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

A new upstream activating sequence (UAS) 5"-GGTGGCAAA-3" was detected in the promoters of 27 out of the 33 proteasomal genes and of several genes related to the ubiquitin-proteasomal system of Saccharomyces cerevisiae. The sequence was termed proteasome-associated control element (PACE). Gel retardation assay revealed specific binding of PACE with an extracted protein. The protein (64K) was purified by affinity chromatography and was homogeneous by SDS-PAGE. Microsequencing showed that the protein is Rpn4p. The ability of Rpn4p to activate transcription was demonstrated with constructs containing fragments of the RPN5and CDC48gene promoters and reporter cat. Binding to PACE, Rpn4p may act as a common transcription factor on the proteasomal and proteasome-related genes.

Isolation and Identification of PACE-Binding Protein Rpn4, a New Transcriptional Activator Regulating 26S-Proteasomal and Other Genes

Molecular analysis of the yeast Ty4 element: homology with Ty1, copia, and plant retrotransposons.

In the framework of the EC programme for sequencing yeast chromosome II, we have determined the nucleotide sequence of a 70 kb region. Subsequent analysis revealed 35 open reading frames, 14 of which correspond to known yeast genes. 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 1.98 kb. Coding regions occupy 75% 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 70 kb segment as depicted in Figure 1 has been deposited in the EMBL data library under Accession Number X78993.

Analysis of a 70 kb region on the right arm of yeast chromosome II.

We have analysed a region some 30 kb centromere distal form PHO5 on the right arm of yeast chromosome II and determined the nucleotide sequence of a 8.95 kb DNA segment from this region. By this analysis we were able to derive the precise location and the transcriptional orientation of CMD1, ALG1, SSN6 and LYS2. An open reading frame of 2370 bp was locatlized between SSN6 and LYS2, which has recently been identified (Schild et al., 1991) to be the RAD16 gene. The putative gene product, 790 amino acids in length, reveals several interesting freatures. It contains a nuclear target singnature and shares several blocks of similarity with the yeast recombinational repair protein RAD54 and the nuclear factor SNF2 (SW12), which is required for teh transcriptioal activation of a number of yeast genes. The similarity blocks in these three proteins are reminiscent of those found in the helicase superfamily. Furthrmore, RAD16 contains a novel ‘double-finger’ motif, which has been encountered in a variety of proteins from different organisms that are suggested to interact with DNA and are involved in diverse functions including site-specific recombination, DNA repair, and transcriptional regulation. The putative gene product of RAD16 then is the first example of a proteins in which the novel double-finger motif is found to be combined with a poteintial DNA helicase framework.

Molecular analysis of yeast chromosome II between CMD1 and LYS2: the excision repair gene RAD16 located in this region belongs to a novel group of double-finger proteins.

THR1, the gene from Saccharomyces cerevisiae, encoding homoserine kinase, one of the threonine biosynthetic enzymes, has been cloned by complementation. The nucleotide sequence of a 3.1-kb region carrying this gene reveals an open reading frame of 356 codons, corresponding to about 40 kDa for the encoded protein. The presence of three canonical GCN4 regulatory sequences in the upstream flanking region suggests that the expression of THR1 is under the general amino acid control. In parallel, the enzyme was purified by four consecutive column chromatographies, monitoring homoserine kinase activity. In SDS gel electrophoresis, homoserine kinase migrates like a 40-kDa protein; the native enzyme appears to be a homodimer. The sequence of the first 15 NH2-terminal amino acids, as determined by automated Edman degradation, is in accordance with the amino acid sequence deduced from the nucleotide sequence. Computer-assisted comparison of the yeast enzyme with the corresponding activities from bacterial sources showed that several segments among these proteins are highly conserved. Further more, the observed homology patterns suggest that the ancestral sequences might have been composed from separate (functional) domains. A block of very similar amino acids is found in the homoserine kinases towards the carboxy terminus that is also present in many other proteins involved in threonine (or serine) metabolism: this motif, therefore, may represent the binding site for the hydroxyamino acids. Limited similarity was detected between a motif conserved among the homoserine kinases and consensus sequences found in other mono- or dinucleotide-binding proteins.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1990.tb19100.x

Yeast homoserine kinase. Characteristics of the corresponding gene, THR1, and the purified enzyme, and evolutionary relationships with other enzymes of threonine metabolism.

Horst Feldmann

Papers

Isolation and Identification of PACE-Binding Protein Rpn4, a New Transcriptional Activator Regulating 26S-Proteasomal and Other Genes

Molecular analysis of the yeast Ty4 element: homology with Ty1, copia, and plant retrotransposons.

Analysis of a 70 kb region on the right arm of yeast chromosome II.

Molecular analysis of yeast chromosome II between CMD1 and LYS2: the excision repair gene RAD16 located in this region belongs to a novel group of double-finger proteins.

Yeast homoserine kinase. Characteristics of the corresponding gene, THR1, and the purified enzyme, and evolutionary relationships with other enzymes of threonine metabolism.