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Protein-protein interaction networks (PPINs) have been employed to identify potential novel interconnections between proteins as well as crucial cellular functions. In this study we identify fundamental principles of PPIN topologies by analysing network motifs of short loops, which are small cyclic interactions of between 3 and 6 proteins. We compared 30 PPINs with corresponding randomised null models and examined the occurrence of common biological functions in loops extracted from a cross-validated high-confidence dataset of 622 human protein complexes. We demonstrate that loops are an intrinsic feature of PPINs and that specific cell functions are predominantly performed by loops of different lengths. Topologically, we find that loops are strongly related to the accuracy of PPINs and define a core of interactions with high resilience. The identification of this core and the analysis of loop composition are promising tools to assess PPIN quality and to uncover possible biases from experimental detection methods. More than 96% of loops share at least one biological function, with enrichment of cellular functions related to mRNA metabolic processing and the cell cycle. Our analyses suggest that these motifs can be used in the design of targeted experiments for functional phenotype detection.

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P<0.0002), no association was noted between the type of germline and somatic NF1 lesion within the same individual.

We report a large series of 173 patients with physical and/or neurological abnormalities and a de novo imbalance identified by array CGH. Breakpoint intervals were screened for the presence of low copy repeats (LCRs) to distinguish between rearrangements formed by non-allelic homologous recombination (NAHR) and rearrangements formed by other mechanisms. We identified significant differences in size and parental origin between the LCR-mediated and non-LCR groups. Non-LCR imbalances were evenly distributed among the four size intervals we defined, whereas LCR-mediated rearrangements had a narrow size distribution, predominantly between 1 and 5 Mb (P = 0.001). Among the LCR-mediated rearrangements there were equal numbers of maternally and paternally derived cases. In contrast, for the non-LCR rearrangements there was a significant excess of paternal cases (P = 0.024) over a wide size range including below 1 Mb. Our results provide novel evidence that unbalanced chromosome rearrangements are not only more frequent in males, but may also arise through different mechanisms than those seen in females. Although the paternal imbalances identified in our study are evenly distributed throughout the four size groups, there are very few maternal imbalances either <1 Mb or >10 Mb. Furthermore, a lower proportion of paternal imbalances are LCR mediated (13/71) compared with the maternal imbalances (12/30). We hypothesise that imbalances of maternal origin arise predominantly through NAHR during meiosis, while the majority of imbalances of paternal origin arise through male-specific mechanisms other than NAHR. Our data suggest that mitotic mechanisms could be important for the formation of chromosome imbalances; however, we found no association with increased paternal age.

DNA replication is tightly regulated, but paradoxically there is reported to be an excess of MCM DNA replication proteins over the number of replication origins. Here, we show that MCM levels in primary human T cells are induced during the G(0)-->G(1) transition and are not in excess in proliferating cells. The level of induction is critical as we show that a 50% reduction leads to increased centromere separation, premature chromatid separation (PCS) and gross chromosomal abnormalities typical of genomic instability syndromes. We investigated the mechanisms involved and show that a reduction in MCM levels causes dose-dependent DNA damage involving activation of ATR & ATM and Chk1 & Chk2. There is increased DNA mis-repair by non-homologous end joining (NHEJ) and both NHEJ and homologous recombination are necessary for Mcm7-depleted cells to progress to metaphase. Therefore, a simple reduction in MCM loading onto DNA, which occurs in cancers as a result of aberrant cell cycle control, is sufficient to cause PCS and gross genomic instability within one cell cycle.

Duplications of distal 8p with and without significant clinical phenotypes have been reported and are often associated with an unusual degree of structural complexity. Here, we present a duplication of 8p23.1-8p23.2 ascertained in a child with speech delay and a diagnosis of ICD-10 autism. The same duplication was found in his mother who had epilepsy and learning problems. A combination of cytogenetic, FISH, microsatellite, MLPA and oaCGH analysis was used to show that the duplication extended over a minimum of 6.8 Mb between 3 539 893 and 10 323 426 bp. This interval contains 32 novel and 41 known genes, of which only microcephalin (MCPH1) is a plausible candidate gene for autism at present. The distal breakpoint of the duplicated region interrupts the CSMD1 gene in 8p23.2 and the medial breakpoint lies between the MSRA and RP1L1 genes in 8p23.1.An interchromosomal insertion between a normal and polymorphically inverted chromosome 8 is proposed to explain the origin of this duplication. Further mapped imbalances of distal 8p are needed to determine whether the autistic component of the phenotype in this family results from the cumulative imbalance of many genes or dosage imbalance of an individual susceptibility gene.

Non-allelic homologous recombination between chromosome-specific LCRs is the most common mechanism leading to recurrent microdeletions and duplications. To look for locus-specific differences, we have used microsatellites to determine the parental and chromosomal origins of a large series of patients with de novo deletions of chromosome 7q11.23 (Williams syndrome), 15q11-q13 (Angelman syndrome, Prader-Willi syndrome) and 22q11 (Di George syndrome) and duplications of 15q11-q13. Overall the majority of rearrangements were interchromosomal, so arising from unequal meiotic exchange, and there were approximately equal numbers of maternal and paternal deletions. Duplications and deletions of 15q11-q13 appear to be reciprocal products that arise by the same mechanisms. The proportion arising from interchromosomal exchanges varied among deletions with 22q11 the highest and 15q11-q13 the lowest. However, parental and chromosomal origins were not always independent. For 15q11-q13, maternal deletions tended to be interchromosomal while paternal deletions tended to be intrachromosomal; for 22q11 there was a possible excess of maternal cases among intrachromosomal deletions. Several factors are likely to be involved in the formation of recurrent rearrangements and the relative importance of these appear to be locus-specific.

In this study, we have used the human BV173 and the mouse BaF3/Bcr-Abl-expressing cell lines as model systems to investigate the molecular mechanisms whereby STI571 and FoxO3a regulate Bim expression and apoptosis. FoxO3a lies downstream of Bcr-Abl signalling and is constitutively phosphorylated in the Bcr-Abl-positive BV173 and BaF3/Bcr-Abl cells. Inhibition of Bcr-Abl kinase by STI571 results in FoxO3a activation, induction of Bim expression and apoptosis. Using reporter gene assays, we demonstrate that STI571 and FoxO3a activate Bim transcription through a FoxO-binding site (FHRE) located within the promoter. This was verified by DNA pull-down and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to induction of Bim expression and apoptosis. Conversely, silencing of FoxO3a in Bcr-Abl-expressing cells abolishes STI571-mediated Bim induction and apoptosis. Together, the results presented clearly confirm FoxO3a as a key regulator of apoptosis induced by STI571, and show that Bim is a direct transcriptional target of FoxO3a that mediates the STI571-induced apoptosis. Thus, STI571 induces an accumulation of FoxO3a activity which in turn binds directly to an FHRE in the promoter to activate Bim expression and apoptosis.

Segmental aneuploidy usually has phenotypic consequences but unbalanced rearrangements without phenotypic consequences have also been reported. In particular, harmless deletions of G-dark bands 5p14 and 16q21 have each been found in more than one independent family. Here, we report two families that were ascertained at prenatal diagnosis and had similar overlapping deletions that removed most of the gene poor G-dark band 2p12. PCR mapping showed that the deletions had a minimum size of 6.1 and 6.9 Mb with at least 13 hemizygous loci including a cluster of six pancreatic islet-regenerating genes. These deletions had no apparent phenotypic consequences in eight family members. In contrast, a third family was ascertained through a child with Wilm's tumour; both the child and his mother had more proximal deletions, developmental delay and some dysmorphic features. The deletion had a minimum size of 5.7 Mb and extended into the gene-rich area of 2p11.2. These results are consistent with the idea that there may be segments of the genome that are consistently haplosufficient. The introduction of higher resolution methods of dosage analysis into diagnostic laboratories is already revealing more transmitted abnormalities of uncertain significance. As a result, published cases of transmitted imbalances have been collected as a guide to the possible significance of such findings in the future (see the 'Chromosome Anomaly Collection' at www.som.soton.ac.uk/research/geneticsdiv).

Translocations occur in a proportion of couples affected by recurrent miscarriages. We describe two such families in which the underlying cause was a cryptic subtelomeric 11p;17p translocation detected only after the birth of an affected child carrying an unbalanced form of the rearrangement. Unbalanced subtelomeric rearrangements are now recognised as a significant cause of mental impairment and we believe that these rearrangements may also be an important cause of recurrent miscarriages. In these two families the translocation is most likely to have arisen from a single ancestral event because all translocation carriers shared almost identical haplotypes around the breakpoints on both chromosomes.

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

The retinoblastoma protein (pRB) is thought to act as a tumour suppressor which is inactivated by phosphorylation. In quiescent (G0) cells pRB exists in a hypophosphorylated form (pRB110), but proliferating cells in G1 contain a significant proportion of phosphorylated pRB (pRB112-114). Studies of synchronized or elutriated cells have suggested that the phosphorylated forms of pRB disappear as cells pass from G2/M to G0/G1 and that pRB is phosphorylated again to pRB114 at the G1/S border. In this study we used two-parameter flow cytometry and cell sorting to isolate cycling cells in early and late G1 (G1A and G1B), and we show that partially phosphorylated pRB is present in cycling human lymphoid cells even in G1A. These G1A cells contain intermediate forms of pRB which become further phosphorylated to pRB112-114 as cells pass into G1B. Therefore pRB is at least partially phosphorylated from early G1 onwards. Cell cycle arrest by alpha-interferon (alpha-IFN) results in an accumulation of cells in both G1A and G1B, and these cells contain mainly pRB110. Since pRB110 is thought to prevent cell proliferation, the cytostatic effect of alpha-IFN may therefore occur by preventing the initial phosphorylation of pRB during or prior to G1A.

The product of the retinoblastoma gene (RB) is a nuclear phosphoprotein which is thought to regulate the proliferation of cells. Its phosphorylation state changes with passage through the cell cycle and it has been proposed that RB protein in its hypo-phosphorylated form prevents cells proliferating. We have investigated the phosphorylation state of the RB protein in an actively-dividing human B-lymphoblastoid cell line and after cell cycle arrest caused by alpha-Interferon (alpha-IFN). We show that the phosphorylation state of the RB protein in cells with 2N DNA content depends on whether the cells are actively cycling. Our data is compatible with the proposal that dephosphorylation of the RB protein allows cells to enter a quiescent state. This study sheds light on the molecular mechanisms which may mediate the cytostatic effects of alpha-IFN.