Selecting breast cancer patients for chemotherapy: the opening of the UK OPTIMA trial.

The mortality from breast cancer has improved steadily over the past two decades, in part because of the increased use of more effective adjuvant therapies. Thousands of women are routinely treated with intensive chemotherapy, which can be unpleasant, is expensive and is occasionally hazardous. Oncologists have long known that some of these women may not need treatment, either because they have a low risk of relapse or because they have tumour biology that makes them less sensitive to chemotherapy and more suitable for early adjuvant endocrine therapy. There is an urgent need to improve patient selection so that chemotherapy is restricted to those patients who will benefit from it. Here we review the emerging technologies that are available for improving patient selection for chemotherapy. We describe the OPTIMA trial, which has just opened to recruitment in the UK, is the latest addition to trials in this area, and is the first to focus on the relative cost-effectiveness of alternate predictive assays.

Changes in gene expression associated with developmental arrest and longevity in Caenorhabditis elegans.

Gene expression in a developmentally arrested, long-lived dauer population of Caenorhabditis elegans was compared with a nondauer (mixed-stage) population by using serial analysis of gene expression (SAGE). Dauer (152,314) and nondauer (148,324) SAGE tags identified 11,130 of the predicted 19,100 C. elegans genes. Genes implicated previously in longevity were expressed abundantly in the dauer library, and new genes potentially important in dauer biology were discovered. Two thousand six hundred eighteen genes were detected only in the nondauer population, whereas 2016 genes were detected only in the dauer, showing that dauer larvae show a surprisingly complex gene expression profile. Evidence for differentially expressed gene transcript isoforms was obtained for 162 genes. H1 histones were differentially expressed, raising the possibility of alternative chromatin packaging. The most abundant tag from dauer larvae (20-fold more abundant than in the nondauer profile) corresponds to a new, unpredicted gene we have named tts-1 (transcribed telomere-like sequence), which may interact with telomeres or telomere-associated proteins. Abundant antisense mitochondrial transcripts (2% of all tags), suggest the existence of an antisense-mediated regulatory mechanism in C. elegans mitochondria. In addition to providing a robust tool for gene expression studies, the SAGE approach already has provided the advantage of new gene/transcript discovery in a metazoan.

Surveying Saccharomyces genomes to identify functional elements by comparative DNA sequence analysis.

Comparative sequence analysis has facilitated the discovery of protein coding genes and important functional sequences within proteins, but has been less useful for identifying functional sequence elements in nonprotein-coding DNA because the relatively rapid rate of change of nonprotein-coding sequences and the relative simplicity of non-coding regulatory sequence elements necessitates the comparison of sequences of relatively closely related species. We tested the use of comparative DNA sequence analysis to aid identification of promoter regulatory elements, nonprotein-coding RNA genes, and small protein-coding genes by surveying random DNA sequences of several Saccharomyces yeast species, with the goal of learning which species are best suited for comparisons with S. cerevisiae. We also determined the DNA sequence of a few specific promoters and RNA genes of several Saccharomyces species to determine the degree of conservation of known functional elements within the genome. Our results lead us to conclude that comparative DNA sequence analysis will enable identification of functionally conserved elements within the yeast genome, and suggest a path for obtaining this information.

Gendered (s)explorations among same-sex attracted young people in Australia.

This paper seeks to import a more complex understanding of gendered subjectivity into discussions of young people and homosexuality, and is based on an Australian national survey (n=749) of same-sex attracted youth (SSAY) aged between 14 and 21. Results revealed significant gender differences with regard to patterns of sexual attraction, behaviour and identity labels among participants. For the young men in the study, there was more congruence between feelings of gender a-typicality, same-sex attractions and same-sex behaviours. Overall, young women displayed more fluidity with regard to their sexual feelings, behaviours and identities. Young women were more likely to be engaged in private explorations of lesbianism, concurrent with participation in heterosexual sex and relationships. Young women were also grappling with more limited and emotionally risky opportunities for sex with other girls who were already known to them as friends. The invisibility of lesbianism as an identity or practice led to confusion about what feelings meant for the future in the arena of lived experience. The paper concludes that more research is needed into the impact of gender on the development of young people's experiences of homosexuality, particularly the manner in which invisibility and lack of social acceptance of a full spectrum of sexual diversity may disadvantage young women's emotional health and well-being.

A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms.

We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Theories and applications for sequencing randomly selected clones.

Theory is developed for the process of sequencing randomly selected large-insert clones. Genome size, library depth, clone size, and clone distribution are considered relevant properties and perfect overlap detection for contig assembly is assumed. Genome-specific and nonrandom effects are neglected. Order of magnitude analysis indicates library depth is of secondary importance compared to the other variables, especially as clone size diminishes. In such cases, the well-known Poisson coverage law is a good approximation. Parameters derived from these models are used to examine performance for the specific case of sequencing random human BAC clones. We compare coverage and redundancy rates for libraries possessing uniform and nonuniform clone distributions. Results are measured against data from map-based human-chromosome-2 sequencing. We conclude that the map-based approach outperforms random clone sequencing, except early in a project. However, simultaneous use of both strategies can be beneficial if a performance-based estimate for halting random clone sequencing is made. Results further show that the random approach yields maximum effectiveness using nonbiased rather than biased libraries.

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

A general approach to single-nucleotide polymorphism discovery.

Single-nucleotide polymorphisms (SNPs) are the most abundant form of human genetic variation and a resource for mapping complex genetic traits. The large volume of data produced by high-throughput sequencing projects is a rich and largely untapped source of SNPs (refs 2, 3, 4, 5). We present here a unified approach to the discovery of variations in genetic sequence data of arbitrary DNA sources. We propose to use the rapidly emerging genomic sequence as a template on which to layer often unmapped, fragmentary sequence data and to use base quality values to discern true allelic variations from sequencing errors. By taking advantage of the genomic sequence we are able to use simpler yet more accurate methods for sequence organization: fragment clustering, paralogue identification and multiple alignment. We analyse these sequences with a novel, Bayesian inference engine, POLYBAYES, to calculate the probability that a given site is polymorphic. Rigorous treatment of base quality permits completely automated evaluation of the full length of all sequences, without limitations on alignment depth. We demonstrate this approach by accurate SNP predictions in human ESTs aligned to finished and working-draft quality genomic sequences, a data set representative of the typical challenges of sequence-based SNP discovery.

High-throughput plasmid DNA purification for 3 cents per sample.

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.

zA map for sequence analysis of the Arabidopsis thaliana genome.

Arabidopsis thaliana has emerged as a model system for studies of plant genetics and development, and its genome has been targeted for sequencing by an international consortium (the Arabidopsis Genome Initiative; http://genome-www. stanford.edu/Arabidopsis/agi.html). To support the genome-sequencing effort, we fingerprinted more than 20,000 BACs (ref. 2) from two high-quality publicly available libraries, generating an estimated 17-fold redundant coverage of the genome, and used the fingerprints to nucleate assembly of the data by computer. Subsequent manual revision of the assemblies resulted in the incorporation of 19,661 fingerprinted BACs into 169 ordered sets of overlapping clones ('contigs'), each containing at least 3 clones. These contigs are ideal for parallel selection of BACs for large-scale sequencing and have supported the generation of more than 5.8 Mb of finished genome sequence submitted to GenBank; analysis of the sequence has confirmed the integrity of contigs constructed using this fingerprint data. Placement of contigs onto chromosomes can now be performed, and is being pursued by groups involved in both sequencing and positional cloning studies. To our knowledge, these data provide the first example of whole-genome random BAC fingerprint analysis of a eucaryote, and have provided a model essential to efforts aimed at generating similar databases of fingerprint contigs to support sequencing of other complex genomes, including that of human.

DNA sequence chromatogram browsing using JAVA and CORBA.

DNA sequence chromatograms (traces) are the primary data source for all large-scale genomic and expressed sequence tags (ESTs) sequencing projects. Access to the sequencing trace assists many later analyses, for example contig assembly and polymorphism detection, but obtaining and using traces is problematic. Traces are not collected and published centrally, they are much larger than the base calls derived from them, and viewing them requires the interactivity of a local graphical client with local data. To provide efficient global access to DNA traces, we developed a client/server system based on flexible Java components integrated into other applications including an applet for use in a WWW browser and a stand-alone trace viewer. Client/server interaction is facilitated by CORBA middleware which provides a well-defined interface, a naming service, and location independence. [The software is packaged as a Jar file available from the following URL: http://www.ebi.ac.uk/jparsons. Links to working examples of the trace viewers can be found at http://corba.ebi.ac.uk/EST. All the Washington University mouse EST traces are available for browsing at the same URL.]

An encyclopedia of mouse genes.

The laboratory mouse is the premier model system for studies of mammalian development due to the powerful classical genetic analysis possible (see also the Jackson Laboratory web site, http://www.jax.org/) and the ever-expanding collection of molecular tools. To enhance the utility of the mouse system, we initiated a program to generate a large database of expressed sequence tags (ESTs) that can provide rapid access to genes. Of particular significance was the possibility that cDNA libraries could be prepared from very early stages of development, a situation unrealized in human EST projects. We report here the development of a comprehensive database of ESTs for the mouse. The project, initiated in March 1996, has focused on 5' end sequences from directionally cloned, oligo-dT primed cDNA libraries. As of 23 October 1998, 352,040 sequences had been generated, annotated and deposited in dbEST, where they comprised 93% of the total ESTs available for mouse. EST data are versatile and have been applied to gene identification, comparative sequence analysis, comparative gene mapping and candidate disease gene identification, genome sequence annotation, microarray development and the development of gene-based map resources.

"I'd never share a needle" -- [but I often have unsafe sex]: considering the paradox of young people's sex and drugs talk.

PIP: This paper presents an analysis of the self-reported sexual and drug injecting behavior of 178 young people and the qualitative accounts of injecting drug use and sexual behavior of a smaller group of 30 who are interviewed as part of the larger sample. The research was conducted in the Australian state of Victoria and Queensland through 89 youth specific health and housing services. Results revealed that young people's stories of the culture and practices surrounding injecting drug use were encouraging with regards to the attention given to notions of responsibility and care for others, and a coherent discourse of belief in safe injecting practice was also evident. A notion of the 'druggie' or responsible drug user was contrasted to a 'junkie' or irresponsible user, within the culture of drug use described. Conversely, reported sexual behaviors revealed neither consistent discourses about safe sex, nor an unambiguously expressed personal commitment to safe sex.^ieng

Single nucleotide polymorphism hunting in cyberspace.

Large-scale sequencing of human cDNA and genomic DNA libraries has produced a large collection of sequence data in public databases. To date, >900,000 human expressed sequence tag (EST) sequences and >80,000,000 bases of genomic DNA sequence have been deposited in Genbank. This ever-expanding data set is a rich source of gene-associated and anonymous single nucleotide polymorphisms (SNPs). DNA sequence variations can be found by comparing the sequences of redundant ESTs and by comparing sequences from overlapping genomic clones. Initial studies have shown that, with proper computer screening, informative SNP markers can be developed from these DNA databases in an efficient and cost-effective manner. Complete public access to these databases will allow individual investigators to add biological value to the human sequence data generated by large-scale sequencing centers.