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.

/pdf/gene-ontology-tool-for-the-unification-of-biology-ppsktrcye3.pdf

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.

/pdf/initial-sequencing-and-analysis-of-the-human-genome-5dapk1rsx1.pdf

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

The gene encoding threonine synthase (THR4) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr4 mutant. This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome III inserted in the vector lambdaMG3. The THR4 gene encodes a protein of 514 amino acids (M.W. 58 kDa), which has extensive homologies with E. coli threonine synthase (thrC) and B subtilis threonine synthase. The 5' flanking region of the gene contains three regulatory sequences [TGACT(C)] for the general amino acid control (GCN). About 130 bp downstream of the THR4 gene another large open reading frame (563 amino acids) is found in the opposite orientation. This may imply that this open reading frame, called CTR86, shares a terminator region with THR4. The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site suggests that the gene product may also be involved in amino acid biosynthesis.

Analysis of the THR4 region on chromosome III of the yeast Saccharomyces cerevisiae.

Chromatography on BD-cellulose columns with a salt gradient and formamide separates cellular DNA into two fractions (fraction I eluted within the salt gradient, fraction II with formamide), the proportions of these two fractions (ca. 2:1) being similar for DNA from a number of eucaryotic organisms. Yeast DNA was chosen for a detailed study of the mode of fractionation. Several physicochemical parameters, binding to nitrocellulose filters, sensitivity towards nuclease S1, labelling properties in vivo, and hybridization properties of the two DNA fractions were compared. It was shown that both fractions are native DNA and that the fractionation does not depend on the size or the (G + C) content of the DNA. Fraction I DNA contains only a small portion of molecules having single-stranded ends. Fraction II DNA is a heterogeneous population, containing molecules with peculiar structural characteristics: (a) It contains DNA molecules with single-stranded ends and/or gaps sensitive to nuclease S1; labeling experiments suggested that these are molecules undergoing repair and replication. (b) Another portion of fraction II is molecules sensitive to nuclease S1 in regions which are not single-stranded. (c) A third portion is DNA which, after treatment with nuclease S1, is still strongly bound to the resin. Indications that the segregation may be due to the presence of specific DNA sequences comes from the above experiments and from the finding that fraction I DNA is enriched in ribosomal genes and fraction II DNA in tRNA genes.

Characteristics of DNA fractionated on benzoylated DEAE-cellulose.

In the framework of the EC programme for sequencing yeast chromosome XV, we have determined the nucleotide sequence of a 26 kb region. Subsequent analysis revealed 13 non-overlapping open reading frames, three of which correspond to known yeast genes. A pair of tRNA genes associated with remnant Ty elements were localized in this region. From structural parameters and/or similarity searches with entries in the current data libraries, a preliminary functional assessment of several of the putative novel gene products can be made. The gene density in this region amounts to one gene in 2 kb. Protein coding regions occupy 61% of the total DNA sequence. Within the intergenic regions, potential regulatory elements can be predicted. The data obtained here may serve as a basis for a more detailed biochemical analysis of the novel genes. The complete nucleotide sequence of the 26 kb segment as depicted in Figure 1 has been deposited at the EBI data library under Accession Number X91067.

Analysis of a 26 kb region on the left arm of yeast chromosome XV.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2007.05.061

Vienna special issue: Molecular machines

Background: Experimental and clinical studies have shown that a prolonged overall treatment time is associated with a decrease in local tumor control. Methods: In a brief review, the clinical data of radiotherapy with split-course or treatment interruption were collected. Results and Conclusion: There was a detrimental effect of the treatment interruption on overall or disease-specific survival in patients with locally advanced head and neck cancer or non-small-cell lung cancer in particular in patients with good prognostic factors and well to moderate differentiated tumors. Therefore, interruption of radiotherapy in patients with curative treatment should be avoided.

Horst Feldmann

Papers

Analysis of the THR4 region on chromosome III of the yeast Saccharomyces cerevisiae.

Characteristics of DNA fractionated on benzoylated DEAE-cellulose.

Analysis of a 26 kb region on the left arm of yeast chromosome XV.

Vienna special issue: Molecular machines

Die Bedeutung der Bestrahlungspause