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Rationale: Amplification of distal 3q is the most common genomic aberration in squamous lung cancer (SQC). SQC develops in a multistage progression from normal bronchial epithelium through dysplasia to invasive disease. Identifying the key driver events in the early pathogenesis of SQC will facilitate the search for predictive molecular biomarkers and the identification of novel molecular targets for chemoprevention and therapeutic strategies. For technical reasons, previous attempts to analyze 3q amplification in preinvasive lesions have focused on small numbers of predetermined candidate loci rather than an unbiased survey of copy-number variation.Objectives: To perform a detailed analysis of the 3q amplicon in bronchial dysplasia of different histological grades.Methods: We use molecular copy-number counting (MCC) to analyze the structure of chromosome 3 in 19 preinvasive bronchial biopsy specimens from 15 patients and sequential biopsy specimens from 3 individuals.Measurements and Main Results: We demonstrate that no low-grade lesions, but all high-grade lesions, have 3q amplification. None of seven low-grade lesions progressed clinically, whereas 8 of 10 patients with high-grade disease progressed to cancer. We identify a minimum commonly amplified region on chromosome 3 consisting of 17 genes, including 2 known oncogenes, SOX2 and PIK3CA. We confirm that both genes are amplified in all high-grade dysplastic lesions tested. We further demonstrate, in three individuals, that the clinical progression of high-grade preinvasive disease is associated with incremental amplification of SOX2, suggesting this promotes malignant progression.Conclusions: These findings demonstrate progressive 3q amplification in the evolution of preinvasive SQC and implicate SOX2 as a key target of this dynamic process. [ABSTRACT FROM AUTHOR]

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics., ((c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

We previously detected 368 expressed sequence tags showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo. In the present study 7 (presumed expressed sequence tags for HYPC, SPTBN1 and TNNC2, and four expressed sequence tags for unknown novel genes) out of the 368 expressed sequence tags were mapped to bovine chromosomes by analyzing deoxyribonucleic acids of bovine/murine somatic cell hybrid panel with polymerase chain reaction using primers specific for those bovine genes.

Many methods exist for genotyping--revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping--determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a 'tiling' approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.

Eight expressed sequence tags for unknown novel genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo were assigned to bovine chromosomes using deoxyribonucleic acids (DNAs) of bovine/murine somatic cell hybrid panel.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and approximately 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.

Chromosomal mapping of expressed sequence tags for HSPCB and MYL1 expressed abundantly in the bovine fetus was performed by analyzing bovine/murine somatic cell hybrid DNAs with polymerase chain reaction (PCR) using primers specific for those 3'-untranslated regions. HSPCB and MYL1 were assigned to bovine chromosomes 23 and 2, respectively.

Chromosomal mapping of the bovine calmodulin 1 and alpha-globin 1 genes was performed by analyzing bovine/murine somatic cell hybrid DNAs with PCR using primers specific for 3'-untranslated regions of those bovine genes. The calmodulin 1 and alpha-globin 1 genes were assigned to bovine chromosomes 25 and 29, respectively. Results from the present study should contribute to improvement in map resolution of bovine chromosomes and increase comparative information available on bovine chromosomes.

We have made a high-resolution HAPPY map of chromosome 6 of Dictyostelium discoideum consisting of 300 sequence-tagged sites with an average spacing of 14 kb along the approximately 4-Mb chromosome. The majority of the marker sequences were derived from randomly chosen clones from four different chromosome 6-enriched plasmid libraries or from subclones of YACs previously mapped to chromosome 6. The map appears to span the entire chromosome, although marker density is greater in some regions than in others and is lowest within the telomeric region. Our map largely supports previous gene-based maps of this chromosome but reveals a number of errors in the physical map. In addition, we find that a high proportion of the plasmid sequences derived from gel-enriched chromosome 6 (and that form the basis of a chromosome-specific sequencing project) originates from other chromosomes.

In order to assess the extent of DNA sequence variation in cattle, introns and exons from both the leptin and Amyloid Precursor Protein (APP) genes have been sequenced in a panel of DNAs derived from 22 diverse animals. Direct DNA sequencing of PCR products was used; thus, 44 chromosomes were studied. Polymorphisms were identified by manual scanning of sequence chromatograms and computerized sequence analysis. Twenty Single Nucleotide Polymorphisms (SNPs) were detected in 1788 bp sequenced from the leptin gene, giving a frequency of 1 SNP per 89 bp. Twenty-four SNPs were detected in a 458-bp fragment of the APP gene; 23 of the polymorphisms were contained in a 302-bp intron 16 fragment. This equates to an SNP frequency of 1 per 13 bp for the intron. We can thus conclude that this portion of the bovine APP gene constitutes a hypermutable region. Nucleotide sequence diversity values of 0.019 and 0.0026 were obtained for APP and leptin respectively.

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

A bovine/murine hybrid cell panel consisting of 57 cell lines was typed with 124 markers by PCR. Southern hybridisation and isozyme analysis in order to establish its utility as a resource for genome mapping. All bovine chromosomes, including the sex chromosomes were represented in the panel. Computerised analysis of syntenies indicated that there are no cell lines containing only a single bovine chromosome. The panel was used to map 10 new bovine microsatellite markers, and the MYL6 and CPE genes. This panel is informative for all bovine chromosomes other than the sex-specific region of the X chromosome and can be used in synteny mapping studies. At present, due to the relatively small number of markers typed, the resolution of the panel does not go beyond the chromosomal level.

In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.

To identify pig chromosomes in pig-mouse somatic cell hybrids, dual-laser flow karyotypes and GTG-banded metaphase spreads of pig, mouse, and 7 pig-mouse hybrid cell lines were compared. Pig chromosomes no. 1, 2, 5, 6, 10, 11, 13, 14, 16, 18, X and Y were tentatively assigned to individual peaks in the pig flow karyotype on the basis of DNA content vs. relative chromosome length. In the 7 hybrid cell lines, 7 out of 8 peaks distinct from those of the mouse cell line could be correlated with the presence of pig chromosomes no. 5, 9, 10, 11 or 16, 14, 15, and 18, whereas 1 peak appeared to correspond to the presence of 1 middle-size chromosome (3, 4, or 7). Other pig chromosomes present in the hybrids could not be detected with certainty due to superposition with mouse peaks and mouse chromosome rearrangements.

A panel of bovine-murine hybrid cell lines was analysed for 10 loci, including three (IGF1, IGHG2 and the calcium release channel gene [CRC]) that have previously been mapped in man, but not in cattle. The IGF and CRC genes were indirectly mapped to chromosomes 5 and 18 respectively and the syntenies of the HOX2 and GH genes and of the NP and FOS genes were confirmed. The results also show that the IGHG2 locus, which is linked to NP and FOS on human chromosome 14, is separated from these genes in cattle. By showing synteny of the IGHG2 and MPI loci, the IGHG2 locus has been indirectly mapped to chromosome 21.