Expressed sequence tag (EST) databases represent a large volume of information on expressed genes including tissue type, expression profile and exon structure. In this study we create an extensive data set of human alternative splicing. We report the analysis of 7867 non-redundant mRNAs, 3011 of which contained alternative splice forms (38% of all mRNAs analysed). From a total of 12 572 ESTs 4560 different possible alternative splice forms were detected. Interestingly, 70% of the alternative splice forms correspond to exon deletion events with only 30% exonic insertions. We experimentally verified 19 different splice forms from 16 genes in a total subset of 20 studied; all of the respective genes are of medical relevance.z 2000 Federation of European Biochemical Societies.

We analyzed data of 76 consecutive patients with myelodysplastic syndrome (MDS) and isolated del(5q) (n ¼ 66) or del(5q) plus one additional chromosomal abnormality (n ¼ 10) included in our MDS database over the last 26 years. The median age of our patient population was 66.8 years. The male to female ratio was 1:1.7. In all, 14 patients (18%) had advanced MDS with an increased medullary blast count. A total of 17 patients (22%) had significant dysplasia in the nonmegakaryocytic cell lines. Nearly half of the study population showed erythroid hypoplasia in the bone marrow. The projected median survival of patients with isolated del(5q) is 146 months for a median followup of 67 months. Patients with an increased medullary blast count and those with an additional chromosomal abnormality have a significantly shorter overall survival (24 and 45 months, respectively) than patients with isolated del(5q). We did not find survival differences for different cytogenetic breakpoints, nor did the amount of dysplasia have an impact on survival in our population. In total, 29 patients have died. Deaths occurred primarily due to transformation into acute leukemia, infection, or cardiac failure. Our data support the current definition of a separate entity of MDS with del(5q) that has been suggested by the World Health Organization.

Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as "nonclassical protein secretion." Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.

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numbers of double-sorted HSC from Npm1 þ / þ and Npm1 þ /À mice (CD45.1 þ CD45.2 þ ) were transplanted into lethally irradiated C57BL/6 (CD45.2) recipients together with competitor cells (CD45.2). Short-term (2 months) and long-term (4 months) competitive repopulating capability was studied by monitoring peripheral blood donor chimerism of recipient mice by flow cytometry. Mice transplanted with Npm1 þ /À HSC exhibited markedly lower engraftment than the recipients of Npm1 þ / þ HSC as measured by total chimerism (threefold; Figure 2a, Po0.03). The percentage total and myeloid chimerism in individual transplanted mice did not differ significantly at 2 and 4 months (data not shown). We also quantified donor chimerism in the bone marrow of the recipient mice 4 months after transplantation (Figure 2b). Similar to the results in the peripheral blood, the competitive repopulating ability of the HSC from Npm1 þ /À mice was significantly reduced (8.5-fold; Figure 2b, Po0.02) in the bone marrow of the recipient mice. Although the engraftment potential of Npm1 þ /À HSC was reduced, we did not observe any effects on the composition of donor-derived mature hematopoietic cells in the peripheral blood of recipient mice (Supplementary Figure 5). Thus, the level of NPM1 expression has a direct effect on HSC repopulating ability in vivo, but has no effect on hematopoietic fate commitment.In summary, our Npm1 þ /À mouse model indicates that NPM1 has a role in maintaining HSC number and in preserving the functional integrity of these cells in the context of competitive transplantation. However, it does not appear to have a role in regulating HSC differentiation. Npm1 haploinsufficiency did not result in significant alterations in mature blood cell numbers or significant dysplasia in our mice. These results are at some variance with a previous report, in which Npm1 heterozygosity was associated with an MDS-like phenotype in older (6-10 months) mice. 4 We cannot completely exclude the possibility that the different techniques used to generate these mutant mice may explain the differences in our results; however, both models are associated with homozygous null embryonic lethality as well as decreased Npm1 mRNA (data not shown) and protein levels in Npm1 þ /À mice. Our study did use mice in which the targeted Npm1 allele was functionally eliminated by a single-gene trap event in the NPM1 locus between exons 7 and 8 (genetic background 129/SvEvBrd  C57BL/6J), whereas in the previous study exons 2-7 were replaced by green fluorescent protein cassette introducing the stop codon after exon 2 (genetic background 129/Sv  C57BL/6). Minor genetic variability between the two different substrains used in these studies (129/Sv vs 129/SvEvBrd) might also influence their respective phenotypes. 7 Although we have no definitive explanation for the difference in phenotypes, only our study directly assessed HSC function. We speculate that the increased number of HSC in Npm þ /À mice could be an important early step in myeloid pathogenesis, as thes...