Using an in vivo phagemid system to identify non-compatible loxP sequences.

The site-specific recombination system of bacteriophage P1 is composed of the Cre recombinase that recognizes a 34-bp loxP site. The Cre/loxP system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. The creation of additional heterologous loxP sequences potentially expands the utility of this system, but only if these loxP sequences do not recombine with one another. We have developed a stringent in vivo assay to examine the degree of recombination between all combinations of each previously published heterologous loxP sequence. As expected, homologous loxP sequences efficiently underwent Cre-mediated recombination. However, many of the heterologous loxP pairs were able to support recombination with rates varying from 5 to 100%. Some of these loxP sequences have previously been reported to be non-compatible with one another. Our study also confirmed other heterologous loxP pairs that had previously been shown to be non-compatible, as well as defined additional combinations that could be used in designing new recombination vectors.     

利用Cre/loxP系统删除转Bt基因水稻中的选择标记基因

来源于噬菌体P1的Cre/loxP位点特异性重组系统是目前在植物遗传转化中应用较多,较成熟的一个标记基因删除系统。在这个系统中,Cre酶可以特异性的识别和切割位于两个lox位点之间的标记基因,整个系统重组仅需Cre和lox识别位点即可完成而无需其它辅因子的参加。利用农杆菌介导法成功地将cre基因导入供试材料"皖粳97",得到转hpt-cre基因水稻植株;将其与先期转基因育成的携带loxp-hpt-loxp-bt基因的"皖粳97"株系进行田间杂交,通过PCR分析,Cre/loxP重组系统定向删除了潮霉素抗性筛选标记基因。     

Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.     

In vivo insertional mutagenesis in Corynebacterium pseudotuberculosis: an efficient means to identify DNA sequences encoding exported proteins

The reporter transposon-based system TnFuZ was used to identify exported proteins of the animal pathogen Corynebacterium pseudotuberculosis. Thirty-four out of 1,500 mutants had detectable alkaline phosphatase (PhoZ) activity. This activity was from 21 C. pseudotuberculosis loci that code for fimbrial and transport subunits and for hypothetical and unknown-function proteins.     

Using an affinity analysis to identify phytoplankton associations

Phytoplankton functional traits can represent particular environmental conditions in complex aquatic ecosystems. Categorizing phytoplankton species into functional groups is challenging and time‐consuming, and requires high‐level expertise in species autecology. In this study, we introduced an affinity analysis to aid the identification of candidate associations of phytoplankton from two data sets comprised of phytoplankton and environmental information. In the Huaihe River Basin with a drainage area of 270,000 km² in China, samples were collected from 217 selected sites during the low‐water period in May 2013; monthly samples were collected during 2006–2011 in a man‐made pond, Dishui Lake. Our results indicated that the affinity analysis can be used to define some meaningful functional groups. The identified phytoplankton associations reflect the ecological preferences of phytoplankton in terms of light and nutrient acquisition. Advantages and disadvantages of applying the affinity analysis to identify phytoplankton associations are discussed with perspectives on their utility in ecological assessment.     

Genomic strategies to identify mammalian regulatory sequences

With the continuing accomplishments of the human genome project, high-throughput strategies to identify DNA sequences that are important in mammalian gene regulation are becoming increasingly feasible. In contrast to the historic, labour-intensive, wet-laboratory methods for identifying regulatory sequences, many modern approaches are heavily focused on the computational analysis of large genomic data sets. Data from inter-species genomic sequence comparisons and genome-wide expression profiling, integrated with various computational tools, are poised to contribute to the decoding of genomic sequence and to the identification of those sequences that orchestrate gene regulation. In this review, we highlight several genomic approaches that are being used to identify regulatory sequences in mammalian genomes.     

Inducible gene trapping with drug-selectable markers and Cre/loxP to identify developmentally regulated genes

Gene trapping in mouse embryonic stem cells is an important genetic approach that allows simultaneous mutation of genes and generation of corresponding mutant mice. We designed a selection scheme with drug selection markers and Cre/loxP technology which allows screening of gene trap events that responded to a signaling molecule in a 96-well format. Nine hundred twenty gene trap clones were assayed, and 258 were classified as gene traps induced by in vitro differentiation. Sixty-five of the in vitro differentiation-inducible gene traps were also responsive to retinoic acid treatment. In vivo analysis revealed that 85% of the retinoic acid-inducible gene traps trapped developmentally regulated genes, consistent with the observation that genes induced by retinoic acid treatment are likely to be developmentally regulated. Our results demonstrate that the inducible gene trapping system described here can be used to enrich in vitro for traps in genes of interest. Furthermore, we demonstrate that the cre reporter is extremely sensitive and can be used to explore chromosomal regions that are not detectable with neo as a selection cassette.     

Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination

The Cre recombinase mediates precise site-specific recombination between a pair of loxP sequences through an intermediate containing Holiday junction. The recombination junction in the loxP sequence is located within the asymmetric 8-nucleotide spacer region. To examine the role of each nucleotide sequence of the spacer region in the recombination process, we synthesized a complete set of 24 loxP spacer mutants with single-base substitutions and 30 loxP spacer mutants with double-base substitutions. Each synthesized loxP mutant was ligated at both ends of a linear DNA or to one end of a DNA-containing wild-type loxP at the other end and their recombination efficiencies were analyzed with an in vitro system. The sequence identity of the right two nucleotides and left four nucleotides in the central six bases of the spacer region was found to be essential for formation and resolution, respectively, of the intermediate product. Furthermore, even when homology was maintained, the recombination efficiencies were lower than that of wild-type loxP and varied among mutants. Based on this knowledge, we identified two loxP mutants with double-base substitutions, mutants 5171 and 2272, which recombine efficiently with an identical mutant but not with the other mutant or wild-type loxP.     

Limited use of the Cre/loxP recombination system in efficient production of loxP-containing minicircles in vivo

The Cre/loxP recombination system of bacteriophage P1 is one of the most powerful tools in genome engineering. We report, however, that the activity of the Cre/loxP system interferes with the stability of the multicopy loxP-bearing plasmids in Escherichia coli recA bacteria. Due to the predominantly unidirectional Cre-mediated high-order multimer formation of these plasmids, the number of their copies (overall yield) gradually decreases. Intermolecular recombination reduces the copy number of plasmids and eventually increases their segregational instability. We have found that in the presence of even the slightest amount of Cre activity, loxP-bearing plasmids continuously undergo multimerization, which very rapidly leads to loxP-plasmid free cells. Our results are compatible with the hypothesis of the multimer catastrophe [Cell, 1984 (36), 1097].     

Correlation approach to identify coding regions in DNA sequences.

Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations. We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions. The algorithm is particularly successful in predicting the location of lengthy coding regions. For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides. The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences. This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences.     

An exploratory algorithm to identify intra-host recombinant viral sequences

Since recombination leads to the generation of mosaic genomes that violate the assumption of traditional phylogenetic methods that sequence evolution can be accurately described by a single tree, results and conclusions based on phylogenetic analysis of data sets including recombinant sequences can be severely misleading. Many methods are able to adequately detect recombination between diverse sequences, for example between different HIV-1 subtypes. More problematic is the identification of recombinants among closely related sequences such as a viral population within a host. We describe a simple algorithmic procedure that enables detection of intra-host recombinants based on split-decomposition networks and a robust statistical test for recombination. By applying this algorithm to several published HIV-1 data sets we conclude that intra-host recombination was significantly underestimated in previous studies and that up to one-third of the env sequences longitudinally sampled from a given subject can be of recombinant origin. The results show that our procedure can be a valuable exploratory tool for detection of recombinant sequences before phylogenetic analysis, and also suggest that HIV-1 recombination in vivo is far more frequent and significant than previously thought.     

Using antibodies against Dictyostelium membranes to identify an actin- binding membrane protein

Polyclonal antibodies made against Dictyostelium discoideum membranes were used to block the interaction of those membranes with actin. As expected, actin interacted mostly with the internal surface of the membrane, demonstrated by the fact that whole cells could only absorb out a minor fraction of the blocking antibody. The antibody was used to show that the membrane component(s) which interacted with actin were probably integral; they could be extracted with detergent but not with solutions designed to extract peripheral membrane proteins. To identify the responsible protein(s), Western transfers of membranes were cut into fractions which were tested for their ability to absorb out the blocking activity of the antibody. We observed a single peak at a molecular weight of approximately 20,000, and thus conclude that a 20,000-mol-wt protein is a major integral membrane actin-binding protein in Dictyostelium. This approach to the identification of proteins involved in actin-membrane interaction has allowed us to make the first identification of an actin-binding membrane protein which is based on its activity in native membranes.     

Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo

Membrane proteins are crucial for many essential cellular processes. As membrane proteins function in complexes, methods to detect and to characterize membrane protein-protein interactions are undoubtedly required. Therefore, we developed the "Membrane-Strep-tagged protein interaction experiment" (Membrane-SPINE) that combines the specific purification of a Strep-tagged membrane protein with the reversible fixation of protein complexes by formaldehyde cross-linking. In combination with MS analysis, we suggest Membrane-SPINE as a powerful tool to identify unknown interaction partners of membrane proteins in vivo.     

A computational approach to identify genes for functional RNAs in genomic sequences

Currently there is no successful computational approach for identification of genes encoding novel functional RNAs (fRNAs) in genomic sequences. We have developed a machine learning approach using neural networks and support vector machines to extract common features among known RNAs for prediction of new RNA genes in the unannotated regions of prokaryotic and archaeal genomes. The Escherichia coli genome was used for development, but we have applied this method to several other bacterial and archaeal genomes. Networks based on nucleotide composition were 80–90% accurate in jackknife testing experiments for bacteria and 90–99% for hyperthermophilic archaea. We also achieved a significant improvement in accuracy by combining these predictions with those obtained using a second set of parameters consisting of known RNA sequence motifs and the calculated free energy of folding. Several known fRNAs not included in the training datasets were identified as well as several hundred predicted novel RNAs. These studies indicate that there are many unidentified RNAs in simple genomes that can be predicted computationally as a precursor to experimental study. Public access to our RNA gene predictions and an interface for user predictions is available via the web.     

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.