These data indicate that during reduction of dioxygen protons are transferred through the D pathway, via Glu243, to the catalytic site in the yeast mitochondrial oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

A high risk phenotype of hypertrophic cardiomyopathy associated with a compound genotype of two mutated β-myosin heavy chain genes

Hypertrophic cardiomyopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that is in most cases familial and transmitted in a dominant fashion. The most frequently affected gene codes for the cardiac (ventricular) β-myosin heavy chain. We have investigated the genetic cause of an isolated case of HCM, which was marked by an extremely severe phenotype and a very early age of onset. HCM is normally not a disease of small children. The proband was a boy who had suffered cardiac arrest at the age of 6.5years (resuscitation by cardioconversion). Upon screening of the β-myosin heavy chain gene as a candidate, two missense mutations, one in exon19 (Arg719Trp) and a second in exon12 (Met349Thr), were identified. The Arg719Trp mutation was de novo, as it was not found in the parents. In contrast, the Met349Thr mutation was inherited through the maternal grandmother. Six family members were carriers of this mutation but only the proband was clinically affected. Segregation and molecular analysis allowed us to assign the Met349Thr mutation to the maternal and the Arg719Trp de novo mutation to the paternal β-myosin allele. Thus, the patient has no normal myosin. We interpret these findings in terms of compound heterozygosity of a dominant (Arg719Trp) and a recessive (Met349Thr) mutation. Whereas a single mutated Arg719Trp allele would be sufficient to cause HCM, the concurrent Met349Thr mutation alone does not apparently induce the disease. Nevertheless, it conceivably contributes to the particularly severe phenotype. Received: 15 September 1997 / Accepted: 26 November 1997     

Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated Escherichia coli: Requirement for DNA End Processing

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m²). The activity decrease was seen by an increase in survival of phage T4 2⁻ of about 200-fold (phage T4 2⁻ has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb⁺) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.     

The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney

Summary Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three -or even four-fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type.The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.Investigations supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs. Saliha Sabanovic is gratefully acknowledged     

Nitrogen turnover in soil and global change

Nitrogen management in soils has been considered as key to the sustainable use of terrestrial ecosystems and a protection of major ecosystem services. However, the microorganisms driving processes like nitrification, denitrification, N-fixation and mineralization are highly influenced by changing climatic conditions, intensification of agriculture and the application of new chemicals to a so far unknown extent. In this review, the current knowledge concerning the influence of selected scenarios of global change on the abundance, diversity and activity of microorganisms involved in nitrogen turnover, notably in agricultural and grassland soils, is summarized and linked to the corresponding processes. In this context, data are presented on nitrogen-cycling processes and the corresponding microbial key players during ecosystem development and changes in functional diversity patterns during shifts in land use. Furthermore, the impact of increased temperature, carbon dioxide and changes in precipitation regimes on microbial nitrogen turnover is discussed. Finally, some examples of the effects of pesticides and antibiotics after application to soil for selected processes of nitrogen transformation are also shown.