The analysis of a human thyroid serial analysis of gene expression (SAGE) library shows the presence of an abundant SAGE tag corresponding to the mRNA of thyroglobulin (TG). Additional, less abundant tags are present that can not be linked to any other known gene, but show considerable homology to the wild-type TG tag. To determine whether these tags represent TG mRNA molecules with alternative cleavage, 3′-RACE clones were sequenced. The results show that the three putative TG SAGE tags can be attributed to TG transcripts and reflect the use of alternative polyadenylation cleavage sites downstream of a single polyadenylation signal in vivo. By screening more than 300 000 sequences corresponding to human, mouse and rat transcripts for this phenomenon we show that a considerable percentage of mRNA transcripts (44% human, 22% mouse and 22% rat) show cleavage site heterogeneity. When analyzing SAGE-generated expression data, this phenomenon should be considered, since, according to our calculations, 2.8% of human transcripts show two or more different SAGE tags corresponding to a single gene because of alternative cleavage site selection. Both experimental and in silico data show that the selection of the specific cleavage site for poly(A) addition using a given polyadenylation signal is more variable than was previously thought.

Bacteria live in an ever-changing environment and must alter protein expression promptly to adapt to these changes and survive. Specific response genes that are regulated by a subset of alternative σ70-like transcription factors have evolved in order to respond to this changing environment. Recently, we have described the existence of a σE regulon including the anti-σ-factor MseR in the obligate human bacterial pathogen Neisseria meningitidis. To unravel the complete σE regulon in N. meningitidis, we sequenced total RNA transcriptional content of wild type meningococci and compared it with that of mseR mutant cells (ΔmseR) in which σE is highly expressed. Eleven coding genes and one non-coding gene were found to be differentially expressed between H44/76 wildtype and H44/76ΔmseR cells. Five of the 6 genes of the σE operon, msrA/msrB, and the gene encoding a pepSY-associated TM helix family protein showed enhanced transcription, whilst aniA encoding a nitrite reductase and nspA encoding the vaccine candidate Neisserial surface protein A showed decreased transcription. Analysis of differential expression in IGRs showed enhanced transcription of a non-coding RNA molecule, identifying a σE dependent small non-coding RNA. Together this constitutes the first complete exploration of an alternative σ-factor regulon in N. meningitidis. The results direct to a relatively small regulon indicative for a strictly defined response consistent with a relatively stable niche, the human throat, where N. meningitidis resides.

Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.Neisseria meningitidis resides in the nasopharynx of approximately 10 to 30% of the human population. However, in some individuals this pathogen results in invasive disease, leading to life-threatening septicemia and meningitis (4,14). The factors triggering meningococcal pathogenic outbreaks have not yet been elucidated, but several host and bacterium factors have been proposed (2,8,13).To date seven complete N. meningitidis genomes have been deposited with GenBank (1, 10-12, 15), of which only one is serogroup B. Here we present the sequence of N. meningitidis serogroup B strain H44/76, which is related to the sequenced serogroup B strain MC58 (GenBank accession no. AE002098) and belongs to the same clonal complex 32 (16). N. meningitidis H44/76 is widely used in molecular genetics studies, including those related to serogroup B vaccine development, because of its favorable natural transformation efficiency.Whole genomic DNA of an H44/76 culture that only had six plate culture passages since its isolation from a patient in Norway in 1976 was sequenced (5). Sequencing was performed using shotgun 454 Titanium (Roche) pyrosequencing according to the manufacturer's recommendations. The coverage was 20-fold with a median read length of 403 nucleotides. De novo and reference mapping assemblies were performed with Newbler (version 2.3; 454 Life Sciences, Branford, CT). The final assembly, containing 46 contigs, was obtained by combining both assemblies using Aligner software (version 3.5; CodonCode Corp., Dedham, MA). Investigation of the regions surrounding contig boundaries showed that further reduction of the number of contigs was hampered due to misassembly of repetitive sequences and duplicated genes, like transposases, rRNA/tRNA regions, two-partner secretion systems, and type IV pili (9).Annotation was performed using the annotation engine at the Institute for Genome Sciences (University of Maryland, Baltimore, MD [http://ae.igs.umaryland.edu/cgi/index.cgi]). We compared the sequences of 21 gene fragments of abcZ, adk, aroE, fumC, gdh, pdhC, pgm, aspA, carB, dhpS, glnA, gpm, mtgA, pilA, pip, ppk, pykA, rpiA, serC, talA, porA, and porB of the currently sequenced H44/76 genome with those sequences of H44/76 previously submitted to the Neisseria Multi Locus Sequence Typing database (6). All 11,549 compared nucleotides were identical, indicating a high quality of our H44/76 genome. Comparative genome analysis of strain H44/76 and MC58 (recently reannotated [12]) was performed using BLAT (7) and progressiveMauve (3).The genome size of H44/76 is 2.18 Mb, and 2,480 open reading frames (ORFs) were annotated, compared to 2.27 Mb and 2...