The primary transcriptome of barley chloroplasts: numerous noncoding RNAs and the dominating role of the plastid-encoded RNA polymerase.

Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here, we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identification of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons, indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions and opposite to annotated genes, demonstrating the existence of numerous noncoding RNA candidates.

Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts.

Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5'- and 3'-exonucleases, resulting in processed RNAs with PPR10 bound at the 5'- or 3'-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5'- or 3'-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation.

Genetic-parameter estimation of milk yield in White Maritza sheep breed using different test day models.

The aims of this study were to estimate the genetic parameters of the test day milk yield (TDMY) of the White Maritza sheep breed population and to choose the most appropriate linear models for genetic-parameter estimation of test day milk yield. The White Maritza sheep breed is a multipurpose native sheep breed in Bulgaria. Test day milk yield data were collected from 1992 to 2015 (24 years). Milk yield recordings were made in 18 flocks according to the AC method (official milk recording by ICAR regulations). The database includes 8768 test day milk yield records belonging to 987 ewes. The pedigree file includes 1937 animals. Nine test day models (TDMs) were formulated and tested for the estimation of the genetic parameters of milk yield. The first three models were repeatability models (REP models), the second three were random regression models (RRMs), and the last three models were also random regression models with an added Ali and Schaeffer regression to describe the lactation curve using first-, second- and third-order polynomials. The average TDMY was 764.47 mL. There were no significant differences in the values of heritability (h2) calculated by the three REP models: REP1 0.355 ±â€¯0.060, REP2 0.344 ±â€¯0.047 and REP3 0.347 ±â€¯0.060. The same applied to the repeatability coefficients, which, for the three REP models, were 0.384 ±â€¯0.065, 0.376 ±â€¯0.051 and 0.378 ±â€¯0.065, respectively. Based on REP model 1, three models with random regression RRM1, RRM2 and RRM3 were constructed, which is associated with the use of first-, second- and third-order polynomials (for the random effects of both the animal and the permanent environment). The trajectories of h2 calculated by the three RRMs were not similar and demonstrated some differences, both at the beginning and in the middle of the milking period. The RRM with third-order polynomials demonstrated more genetic diversity until the 165th day of lactation, but Akaike information criterion (AIC), Bayesian information criterion (BIC) and log-likelihood (LogL) estimates were higher. The regression models with first- and second-degree polynomials were insufficient to reveal genetic diversity to a higher degree than REP model 1. The trend in the trajectories of h2 calculated by the three random regression models with Ali and Schaeffer regression models (ASRMs) was similar to that of random regression models without the Ali and Schaeffer regression incorporated. Although the noted advantages of the random regression models revealed, to a greater extent, the genetic diversity of test day milk yield, AIC, BIC and LogL estimates indicated that repeatability models achieved a better balance between complexity and fitness and a smaller prediction error compared to random regression models.

Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU.

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography.

Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.