The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein.

The recurrent translocation t(8;16)(p11;p13) is a cytogenetic hallmark for the M4/M5 subtype of acute myeloid leukaemia. Here we identify the breakpoint-associated genes. Positional cloning on chromosome 16 implicates the CREB-binding protein (CBP), a transcriptional adaptor/coactivator protein. At the chromosome 8 breakpoint we identify a novel gene, MOZ, which encodes a 2,004-amino-acid protein characterized by two C4HC3 zinc fingers and a single C2HC zinc finger in conjunction with a putative acetyltransferase signature. In-frame MOZ-CBP fusion transcripts combine the MOZ finger motifs and putative acetyltransferase domain with a largely intact CBP. We suggest that MOZ may represent a chromatin-associated acetyltransferase, and raise the possibility that a dominant MOZ-CBP fusion protein could mediate leukaemogenesis via aberrant chromatin acetylation.

Lambda replacement vectors carrying polylinker sequences.

To simplify the construction and screening of genomic libraries, we have made a new family of lambda replacement vectors (EMBL1, EMBL2, EMBL3, EMBL4) and derivatives containing amber mutations (EMBL3 Sam, EMBL3 AamBam, EMBL3 AamSam). These vectors have a large capacity and polylinker sequences flanking the middle fragment. The polylinkers allow a choice of cloning enzymes and, especially useful in the case of cloning of Sau3A partial digests, the excision of the entire insert by flanking SalI (EMBL3) or EcoRI (EMBL4) sites. Phages with inserts can be selected either biochemically (particularly EMBL3) or genetically by their Spi- phenotype. Amber derivatives of the EMBL3 vector allow the application of genetic screening procedures based on selection for the products of homologous recombination events, and for the selective cloning of DNA sequences linked to supF genes.

A high-resolution radiation hybrid map of the region surrounding the Gorlin syndrome gene.

We have constructed a set of hybrid cell lines by irradiation of GM 64063 (human chromosome 9q only on hamster background) and fusion to hamster A23 Tk-, 109 independent lines were tested by PCR with 24 markers from chromosome 9q. The marker density is highest in the 9q22.3-q31 region containing the gene for Gorlin syndrome, a familial predisposition to basal cell carcinoma. The resolution of our map in this region is significantly higher than other published maps and will enable accurate placing of new markers and genes within the 9q22.3-q3.1 region. This is important since yeast artificial chromosomes from the region are likely to contain deletions.

Rapid restriction mapping of DNA cloned in lambda phage vectors.

A protocol for the rapid restriction mapping of phage lambda clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive lambda DNA termini by hybridisation with [32P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the "ladder" of partial digestion products.

A family of cosmid vectors with the multi-copy R6K replication origin.

A family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid R6K, a kanamycin resistance gene and two cos sites, permitting efficient library construction. Additional features of later constructs are (i) the presence of NotI sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated R6K ori leading to an increased copy number.

Cloning, mapping and expression of UBL3, a novel ubiquitin-like gene.

A novel Drosophila melanogaster gene UBL3 was characterized and shown to be highly conserved in man and Caenorhabditis elegans (C. elegans). The human and mouse homologues were cloned and sequenced. UBL3 is a ubiquitin-like protein of unknown function with no conserved homologues in yeast. Mapping of the human and mouse UBL3 genes places them within a region of shared gene order between human and mouse chromosomes on human chromosome 13q12-13 and telomeric mouse chromosome 5 (MMU5).

Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation.

Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling.

Cooperation between GLI and JUN enhances transcription of JUN and selected GLI target genes.

Sustained Hedgehog (HH) signaling is implicated in basal cell carcinoma of the skin and other types of cancer. Here we show that GLI1 and GLI2, the main transcriptional activators of the HH pathway, directly regulate expression of the activator protein 1 (AP-1) family member JUN, a transcription factor controlling keratinocyte proliferation and skin homeostasis. Activation of the JUN promoter by GLI is dependent on a GLI-binding site and the AP-1 sites known to be involved in self-activation of JUN. Transcription of JUN is greatly enhanced in the presence of GLI and requires activated JUN protein. GLI2act is a more potent activator than GLI1 in these experiments and physical interaction with phosphorylated JUN was only detected for GLI2act. The synergistic effect of GLI and JUN extends to the activation of further GLI target genes as shown by shRNA-mediated knockdown of JUN in human keratinocytes. Some of these cooperatively activated genes are involved in cell-cycle progression, which is consistent with a significant reduction of the proliferative potential of GLI in the absence of JUN. These results suggest a novel connection between HH/GLI pathway activity and JUN, which may contribute to the oncogenic activity of HH/GLI signaling in skin.

Cloning and mapping of a human and mouse gene with homology to ecto-ATPase genes.

The human CD39-like-1 gene (CD39L1) was isolated from a selected cDNA library enriched for transcripts from regions of human Chromosome (Chr) 9q. Database searches with sequences of one group of clones from the selected cDNA library showed strong amino acid homology to the lymphoid cell activation antigen CD39, an ecto-apyrase gene from human and mouse. The full-length sequence for CD39L1 identified a putative 472 amino acid protein with greater than 60% identity with the chicken muscle ecto-ATPase protein, as well as homology to a number of other known ecto-ATPases and ecto-apyrases from rat, garden pea, yeast, and Toxoplasma gondii. A high level of amino acid identity suggests that CD39L1 is closely related to the chicken muscle ecto-ATPase. The presence of the human ABC2 gene on an overlapping cosmid and hybridizations to somatic cell mapping panels suggest that CD39L1 maps to human Chr 9q34. A mouse homolog was isolated (showing greater than 78% nucleotide sequence identity) and mapped by FISH to mouse Chr 2, the syntenic region of human 9q34. The genomic structure of CD39L1 reveals 9 exons covering less than 7 kb.

The CD39-like gene family: identification of three new human members (CD39L2, CD39L3, and CD39L4), their murine homologues, and a member of the gene family from Drosophila melanogaster.

The human lymphoid cell activation antigen CD39 is a known E-type apyrase that hydrolyzes extracellular ATP and ADP, a function important in homotypic adhesion, platelet aggregation, and removal by activated lymphocytes of the lytic effect of ATP. The recently identified putative rat homologue of CD39L1 has been shown to have E-type ecto-ATPase activity, by hydrolyzing extracellular ATP. We have characterized three novel CD39-like transcripts, CD39L2, CD39L3, and CD39L4, which share extensive amino acid homology with other nucleotide triphosphatases in vertebrates, invertebrates, and plants, suggesting that these genes also encode proteins with ecto-nucleotidase activity. Isolation and sequencing of full-length cDNA clones for each gene identified putative proteins of 485, 529, and 429 amino acids. The expression pattern of all five human members of the gene family was analyzed. CD39L2, CD39L3, and CD39L4 were mapped on the human genome, and the murine homologues identified with the putative map locations were assigned on the basis of regions of conserved gene order between human and mouse chromosomes. The map location of mcd39l4 places the gene within a region associated with audiogenic seizure susceptibility in mouse. This disorder is characterized by convulsions induced by loud high-frequency sound and has been shown to be associated with increased nucleotide triphosphatase activity.

Molecular tools for the mapping of the human genome.

The rapid progress in molecular cloning and DNA analysis techniques, together with the use of cloned DNA probes from specific chromosomes of the human genome might allow localisation and ultimately identification of genes defined by single mutations. The discrepancy between genetic dimensions expressed in centimorgans, each corresponding to millions of base pairs and distances easily accessible by molecular techniques amounting to at the most hundreds of kilobase pairs may be bridged with some new cloning techniques partially developed at the European Molecular Biology Laboratory. These techniques were designed to allow rapid cloning and analysis of large regions of mammalian genomes.

Procollagen complementary DNA, a probe for messenger RNA purification and the number of type I collagen genes.

Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room temperature. All of 27S rRNA and four-fifths of procollagen mRNAs bind to Sepharose under these conditions, while 18S rRNA and about three-fourths of other poly(A)-containing RNAs do not bind. AMV reverse transcriptase was used to prepare complementary DNA to procollagen mRNA at each purification step. Hybridization studies, in RNA excess, were carried out to establish the enrichment at each step both with respect to total RNA and to poly(A)-containing RNA. While "purified" procollagen mRNA preparations still consist of about 50% 27S rRNA, over 80% of cDNA prepared from it back hybridizes to its template at a log of cr0t1/2 of -1.9. This type I procollagen cDNA hybridizes in DNA excess to DNA isolated from chicken erythrocytes and from embryonic chick calvaria at a log c0t1/2 of 3.1, demonstrating that procollagen cDNA is complementary to unique gene sequences in both tissues and that procollagen genes are not reiterated.

FKHL15, a new human member of the forkhead gene family located on chromosome 9q22.

FKHL15 was isolated from a cDNA library enriched for transcripts from 9q22. Isolation and sequencing of a 3.5-kb cDNA clone identified a putative 376-amino-acid protein with greater than 80% homology over a 100-amino-acid stretch to the forkhead DNA-binding domain. The FKHL15 gene contains a region rich in alanine residues, frequently associated with transcriptional repression. The forkhead genes are believed to play important roles in development and differentiation in many different organisms and have also been implicated in the development of some tumors. The map position of FKHL15 on 9q22 places the gene within the candidate regions for the cancer predisposition syndrome multiple self-healing squamous epitheliomata and the degenerative neurological disorder hereditary sensory neuropathy type I. This is a region frequently lost in squamous cell cancer.

The mouse homolog of PKD1: sequence analysis and alternative splicing.

We have cloned and sequenced the mouse transcript homologous to human polycystic kidney disease 1 (PKD1). The predicted protein is 79% identical to human PKD1 and shows the presence of most of the domains identified in the human sequence. Since the mouse homolog is transcribed from a unique gene and there are no transcribed, closely related copies as has been observed for human PKD1, we have been able to investigate alternative splicing of the transcript. At the junction of exons 12 and 13, several different splicing variants lead to a predicted protein that would be secreted. These forms are predominantly found in newborn brain, while in kidney the transcript homologous to the previously described human RNA predominates.