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Nurgul Sikhayeva,1,2 Yerkebulan Talzhanov,1 Aisha Iskakova,1 Jarkyn Dzharmukhanov,1 Raushan Nugmanova,1 Elena Zholdybaeva,1 Erlan Ramanculov1–3 1National Scientific Laboratory of Biotechnology, National Center for Biotechnology, Astana, Kazakhstan; 2Faculty of Natural Sciences, L.N. Gumilyov Eurasian National University, Astana, Kazakhstan; 3School of Science and Technology, Nazarbayev University, Astana, Kazakhstan Background: Ethnic differences exist in the frequencies of genetic variations that contribute to the risk of common disease. This study aimed to analyse the distribution of several genes, previously associated with susceptibility to type 2 diabetes and obesity-related phenotypes, in a Kazakh population.Methods: A total of 966 individuals belonging to the Kazakh ethnicity were recruited from an outpatient clinic. We genotyped 41 common single nucleotide polymorphisms (SNPs) previously associated with type 2 diabetes in other ethnic groups and 31 of these were in Hardy–Weinberg equilibrium. The obtained allele frequencies were further compared to publicly available data from other ethnic populations. Allele frequencies for other (compared) populations were pooled from the haplotype map (HapMap) database. Principal component analysis (PCA), cluster analysis, and multidimensional scaling (MDS) were used for the analysis of genetic relationship between the populations.Results: Comparative analysis of allele frequencies of the studied SNPs showed significant differentiation among the studied populations. The Kazakh population was grouped with Asian populations according to the cluster analysis and with the Caucasian populations according to PCA. According to MDS, results of the current study show that the Kazakh population holds an intermediate position between Caucasian and Asian populations.Conclusion: A high percentage of population differentiation was observed between Kazakh and world populations. The Kazakh population was clustered with Caucasian populations, and this result may indicate a significant Caucasian component in the Kazakh gene pool. Keywords: single-nucleotide polymorphism, Kazakh population, type 2 diabetes, OpenArray

As a historical nomadic group in Central Asia, Kazaks have mainly inhabited the steppe zone from the Altay Mountains in the East to the Caspian Sea in the West. Fine scale characterization of the genetic profile and population structure of Kazaks would be invaluable for understanding their population history and modeling prehistoric human expansions across the Eurasian steppes. With this mind, we characterized the maternal lineages of 200 Kazaks from Jetisuu at mitochondrial genome level. Our results reveal that Jetisuu Kazaks have unique mtDNA haplotypes including those belonging to the basal branches of both West Eurasian (R0, H, HV) and East Eurasian (A, B, C, D) lineages. The great diversity observed in their maternal lineages may reflect pivotal geographic location of Kazaks in Eurasia and implies a complex history for this population. Comparative analyses of mitochondrial genomes of human populations in Central Eurasia reveal a common maternal genetic ancestry for Turko-Mongolian speakers and their expansion being responsible for the presence of East Eurasian maternal lineages in Central Eurasia. Our analyses further indicate maternal genetic affinity between the Sherpas from the Tibetan Plateau with the Turko-Mongolian speakers., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Askapuli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rabies virus nucleoprotein (N protein) encapsidates genomic RNA of the virus and forms the viral ribonucleoprotein complex. These N proteins represent highly organized structures which activate proliferation of B cells and production antibodies against the N protein. In addition to the B cell, the rabies virus N protein has been shown to induce potent T helper cell responses resulting in a long-lasting and strong humoral immune response. Rabies virus N protein is a molecular target of choice for development of tools to diagnose acute rabies infection. We produced a recombinant immune reactive C-terminal fragment of the rabies virus N protein which contains an antigenic determinant located between positions 360-389. Synthetic gene encoding the N protein was cloned into an expression plasmid to produce the recombinant antigen in Escherichia coli cells BL21 (DE3). SDS-PAGE showed presence of the product with expected molecular weight (44 kDa). The recombinant fragment of the N protein efficiently recognized antibodies in sera from mice immunized with an inactivated rabies virus. Thus produced recombinant antigen of the rabies virus N protein can be used in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of the rabies infection., Competing Interests: The authors declare no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Background: We evaluated the associations between single nucleotide polymorphisms and different clinical parameters related to type 2 diabetes mellitus (T2DM), obesity risk, and metabolic syndrome (MS) in a Kazakh cohort., Methods: A total of 1336 subjects, including 408 T2DM patients and 928 control subjects, were recruited from an outpatient clinic and genotyped for 32 polymorphisms previously associated with T2DM and obesity-related phenotypes in other ethnic groups. For association studies, the chi-squared test or Fisher's exact test for binomial variables were used. Logistic regression was conducted to explore associations between the studied SNPs and the risk of developing T2DM, obesity, and MS, after adjustments for age and sex., Results: After excluding four SNPs due to Hardy-Weinberg disequilibrium, significant associations in age-matched cohorts were found betweenT2DM and the following SNPs: rs9939609 (FTO), rs13266634 (SLC30A8), rs7961581 (TSPAN8/LGR5), and rs1799883 (FABP2). In addition, examination of general unmatched T2DM and control cohorts revealed significant associations between T2DM and SNPsrs1799883 (FABP2) and rs9939609 (FTO). Furthermore, polymorphisms in the FTO gene were associated with increased obesity risk, whereas polymorphisms in the FTO and FABP2 genes were also associated with the risk of developing MS in general unmatched cohorts., Conclusion: We confirmed associations between polymorphisms within the SLC30A8, TSPAN8/LGR5, FABP2, and FTO genes and susceptibility to T2DM in a Kazakh cohort, and revealed significant associations with anthropometric and metabolic traits. In particular, FTO and FABP2 gene polymorphisms were significantly associated with susceptibility to MS and obesity in this cohort.

Brucellosis is a major zoonotic infection in Kazakhstan. However, there is limited data on its incidence in humans and animals, and the genetic diversity of prevalent strains is virtually unstudied. Additionally, there is no detailed overview of Kazakhstan brucellosis control and eradication programs. Here, we analyzed brucellosis epidemiological data, and assessed the effectiveness of eradication strategies employed over the past 70 years to counteract this infection. We also conducted multiple loci variable-number tandem repeat analysis (MLVA) of Brucella abortus strains found in Kazakhstan. We analyzed official data on the incidence of animal brucellosis in Kazakhstan. The records span more than 70 years of anti-brucellosis campaigns, and contain a brief description of the applied control strategies, their effectiveness, and their impact on the incidence in humans. The MLVA-16 method was used to type 94 strains of B. abortus and serial passages of B. abortus 82, a strain used in vaccines. MLVA-8 and MLVA-11 analyses clustered strains into a total of four and seven genotypes, respectively; it is the first time that four of these genotypes have been described. MLVA-16 analysis divided strains into 28 distinct genotypes having genetic similarity coefficient that varies from 60 to100% and a Hunter & Gaston diversity index of 0.871. MST analysis reconstruction revealed clustering into "Kazakhstani-Chinese (Central Asian)", "European" and "American" lines. Detection of multiple genotypes in a single outbreak confirms that poorly controlled trade of livestock plays a crucial role in the spread of infection. Notably, the MLVA-16 profile of the B. abortus 82 strain was unique and did not change during 33 serial passages. MLVA genotyping may thus be useful for epidemiological monitoring of brucellosis, and for tracking the source(s) of infection. We suggest that countrywide application of MLVA genotyping would improve the control of brucellosis in Kazakhstan., Competing Interests: The authors have declared that no competing interests.

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role in the repair of oxidative DNA damage generated by endogenous and host- imposed factors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Brucellosis is an endemic disease in Central Asia characterized by high infection rates in humans and animals. Currently, little is known about the genetic diversity of Brucella spp. circulating in the region, despite the high prevalence of brucellosis. This study aimed to analyze the genetic diversity of Brucella melitensis and Brucella abortus strains circulating in the Republic of Kazakhstan. We genotyped 128 B. melitensis and 124 B. abortus strains collected in regions with the highest prevalence of brucellosis. Genotyping was performed using multi-locus variable-number tandem-repeat analysis (MLVA). Analysis of a subset of 8 loci (MLVA-8) of 128 B. melitensis strains identified genotypes 42 (n=108), 43 (n=2), and 63 (n=19) related to the 'East Mediterranean' group. An MLVA-16 assay sorted 128 B. melitensis strains into 25 different genotypes. Excluding one variable locus, MLVA-15 of B. melitensis was distinct from strains originating in the Mediterranean region; however, 77% of them were identical to strains isolated in China. A minimum spanning tree for B. melitensis using MLVA-15 analysis clustered the local strains together with strains previously collected in China. MLVA-8 analysis of 124 B. abortus strains identified them as genotype 36, suggesting Eurasian distribution of this lineage. Complete MLVA-16 assay analysis clustered the strains into five genotypes, revealing little diversity of B. abortus when compared on the global scale. A minimum spanning tree for B. abortus obtained using MLVA-15 analysis clustered the 2 most prevalent genotypes (n=117) together with strains previously collected in China. Thus, MLVA analysis was used to characterize 252 strains of Brucella collected in Kazakhstan. The analysis revealed genetic homogeneity among the strains. Interestingly, identical MLVA-15 profiles were found in seemingly unrelated outbreaks in China, Turkey, and Kazakhstan. Further analysis is needed for better understanding of the epidemiology of brucellosis in Asia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Introduction: The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular "proteome"). It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI) are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination) or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo., Methods: The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP) was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry., Results: Using several experimental systems (BirA.A + BAP.B), we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments - 3 paralogues of heterochromatin protein HP1α (CBX5), HP1β (CBX1), HP1γ (CBX3), wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA), ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation) and is designed to purify and study the protein composition of chromatin in proximity to the nuclear protein of interest. Using the RAD18 protein as a model, we demonstrated that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2Az has a different pattern of H4 acetylation., Conclusion: Progress in the last decade in cancer drug therapy has led us to the conclusion that the nucleus of eukaryotic cells is an active site for many cellular processes important to the development of cancer. These processes include changes in genetic and epigenetic landscape (e. g. methylation of DNA, modification of histones) and the expression levels of transcription factors, which regulates gene products (e.g. hypoxia-inducible factor 1α (HIF-1α) in chronic anemia, etc.) where protein-protein interactions play important role. Understanding the nature of protein-protein interactions may improve design strategies for small-molecule PPI modulators. PPI assay technologies that closely reflect physiological conditions hold the key to developing specific anti-cancer drugs.

Introduction: ABO blood group genotyping is a new technology in hematology that helps prevent adverse transfusion reactions in patients. Identification of antigens on the surface of red blood cells is based on serology; however, genotyping employs a different strategy and is aimed directly at genes that determine the surface proteins. ABO blood group genotyping by real-time PCR has several crucial advantages over other PCR-based techniques, such as high rapidity and reliability of analysis. The purpose of this study was to examine nucleotide substitutions differences by blood types using a PCR-based method on Kazakh blood donors., Methods: The study was approved by the Ethics Committee of the National Center for Biotechnology. Venous blood samples from 369 healthy Kazakh blood donors, whose blood types had been determined by serological methods, were collected after obtaining informed consent. The phenotypes of the samples included blood group A (n = 99), B (n = 93), O (n = 132), and AB (n = 45). Genomic DNA was extracted using a salting-out method. PCR products of ABO gene were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems). The resulting nucleotide sequences were compared and aligned against reference sequence NM_020469.2. Real-time PCR analysis was performed on CFX96 Touch™ Real-Time PCR Detection System (BioRad)., Results: Direct sequencing of ABO gene in 369 samples revealed that the vast majority of nucleotide substitutions that change the ABO phenotype were limited to exons 6 and 7 of the ABO gene at positions 261, 467, 657, 796, 803, 930 and 1,060. However, genotyping of only three of them (261, 796 and 803) resulted in identification of major ABO genotypes in the Kazakh population. As a result, TaqMan probe based real-time PCR assay for the specific detection of genotypes 261, 796 and 803 was developed. The assay did not take into account several other mutations that may affect the determination of blood group, because they have a low occurrence rate and therefore have not been found in the population sample., Conclusion: Real-time PCR based method for fast and reliable ABO genotyping was developed. This assay may be used as a complement to classic serological blood typing.

Introduction: Phase II xenobiotic biotransformation enzymes perform detoxification of hydrophilic and often toxic Phase I products through glutathionetransferase (GST), UDP-glucuronosyltransferase (UDF), N-acetyltransferase (NAT) families and other enzymes. GST protein families metabolize a large number of electrophilic xenobiotics, by conjugating fusing them with glutathione. Arylamine-N-acetyltransferase (NAT) catalyzes the acetylation of the aromatic and heterocyclic amines., Materials and Methods: This study assesses the frequency of NAT2 and GSTP1 gene polymorphisms in 326 healthy individuals from different regions of Kazakhstan by using Real-Time PCR and direct sequencing methods., Results: The allele frequencies were calculated for NAT2*5 (0.54) and GSTP1 (0.27). GSTP1 alleles were in the Hardy-Weinberg equilibrium (p > 0.05), while NAT2*5 (p = 0.00) were not. The population differences between North, Northeast and South Kazakhstan regions were also analyzed. No statistically significant differences in the frequency of genotypes were found., Conclusion: Allelic polymorphisms of NAT2*5 and GSTP1 genes greatly varied indifferent populations. The Kazakh population was significantly different from the Asian, Caucasoid, African-American and Hispanic populations by NAT2*5 and GSTP1 genes. Allelic variants of the NAT2*5 had a low frequency in Asian populations. Allelic frequency in other world populations varied from 30 to 50%. The differences between Kazakh (0.54) and the world population were statistically significant (p < 0.05). The frequency of GSTP1 (rs1695) in the African American population was 42%. The frequency of GSTP1 in Asian populations varied from 11% to 23%. The frequency in Caucasoid populations was around 30%. The differences between Kazakh population (0.27) and other populations selected were statistically significant (p < 0.05).The study of mutations in GSTP1 and NAT2 genes is necessary in assessing the risk of the development of various diseases, such as cancer. Information on allelic polymorphisms might also be useful for personal perscriptions such as cyclophosphamide, cisplatin, methotrexate, isoniazid, pyrazinamide, and rifampin.

Introduction: The spatiotemporal order plays an important role in cell functioning and is affected in many pathologies such as cancer and neurodegenerative diseases. One of the ultimate goals of molecular biology is reconstruction of the spatiotemporal structure of a living cell at the molecular level. This task includes determination of proximities between different molecular components in the cell and monitoring their time- and physiological state-dependent changes. In many cases, proximity between macromolecules arises due to their interactions; however, the contribution of dynamic self-organization in generation of spatiotemporal order is emerging as another viable possibility. Specifically, in proteomics, this implies that the detection of protein-protein proximity is a more general task than gaining information about physical interactions between proteins, as it could detail aspects of spatial order in vivo that are challenging to reconstitute in binding experiments in vitro ., Methods: In this work, we have developed a method of monitoring protein-protein proximity in vivo . For this purpose, the BirA was fused to one of the interaction partners, whereas the BAP was modified to make the detection of its biotinylation possible by mass spectrometry., Results: Using several experimental systems, we showed that the biotinylation is interaction dependent. In addition, we demonstrated that BAP domains with different primary amino acid structures and thus with different molecular weights can be used in the same experiment, providing the possibility of multiplexing. Alternatively to the changes in primary amino acid structure, the stable isotope format can also be used, providing another way to perform multiplexing experiments. Finally, we also demonstrated that our system could help to overcome another limitation of current methodologies to detect protein-protein proximity. For example, one can follow the state of a protein of interest at a defined time after its interaction with another protein has occurred. This application should be particularly useful for studying multistep intracellular processes, where the proximities between proteins and protein properties typically changed in a sequential manner., Conclusion: This approach has promised in adding temporal dimension in addition to helping reconstruct cell topology in space.

Introduction: Pharmacogenomics is an emerging field of medicine that combines genetics and pharmacology. Pharmacogenomic research is relatively new in Kazahkstan, but, in recent years, significant progress has been made in this field. The National Scientific Laboratory for Biotechnology has launched several government-funded research projects focused on finding genetic markers that determine susceptibility to various drugs. Another goal of pharmacogenetic research in the laboratory is to find the pharmacogenomic markers that target cardiovascular diseases, accounting for allelic frequencies in selected genes in the Kazakh population. In addition, pharmacogenomic testing kits allow patients to choose the drug dosage. For example, the drug Warfarin has been developed within the framework of the "Technology Commercialization Project," funded jointly by the Ministry of Education and Science of the Republic of Kazakhstan and the World Bank., Material and Methods: The pharmacogenomic studies were conducted using the real-time PCR and direct DNA sequencing. DNA was isolated from venous blood or buccal cells, collected from patients., Results: To date, we have identified the most promising areas of research in the field of pharmacogenomics in Kazakhstan. The allelic frequencies of a number of polymorphisms in the Kazakh population have been calculated (CYP2C9, CYP2C19, CYP3A4, VKORC1, CYP4F2, GGCX, CYP2D6, CYP1A2, NAT2, GSTP1, SLC47A1). A unique repository of DNA samples was established and is being replenished during the implementation of aforementioned projects. Development of the testing kit for individual selection of Warfarin dosage is nearing completion. A patent, named "Method of Selection Based Dose Warfarin Genotyping for the Kazakh Population" has been recently obtained. An application for another patent, titled "Express Method of Correction of Warfarin Dosing, Based on Real-time PCR" has received positive evaluation. The results of domestic pharmacogenomic studies will allow a more rational selection of drugs and their dosage regimens specific to the Kazakh population.

Aim: To analyze associations between homocysteine level, MTHFR and FTO rs1477196 polymorphisms and folate status in patients with breast cancer (BC) in order to clarify determinants of hyperhomocysteinemia., Patients and Methods: The study included 315 BC cases and 604 controls., Results: The MTHFRC677T genotype was associated with an increased incidence of BC [Odds ratio (OR)=1.71; 95% Confidential interval (CI)=1.21-2.43]. The MTHFR A1298C genotype was associated with a decreased risk of BC [OR=0.68; 95% CI: 0.49-0.95]. The homocysteine level was not associated with either MTHFR C677T or A1298C, nor with FTO rs1477196, but was inversely correlated with folate status in cancer cases (p<0.0001) and tended to be higher in patients with the MTHFR 677TT genotype. The folate level (p<0.0005) was an independent predictor of hyper-homocysteinemia in patients with BC., Conclusion: These results suggest an important role of homocysteine in breast tumorigenesis. Further studies are warranted to investigate how combined MTHFR genotypes exert their effects on cancer susceptibility.

We have developed an approach termed PUB-NChIP (proximity utilizing biotinylation with native ChIP) to purify and study the protein composition of chromatin in proximity to a nuclear protein of interest. It is based on coexpression of (1) a protein of interest, fused with the bacterial biotin ligase BirA, together with (2) a histone fused to a biotin acceptor peptide (BAP), which is specifically biotinylated by BirA-fusion in the proximity of the protein of interest. Using the RAD18 protein as a model, we demonstrate that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2AZ has a different pattern of H4 acetylation. Finally, biotin pulse-chase experiments show that the H4 acetylation pattern starts to resemble the acetylation pattern of total H4 after the proximity of chromatin to RAD18 has been lost.

Two blind source separation methods (Independent Component Analysis and Non-negative Matrix Factorization), developed initially for signal processing in engineering, found recently a number of applications in analysis of large-scale data in molecular biology. In this short review, we present the common idea behind these methods, describe ways of implementing and applying them and point out to the advantages compared to more traditional statistical approaches. We focus more specifically on the analysis of gene expression in cancer. The review is finalized by listing available software implementations for the methods described., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Many genome-scale studies in molecular biology deliver results in the form of a ranked list of gene names, accordingly to some scoring method. There is always the question how many top-ranked genes to consider for further analysis, for example, in order creating a diagnostic or predictive gene signature for a disease. This question is usually approached from a statistical point of view, without considering any biological properties of top-ranked genes or how they are related to each other functionally. Here we suggest a new method for selecting a number of genes in a ranked gene list such that this set forms the Optimally Functionally Enriched Network (OFTEN), formed by known physical interactions between genes or their products. The method allows associating a network with the gene list, providing easier interpretation of the results and classifying the genes or proteins accordingly to their position in the resulting network. We demonstrate the method on four breast cancer datasets and show that 1) the resulting gene signatures are more reproducible from one dataset to another compared to standard statistical procedures and 2) the overlap of these signatures has significant prognostic potential. The method is implemented in BiNoM Cytoscape plugin (http://binom.curie.fr).

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.

Regulation of gene expression during infection of the thermophilic bacterium Thermus thermophilus HB8 with the bacteriophage P23-45 was investigated. Macroarray analysis revealed host transcription shut-off and identified three temporal classes of phage genes; early, middle and late. Primer extension experiments revealed that the 5' ends of P23-45 early transcripts are preceded by a common sequence motif that likely defines early viral promoters. T. thermophilus HB8 RNA polymerase (RNAP) recognizes middle and late phage promoters in vitro but does not recognize early promoters. In vivo experiments revealed the presence of rifampicin-resistant RNA polymerizing activity in infected cells responsible for early transcription. The product of the P23-45 early gene 64 shows a distant sequence similarity with the largest, catalytic subunits of multisubunit RNAPs and contains the conserved metal-binding motif that is diagnostic of these proteins. We hypothesize that ORF64 encodes rifampicin-resistant phage RNAP that recognizes early phage promoters. Affinity isolation of T. thermophilus HB8 RNAP from P23-45-infected cells identified two phage-encoded proteins, gp39 and gp76, that bind the host RNAP and inhibit in vitro transcription from host promoters, but not from middle or late phage promoters, and may thus control the shift from host to viral gene expression during infection. To our knowledge, gp39 and gp76 are the first characterized bacterial RNAP-binding proteins encoded by a thermophilic phage., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Kazakhstan is one of the 14 countries with a high rate of morbidity due to multidrug-resistant tuberculosis (MDR TB) in WHO European region. The aim of our study was to characterize mutations associated with drug resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates from Kazakhstan. M. tuberculosis strains were isolated from TB patients in different regions of Kazakhstan. A drug susceptibility test was performed on Lowenstein-Jensen medium using the absolute concentration method. Sequencing analysis was performed of the rpoB rifampicin resistance-determining region and the katG gene, the oxyR-ahpC intergenic region, and the inhA promoter region in 259 MDR M. tuberculosis isolates, in 51 isoniazid-resistant isolates, and in 13 rifampicin-resistant isolates. The mutational analysis revealed that the most frequent mutations associated with rifampicin and isoniazid resistance in M. tuberculosis are the substitutions at codons 531 (82.7%) and 315 (98.4%) in the rpoB and katG genes, respectively. In addition, we have found mutations with lower frequency at codon 526 (8.4%), 533 (1.5%), and 516 (1.1%) in the rpoB gene. In 6.2% of the isolates, no mutations were found in the rpoB gene. The findings of this study provide useful data for a better understanding of the mutation spectrum of isoniazid and rifampicin resistance among strains isolated from patients in Kazakhstan. Our results are also useful for the development of diagnostic tests of MDR M. tuberculosis.

The success in driving embryonic stem cells towards hepatic lineage has been confounded by the complexity and cost of differentiation protocols that employ large quantities of expensive growth factors (GFs). Instead of supplementing culture media with soluble GFs, we investigated cultivation and differentiation of mouse embryonic stem cells (mESCs) on printed arrays of GFs. Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP4) were mixed in solution with fibronectin and collagen (I) and then printed onto silane-modified glass slides to form 500 μm diameter protein spots. mESCs were cultured on top of GF spots for up to 12 days and analyzed by RT-PCR and immunostaining at different time points. The stem cells residing on HGF-containing combinations of GFs exhibited requisite features of hepatic differentiation including pronounced loss in pluripotency (Oct4), transient (up and down) expression of endoderm (Sox17) and upregulation of early hepatic markers--albumin and alpha-fetoprotein. The hepatic differentiation was enhanced further by adding hepatic stellate cells to surfaces that already contained mESCs on GF spots. A combination of co-culture with non-parenchymal liver cells and the optimal GF stimulation was found to induce endoderm and hepatic phenotype earlier and to a much greater extent than the GF arrays or micropatterned co-cultures used individually. While this paper investigated hepatic differentiation of mouse ESCs, our findings and stem cell culture approaches are likely to be relevant for human ESC cultivation. Overall, the platform combining printed GF arrays and heterotypic co-cultures will be broadly applicable for identifying the composition of the microenvironment niche for ESC differentiation into various tissue types., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cytokine secretion by leukocytes is an important indicator of immune response to pathogens and therefore has significant implications in disease diagnostics. Given heterogeneity of leukocyte subsets and the ability of multiple cell subsets to secrete the same cytokines, connecting cytokine production to a specific leukocyte subset is a distinct challenge. In the present paper we describe a strategy combining antibody (Ab)-based affinity cell separation and surface plasmon resonance (SPR) for capturing human CD4 T-cells and for label-free detection of cell-secreted interferon (IFN)-gamma--an important inflammatory cytokine. Human blood was introduced into a flow chamber modified with anti-CD4 Abs resulting in capture of CD4(+) T-cells. After mitogenic activation of cells inside the flow chamber, culture medium was routed onto an SPR chip modified with monoclonal IFN-gamma Abs. SPR signal observed in this experiment correlated with cytokine production by T-cells. The strategy of combining SPR detection with cell purification may be used in the future for label-free, sensitive detection of multiple cytokines or proteins secreted by the desired cell subset., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

In this paper, we describe the development of an electrochemical DNA aptamer-based biosensor for detection of interferon (IFN)-γ. A DNA hairpin containing IFN-γ-binding aptamer was thiolated, conjugated with methylene blue (MB) redox tag, and immobilized on a gold electrode by self-assembly. Binding of IFN-γ caused the aptamer hairpin to unfold, pushing MB redox molecules away from the electrode and decreasing electron-transfer efficiency. The change in redox current was quantified using square wave voltammetry (SWV) and was found to be highly sensitive to IFN-γ concentration. The limit of detection for optimized biosensor was 0.06 nM with linear response extending to 10 nM. This aptasensor was specific to IFN-γ in the presence of overabundant serum proteins. Importantly, the same aptasensor could be regenerated by disrupting aptamer-IFN-γ complex in urea buffer and reused multiple times. Unlike standard sandwich immunoassays, the aptasensor described here allowed one to detect IFN-γ binding directly without the need for multiple washing steps and reagents. An electrochemical biosensor for simple and sensitive detection of IFN-γ demonstrated in this paper will have future applications in immunology, cancer research, and infectious disease monitoring.

The goal of the present study was to investigate hepato-protective effects of growth factor (GF) arrays during alcohol injury. Hepatocyte growth factor (HGF) and bone morphogenetic protein (BMP)7 were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 500 microm diameter spots. Primary rat hepatocytes were seeded on top of the arrays forming clusters corresponding in size to the underlying protein spots. Cell arrays were then injured in culture by exposure to 100 mm ethanol for 48 h. Hepatocytes residing on GF spots were found to have less apoptosis then cells cultured on collagen-only spots. Least apoptosis (0.3% as estimated by TUNEL assay) was observed on HGF/BMP7/collagen spots whereas most apoptosis (17.3%) was seen on collagen-only arrays. Interestingly, the extent of alcohol-induced apoptosis in hepatocytes varied based on the concentration of printed GF. In addition to preventing apoptosis, printed GFs contributed to maintenance of epithelial phenotype during alcohol injury as evidenced by higher levels of E-cadherin expression in HGF-protected hepatocytes. Importantly, GF microarrays could be used to investigate heterotypic interactions in the context of liver injury. To highlight this, stellate cells - nonparenchymal liver cells involved in fibrosis - were added to hepatocytes residing on arrays of either HGF/collagen or collagen-only spots. Exposure of these cocultures to ethanol followed by RT-PCR analysis revealed that stellate cells residing alongside HGF-protected hepatocytes were significantly less activated (less fibrotic) compared to controls. Overall, our results demonstrate that GF microarray format can be used to screen anti-fibrotic and anti-apoptotic effects of growth factors as well as to investigate how signals delivered to a specific cell type modulate heterotypic cellular interactions., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In this paper we demonstrate that SWNTs and a covalent immobilization strategy enable very sensitive sensors with excellent long term stability. Organophosphorus hydrolase (OPH) functionalized single and multi-walled carbon nanotube (CNT) conjugates were exploited for direct amperometric detection of paraoxon, a model organophosphate. The catalytic hydrolysis of paraoxon produces equimoles of p-nitrophenol; oxidation was monitored amperometrically in real time under flow-injection (FIA) mode. OPH covalently immobilized on single-walled carbon nanotubes (SWNTs) demonstrated much higher activity than OPH conjugated to multi-walled carbon nanotubes (MWNTs). The dynamic concentration range for SWNT-OPH was 0.5-8.5 micromolL(-1) with a detection limit of 0.01 micromolL(-1) (S/N=3). In addition to this high sensitivity, the immobilized OPH retained a significant degree of enzymatic activity, and displayed remarkable stability with only 25% signal loss over 7 months. These results suggest that covalent immobilization of OPH on CNTs can be used for specific immobilization with advantages of long term stability, high sensitivity, and simplicity., (Published by Elsevier B.V.)

Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.

Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)-based detection of interferon-gamma (IFN-gamma)--an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-gamma (K(d) = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-gamma binding with an aptamer beacon occurred within 15 min of analyte introduction--suggesting dynamic replacement of the quencher-complementary strand by IFN-gamma molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-gamma. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps. The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-gamma. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFN-gamma. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.

The cytokine production by leukocytes correlates with body's ability to mount an immune response and therefore has high diagnostic value. In the present study we employed microfabricated surfaces to capture T-cells from minimally processed human blood, arrange these cells into a single cell array, and then detect interferon (IFN)-gamma released from individual cells. The fabrication of cell capture surfaces started with coating a silane-modified glass slide with a uniform layer of poly(ethylene glycol) (PEG) hydrogel. The hydrogel-coated slide was lyophilized and then incubated with a mixture of monoclonal anti-IFN-gamma and anti-CD4 antibodies (Abs). To define sites for single cell attachment, PEG hydrogel microwells (20 microm diameter) were photolithographically patterned on top of the Ab-containing hydrogel layer. This micropatterning process resulted in fabrication of PEG hydrogel microwells with Ab-decorated bottom and nonfouling walls. To minimize the blood volume requirement and to precisely define shear stress conditions, the engineered surface was enclosed inside a PDMS-based microfluidic device. Introduction of red blood cell (RBC) depleted whole human blood followed by controlled washing led to the isolation of individual CD4 T-cells within PEG microwells. Mitogenic activation and immunofluorescent staining performed inside the microfluidic chamber revealed IFN-gamma cytokine signal colocalized with specific T-cells. The device and process presented here will be expanded in the future to enable multiparametric functional analysis of immune cells organized into high density single cell arrays.

Growth factors are commonly present in soluble form during in vitro cell cultivation experiments in order to provide signals for cellular proliferation or differentiation. In contrast to these traditional experiments, we investigated solid-phase presentation of a hepatocyte growth factor (HGF), a protein important in liver development and regeneration, on microarrays of extracellular matrix (ECM) proteins. In our experiments, HGF was mixed in solution with ECM proteins (collagen (I), (IV) or laminin) and robotically printed onto silane-modified glass slides. Primary rat hepatocytes were seeded onto HGF/ECM protein microarrays and formed cellular clusters that corresponded in size to the dimensions of individual protein spots (500 microm diameter). Analysis of liver-specific products, albumin and alpha1-antitrypsin, revealed several fold higher levels of expression of these proteins in hepatocytes cultured on HGF/ECM microarrays compared to cells cultivated on ECM proteins alone. In addition, cultivation of hepatocytes on HGF/ECM protein spots led to spontaneous reorganization of cellular clusters from a monolayer into three-dimensional spheroids. We also investigated the effects of surface-tethered HGF on hepatocytes co-cultivated with stromal cells and observed a significantly higher level of albumin in co-cultures where hepatocytes were stimulated by HGF/ECM spots compared to co-cultures created on ECM protein islands without the growth factor. In summary, our study suggests that incorporation of HGF into ECM protein microarrays has a profound and long-lasting effect on the morphology and phenotype of primary hepatocytes. In the future, the number of growth factors printed on ECM microarrays will be expanded to enable multiplexed and combinatorial screening of inducers of cellular differentiation or proliferation.

Embryonic stem cells hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, we developed a micropatterned co-culture platform for stimulating hepatic differentiation of mouse embryonic stem cells (mESCs). Studies of mESC and hepatic cell adhesion preferences revealed that mESCs required fibronectin for attachment, while hepatic cells (HepG2) preferred collagen (I) substrate and did not adhere to fibronectin. Printing columns of collagen (I) and fibronectin spots (300 microm diameter), followed by sequential seeding of the two cell types, allowed the positioning of clusters of mESCs adjacent to groups of hepatic cells within the same microarray. These micropatterned co-cultures were maintained for up to two weeks in hepatic differentiation media supplemented. To examine the differentiation, mESCs were selectively extracted from the co-culture using laser microdissection and analyzed using real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). These analyses revealed that mESCs co-cultured with HepG2 cells showed a decrease in pluripotency gene expression concomitant with up-regulation of endodermal genes. In addition, the co-culture format induced a significant increase in the expression of liver genes compared to mESCs cultured alone. In conclusion, micropatterned co-cultures of mESCs and hepatic cells showed a significant promise in driving stem cell differentiation towards hepatic phenotype. In the future, this cell culture platform will be further enhanced to enable efficient conversion of mouse and human ESCs to hepatocytes.

Gut-associated lymphoid tissue is a major target and reservoir of human immunodeficiency virus (HIV)-infected T-cells. Our studies seek to recapitulate, in vitro, interactions between HIV-infected T-lymphocytes and intestinal epithelial cells in order to investigate the mechanisms underlying the disruption of normal epithelial cell and barrier function. Here, we describe a novel approach for creating co-cultures of healthy or HIV-infected T-lymphocytes (Jurkat) and human intestinal epithelial (HT-29) cells where both cell types are positioned on the same surface in a price spatial configuration (micropattern). This co-culture method simplified observation/monitoring of the two cell types and was particularly suited for laser microdissection-based retrieval of the desired cells for downstream gene expressions studies. DNA microarray analysis of epithelial cells retrieved from co-cultures with HIV-1-infected vs. uninfected Jurkat cells revealed that epithelial cells from HIV-infected co-cultures exhibited gene expression patterns consistent with disruption of epithelial barrier formation. Overall, the micropatterned co-culture system described here is envisioned as a valuable new tool for delineating how HIV and other infections contribute to dysfunction of mucosal epithelium.

Cytokines are produced by immune cells in response to viral or bacterial pathogens and therefore have significant diagnostic value. The goal of the present study was to develop a miniature device for detection of interleukin (IL)-2 and interferon (IFN)-gamma cytokines secreted by a small population of CD4 and CD8 T-cells. Microarrays of T-cell- and cytokine-specific Ab spots were printed onto poly(ethylene glycol) (PEG) hydrogel-coated glass slides and enclosed inside a microfluidic device, creating a miniature ( approximately 3 microL) immunoreaction chamber. Introduction of the red blood cell (RBC) depleted whole human blood into the microfluidic device followed by washing at a pre-defined shear stress resulted in isolation of pure CD4 and CD8 T-cells on their respective Ab spots. Importantly, the cells became localized next to anti-IL-2 and -IFN-gamma Ab spots. Mitogenic activation of the captured T-cells was followed by immunofluorescent staining (all steps carried out inside a microfluidic device), revealing concentration gradients of surface-bound cytokine molecules. A microarray scanner was then used to quantify the concentration of IFN-gamma and IL-2 near CD4 and CD8 T-cells. This study represents one of the first demonstrations of a microdevice for capturing desired T-cell subsets from a small blood volume and determining, on-chip, cytokine profiles of the isolated cells. Such a microdevice is envisioned as an immunology tool for multi-parametric analysis of T-cell function with direct applications in diagnosis/monitoring of HIV and other infectious diseases.

RNA interference (RNAi) has emerged as a powerful technology to silence arbitrary genes by designing small RNA constructs based on the targeted messenger RNA sequences. We recently developed a small molecule-controlled RNAi gene switch that combined the molecular recognition by in vitro selected RNA aptamers with versatile gene silencing by small interfering RNAs, and demonstrated for the first time, posttranscriptional modulation of RNAi through direct RNA-small molecule interaction. In this report, we describe the first application of this technology to regulate an endogenous gene in mammalian cells. As a proof-of-concept demonstration we chose to modulate expression of albumin-serum protein produced by the liver. We designed and constructed a theophylline aptamer-fused short hairpin RNA (shRNA) expression vector targeting albumin mRNA in hepatic (HepG2) cells. Transfection of HepG2 cells with the aptamer-shRNA expression vector allowed to control albumin gene expression by adding theophylline into the culture media in dose dependent fashion.

The microarray format is being used extensively for combinatorial screening of cellular interactions with proteins, small molecules, or biomaterials. The utility of microarray-based cell cultivation approaches may be enhanced further by incorporating biosensing elements alongside the cell-adhesive ligands to enable local detection of secreted cellular products. The concept of combining cells and sensing elements in the same microarray is demonstrated in the present paper with hepatocytes serving as a model cellular system. Robotic microarraying was employed to print arrays of 300-mum-diameter collagen (I) spots alongside the antibody (Ab) spots specific to liver proteins: albumin and alpha1-antitrypsin (alpha1-AT). Protein microarrays were printed onto poly(ethylene glycol) hydrogel-coated glass slides, thus eliminating nonspecific adsorption of cells or proteins. When incubated with printed microarrays, hepatocytes became localized on collagen (I) domains but did not attach on Ab spots or elsewhere on hydrogel-coated glass substrates. Liver-specific proteins secreted by hepatocytes were captured on Ab domains in the immediate vicinity of the cells, detected with a sandwich immunofluorescent assay and quantified using a microarray scanner. Importantly, hepatic albumin and alpha1-AT production detected in the microarray was comparable to enzyme-linked immunosorbent assay measurements of these proteins. In the future, the juxtaposition of sensing Ab regions with cell arrays will be particularly useful for the detection of local appearance or loss of phenotype of cells interacting with the printed components of the cellular microenvironment.

This paper describes a novel approach of controlling cell-surface interactions through an electrochemical "switching" of biointerfacial properties of optically transparent microelectrodes. The indium tin oxide (ITO) microelectrodes, fabricated on glass substrates, were modified with poly(ethylene glycol) (PEG) silane to make glass and ITO regions resistant to protein and cell adhesion. Cyclic voltammetry, with potassium ferricyanide serving as a redox reporter molecule, was used to monitor electron transfer across the electrolyte-ITO interface. PEG silane modification of ITO correlated with diminished electron transfer, judged by the disappearance of ferricyanide redox activity. Importantly, application of reductive potential (-1.4 V vs Ag/AgCl reference) corresponded with reappearance of typical ferricyanide redox peaks, thus pointing to desorption of an insulating PEG silane layer. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) characterization of the silanized ITO surfaces after electrical stimulation indicated complete removal of the silane layer. Significantly, electrical stimulation allowed to "switch" chosen electrodes from nonfouling to protein-adhesive while leaving other ITO and glass regions protected by a nonfouling PEG silane layer. The spatial and temporal control of biointerfacial properties afforded by our approach was utilized to micropattern proteins and cells and to construct micropatterned co-cultures. In the future, control of the biointerfacial properties afforded by this novel approach may allow the organization of multiple cell types into precise geometric configurations in order to create better in vitro mimics of cellular complexity of the native tissues.

The genomes of two closely related lytic Thermus thermophilus siphoviruses with exceptionally long (approximately 800 nm) tails, bacteriophages P23-45 and P74-26, were sequenced completely. The P23-45 genome consists of 84,201 bp with 117 putative open reading frames (ORFs), and the P74-26 genome has 83,319 bp and 116 putative ORFs. The two genomes are 92% identical with 113 ORFs shared. Only 25% of phage gene product functions can be predicted from similarities to proteins and protein domains with known functions. The structural genes of P23-45, most of which have no similarity to sequences from public databases, were identified by mass spectrometric analysis of virions. An unusual feature of the P23-45 and P74-26 genomes is the presence, in their largest intergenic regions, of long polypurine-polypyrimidine (R-Y) sequences with mirror repeat symmetry. Such sequences, abundant in eukaryotic genomes but rare in prokaryotes, are known to form stable triple helices that block replication and transcription and induce genetic instability. Comparative analysis of the two phage genomes shows that the area around the triplex-forming elements is enriched in mutational variations. In vitro, phage R-Y sequences form triplexes and block DNA synthesis by Taq DNA polymerase in orientation-dependent manner, suggesting that they may play a regulatory role during P23-45 and P74-26 development.

The genome sequence of bacteriophage phiA1122 has been determined. phiA1122 grows on almost all isolates of Yersinia pestis and is used by the Centers for Disease Control and Prevention as a diagnostic agent for the causative agent of plague. phiA1122 is very closely related to coliphage T7; the two genomes are colinear, and the genome-wide level of nucleotide identity is about 89%. However, a quarter of the phiA1122 genome, one that includes about half of the morphogenetic and maturation functions, is significantly more closely related to coliphage T3 than to T7. It is proposed that the yersiniophage phiA1122 recombined with a close relative of the Y. enterocolitica phage phiYeO3-12 to yield progeny phages, one of which became the classic T3 coliphage of Demerec and Fano (M. Demerec and U. Fano, Genetics 30:119-136, 1945).

Phage T4 effects lysis by its holin T and its endolysin E. Lysis is inhibited (LIN) if the infected cell is subjected to secondary infections by T4 phage particles. The T4 rI gene is required for LIN in all hosts tested. Here, we show that a cloned rI gene can impose a T-specific LIN on T-mediated lysis in the context of the phage lambda infective cycle, in the absence of other T4 genes and without secondary infection by T4. Moreover, it is shown that the T holin accumulates in the membrane during LIN, forming SDS-resistant oligomers. We show by cross-linking experiments that a T-RI heterodimer is formed during LIN, demonstrating that RI belongs to the functional class of antiholins, such as the S107 protein of lambda, which heterodimerizes with its cognate holin, S105. Finally, we show that the addition of Ni(2+) ions to the medium can block lysis by a T protein hexahistidine-tagged at its C-terminus, suggesting that liganding of the periplasmic domain is sufficient to impose lysis inhibition. The results are discussed in terms of a model in which the LIN-inducing signal of the secondary infecting phage influences a conformational equilibrium assumed by RI in the periplasm.

The t protein of bacteriophage T4 shares with other holins the ability to cause the formation of a lethal membrane lesion which allows the phage endolysin to attack the peptidoglycan. Moreover, T, like other holins, acts in a saltatory manner at a precisely programmed time in the vegetative cycle. Unlike other holins, however, T has the unique ability to postpone its lethal function in response to a secondary infection by a T-even phage during the vegetative cycle. A signal transduction system that responds to the secondary infection is thought to be encoded by some of the numerous r genes, defined by mutations that abolish this lysis-inhibition (LIN) response. The primary structure of T differs from two main structural patterns found in more than 30 orthologous groups of holins. Genetic approaches were taken to probe the t sequence for features involved in membrane localization, functional timing and LIN regulation. Gene fusion analysis indicates that T has a single TMD near the N-terminus, with the bulk of the protein residing in the periplasm. Mapping and phenotypic analysis of deletion and point mutations in t indicates that the periplasmic domain of T is the major determinant of the timing mechanism and is involved in the LIN response.