Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.

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Gene Ontology: tool for the unification of biology

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.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

http://science.sciencemag.org/content/sci/291/5507/1304.full.pdf

The sequence of the human genome.

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.

/pdf/the-complete-genome-sequence-of-escherichia-coli-k-12-2lpi4d3nz4.pdf

The Complete Genome Sequence of Escherichia coli K-12

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/403623.pdf

A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae

Regional hyperthermia in deep-seated tumors can be limited by excessive heating of normal tissues, usually associated with pain or local discomfort. In this report, 57 hyperthermia treatments in 8 patients with locally advanced presacral recurrences of colorectal cancer were analyzed with respect to normal tissue temperatures, especially with respect to the perineal fat temperature. In 27 treatments, 1 to 2 catheters had been inserted from the perineal region through a large part of the perirectal and presacral fat into the tumor, so that temperature profiles of the perineal fat could be obtained. The mean maximum temperature (±SD) of the vagina, rectum, bladder, muscle tissue, and perineal fat was 40.8 ± 1.2°C, 40.9 ± 1.6°C, 40.5 ± 1.6°C, 39.8 ± 0.7°C, and 42.6 ± 1.1 °C, respectively. The mean maximum systemic temperature (±SD) was 37.7 ± 0.7°C. In 42% of the treatments, the temperature in the perineal fat ranged between 43 and 46°C and was treatment-limiting. In conclusion, overheating of the perineal fat is a problem in the treatment of eccentrically located tumors of the presacral region when relatively high temperatures in the tumor will be maintained for longer time periods.

Hyperthermia in eccentrically located pelvic tumors: excessive heating of the perineal fat and normal tissue temperatures.

PURPOSE Detection of radiation resistant hypoxic cells in poorly vascularized regions of tumors could facilitate selection of therapeutic schedules for the combination of radiotherapy and hyperthermia. PATIENTS AND METHODS In 29 patients (9 breast carcinomas, 8 soft tissue sarcomas, 8 lymph node metastases of head and neck cancers and 4 different other tumors) the oxygenation status was examined before, during and after radio- and partially thermoradiotherapy. It was registered using a needle electrode under local anesthesia. The pO2-values obtained from about 100 to 200 sites of an individual tumor were displayed as histograms (Eppendorf-pO2-Histograph, Hamburg). RESULTS Hypoxic tissue areas (pO2 < 5 mm Hg) was found more in sarcomas (10.2%) than in lymph node metastases of head and neck cancers (5.8%) and breast carcinomas (3.3%). We observed in the course of therapy increasing pO2 values (reoxygenation) only partially in the groups of breast carcinomas and cervical lymph node metastases, decreasing in the soft tissue sarcomas. CONCLUSIONS In future the selection of patients for hyperthermia could be done on the base of pO2-measurements.

Studies on oxygen partial pressure in tumor tissue under radiotherapy and thermoradiotherapy

The sequence of a 1.9 kb Bam x Hind III fragment from yeast has been determined. This fragment is part of a yeast 6.7 kb Hind III segment cloned into pBR322 (pY20). The fragment carries a single gene for a glutamate tRNA which has no intron. According to genetic analyses [1] this fragment also contains a yeast chromosomal replicator. We have analyzed the sequence for potential open reading frames and for several structural features which are thought to be involved in the initiation of DNA replication. Hybridization studies have revealed that portions of this sequence are repeated within the yeast genome.

Sequence of a yeast DNA fragment containing a chromosomal replicator and a tRNA Glu 3 gene.

Hypoxia is regarded to be an important factor for radioresistance. In general, approximately 2 to 3 times higher radiation doses are needed to kill hypoxic cells when compared with well oxygenated cells (Gray et al. 1953, Evans and Naylor 1963). However, the clinical importance of tumor hypoxia remains uncertain due to methodological problems for routine measurements of intratumoral O2 tensions in patients. Considerable advances have been made in the investigation of tissue oxygenation in patients using a novel technique developed by Fleckenstein (1986). This technique has been shown to be valid in measuring the O2 tension distribution in normal tissues (Weiss and Fleckenstein 1986, Kallinowski et al. 1990) and tumors (Hockel et al. 1991, Vaupel et al.1991).

Horst Feldmann

Papers

Hyperthermia in eccentrically located pelvic tumors: excessive heating of the perineal fat and normal tissue temperatures.

Studies on oxygen partial pressure in tumor tissue under radiotherapy and thermoradiotherapy

Sequence of a yeast DNA fragment containing a chromosomal replicator and a tRNA Glu 3 gene.

Oxygenation of Locally Advanced Recurrent Rectal Cancer, Soft Tissue Sarcoma and Breast Cancer

Thermoradiotherapy in locally advanced deep seated tumours--thermal parameters and treatment results.