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Context We studied the virus component in the microbial food-web in the tropical coastal Sunda Shelf waters. Aims We investigated viral processes, including the lysogenic to lytic shift dynamics with seawater warming and nutrient enrichment. Methods We sampled nearshore and offshore waters of Peninsular Malaysia to provide a natural trophic gradient for correlational analyses. We also conducted laboratory experiments to investigate the changes in virus production rates and lysogeny, when temperature (range: 25–37°C) and nutrient concentrations (range: 0.1–1.0× marine-broth concentration) were manipulated. Key results Observations showed site-dependent differences in dissolved inorganic nutrient concentration, Chl-a (0.21–3.54 μg L−1) and bacterial abundance (4.09 × 105−9.41 × 105 cells mL−1), but not in viral abundance (measured as virus-like particles or VLP) (1.04 × 106−2.39 × 106 VLP mL−1) and virus production rates (0.59 × 105−4.55 × 105 VLP mL−1 h−1). From laboratory experiments, both warming and nutrient enrichment increased virus production (R2 > 0.651), decreased lysogeny (R2 > 0.743), and resulted in an increase of the viral lysis:bacterial production ratio, and a stronger viral control on bacterial production. Conclusions Although both seawater warming and nutrient enrichment increased virus production and reduced lysogeny, nutrient enrichment was a more important factor. Implications The increasing nutrient concentrations in these waters will increase virus production and virus top-down control of bacteria, and drive the ecosystem towards heterotrophy.

Objectives Currently, clinically relevant multigene-based prognostic assessment models for nasopharyngeal carcinoma (NPC) are limited. This paper reports a novel NPC prognosis assessment model based on multiple established NPC-associated biomarkers. Methods We used a modified meta-analysis approach to retrieve eligible studies and analyse the data. Different prognostic biomarkers and hazard ratios (HRs) with 95 % confidence intervals (CIs) of overall survival (OS) data were extracted and tabulated from eligible studies. We then used the formula based on Parmar et al. to determine OS (expressed as HR with 95 % CI). Prognosis assessment risk scores assigned to the logarithm of HR were the basis for interpreting the multigene prognosis assessment model. Finally, we explained the biological significance of this model using a multigenic NPC oncogenesis network system. Results We constructed a multigenic NPC prognosis assessment model consisting of 10 prognostic biomarkers to determine the OS rate in NPC patients. Based on the biomarkers’ expression patterns, the model could determine 1,023 possible OS rates of NPC patients. The risk score derived determines the prognosis status of the NPC patients. The higher the total risk assessment score, the poorer the prognosis. An NPC-associated network involving all ten biomarkers was also derived. Conclusions We provided a novel multigenic NPC prognosis assessment model comprising ten prognostic biomarkers on OS rate in NPC patients. A conceptual molecular-based pathophysiological network of NPC oncogenesis supported the biological relevance of this model.

Fabry disease (FD) is caused by a defective α-galactosidase A (α-GAL A) enzyme responsible for breaking down globotriaosylceramide (Gb

We investigated the appropriateness of faecal indicator bacteria in tropical waters. We compared total coliform (undetectable to 7.2 × 10

Fabry disease (FD) is caused by a defective α-galactosidase A (α-GAL A) enzyme responsible for breaking down globotriaosylceramide (Gb3). To develop affordable therapeutics, more effort is needed to obtain insights into the underlying mechanism of FD and understanding human α-GAL A structure and function in related animal models. We adopted C. elegans as a model to elucidate the sequence and 3D structure of its GANA-1 enzyme and compared it to human α-GAL A. We constructed GANA-1 3D structure by homology modelling and validated the quality of the predicted GANA-1 structure, followed by computational docking of human ligands. The GANA-1 protein shared sequence similarities up to 42.1% with the human α-GAL A in silico and had dual active sites. GANA-1 homology modelling showed that 11 out of 13 amino acids in the first active site of GANA-1 protein overlapped with the human α-GAL A active site, indicating the prospect for substrate cross-reaction. Computational molecular docking using human ligands like Gb3 (first pocket), 4-nitrophenyl-α-D-galactopyranoside (second pocket), α-galactose (second pocket), and N-acetyl-D-galactosamine (second pocket) showed negative binding energy. This revealed that the ligands were able to bind within both GANA-1 active sites, mimicking the human α-GAL A and α-NAGA enzymes. We identified human compounds with adequate docking scores, predicting robust interactions with the GANA-1 active site. Our data suggested that the C. elegans GANA-1 enzyme may possess structural and functional similarities to human α-GAL A, including an intrinsic capability to metabolize Gb3 deposits. Communicated by Ramaswamy H. Sarma

TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.

Ribosomal protein genes encode products that are essential for cellular protein biosynthesis and are major components of ribosomes. Canonically, they are involved in the complex system of ribosome biogenesis pivotal to the catalysis of protein translation. Amid this tightly organised process, some ribosomal proteins have unique spatial and temporal physiological activity giving rise to their extra-ribosomal functions. Many of these extra-ribosomal roles pertain to cellular growth and differentiation, thus implicating the involvement of some ribosomal proteins in organogenesis. Consequently, dysregulated functions of these ribosomal proteins could be linked to oncogenesis or neoplastic transformation of human cells. Their suspected roles in carcinogenesis have been reported but not specifically explained for malignancy of the nasopharynx. This is despite the fact that literature since one and half decade ago have documented the association of ribosomal proteins to nasopharyngeal cancer. In this review, we explain the association and contribution of dysregulated expression among a subset of ribosomal proteins to nasopharyngeal oncogenesis. The relationship of these ribosomal proteins with the cancer are explained. We provide information to indicate that the dysfunctional extra-ribosomal activities of specific ribosomal proteins are tightly involved with the molecular pathogenesis of nasopharyngeal cancer albeit mechanisms yet to be precisely defined. The complete knowledge of this will impact future applications in the effective management of nasopharyngeal cancer.

Consumption of sugars in food and beverages has increased at an alarming rate. While excessive daily sugar intake has been well-associated as the onset of medical complications, additional sugars are still used in manufactured food products just to satisfy the consumers' needs. Hence, there is a need to develop sugar replacers that have low glycemic response without compromising the organoleptic characteristics of food products. This study aimed to determine if SUITENA™, a novel sweetener containing erythritol, xylitol, and Stevia, has low glycemic response upon consumption by human subjects.Six human subjects were randomly chosen and were healthy at the point of experimentation. Capillary blood was collected via finger-prick method to monitor the glycemic response of every individual for 90 min after ingestion of sugar solution.It was found that the mean area under the curve (AUC) of the dextrose standard was 11.8-fold higher (p 0.05) than the AUC of SUITENA™. SUITENA™ was not able to increase blood glucose level for up to 90 min while a spike in blood glucose level was observed from 15 min post-consumption of dextrose solution. We found that SUITENA™ has elicited a glycemic response 8% relative to the standard. Such low glycemic response has been reported by studies on other novel sugars.This preliminary finding suggested that SUITENA™ is a healthier alternative to fast sugars due to its low glycemic response. A larger sampling size is required to confirm the results.

Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to the cell membrane. Standard microscopy techniques however fall short when finer details including membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM) to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging the invasion complexes in high resolution can provide important insights into the behavior of bacterial vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other molecules.

Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to the cell membrane. Standard microscopy techniques however fall short when finer details including membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM) to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging the invasion complexes in high resolution can provide important insights into the behavior of bacterial vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other molecules.

Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting β1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.

Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based high-throughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and cell-dependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host-bacteria interaction.

We sampled the Klang estuary during the inter-monsoon and northeast monsoon period (July–Nov 2011, Oct–Nov 2012), which coincided with higher rainfall and elevated Klang River flow. The increased freshwater inflow into the estuary resulted in water column stratification that was observed during both sampling periods. Dissolved oxygen (DO) dropped below 63 μM, and hypoxia was observed. Elevated river flow also transported dissolved inorganic nutrients, chlorophyll a and bacteria to the estuary. However, bacterial production did not correlate with DO concentration in this study. As hypoxia was probably not due to in situ heterotrophic processes, deoxygenated waters were probably from upstream. We surmised this as DO correlated with salinity (R2 = 0.664, df = 86, p 6.7 h), hypoxia could occur at the Klang estuary. Here, we presented a model that related riverine flow rate to the post-heavy rainfall hypoxia that explicated the episodic hypoxia at Klang estuary. As Klang estuary supports aquaculture and cockle culture, our results could help protect the aquaculture and cockle culture industry here.

The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.

Fabry disease is caused by reduced α-GAL A activity and accumulation of globotriaosylceramide (Gb3). Here, we describe a microplate Gb3 assay using fluorophore-tagged antibody and crude cellular lipid extracts. The assay is able to detect higher Gb3 concentrations in human Fabry cells compared to non-diseased cells. This result was verified by immunofluorescence staining that revealed large amounts of Gb3 deposits in Fabry cell lines, demonstrating the accuracy of this method. This assay may provide the basis for detecting Fabry disease by quantifying Gb3 deposits from human biological samples, for example, from urine and blood.

The association of ribosomal proteins with carcinogenesis of nasopharyngeal carcinoma (NPC) has been established in a limited subset of ribosomal protein genes. To date, three ribosomal protein genes,eL27 (L27),eL41 (L41), andeL43 (L37a), have been found to be differentially expressed in cell lines derived from NPC tumors. This raises the possibility of more ribosomal protein genes that could be associated with NPC. In this study, we investigated the expression profiles of eight ribosomal protein genes,uS8 (S8), uS4 (S9), eS31 (S27a), eL6 (L6), eL18 (L18), uL14 (L23), eL24 (L24), andeL30 (L30), in six NPC-derived cell lines(HONE-1, SUNE1, HK1, TW01, TW04, and C666-1). Their expression levels were compared with that of a nonmalignant nasopharyngeal epithelial cell line (NP69) using quantitative real-time PCR (RT-qPCR) assay. Of the eight genes studied, the expressions of four ribosomal protein genesuS8 (S8), uS4 (S9), eS31 (S27a),anduL14 (L23)were found to be significantly downregulated in NPC cell lines relative to NP69. Our findings provide novel empirical evidence of these four ribosomal protein genes as NPC-associated genetic factors and reinforce the relevance of ribosomal proteins in the carcinogenesis of nasopharyngeal cancer.

The effect of physical shearing on the attachment of six Pseudomonas aeruginosa strains and six Burkholderia cepacia strains to glass, stainless steel, polystyrene and Teflon® was determined. A significant (p < 0.05) decrease in hydrophobicity was apparent for all P. aeruginosa strains (17-36%) and B. cepacia, MS 5 (20%) after shearing. A significant (p < 0.05) decrease in attachment of some P. aeruginosa (0.2-0.5 log CFU/cm2) and B. cepacia (0.2-0.4 log CFU/cm2) strains to some surface types was apparent after shearing. Significant (p < 0.05) correlation was observed for both numbers of flagellated cells and hydrophobicity against attachment to glass, stainless steel and polystyrene for P. aeruginosa while only hydrophobicity showed significant correlation against the same surfaces for B. cepacia. Scanning electron microscopy and protein analysis showed that shearing removed surface proteins from the cells and may have led to the observed changes in hydrophobicity and attachment to abiotic surfaces.

We measured Vibrio spp. distribution and community profile in the tropical estuary of Port Klang and coastal water of Port Dickson, Malaysia. Vibrio spp. abundance ranged from 15 to 2395 colony forming units mL−1, and was driven by salinity and chlorophyll a (Chl a) concentration. However, the effect of salinity was pronounced only when salinity was 4.338, P < 0.05). Co-culture experiments between fast- and slow-growing Vibrio spp. revealed that fast-growing Vibrio spp. (r-strategists) were overwhelmed by slower-growing Vibrio spp. (K-strategists) when nutrient conditions were set towards oligotrophy. In response to resource availability, the intrinsic growth strategy of each Vibrio spp. determined its occurrence and the development of Vibrio spp. community composition.

Apart from their canonical role in ribosome biogenesis, there is increasing evidence of ribosomal protein genes’ involvement in various cancers. A previous study by us revealed significant differential expression of three ribosomal protein genes (RPeL27,RPeL41, andRPeL43) between cell lines derived from tumor and normal nasopharyngeal epithelium. However, the results therein were based on a semiquantitative assay, thus preliminary in nature. Herein, we provide findings of a deeper analysis of these three genes in the context to nasopharyngeal carcinoma (NPC) tumorigenesis. Their expression patterns were analyzed in a more quantitative manner at transcript level. Their protein expression levels were also investigated. We showed results that are contrary to previous report. Rather than downregulation, these genes were significantly overexpressed in NPC cell lines compared to normal control at both transcript and protein levels. Nevertheless, their association with NPC has been established. Immunoprecipitation pulldown assays indicate the plausible interaction of either RPeL27 or RPeL43 with POTEE/TUBA1A and ACTB/ACTBL2 complexes. In addition, RPeL43 is shown to bind with MRAS and EIF2S1 proteins in a NPC cell line (HK1). Our findings supportRPeL27,RPeL41, andRPeL43as potential markers of NPC and provide insights into the interaction targets of RPeL27 and RPeL43 proteins.

Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.

Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.

The effect of physical shearing on the attachment of six Pseudomonas aeruginosa strains and six Burkholderia cepacia strains to glass, stainless steel, polystyrene and Teflon® was determined. A significant (p0.05) decrease in hydrophobicity was apparent for all P. aeruginosa strains (17-36%) and B. cepacia, MS 5 (20%) after shearing. A significant (p0.05) decrease in attachment of some P. aeruginosa (0.2-0.5 log CFU/cm2) and B. cepacia (0.2-0.4 log CFU/cm2) strains to some surface types was apparent after shearing. Significant (p0.05) correlation was observed for both numbers of flagellated cells and hydrophobicity against attachment to glass, stainless steel and polystyrene for P. aeruginosa while only hydrophobicity showed significant correlation against the same surfaces for B. cepacia. Scanning electron microscopy and protein analysis showed that shearing removed surface proteins from the cells and may have led to the observed changes in hydrophobicity and attachment to abiotic surfaces.

Using the size fractionation method, we measured the decay rates of Escherichia coli, Salmonella Typhi and Vibrio parahaemolyticus in the coastal waters of Peninsular Malaysia. The size fractions were total or unfiltered,250 μm,20 μm,2 μm,0.7 μm,0.2 μm and0.02 μm. We also carried out abiotic (inorganic nutrients) and biotic (bacterial abundance, production and protistan bacterivory) measurements at Port Dickson, Klang and Kuantan. Klang had highest nutrient concentrations whereas both bacterial production and protistan bacterivory rates were highest at Kuantan. We observed signs of protist-bacteria coupling via the following correlations: Protistan bacterivory-Bacterial Production: r = 0.773, df = 11, p0.01; Protist-Bacteria: r = 0.586, df = 12, p0.05. However none of the bacterial decay rates were correlated with the biotic variables measured. E. coli and Salmonella decay rates were generally higher in the larger fraction (0.7 μm) than in the smaller fraction (0.7 μm) suggesting the more important role played by protists. E. coli and Salmonella also decreased in the0.02 μm fraction and suggested that these non-halophilic bacteria did not survive well in seawater. In contrast, Vibrio grew well in seawater. There was usually an increase in Vibrio after one day incubation. Our results confirmed that decay or loss rates of E. coli did not match that of Vibrio, and also did not correlate with Salmonella decay rates. However E. coli showed persistence where its decay rates were generally lower than Salmonella.

In this study we isolated and characterized some culturable bacteria from tropical coastal waters of Peninsular Malaysia. We obtained between 0.23 and 1.85 × 103 cfu mL–1 in the Zobell 2216E medium, and cultured 0.04% to 0.12% of total bacterial counts. Different bacterial strains were then selected by 16S rDNA RFLP using four restriction enzymes (DdeI, HhaI, RsaI, and Sau3AI), of which HhaI gave the most RFLP patterns. A total of 54 unique strains were obtained and these were identified by their 16S rDNA gene sequence. These bacterial strains could be divided into five classes: 38 strains of γ-Proteobacteria (61.1%); 3 strains of α-Proteobacteria (5.5%); 2 strains of the Cytophaga-Flavobacterium-Bacteroides group (3.7%); 3 strains of high GC, Gram-positive bacteria (5.5%); and 13 strains of low GC, Gram-positive bacteria (24.1%). These isolates have good potential for further biotechnological studies since about 56% of the isolates exhibited amylase activity, whereas 36% and 18% of the isolates had protease and lipase, respectively. Most (>70%) of the isolates also produced poly-ß-hydroxybutyrate.

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

The phage N15 protelomerase enzyme (TelN) is essential for the replication of its genome by resolution of its telRL domain, located within a telomerase occupancy site (tos), into hairpin telomeres. Isolation of TelN for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. In this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of TelN for at least 4 weeks, greatly simplifying in vitro testing of its activity. This protocol may be extended for functional analysis of other phage and bacterial proteins, particularly DNA-processing enzymes.

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.

Recombineering technology permits flexible engineering of large DNA in Escherichia coli without dependence on suitably placed restriction sites. However, recombineering is limited for modifying highly repetitive DNA because of its potential to trigger instability by uncontrolled self-recombination of the repeats. In this study, induction of the recombineering enzymes and growth condition of the host are optimized to demonstrate intact modification of bacterial artificial chromosomes (BACs) containing long arrays of centromeric alpha satellite repeats. This optimized recombineering protocol may be useful for manipulation of other biologically important repetitive DNAs, including trinucleotide repeat expansions and homologous gene families, to facilitate their functional studies.

Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.

Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development ofin vivohomologous recombination strategies based on recombineering inE. colihas helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACsin vitroandin vivo.

Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.

Recombineering technology in Escherichia coli enables targeting of linear donor DNA to circular recipient DNA using short shared homology sequences. In this work, we demonstrate that recombineering is also able to support recombination between a pair of linear DNA substrates (linear/linear recombineering) in vivo in E. coli. Linear DNA up to 100 kb is accurately modified and remains intact without undergoing rearrangements after recombination. This system will be valuable for direct in vivo manipulation of large linear DNA including the N15 and PY54 prophages and linear animal viruses, and for assembly of linear constructs as artificial chromosome vectors.

The advent of recombineering technology in Escherichia coli has revolutionized the way recombinant DNA molecules are constructed. We present a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli. The N15 telomerase occupancy site was recombined into circular DNA and resolved into individual telomeres by the phage N15 protelomerase enzyme. We demonstrate this technique by assembling linear BACs that replicate stably in their host strain E. coli DH10B. Correct linearization of the BACs was confirmed by restriction mapping using pulsed field gel electrophoresis. The linear BAC DNA can be easily purified using standard plasmid isolation methods and resist degradation from RecBCD nuclease in vitro and in vivo owing to the presence of telomeres. Transfection of a linear 100 kb BAC containing the human beta-globin gene cluster into HT1080 cells produced accurately spliced transcripts, demonstrating that the linear DNA will be useful for subsequent functional studies. This novel recombineering technique may be particularly useful for building large linear constructs for assembling artificial chromosomes with telomeres, and may provide a unique means to clone and study large linear viral genomes that contain hairpin ends.

We investigated the dynamics of attached and free-living bacterial abundance over a period of 18 months in tropical coastal waters of Malaysia. We measured the abundance at both oligotrophic coastal water (Port Dickson) and eutrophic estuary (Klang), and hypothesised that attached bacteria are predominant in eutrophic waters. We found that bacterial abundance was higher at Klang than Port Dickson (Student’s t-test: t=4.87, d.f.=19, P

A large number of mutations have been described in the human β-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human β-globin locus. We report here the use of the GET Recombination system with an Eco RI/Kan R counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG→AAG mutation and the codon 41–42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human β-globin locus. The counterselection cassette was first inserted into the target sequence in the β-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic Eco RI endonuclease gene by expression of lacI q . Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a approximately 200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd(-) rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.