Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, we review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions.

Eight human and six chicken novel alternatively spliced (AS) variants of telomerase reverse transcriptase (TERT) were identified, including a human variant (⌬4-13) containing an in-frame deletion which removed exons 4 through 13, encoding the catalytic domain of telomerase. This variant was expressed in telomerase-negative normal cells and tissues as well as in transformed telomerase-positive cell lines and cells which employ an alternative method to maintain telomere length. The overexpression of the ⌬4-13 variant significantly elevated the proliferation rates of several cell types without enhancing telomerase activity, while decreasing the endogenous expression of this variant by use of small interfering RNA (siRNA) technology reduced cell proliferation. The expression of the ⌬4-13 variant stimulated Wnt signaling. In chicken cells, AS TERT variants containing internal deletions or insertions that eliminated or reduced telomerase activity also enhanced cell proliferation. This is the first report that naturally occurring AS TERT variants which lack telomerase activity stimulate cell proliferation.

Summary Štorchová, H., Hrdličková, R., Chrtek, J., Jr., Tetera, M., Fitze, D. & Fehrer, J.: An improved method of DNA isolation from plants collected in the field and conserved in saturated NaCl/CTAB solution. – Taxon 49: 79‐84. 2000. – ISSN 0040‐0262. A simple method for isolation of genomic DNA from wild plants sampled in remote field areas is presented. The protocol combines NaCl/CTAB leaf preservation with sorbitol extraction of secondary compounds which often contain inhibitors of Taq DNA polymerase activity. The obtained DNA is suitable for random amplified polymorphic DNA (RAPD) analysis of plant populations as well as for specific amplification of chloroplast DNA sequences. The NaCl/CTAB leaf preservation is a powerful alternative to silica gel drying‐based preservation.

No abstract

This manuscript presents the first extensive phylogenetics analysis of a key family of immune regulators, the interferon regulatory factor (IRF) family. The IRF family encodes transcription factors that play important roles in immune defense, stress responses, reproduction, development, and carcinogenesis. Several times during their evolution, the IRF genes have undergone expansion and diversification. These genes were also completely lost on two separate occasions in large groups of metazoans. The origin of the IRF family coincides with the appearance of multicellularity in animals. IRF genes are present in all principal metazoan groups, including sea sponges, placozoans, comb jellies, cnidarians, and bilaterians. Although the number of IRF family members does not exceed two in sponges and placozoans, this number reached five in cnidarians. At least four additional independent expansions lead up to 11 members in different groups of bilaterians. In contrast, the IRF genes either disappeared or mutated beyond recognition in roundworms and insects, the two groups that include most of the metazoan species. The IRF family separated very early into two branches ultimately leading to vertebrate IRF1 and IRF4 supergroups (SGs). Genes encoding the IRF-SGs are present in all bilaterians and cnidarians. The evolution of vertebrate IRF family members further proceeded with at least two additional steps. First, close to the appearance of the first vertebrate, the IRF family probably expanded to four family members, predecessors of the four vertebrate IRF groups (IRF1, 3, 4, 5 groups). In the second step, 10 vertebrate family members evolved from these four genes, likely as a result of the 2-fold duplication of the entire genome. Interestingly, the IRF family coevolved with the Rel/NF-kappaB family with which it shares some important evolutionary characteristics, including roles in defense responses, metazoan specificity, extensive diversification in vertebrates, and elimination of all family members in nematodes.

The cloning and functional characterization of a novel interferon regulatory factor (IRF), IRF-10, are described. IRF-10 is most closely related to IRF-4 but differs in both its constitutive and inducible expression.

The human TERT (hTERT) gene encodes the telomerase catalytic subunit which plays a role in telomerase regulation. Telomerase is activated in more than 90% of all human malignancies and understanding how telomerase is regulated is necessary for implementation of successful anti-cancer therapies. microRNAs (miRNAs) are important regulators of gene expression in eukaryotic cells but evidence of their role in telomerase regulation has not been documented. To determine whether hTERT activity is regulated by multiple miRNAs, eight miRNAs which have putative binding sites in the hTERT 3′UTR together with miR-138-5p were evaluated in luciferase assays with a reporter containing the hTERT 3′UTR. Six miRNAs (let-7g*, miR-133a, miR-138-5p, miR-342-5p, miR-491-5p, and miR-541-3p) specifically inhibited the expression of the reporter luciferase-driven constructs and let-7g*, miR-133a, miR-138-5p, and miR-491-5p also downregulated endogenous telomerase activity in cells. Moreover, all six miRNAs significantly inhibited cell proliferation. miRNAs (miR-133a, miR-138-5p, 342-5p, 491-5p, 541-3p) also have predicted binding sites within the 3′UTR of three genes involved in Wnt signaling (TCF7, MSI1, and PAX5). These miRNAs inhibited the expression of the luciferase reporter constructs containing 3′UTRs of these genes and downregulated protein expression of the TCF7 transcription factor, which mediates the canonical Wnt pathway. Together, these results suggest the existence of a miRNA regulatory network involving the hTERT and Wnt pathway.

The c-rel gene is a member of NF-KBIrel family of transcription factors that regulate expression of a variety of immunoregulatory molecules. The virat oncogene, v-rel, is a truncated and mutated form of the turkey c-rel gene expressed by reticuloendotheliosis virus, strain T. In this study, we demonstrated that three avian immunoregulatory receptors, major histocompatibility (MHC) antigens class I and class II as well as the interleukin-2 receptor (IL-2R), were induced on the surface of splenic tumor cells isolated from chickens infected with reticuloendotheliosis virus, strain T. All cell lines derived from splenic tumors expressed these three proteins. Their expression also correlated with the appearance of endogenous c-rel during a graft-versushost reaction. In vitro, both c-rel and v-rel induced MHC class I, MHC class II, and IL-2R on an avian B-lymphoid cell line, DT95, and a T-lymphoid cell line, MSB-1. Quantitative kinetic analysis demonstrated both the accumulation of MHC class II mRNA and the appearance of surface MHC class II protein in response to the synthesis of either v-rel or c-rel. We show that v-rel induced the expression of MHC class II in the avian B-cell lines DT40 and DT95 more rapidly than c-rel and that, several weeks after infection, v-rel induced MHC class II as much as 50-fold more efficiently than c-rel. Finally, in vitro infection of splenocytes with retroviruses that express v-rel or c-rel induced MHC class I, MHC class II, and IL-2R expression. Quantitative analysis confirmed that p59v-re was consistently more efficient at inducing expression of all three immunoregulatory receptors than exogenous p68c-er. These data suggest that during tumor development, v-rel functions to induce (or suppress) the expression of genes similarly induced (or suppressed) by c-rel. The observations reported in this study are not in agreement with a model in which v-rel promotes tumor development by functioning as a dominant negative mutant of c-rel. In contrast, these findings support the hypothesis that lymphocyte immortalization and tumor development are the result, at least in part, of the capacity of v-rel to function as a dominant positive mutant that induces expression of genes normally regulated by c-rel.