Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides

Circadian rhythms are known to exist in all groups of eukaryotic organisms as well as oxygenic photosynthetic bacteria, cyanobacteria. However, little information is available regarding the existence of rhythmic behaviors in prokaryotes other than cyanobacteria. Here we report biological rhythms of gene expression in a purple bacterium Rhodobacter sphaeroides by using a luciferase reporter gene system. Self-bioluminescent strains of Rb. sphaeroides were constructed, which produced a bacterial luciferase and its substrate, a long chain fatty aldehyde, to sustain the luminescence reaction. After being subjected to a temperature or light entrainment regime, the reporter strains with the luciferase genes driven by an upstream endogenous promoter expressed self-sustained rhythmicity in the constant free-running period. The rhythms were controlled by oxygen and exhibited a circadian period of 20.5 h under aerobic conditions and an ultradian period of 10.6-12.7 h under anaerobic conditions. The data suggest a novel endogenous oscillation mechanism in purple photosynthetic bacteria. Elucidation of the clock-like behavior in purple bacteria has implications in understanding the origin and evolution of circadian rhythms.     

Reprogramming of sheep fibroblasts into pluripotency under a drug-inducible expression of mouse-derived defined factors

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes.     

Multiple GUS expression patterns of a single Arabidopsis gene

Ten independent transposant lines with gene or enhancer traps (ET) inserted into the same gene (At2g01170) were identified in Arabidopsis thaliana . Transposon insertions were confirmed for each line. Only three of five ET lines and only one of the five gene trap (GT) lines displayed uidA (GUS) staining. The GUS (β-glucuronidase) expression patterns of the ET lines were different in all three lines. In the GT line, the GUS expression was restricted to the vascular tissue under all conditions examined. The variation in ET GUS expression suggests that each ET was controlled by different enhancer elements or the different elements of the trapped locus may give rise to different GUS expression patterns. Of five GT lines, three have the GUS gene in the same orientation as the At2g01170 open reading frame, yet only one yielded GUS staining. Regardless of the insertion construct, only those transposants with an insertion at the 3' end of the gene yielded GUS staining. Some transposants displayed a longer root phenotype in the presence of kanamycin that was also observed in 3' insertion sites in At2g01170. Taken together, these data show that insertions in the 5' end of the gene disrupted expression and emphasise the complexity encountered with ET and GT constructs to characterise the expression patterns of genes of interest based solely on GUS expression patterns.     

Determining gene expression on a single pair of microarrays

Background  

In microarray experiments the numbers of replicates are often limited due to factors such as cost, availability of sample or poor hybridization. There are currently few choices for the analysis of a pair of microarrays where N = 1 in each condition. In this paper, we demonstrate the effectiveness of a new algorithm called PINC (PINC is Not Cyber-T) that can analyze Affymetrix microarray experiments.     

Analysis of gene expression in single cells

A cell's structural and functional characteristics are dependent on the specific complement of genes it expresses. The ability to study and compare gene usage at the cellular level will therefore provide valuable insights into cell physiology. Such analyses are complicated by problems associated with sample collection, sample size and the limited sensitivity of expression assays. Advances have been made in approaches to the collection of cellular material and the performance of single-cell gene expression analysis. Recent development in global amplification of mRNA may soon permit expression analyses of single cells to be performed on DNA microarrays.     

Genomic DNA amplification from a single bacterium

Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using phi 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying non-culturable microorganisms.     

Imaging gene expression in single living cells

Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time.     

Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.     

pHUSH: a single vector system for conditional gene expression

Background

Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector.

Results

Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer.

Conclusion

We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.     

Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin

In this paper we describe a method for constructing E. coli plasmids that direct efficient expression of genes that encode eucaryotic or procaryotic proteins. No functional assays for the proteins are needed, and they are produced in their native, unfused state. The only requirement is that the genes be isolable without intervening sequences. We describe as an example the construction of a plasmid that directs the synthesis of about 10,000-15,000 monomers per cell of rabbit beta-globin. The essential steps in a typical construction are as follows. --A region of the gene encoding the amino-terminal portion of the protein is fused to DNA encoding an enzymatically active carboxy terminal fragment of beta-galactosidase. The latter is carried on one of three plasmids designed to facilitate the fusion (the construction of these three plasmids is described in the Appendix). --A "portable promoter" of the lac operon is placed at many positions in front of the fused gene using nucleases in vitro. Those promoter placements that elicit efficient expression of the fused gene are identified by the beta-galactosidase activity that they express. (In the special case we describe, plasmids identified as directing efficient expression of beta-globin were found to bear "hybrid" ribosome binding sites consisting of the Shine-Dalgarno sequence carried on the promoter fragment and the ATG of the beta-globin gene.) --The gene of interest is reconstituted intact, with the portable promoter in place, by recombination in vitro or in vivo.     

Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.     

A single gene that promotes interaction of a phytopathogenic bacterium with its insect vector,Drosophila melanogaster

Insects are major vectors of plant and animal disease, and bacterial phytopathogens are often disseminated by flies. We have previously reported that some isolates of the phytopathogenic bacterial species Erwinia carotovora infect Drosophila and activate an immune response. Using a genetic screen, we have now identified two genes that are required by E. carotovora to infect Drosophila. One of these genes has a regulatory role whereas the other, evf, confers an infectious phenotype: its transfer to non-infectious Erwinia strains or to several enterobacteria improves survival in the gut and triggers the immune response. Overexpression of Erwinia virulence factor (evf) allowed bacteria to colonize the apical side of the gut epithelium and in some cases to spread to the body cavity. Our results demonstrate a specific interaction between plant pathogens and flies that promote their dissemination.     

Effects of biocides on gene expression in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

Although sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris Hildenborough (DvH) are often eradicated in oil and gas operations with biocides, such as glutaraldehyde (Glut), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), and benzalkonium chloride (BAC), their response to these agents is not well known. Whole genome microarrays of D. vulgaris treated with biocides well below the minimum inhibitory concentration showed that 256, 96, and 198 genes were responsive to Glut, THPS, and BAC, respectively, and that these three commonly used biocides affect the physiology of the cell quite differently. Glut induces expression of genes required to degrade or refold proteins inactivated by either chemical modification or heat shock, whereas BAC appears to target ribosomal structure. THPS appears to primarily affect energy metabolism of SRB. Mutants constructed for genes strongly up-regulated by Glut, were killed by Glut to a similar degree as the wild type. Hence, it is difficult to achieve increased sensitivity to this biocide by single gene mutations, because Glut affects so many targets. Our results increase understanding of the biocide's mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment.