A new approach to genome mapping and sequencing: slalom libraries.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.

NotI flanking sequences: a tool for gene discovery and verification of the human genome.

A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera's database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity > or =90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000-20 000 NotI sites, of which 6000-9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.

hUNC93B1: a novel human gene representing a new gene family and encoding an unc-93-like protein.

We have identified a novel human gene UNC93B1 encoding a protein related to unc-93 of Caenorhabditis elegans. The combined sequence derived from several cDNA clones is 2282 bp and comparison with genomic sequence shows that the gene contains 11 exons. The longest open reading frame encodes a deduced sequence of 597 amino acids. Homology analysis shows that the hUNC93B1 gene is highly conserved and related to sequences in Arabidopsis thaliana, C. elegans, Drosophila melanogaster, chicken and mouse. Structural analysis of the deduced amino acid sequence of hUNC93B1 points to possible existence of multiple membrane-spanning domains. hUNC93B1 protein also displays some similarities to the bacterial ABC-2 type transporter signature and to ion transporters of Deinococcus radiodurans and Helicobacter pylori. As revealed by Northern analysis, the level of expression varies significantly between tissues, with the highest level detected in the heart. The gene was mapped to chromosomal band 11q13 by fluorescence in situ hybridization. We suggest that this gene is a member of a novel hUNC93B-related gene family.

High mutability of the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) in cancer.

BACKGROUND: Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS: We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE: This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.

Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21.

BACKGROUND: We modified a tetracycline-regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet-system: the limited range of acceptor cell lines, expressing a tetracycline-controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA-expressing cell lines. METHOD: Two retroviral vectors pLNCtTA-hCMV and pLNCtTA-EF1alpha coding for the tTA were used to engineer cell lines to constitutively express tTA. New expression vectors pETE-Hyg and pETE-Bsd were also created that replicate in episomal form in human cells and facilitate tetracycline-regulated expression of targeted genes. RESULTS: The primate-tropic retroviruses efficiently delivered the regulatory tTA gene into 12 selected human cancer cell lines. Two candidate tumor suppressor genes from the human 3p21-p22 region MAPKAPK3 (3pK) and MLH1 were cloned into the episomal vector and transfected into engineered A9 and KRC/Y cells. The transfectants were subcutaneously grown in SCID mice, and the expression of the transgene was successfully controlled in vivo by tetracycline administered ad libitum in drinking water. The experiments demonstrated that both transgenes did not antagonize the tumorous growth of these cells. CONCLUSIONS: New retroviral and episomal vectors appear particularly suited for tight regulation of genes that cause suppression of cell growth. The generated cell lines can be used in various applications to study the effect of an inducible transgene in human cancer cells.