Detection of V617F mutation of gene jak2 at patients with chronic myeloproliferative neoplasms

The aim of the work was to create a protocol for detecting the V617F mutation of the gene jak2 in samples of patients with chronic myeloproliferative neoplasm which is necessary to unify the procedures of the analysis of blood samples according to WHO criteria for this group of diseases. Methods. Mutation was revealed using reverse transcriptase PCR and direct sequencing of PCR products. Results. Six samples of blood of patients with polycythemia vera were analyzed and the mutation V617F was detected in all six cases. This mutation was not detected in any of RNA samples of healthy donors. A case of simultaneous detection of mutations V617F and fused bcr/abl gene in CML patient was described. Conclusions. The proposed method for detecting the V617F mutation allows molecular genetic differential diagnosis of myeloproliferative neoplasm as well.Мета. Створити протокол, який дозволяє виявляти мутацію V617F гена jak2 у зразках РНК хворих на хронічні мієлопроліферативні неоплазми, що неохідно для уніфікації процедур аналізу зразків крові згідно з чинними критеріями ВОЗ для даної групи захворювань. Методи. Мутацію визначали за допомогою зворотно-транскриптазної полімеразної ланцюгової реакції та прямого секвенування продуктів полімеразної ланцюгової реакції. Результати. Проаналізовано шість зразків крові хворих на справжню поліцитемію і у всіх випадках виявлено мутацію V617F. Дану мутацію не знайдено в жодному з контрольних зразків РНК здорових донорів. Описано випадок одночасного виявлення мутації V617F та злитого гена bcr/abl у хворої на хронічну мієлоїдну лейкемію. Висновки. Запропонований метод дозволяє визначати мутацію V617F, що дає змогу використовувати його для молекулярно-генетичної диференційної діагностики мієлопроліферативних неоплазм.Цель. Создание протокола для виявления мутации V617F гена jak2 в образцах РНК больных с хроническими миелопролиферативными неоплазмами, что необходимо для унификации процедур анализа образцов крови согласно настоящим критериям ВОЗ для данной группы заболеваний. Методы. Мутацию определяли с помощью обратно-транскриптазной полимеразной цепной реакции и прямого секвенирования продуктов ПЦР. Результаты. Проанализированы шесть образцов крови больных истинной полицитемией и во всех случаях обнаружена мутация V617F. Эта мутация не найдена ни в одном из контрольных образцов РНК здоровых доноров. Описан случай одновременного выявления мутации V617F и слитого гена bcr/abl у больной с хронической миелоидной лейкемией. Выводы. Предложенный метод позволяет определять мутацию V617F, его также можно использовать для молекулярно-генетической дифференциальной диагностики миелопролиферативных неоплазм

Improved identification of enriched peptide–RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry

Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein–RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide–RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species

Optimization of obtaining the high label single strain probe based on the M13 phage

A method is proposed to obtain high-label single strain probe on the basis of the M13 phage. This is based on the use of a specific primer and the procedure of purification of the synthesized probe using nitrocellulose.Наведено методику одержання високоміченого одноланцюгового зонда на основі фага М13, що базується на використанні специфічного праймеру та процедурі очищення синтезованого зонду за допомогою нітроцелюлози.Приведена методика получения высокомеченного одноцепочечного зонда на основе фага М13, базирующаяся на использовании специфического праймера и процедуре очистки синтезированного зонда с помощью нитроцеллюлозы

Saturation of front propagation in a reaction-diffusion process describing plasma damage in porous low-k materials

We propose a three-component reaction-diffusion system yielding an asymptotic logarithmic time-dependence for a moving interface. This is naturally related to a Stefan-problem for which both one-sided Dirichlet-type and von Neumann-type boundary conditions are considered. We integrate the dependence of the interface motion on diffusion and reaction parameters and we observe a change from transport behavior and interface motion \sim t^1/2 to logarithmic behavior \sim ln t as a function of time. We apply our theoretical findings to the propagation of carbon depletion in porous dielectrics exposed to a low temperature plasma. This diffusion saturation is reached after about 1 minute in typical experimental situations of plasma damage in microelectronic fabrication. We predict the general dependencies on porosity and reaction rates.Comment: Accepted for publication in Phys. Rev.

Immunocytochemical study of BCR and bcr-abl localization in K562 cells

Aim: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. Methods: The coding sequences of DН and РН domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. Results: The gene constructs containing sequences coding for DН and РН domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. Conclusion: Antibodies against DН and РН domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes

Structural insights into how Prp5 proofreads the pre-mRNA branch site

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing

Dynamic Regulation of Alternative Splicing by Silencers that Modulate 5′ Splice Site Competition

SummaryAlternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with ∼70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5′ splice site located downstream of a weakened 5′ splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5′ splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5′ splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5′ splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate-limiting step(s) in splicing can lead to dramatic shifts in splice site choice

Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices

Development of molecular oncohematology in Ukraine

Disruption of the genetic component of cells are mandatory element of malignant transformation. For the majority of blood neoplasias genetic disorders have been discovered, and they can be used for diagnosis and appropriate therapy. The data obtained by authors about the role of domains of Bcr-Abl protein (the main etiological factor in the pathogenesis of leukemia with Ph-chromosome) are presented in this review as well as approved diagnostic methods for myeloproliferative disorders and acute leukemias.Обов’язковим і характерним елементом злоякісної трансформації є порушення генетичного компонента клітини. Для більшості неоплазій системи крові генетичні порушення є відомими, що дозволяє використовувати їх для діагностики і відповідної терапії. Наведено авторські дані стосовно ролі доменів білка Bcr-Abl (головного етіологічного фактора в патогенезі лейкемій з філадельфійською хромосомою) та представлено апробовані методи діагностики мієлопроліферативних захворювань і гострих лейкемій.Обязательным и характерным элементом злокачественной трансформации являются нарушения генетического компонента клетки. Для большинства неоплазий системы крови известны генетические нарушения, что позволяет использовать их для диагностики и проведения соответствующей терапии. Приведены авторские данные о роли доменов белка Bcr-Abl (главного этиологического фактора в патогенезе лейкемий с филадельфийской хромосомой) и представлены апробированные методы диагностики миелопролиферативных заболеваний и острых лейкемий