The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: a possible coordinate role in ribosome biogenesis.

RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribonuclease originally described in the nucleus that processes RNA transcripts to generate their mature 5' termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2'-O-methyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized that RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.

Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders.

Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD). Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma. other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis. To date, the critical genomic segment and candidate genes involved in these deletions have not been identified. In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization. As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3. In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1). Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment. Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster. This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.

The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain.

The aquaporin family of membrane water transport proteins are expressed in diverse tissues, and in brain the predominant water channel protein is AQP4. Here we report the isolation and characterization of the human AQP4 cDNAs and genomic DNA. Two cDNAs were isolated corresponding to the two initiating methionines (M1 in a 323-aa polypeptide and M23 in a 301-aa polypeptide) previously identified in rat [Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. & Agre, P. (1994) Proc. Natl. Acad. Sci. USA 91, 13052-13056]. Similar to other aquaporins, the AQP4 gene is composed of four exons encoding 127, 55, 27, and 92 amino acids separated by introns of 0.8, 0.3, and 5.2 kb. Unlike other aquaporins, an alternative coding initiation sequence (designated exon 0) was located 2.7 kb upstream of exon 1. When spliced together, M1 and the subsequent 10 amino acids are encoded by exon 0; the next 11 amino acids and M23 are encoded by exon 1. Transcription initiation sites have been mapped in the proximal promoters of exons 0 and 1. RNase protection revealed distinct transcripts corresponding to M1 and M23 mRNAs, and AQP4 immunoblots of cerebellum demonstrated reactive polypeptides of 31 and 34 kDa. Using a P1 and a lambda EMBL subclone, the chromosomal site of the human AQP4 gene was mapped to chromosome 18 at the junction of q11.2 and q12.1 by fluorescence in situ hybridization. These studies may now permit molecular characterization of AQP4 during human development and in clinical disorders.

Ordered tandem arrangement of chromosomes in the sperm heads of monotreme mammals.

A very old unanswered question in classical cytology is whether chromosomes are arranged randomly in sperm or whether they occupy specific positions. Even with modern methods of chromosome painting, it is difficult to resolve this question for the very condensed and almost spherical sperm head of most mammals. We have taken advantage of the unusual fibrillar sperm head of monotreme mammals (echidna and platypus) to examine the position of chromosome landmarks in a two-dimensional array. We used fluorescence and radioactive in situ hybridization to telomeric, rDNA, and unique sequences to show that chromosomes are arranged tandemly and in a defined order in the sperm nucleus.

FHIT gene alterations in head and neck squamous cell carcinomas.

To determine whether the FHIT gene at 3p14.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) showed the presence of multiple cell populations with losses of different portions of FHIT alleles by FISH of FHIT genomic clones to interphase nuclei. When the data obtained by FISH and by reverse transcriptase-PCR analyses are combined, 22 of 26 cell lines showed alterations of at least one allele of the FHIT gene. Our data indicate that the FHIT gene is disrupted in HNSCCs and hence, loss of FHIT function may be important in the development and/or progression of head and neck cancers.

A methylated human 9-kb repetitive sequence on acrocentric chromosomes is homologous to a subtelomeric repeat in chimpanzees.

We have implemented an approach for the detection of DNA alterations in cancer by means of computerized analysis of end-labeled genomic fragments, separated in two dimensions. Analysis of two-dimensional patterns of neuroblastoma tumors, prepared by first digesting DNA with the methylation-sensitive restriction enzyme Not I, yielded a multicopy fragment which was detected in some tumor patterns but not in normal controls. Cloning and sequencing of the fragment, isolated from two-dimensional gels, yielded a sequence with a strong homology to a subtelomeric sequence in chimpanzees and which was previously reported to be undetectable in humans. Fluorescence in situ hybridization indicated the occurrence of this sequence in normal tissue, for the most part in the satellite regions of acrocentric chromosomes. A product containing this sequence was obtained by telomere-anchored PCR using as a primer an oligonucleotide sequence from the cloned fragment. Our data suggest demethylation of cytosines at the cloned Not I site and in neighboring DNA in some tumors, compared with normal tissue, and suggest a greater similarity between human and chimpanzee subtelomeric sequences than was previously reported.

Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes.

We present evidence that the microtubule-associated protein tau is present in oligodendrocytes (OLGs), the central nervous system cells that make myelin. By showing that tau is distributed in a pattern similar to that of myelin basic protein, our results suggest a possible involvement of tau in some aspect of myelination. Tau protein has been identified in OLGs in situ and in vitro. In interfascicular OLGs, tau localization, revealed by monoclonal antibody Tau-5, was confined to the cell somata. However, in cultured ovine OLGs with an exuberant network of processes, tau was detected in cell somata, cellular processes, and membrane expansions at the tips of these processes. Moreover, in such cultures, tau appeared localized adjacent to or coincident with myelin basic protein in membrane expansions along and at the ends of the cellular processes. The presence of tau mRNA was documented using fluorescence in situ hybridization. The distribution of the tau mRNA was similar to that of the tau protein. Western blot analysis of cultured OLGs showed the presence of many tau isoforms. Together, these results demonstrate that tau is a genuine oligodendrocyte protein and pave the way for determining its functional role in these cells.

Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.

A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3.

Deletion of the short arm of human chromosome 1 is the most common cytogenetic abnormality observed in neuroblastoma. To characterize the region of consistent deletion, we performed loss of heterozygosity (LOH) studies on 122 neuroblastoma tumor samples with 30 distal chromosome 1p polymorphisms. LOH was detected in 32 of the 122 tumors (26%). A single region of LOH, marked distally by D1Z2 and proximally by D1S228, was detected in all tumors demonstrating loss. Also, cells from a patient with a constitutional deletion of 1p36, and from a neuroblastoma cell line with a small 1p36 deletion, were analyzed by fluorescence in situ hybridization. Cells from both sources had interstitial deletions of 1p36.2-36.3 which overlapped the consensus region of LOH defined by the tumors. Interstitial deletion in the constitutional case was confirmed by allelic loss studies using the panel of polymorphic markers. Four proposed candidate genes--DAN, ID3 (heir-1), CDC2L1 (p58), and TNFR2--were shown to lie outside of the consensus region of allelic loss, as defined by the above deletions. These results more precisely define the location of a neuroblastoma suppressor gene within 1p36.2-36.3, eliminating 33 centimorgans of proximal 1p36 from consideration. Furthermore, a consensus region of loss, which excludes the four leading candidate genes, was found in all tumors with 1p36 LOH.

Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

(Table T1) Total procaryotes, and living bacteria and archaea in black shales of ODP Leg 207 sites

Subseafloor sediments harbor over half of all prokaryotic cells on Earth (Whitman et al., 1998). This immense number is calculated from numerous microscopic acridine orange direct counts (AODCs) conducted on sediment cores drilled during the Ocean Drilling Program (ODP) (Parkes et al., 1994, doi:10.1038/371410a0, 2000, doi:10.1007/PL00010971). Because these counts cannot differentiate between living and inactive or even dead cells (Kepner and Pratt, 1994; Morita, 1997), the population size of living microorganisms has recently been enumerated for ODP Leg 201 sediment samples from the equatorial Pacific and the Peru margin using ribosomal ribonucleic acid targeting catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) (Schippers et al., 2005, doi:10.1038/nature03302). A large fraction of the subseafloor prokaryotes were alive, even in very old (16 Ma) and deep (>400 m) sediments. In this study, black shale samples from the Demerara Rise (Erbacher, Mosher, Malone, et al., 2004, doi:10.2973/odp.proc.ir.207.2004) were analyzed using AODC and CARD-FISH to find out if black shales also harbor microorganisms.

Centromere identification and inactivation on a neo-Y chromosome fusion in threespine stickleback fish (Gasterosteus aculeatus)

Thesis (Ph.D.)--University of Washington, 2016-05

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.

The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass

The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors. The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength. Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies. In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries. Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters. The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics. Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations. The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater. Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates. (C) 2003 Elsevier Ltd. All rights reserved.