Studies on the Activity of the Long Terminal Repeat of Rous Sarcoma Virus in Animal Cells and in Yeast

Transcription from the Rous sarcoma virus (RSV) Long terminal repeat (LTR) in untransformed rat 3Yl fibroblasts is dependent on the presence of serum. Within an hour of addition of serum to a serum-deprived culture there is a 5 fold stimulation in the level of transcripts initiated at the LTR. This stimulation does not require synthesis of new proteins. Mutations in the RSV LTR revealed that serum-stimulated transcription was mostly dependent on two CCAAT boxes in the LTR, though other upstream sequences may play a secondary role. Serum caused the rapid appearance of a nuclear protein that binds to the two CCAAT boxes. This serum-induced CCAAT factor was also bound by CCAAT sequences from other promoters, e.g. those of human heat shock protein 70, human c-Ha-ras, human histone 1 etc, but not by the adenovirus origin of replication, or the SV40 enhancer core sequence. This data suggests that the serum induced CCAAT factor is related to CPl or CP2 rather than the NFl or CjEBP types of CCAAT binding factors. The abundance of the factor in the nucleus is increased by serum even in the presence of inhibitors of new protein synthesis. The serum dependence of transcription from the RSV L TR is lost in v-src transformed 3Yl, and cross-feeding experiments showed that the mechanism did not involve the production of extra-cellular growth factors. Temperature-sensitive (ts) v-src transformed 3Yl was used to demonstrate that the tyrosine kinase activity of v-src can (a) substitute for the serum-requirement of the RSV LTR, (b) increase the level of transcripts initiated in the LTR even in the presence of serum, (c) exert its serum-sparing effect on the RSV L TR in the absence of new protein synthesis, and (d) increase the amount of CCAAT binding factor in the nucleus. Orthovanadate, an inhibitor of tyrosine-phosphatases, which non-specifically elevates the level of phosphotyrosine in the cells, can mimic the effects of serum and v-src on transcription from the RSV L TR. The RSV LTR directed accurate initiation of transcripts in Saccharomyces cerevisiae, about 60 bases downstream from the TATA box that is used in animal cells. Mutations in the LTR revealed that the authentic TATA box was absolutely necessary for the transcription, and the CCAAT boxes that are responsive to serum in animal cells, were acting as upstream activating sequences . The absence of the TATA box or the CCAAT boxes gave a phenotype to the yeast carrying the mutant derivatives of RSVCAT, making it possible to establish a genetic system for cloning genes for yeast and mammalian transcription factors, and do structure-function analysis on them. The activity of the RSV L TR in yeast is not stimulated by la.ctate, and is not decreased by mutations in the HAP2 or HAP3 genes, suggesting that the UAS activity of the CCAAT boxes is mediated by yeast CCAAT binding factors other than HAP2/HAP3. While a high level of V-STC is toxic to the yeast, expression of very low levels of the oncogene may be stimulating the CAT activity from the RSV L TR about two fold. If this can be seen at the phenotypic level, it may be possible to establish a genetic system to study how v-src influences gene expression in yeast

Identification and characterization of the human ORC6 homolog

A new protein was cloned and identified as the sixth member of the human Origin Recognition Complex (ORC). The newly identified 30-kDa protein hsORC6 is 28% identical and 49% similar to ORC6p from Drosophila melanogaster, which is consistent with the identities and similarities found among the other ORC members reported in the two species. The human ORC6 gene is located on chromosome 16q12. ORC6 protein level did not change through the cell cycle. Like ORC1, ORC6 did not co-immunoprecipitate with other ORC subunits but was localized in the nucleus along with the other ORC subunits. Several cellular proteins co-immunoprecipitated with ORC6, including a 65-kDa protein that was hyperphosphorylated in G1 and dephosphorylated in mitosis. Therefore, unlike the tight stoichiometric association of six yeast ORC subunits in one holo-complex, only a small fraction of human ORC1 and ORC6 is likely to be associated with a subcomplex of ORC2, 3, 4 and 5 suggesting differences in the architecture and regulation of human ORC

LDA-Based Industry Classification

Industry classification is a crucial step for financial analysis. However, existing industry classification schemes have several limitations. In order to overcome these limitations, in this paper, we propose an industry classification methodology on the basis of business commonalities using the topic features learned by the Latent Dirichlet Allocation (LDA) from firms’ business descriptions. Two types of classification – firm-centric classification and industry-centric classification were explored. Preliminary evaluation results showed the effectiveness of our method

APC/C – the master controller of origin licensing?

DNA replication must be tightly controlled to prevent initiation of a second round of replication until mitosis is complete. So far, components of the pre-replicative complex (Cdt1, Cdc6 and geminin) were considered key players in this regulation. In a new study, Machida and Dutta have shown that depletion of Emi1 caused cells to replicate their DNA more than once per cell cycle [1]. This effect was dependent on the ability of Emi1 to inhibit the APC/C. In addition to its role in regulating entry into mitosis, oscillation of APC/C activity regulates pre-RC formation: high APC/C activity in late M/G1 allows pre-RC formation and low APC/C activity in S/G2 prevents pre-RC formation for a second time thereby preventing rereplication. Each redundant pathway to prevent rereplication is dependent on regulating one of the pre-RC components, and all of the pathways are co-regulated by Emi1 through the APC/C. In this commentary we discuss how this new role of Emi1 adds to our understanding of the regulation of replication initiation. We also review the literature to analyze whether APC/C has a role in regulating endoreduplication (a normal state of polyploidy in some differentiated cells). Similarly a role of premature APC/C activation in genomic instability of tumors is discussed

Architecture of the human origin recognition complex

All the human homologs of the six subunits of Saccharomyces cerevisiae origin recognition complex have been reported so far. However, not much has been reported on the nature and the characteristics of the human origin recognition complex. In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found that human ORC2, -3, -4 and -5 form a core complex. HsORC1 and HsORC6 subunits did not enter into this core complex, suggesting that the interaction of these two subunits with the core ORC2–5 complex is extremely labile. We found that the C-terminal region of ORC2 interacts directly with the N-terminal region of ORC3. The C-terminal region of ORC3 was, however, necessary to bring ORC4 and ORC5 into the core complex. A fragment containing the N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited the ORC2-ORC3 interaction. Overexpression of this fragment in U2OS cells blocked the cells in G1, providing the first evidence that a mammalian ORC subunit is important for the G1-S transition in mammalian cells

STUDIES ON VARIATION OF GNSS SIGNAL STRENGTHS FROM INDIA

Commercial GNSS receivers provide the measure of received satellite signal strength in terms of Signal to Noise ratio (SNR) or Carrier to Noise Ratio (C/N0), through in many cases the terms are used interchangeably. The received signal strength affects the receiver performance is one of the measure of usability of satellites for position solution purpose. GNSS signals passes through the atmosphere and are affected by atmosphere, therefore the values are also used for atmospheric research purposes. This papers presents the results of long-term studies on GPS, GLONASS and GALILEO signal strengths and their variation patterns using data from a commercial multi-GNSS receiver operating from Burdwan in eastern India. It may be observed that generally signal strength values increases with decreasing fluctuations for increasing elevation angle of satellites and saturates above certain elevation angle. The three constellations offer slightly different signal strengths and new generation Galileo and GLONASS satellites provides higher satellite signal strengths. The results would be useful in understanding the GNSS signal strength for various purposes

Studies on variation of GNSS signal strengths from India

64-71Commercial Global Navigation Satellite System (GNSS) receivers provide the measure of received satellite signal strength in terms of Signal to Noise ratio (SNR) or Carrier to Noise ratio (C/N0), through in many cases the terms are used interchangeably. The received signal strength affects the receiver performance, as this is one of the measures of usability of satellites for position solution purpose. GNSS signals pass through and are affected by atmosphere, therefore the signal strength values were also used for atmospheric research purposes. This paper presents the results of long-term studies on GPS, GLONASS and Galileo signal strengths and their variation patterns using data from a commercial multi-GNSS receiver operating from Burdwan situated in eastern India. It may be observed that generally signal strength values increase with increasing elevation angle of satellites with decreasing fluctuations and the values saturate above certain elevation angle. The three constellations offer slightly different signal strengths and new generation Galileo and GLONASS satellites provide higher satellite signal strengths. The results would be useful in understanding the usability of GNSS signals for various purposes

Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets

BACKGROUND: tRFs, 14 to 32 nt long single-stranded RNA derived from mature or precursor tRNAs, are a recently discovered class of small RNA that have been found to be present in diverse organisms at read counts comparable to miRNAs. Currently, there is a debate about their biogenesis and function. RESULTS: This is the first meta-analysis of tRFs. Analysis of more than 50 short RNA libraries has revealed that tRFs are precisely generated fragments present in all domains of life (bacteria to humans), and are not produced by the miRNA biogenesis pathway. Human PAR-CLIP data shows a striking preference for tRF-5s and tRF-3s to associate with AGO1, 3 and 4 rather than AGO2, and analysis of positional T to C mutational frequency indicates these tRFs associate with Argonautes in a manner similar to miRNAs. The reverse complements of canonical seed positions in these sequences match cross-link centered regions, suggesting these tRF-5s and tRF-3s interact with RNAs in the cell. Consistent with these results, human AGO1 CLASH data contains thousands of tRF-5 and tRF-3 reads chimeric with mRNAs. CONCLUSIONS: tRFs are an abundant class of small RNA present in all domains of life whose biogenesis is distinct from miRNAs. In human HEK293 cells tRFs associate with Argonautes 1, 3 and 4 and not Argonaute 2 which is the main effector protein of miRNA function, but otherwise have very similar properties to miRNAs, indicating tRFs may play a major role in RNA silencing. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-014-0078-0) contains supplementary material, which is available to authorized users