A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

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The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas (R. Kambadur et al., 1998, Genes Dev. 12, 246-260). Seeking to identify the cellular mechanisms that generate these expression compartments, we studied the lineage development of isolated NBs in culture. We found that the Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. Our results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions generating additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb --> Pdm --> Cas --> Gh expression during NB outgrowth.

The power of experiments aimed at detecting linkage between a quantitative locus and a marker locus, both segregating in the backross or F2 generation of a cross between two inbred lines, is examined. Given that the two lines are close to fixation for alternative alleles of both marker locus and quantitative locus, it is concluded that experiments involving a few thousand offspring should be able to detect close linkages involving quantitative loci (or groups of loci) having rather modest effects (i.e., that contribute, say, 1% of the total phenotypic variance in the F2).

The beige mutation is a murine autosomal recessive disorder, resulting in hypopigmentation, bleeding and immune cell dysfunction. The gene defective in beige is thought to be a homologue of the gene for the human disorder Chediak-Higashi syndrome. We have identified the murine beige gene by in vitro complementation and positional cloning, and confirmed its identification by defining mutations in two independent mutant alleles. The sequence of the beige gene message shows strong nucleotide homology to multiple human ESTs, one or more of which may be associated with the Chediak-Higashi syndrome gene. The amino acid sequence of the Beige protein revealed a novel protein with significant amino acid homology to orphan proteins identified in Saccharomyces cerevisiae, Caenorhabditis elegans and humans.

Previous studies have reported the presence of renin mRNAs in several mouse tissues and angiotensinogen mRNAs in various rat tissues. Clarification as to whether renin and angiotensinogen mRNAs are coexpressed in the same tissues of the same animal species is important for understanding the biology of the tissue renin-angiotensin system. We employed mouse renin cDNA and rat angiotensinogen cDNA to compare tissue distributions of renin and angiotensinogen in RNAs of the rat and mouse. Both cDNA probes readily cross-hybridize with the corresponding mRNA of the other species. Our results demonstrate several patterns of distribution. Renin and angiotensinogen mRNAs are readily detected in kidney and adrenals of both species. In brain and heart, angiotensinogen mRNAs are present in concentrations that far exceed renin mRNA levels in these organs in both species. In mouse and rat livers, angiotensinogen, but not renin, mRNA is demonstrated. In rat testis, only renin mRNA can be detected, whereas in mouse testes both renin and angiotensinogen mRNA are present. In CD-1 male mouse submandibular gland, renin mRNA exists in high concentrations, whereas angiotensinogen mRNA is present in low levels. In contrast, neither renin nor angiotensinogen mRNA could be detected in rat salivary gland. In summary, our study demonstrates the widespread codistribution of renin and angiotensinogen mRNAs in many tissues of both species, allowing for the possibility of local angiotensin production. However, tissue and species differences in these gene expressions also exist. Understanding differential tissue expressions of these genes will provide additional important insight into the biology of the renin-angiotensin system.

Here, we describe a multigenomic DNA sequence-analysis tool, EVOPRINTER, that facilitates the rapid identification of evolutionary conserved sequences within the context of a single species. The EVOPRINTER output identifies multispecies-conserved DNA sequences as they exist in a reference DNA. This identification is accomplished by superimposing multiple reference DNA vs. test-genome pairwise BLAT (BLAST-like alignment tool) readouts of the reference DNA to identify conserved nucleotides that are shared by all orthologous DNAs. EVOPRINTER analysis of well characterized genes reveals that most, if not all, of the conserved sequences are essential for gene function. For example, analysis of orthologous genes that are shared by many vertebrates identifies conserved DNA in both protein-encoding sequences and noncoding cis-regulatory regions, including enhancers and mRNA microRNA binding sites. In Drosophila, the combined mutational histories of five or more species affords near-base pair resolution of conserved transcription factor DNA-binding sites, and essential amino acids are revealed by the nucleotide flexibility of their codon-wobble position(s). Conserved small peptide-encoding genes, which had been undetected by conventional gene-prediction algorithms, are identified by the codon-wobble signatures of invariant amino acids. Also, EVOPRINTER allows one to assess the degree of evolutionary divergence between orthologous DNAs by highlighting differences between a selected species and the other test species.comparative genomics ͉ evolution ͉ gene structure and function D eciphering the regulatory mechanisms that control coordinate gene expression is a long-standing goal of biology. The comparison of orthologous DNA sequences from multiple vertebrate or invertebrate species promises to identify the cisregulatory elements that are central to the dynamic interplay between a gene and its transcriptional regulators (1-3). This cross-species comparison, termed phylogenetic footprinting, is based on the hypothesis that functionally important sequences evolve at a significantly slower rate than nonfunctional DNA (1). Phylogenetic footprinting has been used successfully to discover multispecies-conserved sequences (MCSs) that are critical for gene function (reviewed in refs. 2, 4, and 5). An essential first step in this process is the alignment of multiple orthologous DNAs. Multisequence-alignment programs include THREADED BLOCKSET ALIGNER (6), FOOTPRINTER (7), CONREAL (5), and PHYME (8). The multiDNA alignments are accomplished either by simultaneous or sequential pairwise alignments of input DNAs, with alignment gaps introduced to optimize the overall homology comparisons.Individual genome searches have also been commonly used to initiate MCS searches, and two popular whole-genome search algorithms are BLAST (9) and BLAT (BLAST-like alignment tool) (10). One significant difference between the BLAST and BLAT algorithms is that BLAT keeps an index of a species genome in memory and uses this index to scan linearly through ...