Putting energy into mitochondrial protein import.

The import of proteins into mitochondria occurs in several steps. At least three of these steps require ATP and involve molecular chaperones. This energy requirement has served as a useful tool for elucidating the import pathways into the four mitochondrial compartments.

A whole-genome assembly of Drosophila.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

The genome sequence of Drosophila melanogaster.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

A high-resolution radiation hybrid map of the human genome draft sequence.

We have constructed a physical map of the human genome by using a panel of 90 whole-genome radiation hybrids (the TNG panel) in conjunction with 40,322 sequence-tagged sites (STSs) derived from random genomic sequences as well as expressed sequences. Of 36,678 STSs on the TNG radiation hybrid map, only 3604 (9.8%) were absent from the unassembled draft sequence of the human genome. Of 20,030 STSs ordered on the TNG map as well as the assembled human genome draft sequence and the Celera assembled human genome sequence, 36% of the STSs had a discrepant order between the working draft sequence and the Celera sequence. The TNG map order was identical to one of the two sequence orders in 60% of these discrepant cases.

Protein sorting in mitochondria.

Most polypeptides that are imported into the mitochondrial matrix use a common translocation machinery. By contrast, proteins of the other mitochondrial compartments are imported by a variety of different mechanisms. Some of these proteins completely bypass the common translocation machinery, others use only the outer membrane components of this machinery, and still others use components of this machinery from both the outer and inner membranes. Import to the intermembrane space compartment provides examples of all three possibilities.

Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction.

We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.

The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions.

Cytochrome b2, a protein of the yeast mitochondrial intermembrane space, is synthesized with an 80 residue bipartite presequence. The amino-terminal portion resembles a matrix-targeting signal. The carboxy-terminal portion acts as a 'sorting signal' for the intermembrane space and contains a hydrophobic stretch. In order to define this sorting signal, we fused the first 167 residues of the cytochrome b2 precursor to a passenger protein, expressed the fusion protein in yeast and selected for mutations that caused mislocalization of the passenger protein to the matrix. Most mutations mapped within the first 81 amino-terminal residues of the cytochrome b2 moiety. They were located in three regions, all downstream of the matrix-targeting domain: a cluster of three basic residues upstream of the hydrophobic stretch, the hydrophobic stretch itself and the first residue of mature cytochrome b2. The level of missorting caused by mutations within the hydrophobic stretch did not correlate with their effects on hydrophobicity, but appeared to be related to changes in the conformation of this stretch. We conclude that the intermembrane space sorting signal of cytochrome b2 is decoded by protein-protein interactions rather than by simple partitioning into a lipid bilayer.