Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one T(n) microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the T(n) microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000-56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Antiinflammatory effects of glucocorticoids are critical to treatment of airway inflammation in such common disorders as asthma. There is considerable variation in responsiveness to glucocorticoid, and prolonged exposure can result in glucocorticoid resistance. We cloned LGL2, a glucocorticoid-inducible gene in fetal rat lung. We described the characterization of lgl2 as a nuclear transport protein, classified as importin 13 (IPO13), and demonstrated developmental regulation of IPO13 nucleocytoplasmic shuttling. We now report on the identification of the glucocorticoid receptor (GR) as a cargo substrate for IPO13. Binding of GR and IPO13 was demonstrated by GR-GST pulldown and coimmunoprecipitation. To investigate the role of IPO13 in modulating GR signaling in the lung, we studied IPO13-regulated GR transport in airway epithelial cells. Small interfering RNAs that inhibited IPO13 synthesis prevented nuclear translocation of GR. Silencing of IPO13 also abrogated the ability of cortisol to inhibit synthesis of the inflammatory cytokine IL-8 after stimulation with TNF-alpha. Our findings support a role for IPO13 in promoting nuclear occupancy of GR in a way that strongly potentiates the antiinflammatory effects of glucocorticoids. We speculate that variation in cellular levels of IPO13 and intracellular IPO13 shuttling rates may contribute to glucocorticoid resistance.

The glucocorticoids (GC) and retinoids (RA) modulate branching morphogenesis and cytodifferentiation in the developing lung. We investigated downstream target genes that link glucocorticoid stimulation to the achievement of a mature lung in glucocorticoid receptor (GR) knockout mice. All GR(null) mice and approximately 80% of mice homozygous for a hypomorphic allele (GR(hypo)) die shortly after birth of respiratory failure. cDNA microarray analysis showed organ-specific upregulation of the retinoic acid responsive gene midkine (MK) and its chondroitin-sulfate binding partner PG-M/versican at fetal day 18 and at neonatal day 1 in lungs of GR(hypo) mice, and at neonatal day 1 in lungs of GR(null) mice. By contrast, lung MK and PG-M/versican were downregulated in these mice at fetal day 16.5. In situ hybridization studies showed a dramatic decrease in MK and PG-M/versican RNA between days 16.5 and 17.5 in GR(WT) but not in GR(null) mice. Continued diffuse and robust expression of MK protein was observed in GR(null) mice at neonatal day 1. These findings suggest that MK may contribute to the dysmature lung phenotype in GR-deficient mice. Exposure of cultured day 21 fetal rat lung cells to GC downregulated MK, whereas RA enhanced MK expression. Our findings demonstrate the coincident modulation of expression of MK at the same developmental time point by both GC and RA, providing a potential mechanism for the integration of GC and RA effects on fetal lung development.

The ␤-hexosaminidases (Hex) catalyze the cleavage of terminal amino sugars on a broad spectrum of glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer of ␣ and ␤ subunits (␣␤), and Hex B, a homodimer of ␤ subunits (␤␤), have different substrate specificities. The ␤ subunit (HEXB gene product), hydrolyzes neutral substrates. The ␣ subunit (HEXA gene product), hydrolyzes both neutral and charged substrates. Only Hex A is able to hydrolyze the most important natural substrate, the acidic glycolipid GM 2 ganglioside. Mutations in the HEXA gene cause Tay-Sachs disease (TSD), a GM 2 ganglioside storage disorder. We investigated the role of putative active site residues Asp-␣258, Glu-␣307, Glu-␣323, and Glu-␣462 in the ␣ subunit of Hex A. A mutation at codon 258 which we described was associated with the TSD B1 phenotype, characterized by the presence of normal amounts of mature but catalytically inactive enzyme. TSD-B1 mutations are believed to involve substitutions of residues at the enzyme active site. Glu-␣307, Glu-␣323, and Glu-␣462 were predicted to be active site residues by homology studies and hydrophobic cluster analysis. We used sitedirected mutagenesis and expression in a novel transformed human fetal TSD neuroglial (TSD-NG) cell line (with very low levels of endogenous Hex A activity), to study the effects of mutation at candidate active site residues. Mutant HEXA cDNAs carrying conservative or isofunctional substitutions at these positions were expressed in TSD-NG cells. ␣E323D, ␣E462D, and ␣D258N cDNAs produced normally processed peptide chains with drastically reduced activity toward the ␣ subunitspecific substrate 4MUGS. The ␣E307D cDNA produced a precursor peptide with significant catalytic activity. Kinetic analysis of enzymes carrying mutations at Glu-␣323 and Asp-␣258 (reported earlier by Bayleran, J., Hechtman, P., Kolodny, E., and Kaback, M. (1987) Am. J. Hum. Genet. 41, 532-548) indicated no significant change in substrate binding properties. Our data, viewed in the context of homology studies and modeling, and studies with suicide substrates, suggest that Glu-␣323 and Asp-␣258 are active site residues and that Glu-␣323 is involved in catalysis.

We previously described the cloning of the late gestation lung 1 gene (LGL1), a novel glucocorticoid-inducible gene expressed in the mesenchyme of fetal lung. We report here evidence for a role of the LGL1 gene product (lgl1) in fetal rat lung airway branching morphogenesis, temporal and spatial localization of LGL1 mRNA and lgl1 protein in fetal rat lung, and a correction of the previously published LGL1 sequence. Both the mRNA and protein were detected during fetal lung development. LGL1 mRNA was detected from gestational Day 12 by reverse transcriptase-polymerase chain reaction, and from Day 13 by in situ hybridization. lgl1 protein was detected from Day 18 by Western analysis and from Day 16 by immunohistochemistry. The types of cells expressing LGL1 mRNA and lgl1 protein were assessed by immunohistochemical staining of adjacent serial tissue sections for markers of mesenchymal (vimentin) and smooth muscle (alpha-actin) cells. As gestation advanced, increasing amounts of mRNA and protein were expressed in these cells. In support of a role for lgl1 in airway branching morphogenesis, antisense (but neither sense nor scrambled) oligodeoxynucleotides directed against LGL1 inhibited airway branching in fetal rat lung buds in explant culture, in a dose- and time-dependent manner. The levels of lgl1 protein and LGL1 mRNA expression were decreased in those explants that had inhibited airway branching, compared with the uninhibited controls. Our findings suggest that lgl1 plays an important role in fetal airway branching morphogenesis.

Tay-Sachs disease (TSD) is an autosomal recessive, progressive, and fatal neurodegenerative disorder. Within the last 25 years, the discovery of the enzymatic basis of the disease, the deficiency of the enzyme hexosaminidase A, has made possible both enzymatic diagnosis of TSD and heterozygote identification. TSD is the first genetic condition for which a community-based heterozygote screening program was attempted with the intention of reducing the incidence of a genetic disease. In this article we review the clinical, biochemical, and molecular features of TSD as well as the development of laboratory technology that has been deployed in community genetic screening programs. We describe the assay procedures used and some of the limitations in their accuracy. We consider the impact of DNA-based technology on the process of identification of individuals carrying mutant genes associated with TSD and we discuss the social context within which genetic screening occurs.

Secreted glycoproteins serve a variety of functions related to cell-cell communication in developmental systems. We cloned LGL1, a novel glucocorticoid-inducible gene in foetal lung, and described its temporal and spatial localization in the rat. Disruption of foetal mesenchyme-specific LGL1 expression using antisense oligodeoxynucleotides, which was associated with a 50% decrease in lgl1 protein levels, inhibited airway epithelial branching in foetal rat gestational day 13 lung buds in explant culture. These findings suggested that lgl1 functions as a secreted signalling molecule. We now provide evidence supporting a role for lgl1 in mesenchymal-epithelial interactions that govern lung organogenesis. Lgl1 is a secreted glycoprotein with a conserved N-terminal secretory signal peptide. Using dual immunofluorescence, intracellular lgl1 was found to co-localize with markers of the Golgi apparatus and endoplasmic reticulum, consistent with its association with secretory vesicles. Using pulse-chase studies, we show that lgl1 is a stable protein with a half-life of 11.5 h. Furthermore, at gestational days 20 and 21 (term=22), foetal distal lung epithelial cells import lgl1 protein. Taken together, our findings support distinct roles for lgl1 as a mediator of glucocorticoid-induced mesenchymal-epithelial interactions in early and late foetal lung organogenesis.