Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.     

Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.     

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.     

Fgf8 is required for outgrowth and patterning of the limbs

The expression pattern and activity of fibroblast growth factor-8 (FGF8) in experimental assays indicate that it has important roles in limb development, but early embryonic lethality resulting from mutation of Fgf8 in the germ line of mice has prevented direct assessment of these roles. Here we report that conditional disruption of Fgf8 in the forelimb of developing mice bypasses embryonic lethality and reveals a requirement for Fgf8 in the formation of the stylopod, anterior zeugopod and autopod. Lack of Fgf8 in the apical ectodermal ridge (AER) alters expression of other Fgf genes, Shh and Bmp2.     

A constitutively open potassium channel formed by KCNQ1 and KCNE3

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.