DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.

Loss of sequences from human chromosome 10q has been associated with the progression of human cancer. Medulloblastoma and glioblastoma multiforme are the most common malignant brain tumours in children and adults, respectively. In glioblastoma multiforme, the most aggressive form, 80% of the tumours show loss of 10q. We have used representational difference analysis to identify a homozygous deletion at 10q25.3-26.1 in a medulloblastoma cell line and have cloned a novel gene, DMBT1, spanning this deletion. DMBT1 shows homology to the scavenger receptor cysteine-rich (SRCR) superfamily. Intragenic homozygous deletions has been detected in 2/20 medulloblastomas and in 9/39 glioblastomas multiformes. Lack of DMBT1 expression has been demonstrated in 4/5 brain-tumour cell lines. We suggest that DMBT1 is a putative tumour-suppressor gene implicated in the carcinogenesis of medulloblastoma and glibolastoma multiforme.

X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions.

X-linked recessive dyskeratosis congenita (DKC) is a rare bone-marrow failure disorder linked to Xq28. Hybridization screening with 28 candidate cDNAs resulted in the detection of a 3' deletion in one DKC patient with a cDNA probe (derived from XAP101). Five different missense mutations in five unrelated patients were subsequently identified in XAP101, indicating that it is the gene responsible for X-linked DKC (DKC1). DKC1 is highly conserved across species barriers and is the orthologue of rat NAP57 and Saccharomyces cerevisiae CBF5. The peptide dyskerin contains two TruB pseudouridine (psi) synthase motifs, multiple phosphorylation sites, and a carboxy-terminal lysine-rich repeat domain. By analogy to the function of the known dyskerin orthologues, involvement in the cell cycle and nucleolar function is predicted for the protein.

DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer.

The gene deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain, gastrointestinal, and lung cancer. It codes for a protein of unknown function belonging to the superfamily of scavenger receptor cysteine-rich proteins. We aimed at getting insights into the functions of DMBT1 by expression analyses and studies with a monoclonal antibody against the protein. The DMBT1 mRNA is expressed throughout the immune system, and Western blot studies demonstrated that isoforms of DMBT1 are identical to the collectin-binding protein gp-340, a glycoprotein that is involved in the respiratory immune defense. Immunohistochemical analyses revealed that DMBT1 is produced by both tumor-associated macrophages and tumor cells and that it is deregulated in glioblastoma multiforme in comparison to normal brain tissue. Our data further suggest that the proteins CRP-ductin and hensin, both of which have been implicated in epithelial differentiation, are the DMBT1 orthologs in mice and rabbits, respectively. These findings and the spatial and temporal distribution of DMBT1 in fetal and adult epithelia suggest that DMBT1 further plays a role in epithelial development. Rearrangements of DMBT1 were found in 16 of 18 tumor cell lines, and hemizygous deletions were observed in a subset of normal individuals, indicating that the alterations in tumors may be a result of both pre-existing deletions uncovered by a loss of heterozygosity and secondary changes acquired during tumorigenesis. Thus, DMBT1 is a gene that is highly unstable in cancer and encodes for a protein with at least two different functions, one in the immune defense and a second one in epithelial differentiation.

Deleted in Malignant Brain Tumors 1 is a versatile mucin-like molecule likely to play a differential role in digestive tract cancer.

Deleted in Malignant Brain Tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor gene for brain, lung, and digestive tract cancer. In particular, alterations of the gene and/or a loss of expression have been observed in gastric, colorectal, and esophageal carcinomas. Initial evidence has accumulated that DMBT1 may represent a multifunctional protein. Because the consequences of a loss of DMBT1 function may be different depending on its original function in a particular tissue, we wondered if it is appropriate to assume a uniform role for DMBT1 in digestive tract carcinomas. We hypothesized that a systematic characterization of DMBT1 in the human alimentary tract would be useful to improve the understanding of this molecule and its role in digestive tract carcinomas. Our data indicate that the expression pattern and subcellular distribution of DMBT1 in the human alimentary tract is reminiscent of epithelial mucins. Bovine gallbladder mucin is identified as the DMBT1 homologue in cattle. An elaborate alternative splicing may generate a great variety of DMBT1 isoforms. Monolayered epithelia display transcripts of 6 kb and larger, and generally show a lumenal secretion of DMBT1 indicating a role in mucosal protection. The esophagus is the only tissue displaying an additional smaller transcript of approximately 5 kb. The stratified squamous epithelium of the esophagus is the only epithelium showing a constitutive targeting of DMBT1 to the extracellular matrix (ECM) suggestive of a role in epithelial differentiation. Squamous cell carcinomas of the esophagus show an early loss of DMBT1 expression. In contrast, adenocarcinomas of the esophagus commonly maintain higher DMBT1 expression levels. However, presumably subsequent to a transition from the lumenal secretion to a targeting to the ECM, a loss of DMBT1 expression also takes place in adenocarcinomas. Regarding DMBT1 as a mucin-like molecule is a new perspective that is instructive for its functions and its role in cancer. We conclude that DMBT1 is likely to play a differential role in the genesis of digestive tract carcinomas. However, although DMBT1 originally has divergent functions in monolayered and multilayered epithelia, carcinogenesis possibly converges in a common pathway that requires an inactivation of its functions in the ECM.

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability.

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.

Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts.

A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP-dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis.

Promoter of the gene encoding the bovine catalytic subunit of cAMP-dependent protein kinase isoform C beta 2.

Genomic sequences flanking the 5' end of the cDNA encoding isoform C beta 2 of the catalytic subunit of bovine cAMP-dependent protein kinase were cloned, sequenced and analyzed for promoter activity and transcription initiation sites. A region of 913 bp upstream the translation initiator ATG was amplified from genomic DNA by vectorette polymerase chain reaction. In primer extension reactions and RNase protection assays, residues C (at position -91), T (-71) and G (-70) were found to serve as transcription initiation sites of the gene. Amplification products and sub-fragments thereof were ligated upstream of the reporter gene chloramphenicol acetyltransferase to test for promoter activity. Constructs were transiently transfected into a Chinese hamster ovary cell line which was shown to express endogenous C beta 2 mRNA. The genomic sequence upstream the C beta 2 cDNA does have promoter activity. The region from position -51 to -292 proved sufficient to drive efficient transcription of the reporter gene. The promoter is AT rich (68%), does not contain a TATA box within 50 bp upstream of the first initiation site and possesses putative binding sites for several transcription factors such as PEA-3 and a glucocorticoid receptor.

Epitopes fused to F-pilin are incorporated into functional recombinant pili.

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

MicroRNA-206 functions as a pleiotropic modulator of cell proliferation, invasion and lymphangiogenesis in pancreatic adenocarcinoma by targeting ANXA2 and KRAS genes.

Recent advances in cancer biology have emerged important roles for microRNAs (miRNAs) in regulating tumor responses. However, their function in mediating intercellular communication within the tumor microenvironment is thus far poorly explored. Here, we found miR-206 to be abrogated in human pancreatic ductal adenocarcinoma (PDAC) specimens and cell lines. We show that miR-206 directly targets the oncogenes KRAS and annexin a2 (ANXA2), thereby acting as tumor suppressor in PDAC cells by blocking cell cycle progression, cell proliferation, migration and invasion. Importantly, we identified miR-206 as a negative regulator of oncogenic KRAS-induced nuclear factor-κB transcriptional activity, resulting in a concomitant reduction of the expression and secretion of pro-angiogenic and pro-inflammatory factors including the cytokine interleukin-8, the chemokines (C-X-C motif) ligand 1 and (C-C motif) ligand 2, and the granulocyte macrophage colony-stimulating factor. We further show that miR-206 abrogates the expression and secretion of the potent pro-lymphangiogenic factor vascular endothelial growth factor C in pancreatic cancer cells through an NF-κB-independent mechanism. By using in vitro and in vivo approaches, we reveal that re-expression of miR-206 in PDAC cells is sufficient to inhibit tumor blood and lymphatic vessel formation, thus leading to a significant delay of tumor growth and progression. Taken together, our study sheds light onto the role of miR-206 as a pleiotropic modulator of different hallmarks of cancer, and as such raising the intriguing possibility that miR-206 may be an attractive candidate for miRNA-based anticancer therapies.

Isolation of human and mouse HMG2a cDNAs: evidence for an HMG2a-specific 3' untranslated region.

We have isolated cDNAs of the human gene for high mobility group protein HMG2a, using the method of direct cDNA selection. The gene maps to chromosome band Xq28, and is located within 40 kb from marker DXS1684, at a distance of 5.4 Mb from the telomere. The deduced human HMG2a protein sequence has a length of 199 amino acids and is 97% identical to the sequence of chicken HMG2a. The 3' untranslated regions of the HMG2a gene in both species are highly homologous (87% identical nucleotides), and are even more conserved than the coding sequences (84% identical nucleotides). In addition, a partial cDNA sequence of the putative HMG2a gene from mouse was identified. The 3' untranslated regions from human and mouse are 90% identical. We conclude that the 3' untranslated sequences have been under strong selective pressure during evolution. Whereas expression of the chicken HMG2a gene has previously been demonstrated in liver of newly hatched chicken, the human HMG2a gene is transcribed mainly in placenta.

Construction of a physical map of an autism susceptibility region in 7q32.3-q33.

The fast evolving progress of the human genome mapping and sequencing efforts facilitate the detection of genes also for complex traits. We focus on the detection of susceptibility loci for autism, a prototypical pervasive developmental disorder. Five genome screens worldwide have identified several putative locations of susceptibility genes thus far, with the most common region on chromosome 7q. In order to identify new candidate genes for infantile autism we constructed a physical map of bacterial artificial chromosome, P1-derived artificial chromosome and yeast artificial chromosome clones of a 3 Mb region between D7S1575 and D7S500, including a complete contig of the approximately 1.2 Mb region around D7S2533, the marker with the most significant association result. We developed 16 novel sequence tag sites and mapped 23 genes/expressed sequence tags to the contigs. As this map contains a putative autistic disorder locus this integrated physical and transcript map provides a valuable resource for identification of candidate gene(s).

DelGEF, an RCC1-related protein encoded by a gene on chromosome 11p14 critical for two forms of hereditary deafness.

We have cloned a human cDNA, DELGEF (deafness locus associated putative guanine nucleotide exchange factor), derived from a 225 kb genomic sequence of chromosome 11p14, critical for the Usher 1C syndrome and for DFNB18, a locus for non-syndromic sensorineural deafness. The amino acid sequence of the protein hDelGEF1 is homologous to the nucleotide exchange factor RCCI for the small GTPase Ran. hDelGEF2 is derived from the same DELGEF gene by alternative splicing. In addition, we have identified a murine homologue, mDelGEF. The ubiquitously expressed soluble protein hDelGEF1 is found both in the cyytoplasm and in the nucleus. Overexpressed hDelGEF2 colocalizes with mitochondria.

Improved fluorescent cycle sequencing protocol allows reading nearly 1000 bases.

Recently available thermostable DNA polymerases result in enhanced resolution and accuracy compared to thermal enzymes used previously in fluorescent cycle sequencing. These new enzymes produce less variations in peak intensities, enhance gel resolution and are less sensitive to unspecific termination caused by either DNA structure or impurities in the DNA preparation. Optimization of nucleotide ratios and the usage of high concentrations of detergents in the sequencing reaction result in sequence readings up to 1000 bases and improve overall reliability of the sequencing protocol; this works successfully in about 90% of cases.

Primer design for automated DNA sequencing utilizing T7 DNA polymerase and internal labeling with fluorescein-15-dATP.

We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.

Automated cycle sequencing with Taquenase: protocols for internal labeling, dye primer and "doublex" simultaneous sequencing.

This paper describes automated cycle sequencing protocols for internal labeling, dye primer and "doublex" simultaneous sequencing using Taquenase, a new genetically modified DNA polymerase with increased thermostability. Sequencing performance both with labeled and unlabeled primer yields uniform unambiguous signals up to the resolution limit of the sequencing gels. Primer walking with internal labeling was successfully performed on Pl-derived artificial chromosome (PAC) constructs with 130-kb inserts. Taquenase, a commercially available modified thermostable sequencing enzyme (delta 280, F667Y Taq DNA polymerase), incorporates a variety of fluorescent dNTPs carrying fluorescein isothiocyanate, TexasRed or Cy5 labels during the cycle-sequencing process with higher efficiency than other thermostable DNA polymerases. Comparison to other modified Taq DNA polymerases suggests that the particular N-terminal deletion of Taquenase rather than the presence of the F667Y mutation is responsible for the efficient incorporation and extension of labeled dNTPs. Taquenase makes feasible highly accurate "doublex" simultaneous cylce sequencing on both strands of template DNA with two internal labels or two dye-labeled primers in combination with the EMBL-2-dye DNA sequencing system, ARAKIS, or with two commercial DNA sequencers. It allows up to 2000 bases at > 99% accuracy to be determined in a single reaction.

Automated low-redundancy large-scale DNA sequencing by primer walking.

Low-redundancy automated DNA sequencing by primer walking is described. T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 bases (in the unedited raw data). The low error rate allows efficient sequencing with low (2-3 times) redundancy. Plasmid subclones covering 20 kb of a cosmid insert were sequenced with an overall redundancy of 2.7 in the course of the European community Saccharomyces cerevisiae genome sequencing project. Neighboring plasmid subclones were linked by direct cosmid sequencing. Sets of ten walking primers are synthesized on the EMBL multiple segmental DNA synthesizer at low costs and used for sequencing with greater than 95% efficiency. The accuracy of the directed approach is improved by simultaneous walking on both strands by designing two primers in opposite directions in the same starting region. One primer is used to confirm sequence data on the opposite strand, and the other primer to obtain new sequence data.

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer.

Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis. It has been shown to occur in resistance to a range of cancer therapies, including tamoxifen. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. To investigate the role of miRNAs in the development of resistance to tamoxifen as well as accompanying EMT-like properties, we established a tamoxifen-resistant (TamR) model by continually exposing MCF-7 breast cancer cells to tamoxifen. In addition to the molecular changes known to be involved in acquired tamoxifen resistance, TamR cells displayed mesenchymal features and had increased invasiveness. Genome-wide miRNA microarray analysis revealed that miRNA-375 was among the top downregulated miRNAs in resistant cells. Re-expression of miR-375 was sufficient to sensitize TamR cells to tamoxifen and partly reversed EMT. A combination of mRNA profiling, bioinformatics analysis and experimental validation identified metadherin (MTDH) as a direct target of miR-375. Knockdown of MTDH partially phenocopied the effects of miR-375 on the sensitivity to tamoxifen and the reversal of EMT. We observed an inverse correlation between the expression of miR-375 and its target MTDH in primary breast cancer samples, implying the pathological relevance of targeting. Finally, tamoxifen-treated patients with higher expression of MTDH had a shorter disease-free survival and higher risk of relapse. As most cancer-related deaths occur because of resistance to standard therapies and metastasis, re-expression of miR-375 or targeting MTDH might serve as potential therapeutic approaches for the treatment of TamR breast cancer.