👤 Gregory T Jones

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens. Show less

The most well established molecular markers of poor outcome in Wilms' tumor are loss of heterozygosity at chromosomes 1p and/or 16q, although to date no specific genes at these loci have been identified. We have previously shown a link between genomic gain of chromosome 1q and tumor relapse and sought to further elucidate the role of genes on 1q in treatment failure. Microarray-based comparative genomic hybridization identified a microamplification harboring a single gene (CACNA1E) at 1q25.3 in 6 of 76 (7.9%) Wilms' tumors, correlating with a shorter relapse-free survival (P = 0.0044, log-rank test). Further characterization of this gene was carried out by measuring mRNA and protein expression as well as stable transfection of HEK293 cells. Overexpression of the CACNA1E transcript was associated with DNA copy number (P = 0.0204, ANOVA) and tumor relapse (P = 0.0851, log-rank test). Immunohistochemistry against the protein product Ca(V)2.3 revealed expression localized to the apical membrane in the distal tubules of normal kidney but not to the metanephric blastemal cells of fetal kidney from which Wilms' tumors arise. Nuclear localization in 99 of 160 (61.9%) Wilms' tumor cases correlated with a reduced relapse-free survival, particularly in cases treated with preoperative chemotherapy (P = 0.009, log-rank test). Expression profiling of stably transfected HEK293 cells revealed specific up-regulation of the immediate early response genes EGR1/EGR2/EGR3 and FOS/FOSB, mediated by activation of the MEK/ERK5/Nur77 pathway. These data identify a unique genetic aberration with direct clinical relevance in Wilms' tumor relapse and provide evidence for a potential novel mechanism of treatment resistance in these tumors. Show less

Endocytic pathways are highly dynamic gateways for molecules to enter cells. Functionality and specificity is in part controlled by a number of small GTPases called Rabs. In defined cellular locations, Rabs mediate multiple functions in membrane trafficking via their specific interaction with organelle membranes and a host of affector and effector molecules. On endocytic pathways, Rabs have been shown to control the formation of vesicles on the plasma membrane and the downstream delivery of internalized molecules to a number of cellular locations. As numerous Rabs are located to endocytic pathways, an internalized molecule may traverse a number of Rab specific substations or subdomains en route to its final destination. Rabs 5, 21 and 22 have all been localized to the early endocytic pathway and have been shown to share a number of characteristics to merit their segregation into a single functional endocytic group. In this review, we compare experiments that describe similarities and differences in endosome morphology and function that is mediated by their expression in cells. Show less

Combined hyperlipidemia is a common disorder, characterized by a highly atherogenic lipoprotein profile and a substantially increased risk of coronary heart disease. The purpose of this study was to establish whether variations of apolipoprotein A5 (APOA5), a newly discovered gene of lipid metabolism located 30 kbp downstream of the APOA1/C3/A4 gene cluster, contributes to the transmission of familial combined hyperlipidemia (FCHL). We performed linkage and association tests on 128 families. Two independent alleles, APOA5c.56G and APOC3c.386G, of the APOA1/C3/A4/A5 gene cluster were overtransmitted in FCHL (P=0.004 and 0.007, respectively). This was paired with reduced transmission of the common APOA1/C3/A4/A5 haplotype (frequency 0.4461) to affected subjects (P=0.012). The APOA5c.56G genotype accounted for 7.3% to 13.8% of the variance in plasma triglyceride levels in probands (P<0.004). The APOC3c.386G genotypes accounted for 4.4% to 5.1% of the variance in triglyceride levels in FCHL spouses (P<0.007), suggesting that this allele marks a FCHL quantitative trait as well as representing a susceptibility locus for the condition. A combined linkage and association analysis establishes that variation at the APOA1/C3/A4/A5 gene cluster contributes to FCHL transmission in a substantial proportion of northern European families. Show less

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics. Show less

The osteochondroma is a common, benign, primary tumor of bone. A mechanism for its pathogenesis has not been identified, but loss of function of EXT genes is implicated in sporadic and hereditary multiple osteochondromas. Recent advances in the understanding of other molecular signaling pathways in the physis cast doubt on the latest pathogenetic theories. These advances are reviewed and used as the basis for a revised theory for pathogenesis: A clone of proliferating chondrocytes without functional EXT1 (or EXT2) expression fails to produce heparan sulfate; lack of heparan sulfate at the cell surface disrupts fibroblast growth factor signaling and Indian hedgehog diffusion, leading to focal overproliferation and adjacent bone collar deficiency, respectively; together these effects are proposed to contribute to osteochondroma pathogenesis. Show less

The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription. The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate-dependent chromatinremodeling complexes. Show less

The HP1 class of chromobox (Cbx) genes encode an evolutionarily conserved family of proteins involved in the packaging of chromosomal domains into a repressive heterochromatic state. The murine Cbx5, Cbx1 and Cbx3 genes encode the three mouse HP1 proteins, mHP1 alpha, -beta and -gamma respectively. Here, we report the cloning of the mouse Cbx3/HP1 gamma gene and the chromosomal localisation of Cbx3 and three Cbx3-related pseudogenes. The Cbx3 structural gene is located on mouse Chromosome 6, close to the Hoxa cluster. Two Cbx3 processed pseudogenes are separated by just 300 bp and are arranged in a head-to-tail configuration on Chromosome 13 while a third pseudogene is found on mouse Chromosome 4. The genomic intron-exon arrangement of Cbx3 is different from the conserved organisation of three other mammalian HP1 genes, Cbx1 (mHP1 beta), CBX3 (hHP1 gamma), and Cbx5 (mHP1 alpha) in that Cbx3 lacks an intron that is present in the others. Show less

The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed. Show less

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions. Show less

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts. Show less

Leukaemogenesis correlates with alterations in chromatin structure brought about by the gain or loss of interactive domains from regulatory factors that are disrupted by chromosomal translocations. The gene MLL, a target of such translocation events, forms chimaeric fusion products with a variety of partner genes. While MLL appears to be involved in chromatin-mediated gene regulation, the functions of its partner genes are largely speculative. We report the biochemical analysis of the MLL partner gene AF10 and its possible role in leukaemogenesis. AF10 has been reported to be re-arranged with genes other than MLL leading to the same phenotype, a myeloid leukaemia. We have identified a novel protein-protein interaction motif in the AF10 protein comprising the extended LAP/PHD-finger. This domain mediates homo-oligomerisation of recombinant AF10 and is conserved in several proteins, including MLL itself. AF10 binds cruciform DNA via a specific interaction with an AT-hook motif and is localised to the nucleus by a defined bipartite nuclear localisation signal in the N-terminal region. Show less

We have cloned Af10, the murine homologue of the MLL partner gene AF10. The predicted open reading frame of Af10 contains 1069 aa which are 90% identical to those of AF10. Af10 contains an N-terminal cysteine-rich region with a LAP/PHD finger, a leucine zipper domain and a glutamine-rich region at the C-terminus, features also found in the human proteins AF10 and AF17. A single 5. 5-kb transcript was detected in murine tissues with the highest level of expression in the testes. A polyclonal antibody raised to the cysteine-rich region of AF10 was able to identify a double band of 140 kDa on Western analysis in mouse testicular extracts. After subcellular separation Af10 was identified in both the nuclear and cytoplasmic extracts, again as a double band of 140 kDa in size. In situ hybridisation studies were performed with sense and antisense digoxigenin-labelled oligonucleotides. High levels of expression were noted in postmeiotic germ cells, especially in spermatids from around stage VI to stage VIII. High levels of expression were also seen in the white matter of the cerebellum, extending into the granular layer. The expression in differentiated rather than in proliferating cells suggests that the role of Af10 may lie in the suppression of proliferation rather than in differentiation. Since the LAP/PHD finger domains are lost in the MLL-AF10 fusion, arguably such a function could be carried out by this domain. Show less

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell. Show less

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy. Show less

Deletion analysis offers a powerful alternative to linkage and karyotypic approaches for human chromosome mapping. A panel of deletion hybrids has been derived by mutagenizing J1, a hamster cell line that stably retains chromosome 11 as its only human DNA, and selecting for loss of MIC1, a surface antigen encoded by a gene in band 11p13. A unique, self-consistent map was constructed by analyzing the pattern of marker segregation in 22 derivative cells lines; these carry overlapping deletions of 11p13, but selectively retain a segment near the 11p telomere. The map orders 35 breakpoints and 36 genetic markers, including 3 antigens, 2 isozymes, 12 cloned genes, and 19 anonymous DNA probes. The deletions span the entire short arm, dividing it into more than 20 segments and define a set of reagents that can be used to rapidly locate any newly identified marker on 11p, with greatest resolution in the region surrounding MIC1. The approach we demonstrate can be applied to map any mammalian chromosome. To test the gene order, we examined somatic cell hybrids from five patients, whose reciprocal translocations bisect band 11p13; these include two translocations associated with familial aniridia and two with acute T-cell leukemia. In each patient, the markers segregate in telomeric and centromeric groups as predicted by the deletion map. These data locate the aniridia gene (AN2) and a recurrent T-cell leukemia breakpoint (TCL2) in the marker sequence, on opposite sides of MIC1. To provide additional support, we have characterized the dosage of DNA markers in a patient with Beckwith-Wiedemann syndrome and an 11p15-11pter duplication. Our findings suggest the following gene order: TEL - (HRAS1, MER2, CTSD, TH/INS/IGF2, H19, D11S32) - (RRM1, D11S1, D11S25, D11S26) - D11S12 - (HBBC, D11S30) - D11S20 - (PTH, CALC) - (LDHA, SAA, TRPH, D11S18, D11S21) - D11S31 - D11S17 - HBVS1 - (FSHB, D11S16) - AN2 - MIC1 - TCL2 - delta J - CAT - MIC4 - D11S9 - D11S14 - ACP2 - (D11S33, 14L) - CEN. We have used the deletion map to show the distribution on 11p of two centromeric repetitive elements and the low-order interspersed repeat A36Fc. Finally, we provide evidence for an allelic segregation event in the hamster genome that underlies the stability of chromosome 11 in J1. The deletion map provides a basis to position hereditary disease loci on 11p, to distinguish the pattern of recessive mutations in different forms of cancer and, since many of these genes have been mapped in other mammalian species, to study the evolution of a conserved syntenic group. Show less

Asparaginase II (L-asparagine amidohydrolase, EC 3.5.1.1) activity of cells from stationary phase cultures of Saccharomyces cerevisiae is very low. When these cells are inoculated into minimal medium, asparaginase II specific activity rises rapidly and reaches a maximum after 9-10 h. During the next 2.5-3 h, a rapid decrease in asparaginase II specific activity occurs. The enzyme does not appear to be secreted into the medium or to be reabsorbed into the cell. Addition of protease inhibitors at the time of maximum activity partially or totally prevents the loss of asparaginase II. L-1-Tosylamide-2-phenylethyl chloromethyl ketone decreases the rate of loss. The sulfhydryl reagents p-hydroxymercuribenzoate and iodoacetamide inhibit the loss of asparaginase II. However, addition of EDTA causes a further increase in activity. This increase is due to de novo protein synthesis. The effect of EDTA can be reversed by the addition of Zn2+. The most likely explanation for the rapid loss of asparaginase II is proteolytic degradation by a Zn2+-dependent, thiol protease or peptidase. Show less

A clone panel containing various segments of human chromosome 11 has been selected and use for regional assignment of the gene for human lysosomal acid phosphatase (ACP2) to the short arm of chromosome 11, in the region 11p11 leads to 11p12. Further evidence has also been presented to update the regional assignment of the gene for lactate dehydrogenase A (LDHA) to 11p12 leads to 11p13, and to support a previous assignment of the genes for the two components of the human cell-surface antigens of the SA11 (previously designated AL) group, SA11-1 and SA11-3 (previously designated AL-a1 and AL-a3), to 11pter leads to 11p13. This regional clone panel will be useful for rapid regional mapping of other genes assigned to chromosome 11. Show less