Passenger Genes Research Articles

Finding insights of Pancreas development

Pancreas is organ which contains digestive enzymes that assist the absorption of nutrients to be digested by small intestine which makes this gland as main energy regulator which up regulates expression to counter metabolism or down regulates in promoting or maintaining blood pressure etc. It is made up of exocrine and endocrine parts which have enzymes & millions of cells regulating its tissue specific expression. Multiple pathways too are associated with pancreatic mechanism which is directly epicenter in modulating energy mechanism throughout organism behavior. We in present study, wants to find out mechanism of pancreatic development with its signaling. Pancreatic beta cell, alpha cell, delta cells, pp cells and all endocrine part is mainly develops by differentiation of PDX gene which further divides and interacts with other differentiation factors of genes which promoted maintenance and development of Islets of Langerhans. We have identified Driver genes involved in this mechanism with their interacting passenger genes which are associated in development of Langerhans. Although, we to have created driver genes interactome of all drivers in development with their respective passengers. For analyses of gene expression we have modeled all gene expression to know their function at proteomic level with this we know that what will be the expression scenario of multiple proteins at structure level. We have used many system biological tools like Cytoscape, Modeller, Grommacs with online tools such as Castp, Phyre2, ifold etc.

Identifying potential cancer driver genes by genomic data integration

Cancer is a genomic disease associated with a plethora of gene mutations resulting in a loss of control over vital cellular functions. Among these mutated genes, driver genes are defined as being causally linked to oncogenesis, while passenger genes are thought to be irrelevant for cancer development. With increasing numbers of large-scale genomic datasets available, integrating these genomic data to identify driver genes from aberration regions of cancer genomes becomes an important goal of cancer genome analysis and investigations into mechanisms responsible for cancer development. A computational method, MAXDRIVER, is proposed here to identify potential driver genes on the basis of copy number aberration (CNA) regions of cancer genomes, by integrating publicly available human genomic data. MAXDRIVER employs several optimization strategies to construct a heterogeneous network, by means of combining a fused gene functional similarity network, gene-disease associations and a disease phenotypic similarity network. MAXDRIVER was validated to effectively recall known associations among genes and cancers. Previously identified as well as novel driver genes were detected by scanning CNAs of breast cancer, melanoma and liver carcinoma. Three predicted driver genes (CDKN2A, AKT1, RNF139) were found common in these three cancers by comparative analysis.

Identification of driver genes in microsatellite-unstable colorectal cancers

SUMMARY Somatic mutations in key driver genes are necessary for colorectal tumor formation. Whereas the majority of sporadic colorectal cancer (CRC) cases have chromosomal instability, a sixth of cases generate somatic mutations by a deficiency in the mismatch repair system. This choice of mutational mechanism has a strong impact on which genes in CRC pathways are the most likely to be mutated in an individual tumor. A specific challenge in understanding CRC with microsatellite instability is to discriminate driver from passenger genes given the large number of nucleotide repeats that are frequently found mutated in patient tumors. This review, therefore, discusses strategies for the identification and validation of cancer genes in microsatellite-unstable colorectal cancers.

The Landscape of Candidate Driver Genes Differs between Male and Female Breast Cancer

The rapidly growing collection of diverse genome-scale data from multiple tumor types sheds light on various aspects of the underlying tumor biology. With the objective to identify genes of importance for breast tumorigenesis in men and to enable comparisons with genes important for breast cancer development in women, we applied the computational framework COpy Number and EXpression In Cancer (CONEXIC) to detect candidate driver genes among all altered passenger genes. Unique to this approach is that each driver gene is associated with several gene modules that are believed to be altered by the driver. Thirty candidate drivers were found in the male breast cancers and 67 in the female breast cancers. We identified many known drivers of breast cancer and other types of cancer, in the female dataset (e.g. GATA3, CCNE1, GRB7, CDK4). In contrast, only three known cancer genes were found among male breast cancers; MAP2K4, LHP, and ZNF217. Many of the candidate drivers identified are known to be involved in processes associated with tumorigenesis, including proliferation, invasion and differentiation. One of the modules identified in male breast cancer was regulated by THY1, a gene involved in invasion and related to epithelial-mesenchymal transition. Furthermore, men with THY1 positive breast cancers had significantly inferior survival. THY1 may thus be a promising novel prognostic marker for male breast cancer. Another module identified among male breast cancers, regulated by SPAG5, was closely associated with proliferation. Our data indicate that male and female breast cancers display highly different landscapes of candidate driver genes, as only a few genes were found in common between the two. Consequently, the pathobiology of male breast cancer may differ from that of female breast cancer and can be associated with differences in prognosis; men diagnosed with breast cancer may consequently require different management and treatment strategies than women.

Abstract 801: The landscape of candidate driver genes differs between male and female breast cancers.

Abstract Male breast cancer (MBC) is a rare disease, accounting for <1% of all breast cancer cases. Although it shares many similarities with female breast cancer (FBC), distinct differences in age distribution, levels of hormone receptors, prognosis, as well as on the transcriptional and genomic levels, have been reported. Due to the rarity of the disease, no large comprehensive studies of the etiology of the disease or randomized trials for optimizing patient management have been conducted. Thus, recommendations for managing MBC are extrapolated from prior knowledge and guidelines for FBC. The aim of this study was to identify candidate driver genes and distinguish them from passenger genes in MBC, an approach that has not been comprehensively explored before. We further compared the landscape of candidate drivers between MBC and FBC, to determine similarities/differences. To this end, we used the computational framework CONEXIC that integrates array-based comparative genomic hybridization (aCGH) data and gene expression (GEX) data for detecting candidate driver genes. Two datasets with matched aCGH and GEX data were used; one containing 53 MBC tumors and the other containing 359 FBC tumors. Male and female breast cancers displayed different landscapes of candidate drivers. We correctly identified many of the known drivers for breast cancer and other types of cancer in the FBC dataset (e.g. GATA3, CCNE1, CDK4, GRB7); however these were not replicated among the MBC tumors. Additionally, we identified novel candidate drivers for breast cancer within both the MBC and FBC datasets. When candidate drivers were investigated separately in the intrinsic subgroups of FBC, differences in the landscapes of drivers were observed, with only a few genes overlapping across the subgroups. Interestingly, Rho GTPase activating protein 30, ARHGAP30, was ranked as a top candidate driver for the HER2 and basal FBC subgroups, as well as for MBC. In fact, this was the only candidate driver gene shared between female and male breast cancers. ARHGAP30 binds to the GTPases RhoU/Wrch-1 and overexpression of RhoU is required for migration. The finding that MBC shares a candidate driver gene with the estrogen receptor (ER) negative HER2 and basal FBC subgroups is surprising, given that the majority of the MBC tumors are ER positive. In summary, the combination of aCGH and GEX data revealed novel candidate driver genes among MBCs, as well as vast differences in the landscapes of candidate drivers between male and female breast cancers. Consequently, the pathobiology of MBC may be very distinct from that of FBC, and men diagnosed with breast cancer may hence require different management and treatment strategies than women. Citation Format: Ida Johansson, Markus Ringnér, Ingrid Hedenfalk. The landscape of candidate driver genes differs between male and female breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2013-801

Abstract A81: Global gene regulatory and protein interaction networks in breast cancer metastasis

Abstract Purpose: Invasion-metastasis cascades that underlie macroscopic metastases remain poorly understood. Tackling such a complexity demands systems biology approaches by means of filtering and integrating myriad data. To gain a fine resolution of mechanisms underlying the process of metastasis, here we reconstruct global gene regulatory and protein interaction networks that potentially drive breast cancer metastasis. Procedures: A systematic literature review and a comprehensive data mining of well-curated cancer gene datasets were carried out to uncover a core metastasis gene set (CMGS) using an in house Boolean logic framework. Meanwhile, a gene regulatory network (GRN) was built up to distinguish between key cancer driver genes and passenger genes through integration of multiple large-scale genomic studies of breast cancer. Furthermore, CMGS was projected onto the latest HRPD protein interaction network (PIN) and the GRN to derive metastasis-specific protein interaction network (mPIN) and gene regulatory network (mGRN), respectively. To gain mechanistic insights of mPIN and mGRN, a key network driver analysis was employed to identify critical hub nodes of the networks as key regulators. Results: We demonstrate that CMGS is of significantly higher connectivity in both PIN and GRN than those not in CMGS, suggesting its critical controlling power to the system. As expected, both mPIN and mGRN are most enriched for angiogenesis, integrin signaling, and p53 pathways. Moreover, these metastasis specific networks are also enriched for well-known metastasis-related pathways such as Wnt, TGF-beta, and FGF pathways. Surprisingly, inflammatory pathways, such as chemokine signaling and B-cell activation pathways are also over-represented in the networks. The top hub genes of mPIN include AR, ABL1, ESR1, AKT1, SMAD4, TP53, CSNK2A1, MAPK1, PIK3R1 and SMAD3. This hub gene set is distinct from the top key regulators of mGRN including ACTA2, ACTL6A, ADM, AEBP1, ARF1, ARPC4, ATM, AURKA and BIRC5. Notably, all the top ten drivers of mGRN except ATM and AURKA are not in CMGS, suggesting the novel targets of metastasis uncovered through our integrative network analysis. Indeed, ARF1, encoding the GTPase ADP-ribosylation factor 1, has been shown to play an important role in both cancer cell proliferation and migration. Moreover, the subnetwork regulated by ARF1 clearly links to inflammatory key players, especially via microvesicle related biological process. Importantly, both driver gene sets are tightly connected by TP53 and MAPK8 physically and genetically, suggesting the strong connection between p53 pathway and inflammatory response as MAPK8 activation is pivotal for chemokine mediated inflammation processes especially for TNF-alpha. Conclusion: Taken together, our global metastasis-specific protein interaction and gene regulation networks as well as their key regulators shed light on a potential trajectory of invasion-metastasis cascades that enable the progress of breast cancer metastasis, underscoring the instrumental role of a novel signaling network involving p53, microvesicle associated ARF1, and TNF-alpha mediated inflammatory response pathways. Citation Format: Bin Zhang, Yongzhong Zhao, Jun Zhu. Global gene regulatory and protein interaction networks in breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A81.

Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites

Metabolic engineering of Escherichia coli using plasmids is problematic, which is addressed by developing a toolbox for genomic engineering of E. coli. This toolbox includes the attP site-based integration vectors and the attB site-based template vectors, equipped with mutant loxP sites (i.e., LE* and RE*). The former vectors facilitate integration of passenger genes into attB sites while the latter allows creation of new attB sites. Consequently, the inserted vector backbone is flanked by LE* and RE* sites and can be rescued by Cre. Based on this approach, the biosynthetic pathway of poly(3-hydroxybutyric acid) was first built in E. coli. By scoring the observable phenotype of integrants, the result revealed that the efficiency of gene integration could reach 100%. In addition, reconstruction of the n-butanol-synthesizing pathway in E. coli resulted in a plasmid-free producer strain. As a consequence, the producer strain was able to stably overproduce n-butanol (3.7g/L) from glucose (20g/L). Finally, exoglucanase was overexpressed in E. coli that carried multiple genomic copies of the celY gene. Overall, it indicates a promise of our method for cycling improvement of E. coli.

Passenger deletions generate therapeutic vulnerabilities in cancer

Inactivation of tumor suppressor genes via homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighboring genes. We hypothesized that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities in the case where the collaterally deleted gene is a member of a functionally redundant family of genes exercising an essential function. The glycolytic gene Enolase 1 (ENO1) in the 1p36 locus is deleted in Glioblastoma (GBM), which is tolerated by expression of ENO2. We demonstrate that shRNA-mediated extinction of ENO2 selectively inhibits growth, survival, and tumorigenic potential of ENO1-deleted GBM cells and that the enolase inhibitor phosphonoacetohydroxamate (PhAH) is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger deleted genes encoding functionally-redundant essential activities and provide an effective treatment strategy for cancers harboring such genomic events.

Identification of novel driver tumor suppressors through functional interrogation of putative passenger mutations in colorectal cancer

Cancer genome sequencing efforts are leading to the identification of genetic mutations in many types of malignancy. However, the majority of these genetic alterations have been considered random passengers that do not directly contribute to tumorigenesis. We have previously conducted a soft agar-based short hairpin RNA (shRNA) screen within colorectal cancer (CRC) candidate driver genes (CAN-genes) using a karyotypically diploid hTERT- and CDK4-immortalized human colonic epithelial cell (HCEC) model and discovered that depletion of 65 of the 151 CAN-genes enhanced anchorage-independent growth in HCECs with ectopic expression of K-Ras(V12) and/or TP53 knockdown. We now constructed an interaction map of the confirmed CAN-genes with CRC non-CAN-genes and screened for functional tumor suppressors. Remarkably, depletion of 15 out of 25 presumed passenger genes that interact with confirmed CAN-genes (60%) promoted soft agar growth in HCECs with TP53 knockdown compared to only 7 out of 55 (12.5%) of presumed passenger genes that do not interact. We have thus demonstrated a pool of driver mutations among the putative CRC passenger/incidental mutations, establishing the importance of employing biological filters, in addition to bioinformatics, to identify driver mutations.

A Sparse Regulatory Network of Copy-Number Driven Gene Expression Reveals Putative Breast Cancer Oncogenes

Copy number aberrations are recognized to be important in cancer as they may localize to regions harboring oncogenes or tumor suppressors. Such genomic alterations mediate phenotypic changes through their impact on expression. Both cis- and transacting alterations are important since they may help to elucidate putative cancer genes. However, amidst numerous passenger genes, trans-effects are less well studied due to the computational difficulty in detecting weak and sparse signals in the data, and yet may influence multiple genes on a global scale. We propose an integrative approach to learn a sparse interaction network of DNA copy-number regions with their downstream transcriptional targets in breast cancer. With respect to goodness of fit on both simulated and real data, the performance of sparse network inference is no worse than other state-of-the-art models but with the advantage of simultaneous feature selection and efficiency. The DNA-RNA interaction network helps to distinguish copy-number driven expression alterations from those that are copy-number independent. Further, our approach yields a quantitative copy-number dependency score, which distinguishes cis- versus trans-effects. When applied to a breast cancer data set, numerous expression profiles were impacted by cis-acting copy-number alterations, including several known oncogenes such as GRB7, ERBB2, and LSM1. Several trans-acting alterations were also identified, impacting genes such as ADAM2 and BAGE, which warrant further investigation. An R package named lol is available from www.markowetzlab.org/software/lol.html.

Deciphering causal and statistical relations of molecular aberrations and gene expressions in NCI-60 cell lines.

BackgroundCancer cells harbor a large number of molecular alterations such as mutations, amplifications and deletions on DNA sequences and epigenetic changes on DNA methylations. These aberrations may dysregulate gene expressions, which in turn drive the malignancy of tumors. Deciphering the causal and statistical relations of molecular aberrations and gene expressions is critical for understanding the molecular mechanisms of clinical phenotypes.ResultsIn this work, we proposed a computational method to reconstruct association modules containing driver aberrations, passenger mRNA or microRNA expressions, and putative regulators that mediate the effects from drivers to passengers. By applying the module-finding algorithm to the integrated datasets of NCI-60 cancer cell lines, we found that gene expressions were driven by diverse molecular aberrations including chromosomal segments' copy number variations, gene mutations and DNA methylations, microRNA expressions, and the expressions of transcription factors. In-silico validation indicated that passenger genes were enriched with the regulator binding motifs, functional categories or pathways where the drivers were involved, and co-citations with the driver/regulator genes. Moreover, 6 of 11 predicted MYB targets were down-regulated in an MYB-siRNA treated leukemia cell line. In addition, microRNA expressions were driven by distinct mechanisms from mRNA expressions.ConclusionsThe results provide rich mechanistic information regarding molecular aberrations and gene expressions in cancer genomes. This kind of integrative analysis will become an important tool for the diagnosis and treatment of cancer in the era of personalized medicine.

Inferring causal genomic alterations in breast cancer using gene expression data

BackgroundOne of the primary objectives in cancer research is to identify causal genomic alterations, such as somatic copy number variation (CNV) and somatic mutations, during tumor development. Many valuable studies lack genomic data to detect CNV; therefore, methods that are able to infer CNVs from gene expression data would help maximize the value of these studies.ResultsWe developed a framework for identifying recurrent regions of CNV and distinguishing the cancer driver genes from the passenger genes in the regions. By inferring CNV regions across many datasets we were able to identify 109 recurrent amplified/deleted CNV regions. Many of these regions are enriched for genes involved in many important processes associated with tumorigenesis and cancer progression. Genes in these recurrent CNV regions were then examined in the context of gene regulatory networks to prioritize putative cancer driver genes. The cancer driver genes uncovered by the framework include not only well-known oncogenes but also a number of novel cancer susceptibility genes validated via siRNA experiments.ConclusionsTo our knowledge, this is the first effort to systematically identify and validate drivers for expression based CNV regions in breast cancer. The framework where the wavelet analysis of copy number alteration based on expression coupled with the gene regulatory network analysis, provides a blueprint for leveraging genomic data to identify key regulatory components and gene targets. This integrative approach can be applied to many other large-scale gene expression studies and other novel types of cancer data such as next-generation sequencing based expression (RNA-Seq) as well as CNV data.

Identification and Characterization of a Promoter Cassette Conferring Adipocyte-Specific Gene Expression

The adipocyte-specific secretory molecule adiponectin has found widespread acceptance as a systemic marker that effectively integrates a number of signals associated with metabolic dysfunction at the level of adipose tissue. The widely used aP2 promoter cassette, which is frequently chosen to achieve adipocyte-specific expression of transgenes, conveys transcription in cell types other than adipocytes, such as macrophages and cardiomyocytes. To improve our ability to drive transgene expression in a more adipocyte-specific way, we aimed to define the minimal promoter segment from the adiponectin genomic locus. We generated a series of transgenic animals in which the expression of reporter genes and Cre recombinase was driven by 2, 4.9, and 5.4 kb of adiponectin promoter sequences. We found that the 5.4-kb adiponectin promoter fragment is the most effective cassette conveying adipocyte-specific expression of target genes. We therefore define a novel promoter cassette that ensures adipocyte-specific expression of passenger genes and may be used in the generation of transgenic mouse models to study gene function in vivo.

Route 66: investigations into the organisation and distribution of the IS 66 family of prokaryotic insertion sequences

We have analysed a large number of members of the IS 66 family of prokaryotic insertion sequences which include three orfs (A, B and C) and we show that these are closely related to a second group (IS Bst12) which includes only orfC, encoding the transposase TnpC. These two IS groups are also related by their terminal inverted repeats (IR) which show significant conservation. We propose that both groups be included in the same family, the IS 66 family. We have also identified members of both IS groups which include passenger genes and which, together with those already identified in the IS 1595 family, suggest that such structures are quite common. We observe that the classical elements are largely limited to the Proteobacteria, although one clade contains only Firmicutes and another is limited to the Bacteriodetes/Chlorobi. While also present in the Proteobacteria, IS Bst12 group elements are much more widely spread among phyla. They are present in the Actinobacteria, Cyanobacteria, Deinococcus/Thermus, Firmicutes and Planctomycetes. Moreover, they are also found in the Euryarchaeota phylum of archaea (but not in the Crenarchaeota). All Euryarchaeota members are contained within a clade together with those from the Actinobacteria, Deinococcus/Thermus and Firmicutes. The specificity of both classical IS 66 and IS Bst12 clades may be useful in identifying specific bacterial and archaeal phyla in complex metagenomes.

Abstract A51: Development and initial characterization of isogenic cell lines stably overexpressing prostate cancer candidate genes

Abstract Microarray analyses of global changes in gene expression patterns have recovered a large number of genes, which are deregulated in prostate cancer, when compared to normal prostate tissue. In order to successfully translate these data into clinical intervention strategies, it is crucial to determine genes that truly contribute to tumor development (which may be referred to as ‘driver genes’) and to separate them from the majority of secondary anomalies in expression levels (‘passenger genes’). To obtain the missing functional information, we construct isogenic cell line libraries derived from commonly used prostate (RWPE-1) and prostate cancer (LNCaP, PC-3) cell lines and subject them to a range of functional assays. Using a system for rapid generation of stably-transfected isogenic cell lines, that was developed in our group, we create a prostate cancer library in a two-step procedure. At first, acceptor clones are generated from cancer and normal prostate cell lines by stable integration of a plasmid carrying a recombinase target sequence. Incorporation of a second expression plasmid with the gene of interest is then mediated by site-specific recombination. This procedure allows a facilitated transgene integration into a predefined, transcriptionally active locus. It also minimizes the influence of genetic background, as clones generated within such a library differ among each other only by the presence of the gene of interest in the same chromosomal location. Therefore, in contrast to traditional knock-in strategies, our system ensures that readily interpretable phenotypes are obtained and thereby provides a highly standardized resource for functional gene analysis. The technology is well suited for medium throughput and was designed to link data from high-throughput genomic analyses with functional gene analysis in animal models. LNCaP and PC-3 acceptor cell lines have been generated and characterized. Recombination of PC-3 with several well-described, cancer-relevant genes under the control of an inducible promoter indicated the feasibility of the method. The next step will include initial screening of the library of roughly 100 recombinants for changes in the cell viability and proliferation. The most promising effectors from the primary screen will be followed with an expanded range of functional assays, addressing their impact on cell cycle, apoptosis and cell migration. This can then serve as a starting point for an individual characterization of the novel prostate cancer genes. Citation Information: Cancer Res 2009;69(23 Suppl):A51.

Random transposon vectors pUTTns for the markerless integration of exogenous genes into gram-negative eubacteria chromosomes

A set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS 50 R transposase tnp gene in cis, but they are external to the mobile element. The vectors' conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker ( sacB), a 430-bp repeat of the sacB gene 3′ end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene ( mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene ( oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria.

The genetic and epigenetic basis of ependymoma

Although ependymoma is the third most common pediatric brain tumor, we know little about the genetic/epigenetic basis of its initiation, maintenance, or progression. This is due in part to the heterogeneity of the disease, as well as the small sample size of the cohorts analyzed in most studies. Many of the genetic aberrations identified to date are large genomic regions, making the differentiation between passenger and driver genes difficult. The finding of a balanced karyotype in a significant subset of pediatric posterior fossa ependymomas increases the difficulty of identifying targets for rationale therapy. The paucity of in vitro and in vivo model systems for ependymoma compound the difficulties outlined above. In this review, we discuss the published literature on ependymoma genetics and epigenetics and discuss possible future directions for the field.

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites.

The new IS 1595 family, its relation to IS 1 and the frontier between insertion sequences and transposons

A picture of insertion sequence (IS) diversity is emerging in which previously well-defined groups or families, while remaining tightly clustered, no longer have defined borders but tend to be joined by ISs with partially shared characteristics. This is to some extent due to the presence or absence of different structurally defined transposase domains (and their spacing) and sequence similarities between IS ends. A surprising result arising from the detailed analysis of ISs in various bacterial genomes is the presence of close relatives carrying passenger genes (e.g. antibiotic resistances, methyltransferases, or transcriptional regulators as well as unknown functions). This is beginning to obscure the previously defined line between ISs (no additional orfs) and transposons. We include the lowercase prefix “t” to distinguish them from classical ISs. This is illustrated here by the IS 1595 family, distantly related to IS 1, which can be subdivided into several groups based on BLAST analysis of transposases, the genetic organization (number and position of the orfs) and the inverted repeats at their ends. The classification was subsequently confirmed using MCL (Markov cluster algorithm) software with parameters derived from the well-defined IS 3 family. Many new ISs were identified from the public databases using a reiterative BLAST approach.

Tackling the methylome: recent methodological advances in genome-wide methylation profiling

DNA methylation of promoter CpG islands is strongly associated with gene silencing and is known as a frequent cause of loss of expression of tumor suppressor genes, as well as other genes involved in tumor formation. DNA methylation of driver genes is very likely outnumbered by the number of methylated passenger genes, though these can be useful as tumor markers. Much of what is known about the importance of DNA methylation in cancer was gained through small- and moderate-scale analysis of gene promoters and tumor samples. A much better understanding of the role of DNA methylation in cancer, either as a marker of disease or as an active driver of tumorigenesis, will likely be gained from genome-wide studies of this modification in normal and malignant cells. This goal has become more attainable with the recent introduction of large-scale genome analysis methodologies and these have been modified to allow for investigation of DNA methylation. Several research groups have been formed to coordinate efforts and apply these methodologies to decipher the methylome of healthy and diseased tissues. In this article we review technological advances in genome-wide methylation profiling.