Identification of metastasis-associated genes by transcriptional profiling of metastatic versus non-metastatic colon cancer cell lines.

In order to define genes which mediate liver tropism of colon cancer metastasis we have compared the transcriptional profile of 5600 full-length genes using the Affymetrix HuGene FL array technology of the non-metastatic colon cancer cell lines KM12C and the two metastatic cell lines, KM12SM and KM12L4A, which are derived from KM12C. We present data on genes which are up- and downregulated in the metastatic cell line and those which are selectively upregulated in one of the metastatic cell lines. We have sub-grouped the deregulated genes into different categories, such as immune response, modulation of transcription, enzymes, cell cycle/apoptosis, interferon- and tumor necrosis factor-regulated genes, tumor antigens and transmembrane receptors, intracellular signaling, cytoskeleton and extracellular matrix associated proteins, 'others' and genes of unknown function.

Identification of metastasis-associated genes by transcriptional profiling of a metastasizing versus a non-metastasizing human melanoma cell line.

In order to identify genes associated with the metastatic phenotype we have compared the expression pattern of 6800 genes in a metastatic (NMCL-1) versus a non-metastatic (530) human melanoma cell line making use of DNA microarrays. The differentially expressed genes identified are involved in control of transcription, regulation of the cell-cycle, proteolysis, cell adhesion, immune response and signaling. A remarkable feature of the system under investigation is the consistent down-regulation of MHC-related and cell adhesion mediating genes in the metastatic cell line.

Identification of metastasis-associated genes by transcriptional profiling of a pair of metastatic versus non-metastatic human mammary carcinoma cell lines.

Cell lines 4A4 and 2C5 are the respective metastatic and non-metastatic variants of the human mammary carcinoma cell line MDA-MB-435 in the nude mouse system. We compared the transcriptional profile of approximately 5000 full-length genies using the Affymetrix HuGene FL Array technology. We have shown that the metastatic phenotype is mediated by different functional categories of genes, e.g. genes involved in immune response, genes responsible for tumor antigens, genes involved in migration and invasion, genes involved in mediating signal transduction, genes responsible for transcription factors, genes involved in phospholipid signaling, genes involved in modulation of extracellular matrix and cytoskeleton, genes with a cell-type specific mode of expression and genes which do not fit into the subclasses as defined above. Our results suggest an important role of Autocrine Motility Factor (AMF) as a mediator of metastasis in this system.

The transcriptional program of a human B cell line in response to Myc.

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Identification of breast cancer metastasis-associated genes by chip technology.

In order to identify genes associated with metastasis of mammary carcinoma, we compared the transcriptional profile (Affymetrix chip technology) of two cell lines derived from primary mammary carcinoma, three cell lines derived from bone marrow micrometastasis, a cell line derived from a lymph node metastasis as well as a cell line derived from malignant ascites. We found that 11 genes (0.16%) were up-regulated in all five cell lines derived from metastasis and 32 genes (0.45%) were up-regulated in four of these cell lines. Sixteen genes (0.23%) were down-regulated in the five metastatic cell lines, while 24 genes (0.34%) were down-regulated in four of the metastatic cell lines. The usefulness of our system for the identification of genes associated with metastasis of mammary carcinoma is demonstrated by the identification of genes which have already been implicated in metastasis of mammary carcinoma. This suggests that further evaluation of identified de-regulated genes, which until now have not been seen in context with metastasis of mammary carcinoma, should be undertaken.

Transcriptional profiling of cell lines derived from an orthotopic pancreatic tumor model reveals metastasis-associated genes.

In order to identify genes associated with metastasis of ductal pancreatic adenocarcinoma we investigated pancreatic tumor cell lines derived from an orthotopic pancreatic tumor model in SCID mice. Transcriptional profiling (Affymetrix Gene Chip Technology) was performed with cell lines derived from the primary tumor and metastatic lesions such as mesentery, liver and lungs. We scored for genes commonly deregulated in the cell lines derived from the metastatic lesions. Of 7070 genes investigated, 59 (0.83%) were found to be deregulated in the cell lines derived from the metastatic lesions. We grouped these genes into different categories such as transcription, translation, cytoskeleton, cell adhesion, chromosome instability, tumor suppressor genes, enzymes and "others". The most remarkable features of the system are the up-regulation of high mobility group protein HMG-I (Y), twenty-one ribosomal proteins, GAPDH and the laminin receptor in the cell lines derived from the metastatic lesions, whereas tumor suppressor genes such as maspin and RB1 were down-regulated. Inhibition or reconstitution of the activity of these targets are an emerging strategy for inhibition of metastasis in this system.

Alternative regulation principles for the production of recombinant proteins in Escherichia coli.

Established expression vectors exploiting regulated promoters such as the lac or tac promoters have economic and technical limitations when used for the industrial production of recombinant proteins. Consequently, alternative expression systems are being developed that can be more readily manipulated while maintaining high yields of protein. Several suitable expression vectors have been described for use in Escherichia coli that are based on promoters the activity of which is under metabolic control. This article discusses the advantages and disadvantages of a cross-section of these expression systems, how they compare with established systems and how they can be applied to the industrial-scale production of recombinant proteins.

Cloning and characterization of the MamI restriction-modification system from Microbacterium ammoniaphilum in Escherichia coli.

The genes encoding a class-IIN restriction-modification (R-M) system (MamI, sequence specificity [symbol: see text] from Microbacterium ammoniaphilum have been cloned in Escherichia coli. The vector used for cloning was plasmid pUC18 modified by the inclusion of three MamI recognition sites. Recombinant clones containing the mamIM gene in its genomic context became fully methylated in vivo and remained completely resistant against digestion with the R.MamI restriction endonuclease (ENase). Determination of the nucleotide (nt) sequence revealed three open reading frames with lengths of 1089 bp (ORF1), 276 bp (ORFc) and 927 bp (ORF2). On the basis of expression and deletion experiments, the 1089-bp ORF1 was assigned to mamIM encoding the M.MamI DNA methyltransferase (MTase). By amino acid sequencing of the N terminus of R.MamI and comparison of the deduced nt sequence with ORF2, the 927-bp ORF2 was identified as the mamIR gene encoding R.MamI. The 276-bp ORFc, located between mamIR and mamIM, is part of the DNA sequence downstream from mamIM shown to be necessary for controlled mamIM expression.

Mutation in dipZ leads to reduced production of active human placental alkaline phosphatase in Escherichia coli.

We have tested the expression of alkaline phosphatases in a mutant strain of Escherichia coli deficient in dipZ, a gene coding for a protein involved in cytochrome c biogenesis, and the isogenic wild-type strain. The yield of soluble and active human placental alkaline phosphatase was significantly reduced in the mutant but could be fully recovered by expression of dipZ subcloned in a vector with low copy number. Overexpression of E. coli alkaline phosphatase was unaffected in the mutant with or without dipZ co-expression.

A complex family of class-II restriction endonucleases, DsaI-VI, in Dactylococcopsis salina.

A series of class-II restriction endonucleases (ENases) was discovered in the halophilic, phototrophic, gas-vacuolated cyanobacterium Dactylococcopsis salina sp. nov. The six novel enzymes are characterized by the following recognition sequences and cut positions: 5'-C decreases CRYGG-3' (DsaI); 5'-GG decreases CC-3' (DsaII); 5'-R decreases GATCY-3' (DsaIII); 5'-G decreases GWCC-3' (DsaIV); 5'-decreases CCNGG-3' (DsaV); and 5'-GTMKAC-3' (DsaVI), where W = A or T, M = A or C, K = G or T, and N = A, G, C or T. In addition, traces of further possible activity were detected. DsaI has a novel sequence specificity and DsaV is an isoschizomer of ScrFI, but with a novel cut specificity. A purification procedure was established to separate all six ENases, resulting in their isolation free of contaminating nuclease activities. DsaI cleavage is influenced by N6-methyladenine residues [derived from the Escherichia coli-encoded DNA methyltransferase (MTase) M.Eco damI] within the overlapping sequence, 5'-CCRYMGGATC-3'; DsaV hydrolysis is inhibited by a C-5-methylcytosine residue in its recognition sequence (5'-CMCNGG-3'), generated in some DsaV sites by the E. coli-encoded MTase, M.Eco dcmI.

MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'.

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'.

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.