Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65).
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U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing. (+info)
Chromatin modification by DNA tracking.
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In general, the transcriptional competence of a chromatin domain is correlated with increased sensitivity to DNase I cleavage. A recent observation that actively transcribing RNA polymerase II piggybacks a histone acetyltranferase activity [Wittschieben, B., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Ohba, R., Li, Y., Allis, C. D., Tempst, P. & Svejstrup, J. Q. (1999) Mol. Cell 4, 123-128] implies that the state of histone acetylation, and hence the ability of chromatin to fold, can be altered by a processive mechanism. In this article, it is proposed that tracking-mediated chromatin modification could create and/or maintain an open configuration in a complete chromatin domain including both intra- and extragenic regions. This mechanism suggests a putative functional role for the extragenic transcription observed at the beta-globin and other loci in vertebrate cells. (+info)
Globulins in protein supplements promote the development of preimplantation embryos.
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PURPOSE: Our purpose was to investigate the effect of alpha- and beta-globulins contained in protein supplements on the development of preimplantation embryos. METHODS: Mouse one-cell embryos were cultured in medium supplemented with 4 mg/ml human serum albumin (HSA), 4 mg/ml HSA plus human globulins (0.2, 0.4, 0.8, and 1.6 mg/ml) that consisted predominantly of alpha- and beta-globulins, or 10% Plasmanate Cutter (PC). Blastocysts developed in media supplemented with these various protein sources were stained with Hoechst 33342 to determine the number of cells. RESULTS: Supplementation with 0.4 to 1.6 mg/ml globulins or PC significantly increased the rate of blastocyst development compared with that observed with the addition of HSA. Supplementation with globulins significantly increased the hatching rate in a dose-dependent manner. The number of cells in the blastocysts was significantly increased when the embryos were cultured with 0.8 mg/ml of the globulins or PC. CONCLUSIONS: The present observations suggest that alpha- and beta-globulins in protein supplements promote embryo development and hatching. (+info)
Influence of histochemical and immunohistochemical stains on polymerase chain reaction.
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The polymerase chain reaction (PCR) analysis of DNA extracted from tissue sections can be applied to a variety of research and diagnostic protocols. To analyze selectively the specific areas of tissue, a direct microdissection of histochemically or immunohistochemically stained sections, if satisfactory for PCR, is helpful. However, the influence of various staining methods on PCR has been poorly investigated. In this study, paraffin sections of formalin-fixed lymph node samples were histochemically stained with Mayer's hematoxylin, eosin Y, methyl green, or May-Grunwald solution and immunostained for CD45 using 3,3'-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuchsin as the chromogen. In addition, unstained sections were treated with trypsin, microwave, or pressure cooker, the techniques frequently used in immunostains for antigen unmasking. DNA was extracted from each section, and the PCR efficiency in amplifying a 110 bp portion of the beta-globin gene was evaluated by two parameters: the cycle count in which the first visible band was obtained (CYCLE(min)) and the maximum amount of PCR products (CONC(max)). The hematoxylin stain showed a significantly prolonged CYCLE(min) (P < .01) and lower CONC(max) (P < .05) in comparison with unstained and untreated control sections. The May-Grunwald stain showed a prolonged CYCLE(min) (P < .01), although the CONC(max) was not significantly different from that of the control (P = .051). The eosin and methyl green stains showed no effects against PCR. In immunostains, the DAB-Co method showed a lower CONC(max) (P < .05), whereas the CYCLE(min) was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigen-unmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLE(min) (P < .01), and the PCR amplification did not reach the "plateau" level with a maximum of 60 cycles. The PCR efficiency was worse in microwave or pressure cooker treatment, with neither CYCLE(min) nor CONC(max) being obtained. When target areas from sections for subsequent PCR amplification are microdissected, methyl green is most suitable as a dye for nuclear staining. The immunohistochemical visualization with DAB or new fuchsin yields no unfavorable effects. A successful PCR amplification may not be expected in sections that are pretreated in a microwave oven or pressure cooker. (+info)
Suppression of metastasis by thymidine phosphorylase inhibitor.
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We developed a novel inhibitor of thymidine phosphorylase (TP), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI), that is about 1000-fold more active than 6-amino-5-chlorouracil, one of the most potent TP inhibitors. TPI inhibited the high chemotactic motility and basement membrane invasion of KB/TP cells, a TP-positive clone transfected with Rous sarcoma virus (RSV)/TP, to the levels seen in KB/CV cells, a control clone transfected with RSV. In nude mice, oral administration of TPI suppressed not only macroscopic liver metastases of highly metastatic KB/TP cells but also the level of human beta-globin as a molecular marker of micrometastases in the livers of the mice. These findings demonstrate that TP plays a key role in the invasiveness and metastasis of TP-expressing solid tumors and suggest that TPI might be a novel antimetastatic agent for blood-borne metastasis. (+info)
Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy.
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BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy. (+info)
Fluorescence PCR quantification of cyclin D1 expression.
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We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A beta-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL. (+info)
Y14 and hUpf3b form an NMD-activating complex.
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Messenger RNAs with premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). In mammals, PTCs are discriminated from physiological stop codons by a process thought to involve the splicing-dependent deposition of an exon junction complex (EJC), EJC-mediated recruitment of Upf3, and Upf2 binding to the N terminus of Upf3. Here, we identify a conserved domain of hUpf3b that mediates an interaction with the EJC protein Y14. Tethered function analysis shows that the Y14/hUpf3b interaction is essential for NMD, while surprisingly the interaction between hUpf3b and hUpf2 is not. Nonetheless, hUpf2 is necessary for NMD mediated by tethered Y14. RNAi-induced knockdown and Y14 repletion of siRNA-treated cells implicates Y14 in the degradation of beta-globin NS39 mRNA and demonstrates that Y14 is required for NMD induced by tethered hUpf3b. These results uncover a direct role of Y14 in NMD and suggest an unexpected hierarchy in the assembly of NMD complexes. (+info)