The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

B and T lymphocytes are the mediators of immunity, but their function is under the control of dendritic cells. Dendritic cells in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. They not only activate lymphocytes, they also tolerize T cells to antigens that are innate to the body (self-antigens), thereby minimizing autoimmune reactions. Once a neglected cell type, dendritic cells can now be readily obtained in sufficient quantities to allow molecular and cell biological analysis. With knowledge comes the realization that these cells are a powerful tool for manipulating the immune system.

Dendritic cells and the control of immunity

We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other

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Mapping and quantifying mammalian transcriptomes by RNA-Seq.

BACKGROUND Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

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Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib

Methods of using a transposon as an insertional mutagen are provided. Also provided is a transgenic animal that includes polynucleotides encoding a transposon and transposase that can be used to identify genomic sequences. The methods and transgenic animals may be used to detect cancer-related genes by identifying common insertion sites in tumor cells.

Methods and compositions for identification of genomic sequences

Genomic Organization and Chromosomal Localization of the Mouse Telencephalin Gene, a Neuronal Member of the ICAM Family ☆

Work in many laboratories has provided numerous examples of the conservation of genetic pathways during evolution. Although the morphogenetic changes that result in mesoderm formation vary greatly among individuals of different phyla, recent work has shown that several genes involved in mesoderm formation have been evolutionarily conserved. The Drosophila gene snail encodes a protein with five zinc fingers (Boulay et al. 1987). snail is one of two genes required zygotically for mesoderm formation during Drosophila embryogenesis. Embryos homozygous for a null mutation of snail form no mesodermal germ layer and die late in embryogenesis (reviewed in Leptin et al. 1992). The SNAIL protein is a transcriptional repressor and acts to maintain proper germ layer boundaries by repressing the expression within the mesoderm of regulatory genes involved in ectodermal development (Leptin 1991). Several groups have isolated vertebrate homologs of snail (Sargent and Bennett 1990; Hammerschmidt and NtissleinVolhard 1993; Thisse et al. 1994, 1995; Nieto et al. 1994). We and others have described the isolation of cDNA clones of the Sna gene, a mouse homolog of snail (Nieto et al. 1992; Smith et al. 1992). Sna encodes a 264-amino acid protein containing four zinc fingers. Analysis of the RN, A expression pattem of the Sna gene during embryonic development in mice revealed that it is expressed early durifig mesoderm formation and is later expressed in all mesenchymal cells, whether of mesodermal or neural crest origin (Nieto et al. 1992; Smith et al. 1992). In this paper we describe the genomic organization of the mouse Sna gene and map it to the distal portion of mouse Chromosome (Chr) 2. A strain 129/Sv mouse genomic phage library was screened with a mouse Sna cDNA clone. Hybridizing phage clones were plaque-purified, and an 18.5-kb SalI restriction fragment that hybridized to the Sna cDNA was subcloned. This genomic subclone was further mapped and was hybridized with oligonucleotides corresponding to different regions of the Sna cDNA. Exons and exonintron boundaries were sequenced with oligonucleotides corresponding to the Sna cDNA as primers. The sequence of the region upstream of the first exon contains consensus sequences for DNA binding sites of several transcription factors (data not shown; GenBank accession number U95961). The localization of the exons in the genomic DNA map was done by PCR, with oligonucleotides corresponding to sequences of the different exons used as primers. The resulting amplified bands were then analyzed by restriction enzyme digestion. The genomic organization of the mouse Sna gene is shown in Fig. 1. The gene is comprised of three exons spanning approximately 5 kb. Nucleotide sequences flanking exon-intron borders

Genomic organization and chromosomal localization of the mouse snail (Sna) gene

Assignment of the Glial Inwardly Rectifying Potassium Channel KAB-2/Kir4.1 (Kcnj10) Gene to the Distal Region of Mouse Chromosome 1

Nature 349, 709-713 (1991) WE have discovered frameshift errors made in the assembly of our published cDNA sequence affecting only the region of nucleotides 1138-1180, corresponding to amino acid residues 367-379. The correct sequence is available on the EMBL database under accession number X57377, release 28.

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Nancy A. Jenkins

Papers

Methods and compositions for identification of genomic sequences

Genomic Organization and Chromosomal Localization of the Mouse Telencephalin Gene, a Neuronal Member of the ICAM Family ☆

Genomic organization and chromosomal localization of the mouse snail (Sna) gene

Assignment of the Glial Inwardly Rectifying Potassium Channel KAB-2/Kir4.1 (Kcnj10) Gene to the Distal Region of Mouse Chromosome 1

Erratum: Novel myosin heavy chain encoded by murine dilute coat colour locus (Nature (1991) 349 (709-713))