Summary Eukaryotic cells make many types of primary and processed RNAs that are found either in specific sub-cellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic sub-cellular localizations are also poorly understood. Since RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modifications and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations taken together prompt to a redefinition of the concept of a gene.

Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.

BackgroundThe brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines.Methodology/Principal FindingsTo examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies.Conclusions/SignificanceThe results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

-Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manB core (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB core to distinguish it from the wbk manB O-Ag . The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.Brucellosis is a zoonotic disease that causes heavy economic losses and human suffering. Under most conditions, vaccination and serological identification and culling of infected animals are the only practical means to achieve its eradication, but the best vaccines available (Brucella abortus S19 for cattle and B. melitensis Rev1 for sheep and goats) may induce abortions when used in pregnant animals and are virulent for humans. Moreover, like field strains, they carry a cell surface smoothtype lipopolysaccharide (S-LPS) whose immunodominant section (the N-formylperosamine O-polysaccharide) induces an antibody response that may be difficult to distinguish from that resulting from a true infection (25,48). This complicates serodiagnosis because the tests currently used detect antibodies to the O-polysaccharide.To overcome these problems, several strategies are possible. The early observation that rough (R) B. abortus strains are attenuated and do not agglutinate with antibody elicited by S bacteria (63) soon led to the concept of Brucella R vaccines and, more than 50 years ago, the spontaneous R mutant B. abortus 45/20 was studied for this purpose. However, strain 45/20 was unstable, and its use was abandoned (1, 48). The same strategy was followed to devel...