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Scheduled and unscheduled DNA synthesis in human leukemic cell lines positive and negative for terminal deoxynucleotidyl transferase
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Active polypeptide fragments common to prokaryotic, eukaryotic, and mitochondrial DNA polymerases
No full textWith a procedure that allows the renaturation of the DNA polymerase catalytic activity in situ after SDS-polyacrylamide gel electrophoresis, we have compared the active polypeptides present in extracts from organisms covering a wide evolutionary range from prokaryotes to eukaryotes, namely: Escherichia coli, Oryza sativa, Daucus carota , Neurospora crassa, Dictyostelium discoideum, Saccharomyces cerevisiae, Ceratitis capitata, Leucophaea maderae , Xenopus laevis, rat tissues and human lymphoblastoid cells. Two main clusters of active peptides are visible in mammalian and adult insect tissues, characterized by a mol. wt. greater than 70000 and less than 50000, respectively. High mol. wt. peptides are heterogeneous in size and correspond to active fragments of DNA polymerase alpha, whereas low mol. wt. peptides show the same migration rate as purified DNA polymerase beta and are not generated by proteolysis of the high mol. wt. cluster, In the three species of fungi studied, only high mol. wt. peptides are found. The same is true in plant cells, where no DNA polymerase beta activity is detectable and the pattern of the high mol. wt. cluster is similar to that observed in E. coli extracts (which also lack low mol. wt. peptides). Also in mitochondria from higher and lower eukaryotes only high mol. wt. species are observed, and the active band(s) range from 70000 to 145000 daltons. Our results indicate that the structure of DNA polymerase has been highly conserved during evolution so that an active fragment of mol. wt. greater than or equal to 70 000 is always found in prokaryotic enzymes and in the replicative species of eukaryotic and mitochondrial DNA polymerases; at a certain stage in evolution, another species of low mol. wt. DNA polymerase (beta or beta-like) appears
Complex pattern of HTLV-2 splicing and expression in PBMCs from infected patients
No full textHTLV express multiple gene products from the same coding region by employing different strategies, including alternative splicing. Our investigations were focused on the analysis of the levels of expression of different HTLV-2 transcripts, to test their temporal expression at different stages of viral cycle and their quantitation by real time RT-PCR, in infected cell lines and in primary cultures of PBMCs from infected subjects.
An early transcription of tax/rex regulatory mRNA was observed, followed by a gradual and steady increase of gag/pol and env structural transcripts and of other accessory mRNAs. We identified a novel 3’ splice acceptor site, used by both HTLV-2 A and B subtypes to generate alternative doubly spliced mRNAs within the pX region and also a novel env isoform preferentially expressed in 2B subtypes and behaving as a late gene. Among the singly spliced mRNAs coding for other accessory proteins, the p28, p22/p20-II isoform was found to be highly expressed as compared to its alternative p28, p22/p20-I form. The APH-2 transcript from the negative strand behaved as a late gene in stably infected cells, while in ex-vivo PBMCs its kinetics appeared to be variable so that a clear pattern of expression was not yet assessed.
In conclusion, the temporal transcription of different HTLV-2 transcripts follows a distinct expression pattern: tax/rex is the first mRNA to be expressed, thus indicating that it is necessary at the beginning of the infection cycle to transactivate and regulate viral and cellular transcripts, while gag/pol and env structural genes are expressed later in the viral cycle
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The MHC-II Transactivator CIITA, a Viral Restriction Factor Targeting Human T-Cell Lymphotropic Virus Type 1 Tax-1 Function and Inhibiting Viral Replication
No full textHuman T-cell Lymphotropic Virus type-1 (HTLV-1) is the causative agent of Adult T cell Leukemia-Lymphoma (ATLL). The viral transactivator Tax-1 protein plays a key role in the pathiogenesis of ATLL. We demonstrate that CIITA, the master regulator of MHC class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 aminoacids of Tax-1 and two CIITA adjacent regions (1-252 and 253-410). Interestingly only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64-124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular co-activators PCAF, CREB and ATF1 which are required for the optimal activation of HTLV-1 promoter. Accordingly, the over-expression of PCAF, CREB and ATF1 restored Tax-1-dependent transactivation of the viral LTR promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreadin
Complex pattern of HTLV-2 temporal expression in infected cell lines and patient PBMCs and identification of a novel splicing acceptor site
No full textBackground: Similarly to other retroviruses, HTLV-2 expresses multiple gene products from the same coding region by means of different strategies, including a complex pattern of alternative splicing.
Methods: Our investigation has been focused on the analysis of the levels of expression of different HTLV-2 transcripts, to highlight the kinetics of transcription at different stages of virus gene expression and their quantitation, by real time RT-PCR, in infected cell lines and in short term cultures of PBMCs from infected patients.
Results: Results obtained show an early transcription of tax/rex regulatory mRNA, followed by a gradual and steady increase of gag/pol and env structural transcripts and of other accessory mRNAs.Data demostrate that the expression of different HTLV-2 genes follows a distinct timing, both in chronically infected cells and in PBMCs isolated from infected patients. The regulatory transcript tax/rex is the first one to be expressed whereas the structural and accessory mRNAs are expressed at a later time point.
Further studies aimed at better understanding the complex pattern of splicing, allowed us to identify a novel 3’ acceptor site of splicing for the second exon of HTLV-2. This new 3’ acceptor site is capable of expressing an isoform of the singly spliced env mRNA. In addition, it could also be used as an alternative splicing system within the X region for the tax/rex, p10/p11 and p? transcripts.
Conclusions: This study demostrated that the expression of HTLV-2 transcripts is presenting distinct patterns, indicating that the control of viral gene expression is highly regulated both in its kinetics and expression level. Moreover, the use of multiple acceptor sites is likely to play an important role for the preferential expression of specific proteins at different phases of the viral cycle
Analysis of viral transcripts of Human T-cell Leukemia Virus type 2 (HTLV-2) in human T- and B- infected cells and PBMCs from infected subjects
No full textRecent studies carried out on HTLV-1-infected cells have provided information on the relative abundance and timing of expression of different viral transcripts. In the case of HTLV-2, information on the profile and temporal regulation of viral gene expression is still incomplete.
To address this point, we set up a splice-junction-specific real-time RT-PCR to evaluate the quantitative level of individual HTLV-2 transcripts. Results obtained led to the quantitation of all the HTLV-2 mRNAs described so far, namely gag/pol, env, tax/rex, p28,p22/p20rex-1, p28,p22/p20rex-2 and p10/p11. This analysis was carried out first on chronically infected lines: T-cells infected with the HTLV-2A subtype (Mo-T and C344) or B-cells infected with the HTLV-2B subtype (BJAB-Gu cells). Results showed different levels of expression of env and tax/rex transcripts in T- and B-cells. The expression profile and kinetics of expression of the different transcripts was analyzed in PBMC obtained ex vivo from infected individuals and cultured for several hours. Current experiments are also aimed at testing the possible connections between the pattern of HTLV-2 gene expression and the different phases of the cell cycle
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