Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

• Klíčová slova
• androgenesis barley, double haploid, embryo culture, transformation,
• Publikační typ
• časopisecké články MeSH

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

• Klíčová slova
• RNA-seq, SW55Ti rotor, mouse early embryo, mouse oocyte, mouse zygote, polysome fractionation, polysome profiling, translatome,
• MeSH
• jaderný obal genetika   metabolismus   MeSH
• meióza genetika   MeSH
• myši MeSH
• oocyty růst a vývoj   metabolismus   MeSH
• polyribozomy genetika   MeSH
• proteiny vázající RNA genetika   MeSH
• RNA messenger skladovaná genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• RNA ribozomální 28S genetika   MeSH
• sekvenování transkriptomu MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny vázající RNA MeSH
• RNA messenger skladovaná MeSH
• RNA ribozomální 18S MeSH
• RNA ribozomální 28S MeSH

We employed virus-like elements (VLEs) pGKL1,2 from Kluyveromyces lactis as a model to investigate the previously neglected transcriptome of the broader group of yeast cytoplasmic linear dsDNA VLEs. We performed 5' and 3' RACE analyses of all pGKL1,2 mRNAs and found them not 3' polyadenylated and containing frequently uncapped 5' poly(A) leaders that are not complementary to VLE genomic DNA. The degree of 5' capping and/or 5' mRNA polyadenylation is specific to each gene and is controlled by the corresponding promoter region. The expression of pGKL1,2 transcripts is independent of eIF4E and Pab1 and is enhanced in lsm1Δ and pab1Δ strains. We suggest a model of primitive pGKL1,2 gene expression regulation in which the degree of 5' mRNA capping and 5' non-template polyadenylation, together with the presence of negative regulators such as Pab1 and Lsm1, play important roles. Our data also support a hypothesis of a close relationship between yeast linear VLEs and poxviruses.

• Klíčová slova
• Lsm1, Pab1, eIF4E, linear cytoplasmic plasmid, pGKL, poly(A) leader, poxvirus, virus-like element,
• Publikační typ
• časopisecké články MeSH

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

• MeSH
• cytoplazma MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• fungální proteiny genetika   MeSH
• genetická transkripce * MeSH
• Kluyveromyces genetika   MeSH
• konformace nukleové kyseliny MeSH
• molekulární evoluce * MeSH
• polyadenylace MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u hub MeSH
• responzivní elementy * MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie MeSH
• stabilita RNA MeSH
• viry genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• fungální proteiny MeSH

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.

• Klíčová slova
• 3′RACE, 4EHP, Alternative polyadenylation, Polyadenylation signal, Translation, eIF4E, eIF4E2, eIF4E3,
• MeSH
• 3' nepřekládaná oblast MeSH
• buněčné linie MeSH
• eukaryotický iniciační faktor 4E genetika   metabolismus   MeSH
• exprimované sekvenční adresy MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mozek metabolismus   MeSH
• placenta metabolismus   MeSH
• polyadenylace genetika   MeSH
• proteiny vázající čepičku mRNA genetika   MeSH
• regulace genové exprese MeSH
• sestřih RNA genetika   MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• EIF4E2 protein, human MeSH Prohlížeč
• eIF4E3 protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 4E MeSH
• messenger RNA MeSH
• proteiny vázající čepičku mRNA MeSH

A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10-16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.

• MeSH
• buněčné linie MeSH
• genetická transkripce * MeSH
• Hepacivirus genetika   MeSH
• komplementární DNA genetika   MeSH
• lidé MeSH
• luciferasy světlušek genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• komplementární DNA MeSH
• luciferasy světlušek MeSH