CBP/p300 as a co-factor for the Microphthalmia transcription factor.

The Microphthalmia basic-Helix-Loop-Helix-Leucine Zipper (bHLH-LZ) transcription factor (Mi) plays a crucial role in the genesis of melanocytes; mice deficient for a functional (Microphthalmia) gene product lack all pigment cells. We show here that the Mi activation domain resides N-terminal to the DNA-binding domain and that as little as 18 amino acids are sufficient to mediate transcription activation. The minimal activation region of Mi is highly conserved in the related transcription factor TFE3 and is predicted to adopt an amphipathic alpha-helical conformation.

A novel candidate tumour suppressor locus at 9q32-33 in bladder cancer: localization of the candidate region within a single 840 kb YAC.

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, implicating the presence of a tumour suppressor gene or genes on 9q. To define the location of a tumour suppressor locus on 9q in TCC, we screened 156 TCCs of the bladder and upper urinary tract by detailed deletion mapping using 31 microsatellite markers on 9q. Partial deletions of 9q were found in 10 TCCs (6%), and LOH at all informative loci on 9q was found in 77 TCCs (49%).

A single serine residue at position 375 of VP16 is critical for complex assembly with Oct-1 and HCF and is a target of phosphorylation by casein kinase II.

We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines towards the C-terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C-terminal boundary for complex formation with Oct-1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375.

Molecular and cellular characterization of CRP1, a Drosophila chromatin decondensation protein.

CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins revealed strong homologies to Xenopus nucleoplasmin and NO38. CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3.

Mutation analysis of 8p genes POLB and PPP2CB in bladder cancer.

The DNA polymerase beta gene (POLB), which encodes a DNA polymerase believed to be involved in short gap-filling DNA synthesis, has been mapped to the proximal region of 8p (8p12-p11), a region commonly deleted in bladder carcinoma and a wide variety of other neoplasms. Also mapped to this region (8p12-p11.2) is the gene encoding the beta isoform of the catalytic subunit of protein phosphatase 2A (PPP2CB), a major serine/threonine phosphatase thought to play a regulatory role in many cellular pathways.

Intercellular trafficking and protein delivery by a herpesvirus structural protein.

We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism.

Phosphorylation of the herpes simplex virus type 1 tegument protein VP22.

The herpes simplex virus type 1 tegument protein VP22 is known to be highly phosphorylated during infection. Here we show that two electrophoretic forms of VP22 can be identified in infected cell extracts and that this heterogeneity is accounted for by phosphorylation. Furthermore, the nonphosphorylated form of VP22 appears to be specifically incorporated into virions.

Fluorescence in situ hybridization deletion mapping at 4p16.3 in bladder cancer cell lines refines the localisation of the critical interval to 30 kb.

An allelotype analysis of transitional cell carcinoma of the bladder identified loss of heterozygosity (LOH) on chromosome arm 4p in 22% of tumours. In a more detailed LOH study of 178 bladder carcinomas, a 750 kb common region of deletion was identified between the markers D4S43 and D4S127 just telomeric to the Huntington disease locus. To refine this region of deletion at 4p16.

Weak and strong states of kinesin and ncd.

Kinesin superfamily molecular motors step along microtubules (MTs) via a cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (K(kin)d approximately 0.

Deletion mapping implicates two tumor suppressor genes on chromosome 8p in the development of bladder cancer.

Chromosome 8 loss of heterozygosity (LOH) in cancer of the urinary bladder is associated with high tumour grade and stage. We have screened 193 cases of transitional cell carcinoma (TCC) of the bladder using 30 microsatellite polymorphisms on chromosome 8. Forty three cases (22%) showed LOH on 8p, the majority of which (72%) had lost the entire short arm.

Kinetics and motility of the Eg5 microtubule motor.

We have investigated the kinetic properties of the slow plus end directed microtubule (MT) motor Eg5. The recombinantly expressed fusion protein E437GST, containing residues 12-437 of Eg5 fused to the N-terminus of glutathione S-transferase (GST), is dimeric and motile, translocating MTs at an average speed of 0.063 (+/-0.

Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA-binding proteins.

We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A. This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction. The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity.

VP16 interacts via its activation domain with VP22, a tegument protein of herpes simplex virus, and is relocated to a novel macromolecular assembly in coexpressing cells.

In addition to its function as a powerful transactivator of viral immediate-early transcription, VP16 is an essential component of the herpes simplex virus (HSV) virion. As such, VP16 is introduced into cells, to effect its function in transactivation, as part of the virus tegument. Here we examine the potential for VP16 protein-protein interactions specific to virus-infected cells and show that VP16 copurifies in a highly enriched fraction with a single major polypeptide which we identify as the virus-encoded structural protein VP22.

The POU domain transcription factor Brn-2: elevated expression in malignant melanoma and regulation of melanocyte-specific gene expression.

Previous work has shown that melanoma cell lines express a distinct octamer binding protein. Given the role of octamer-binding proteins in cell differentiation and development, the role this factor is a key issue in understanding melanocyte differentiation and transformation. Using a proteolytic clipping assay, we show that the melanoma-specific octamer factor is Brn-2/N-Oct3, a POU domain protein previously known to be expressed in adult brain and in the developing nervous system.

Purification and characterisation of a DNA helicase, dheI I, from Drosophila melanogaster embryos.

We have purified a DNA helicase (dhel l) from early Drosophila embryos. dhel l co-purifies with the single-stranded DNA binding protein dRP-A over two purification steps, however, the proteins can be separated by their different native molecular weight, with dhel l activity co-sedimenting with a polypeptide of approximately 200 kDa and a sedimentation coefficient of 8.6 S.

pWITCH: a versatile two-hybrid assay vector for the production of epitope/activation domain-tagged proteins both in vitro and in yeast.

We describe the construction of a new vector, pWITCH, designed to facilitate the characterisation of proteins encoded by novel cDNAs isolated using either a one- or two-hybrid assay. Expression of directionally cloned cDNAs is directed in vivo in Saccharomyces cerevisiae from the inducible GAL10 promoter and in vitro from the T7 promoter, while translation of the expressed cDNAs results in proteins which are tagged in vitro with a specific epitope and in vivo with both the epitope and the VP16 transcription activation domain. The principle of using multiple promoters each able to operate under different conditions to express different combinations of protein domains without the need for subcloning should be generally applicable.

Equine herpesvirus 1 gene 12, the functional homologue of herpes simplex virus VP16, transactivates via octamer sequences in the equine herpesvirus IE gene promoter.

The HSV-1 transactivator of immediate-early (IE) gene expression, VP16, has several functional homologues among the alphaherpesviruses which have not yet been extensively studied in relation to their modes of action. To date, nothing is known of the exact sites or mechanism of interaction of the equine herpesvirus type 1 (EHV-1) homologue, the gene 12 protein, with the EHV-1 IE promoter. We show that the gene 12 protein utilises the promoter proximal region of the IE gene to induce activation and identify four potential octamer DNA binding sites within that region.

Detailed deletion mapping of chromosome 9q in bladder cancer: evidence for two tumour suppressor loci.

Loss of heterozygosity (LOH) at loci on chromosome 9p and/or 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder. However, localisation of the tumour suppressor locus or loci on 9q has been hampered by the relative infrequency of tumours with subchromosomal deletions. We have used 24 microsatellite markers to examine LOH in 70 new cases of TCC of the bladder and upper urinary tract.

Mitochondrial single-stranded DNA-binding protein from Drosophila embryos. Physical and biochemical characterization.

Using a stringent purification procedure on single-stranded DNA cellulose, we have isolated the mitochondrial single-stranded DNA-binding protein from Drosophila melanogaster embryos. Its identity is demonstrated by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial protein is immunologically and biochemically distinct from the previously characterized nuclear replication protein A from Drosophila (Mitsis, P.

p16 (CDKN2) is a major deletion target at 9p21 in bladder cancer.

The p16 gene has been identified as a candidate tumour suppressor gene at 9p21, a region commonly deleted in bladder cancer. We screened 140 bladder tumours and 16 cell lines for deletions and sequence variants of p16. Eight cell lines showed homozygous deletion of p16 and two had small sequence variations.

ADP release is the rate-limiting step of the MT activated ATPase of non-claret disjunctional and kinesin.

The motor protein non-claret disjunctional (ncd) moves towards the minus ends of microtubules (MTs), whereas its close relative kinesin moves in the opposite direction towards the plus ends of MTs. The mechanisms of movement and directional reversal for these motor proteins are unknown. Here we report the rate constants for MT activated ADP release from a recombinant double-headed ncd protein, GST-MC5, and a recombinant double-headed kinesin protein, K delta 401, measured using the fluorescent nucleotide analogues methylanthranilyol ATP (mantATP) and mantADP.

Kinesin and ncd bind through a single head to microtubules and compete for a shared MT binding site.

Kinesin and non claret disjunctional are closely related molecular motors that move in opposite directions along microtubules. We have used recombinant single-headed and double-headed constructs of both rat kinesin heavy chain and non claret disjunctional to investigate the interactions of these motor proteins with microtubules. At saturation the stoichiometry of binding for non claret disjunctional and kinesin to microtubules is one molecule (single or double-headed) per tubulin heterodimer.

Deletion mapping of chromosome 11 in carcinoma of the bladder.

Deletions of the short arm of chromosome 11 have been identified by both cytogenetic and molecular criteria in bladder and other types of solid tumor, indicating the presence of one or more suppressor loci in this region. To localize the 11p deletion target(s) more precisely and to screen for loss of heterozygosity (LOH) on the long arm of the chromosome, 100 bladder tumors were analyzed for LOH on chromosome 11 using restriction fragment length polymorphisms (RFLPs) and microsatellite markers mapped to both 11p and 11q. Thirty-four tumors were found to have LOH at 1 or more loci.