Loss of heterozygosity in tumor cells requires re-evaluation: the data are biased by the size-dependent differential sensitivity of allele detection

Normal tissue contamination of tumors may eclipse the detection of loss of heterozygosity (LOH) by microsatellite analysis and may also hamper isolation of tumor suppressor genes. To test the potential impact of this problem, we prepared artificial mixtures of mouse-human microcell hybrid lines that carried different alleles of the same chromosome 3 marker. After performing an allele titration assay, we found a consistent difference between the LOH of a high molecular weight (H) allele and the LOH of a low molecular weight (L) allele of the same CA repeat marker. It follows that normal tissue admixtures will be less of a problem when LOH affects a H allele than with a L allele. Random screening of 100 papers published between 1994 and 1999 revealed that the loss of a L allele was recorded at about half the frequency (52%) of loss of a H allele. To avoid this bias, we have developed rules for the evaluation of LOH data. We suggest that the loss of a L allele should be given more weight than the loss of a H allele in LOH studies using microsatellite markers.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

NotI passporting to identify species composition of complex microbial systems

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.     

Mapping the amino acid properties of constituent nucleoporins onto the yeast nuclear pore complex

Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules. Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the authors.     

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 ~46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of ~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.     

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.     

Cloning of polymorphisms (COP): enrichment of polymorphic sequences from complex genomes

Here we describe a new procedure (cloning of polymorphisms, COP) for enrichment of single nucleotide polymorphisms (SNPs) that represent restriction fragment length polymorphisms (RFLPs). COP would be applicable to the isolation of SNPs from particular regions of the genome, e.g. CpG islands, chromosomal bands, YACs or PAC contigs. A combination of digestion with restriction enzymes, treatment with uracil-DNA glycosylase and mung bean nuclease, PCR amplification and purification with streptavidin magnetic beads was used to isolate polymorphic sequences from the genomes of two human samples. After only two cycles of enrichment, 80% of the isolated clones were found to contain RFLPs. A simple method for the PCR detection of these polymorphisms was also developed.     

NotI clones in the analysis of the human genome

NotI linking clones contain sequences flanking NotI recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of NotI clones in genome research, high density grids with 50 000 NotI linking clones originating from six representative NotI linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of NotI sites in the human genome. A total of 3437 sequences flanking NotI sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity ≤ 90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of NotI sites with the first exon and suggest that the so-called 3′ ESTs can actually be generated from 5′-ends of genes that contain NotI sites in their first exon.