The engrailed locus of Drosophila: structural analysis of an embryonic transcript

cDNA clones originating from the engrailed gene of Drosophila have been isolated from recombinant phage libraries that were made using poly(A)+ RNA extracted from early embryos. The DNA sequence of one of these clones includes a homeo box, a 180 bp sequence present in several other Drosophila genes important in formation of body pattern during development. The homeo boxes found in the other Drosophila genes, as well as in cognate sequences from a wide range of segmented animals, including higher vertebrates, are highly conserved. By contrast, the homeo box within the engrailed gene diverges substantially and, unlike the other homeo boxes, is interrupted by an intervening sequence. The engrailed homeo box is located near the 3' end of a 1700 bp open reading frame. If translated, this sequence would produce a protein of unusual composition. We also show that a neighboring gene has a large region with strong homology to engrailed, and that it also contains a homeo box.     

Low genetic diversity of a high mountain burnet moth species in the Pyrenees

The burnet moth Zygaena anthyllidis, endemic to the high elevations of the Pyrenees, is vulnerable to land-use. In order to identify conservation priorities based on an assessment of genetic diversity within populations and gene flow among populations, we examined Z. anthyllidis’ genetic variability and differentiation based on allozyme electrophoresis from seven populations scattered across its entire range. In comparison to other mountain Lepidoptera, the populations studied exhibit a low level of genetic diversity. Remarkable between-population differentiation (F _ST = 0.053), the presence of private alleles, and the lack of significant isolation-by-distance pattern characterises the genetic make-up of the species. We interpreted the pattern of genetic differentiation as a consequence of low dispersal power in combination with insufficient landscape connectivity. Ongoing land-use change might reinforce genetic differentiation due to habitat fragmentation and additionally affect negatively allozyme variability at shifting range margins, i.e. the capacity to adapt to changing environments. We therefore suggest creating a network of suitable habitats at the landscape scale to facilitate genetic exchange and to conserve the species’ overall genetic variability.     

Molecular dissection of zyxin function reveals its involvement in cell motility

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.     

A RecA filament capping mechanism for RecX protein

The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA. Based on multiple observations, we propose that RecX has a RecA filament capping activity. This activity has predictable effects on the formation and disassembly of RecA filaments. In vivo, the RecX protein may limit the length of RecA filaments formed during recombinational DNA repair and other activities. RecX protein interacts directly with RecA protein, but appears to interact in a functionally significant manner only with RecA filaments bound to DNA.     

Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-P(i) cotransporter during ontogeny

We sought to characterize expression of an apically expressed intestinal Na-P(i) cotransporter (Na-P(i)-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-P(i) uptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8-9 mo of age. Na-P(i)-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-P(i) uptake and Na-P(i)-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-P(i) uptake and Na-P(i)-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-P(i) uptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-P(i)-IIb mRNA and protein expression. We also demonstrate that Na-P(i)-IIb is an N-linked glycoprotein and that glycosylation is age dependent.     

Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid Southwestern soils

Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. However, little is known about the distribution of biosurfactant-producing bacteria in the environment. The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites. A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R(2)A agar. The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose. Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested. The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates. Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a Flavobacterium sp. Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates). Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions. These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates. In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay. Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils.     

Neoglycolipids derived from phosphatidylethanolamine serve as probes in cell culture studies on glycolipid metabolism

The neoglycolipid (NeoGL) N-acetyl-1-deoxy-1-phosphatidylethanolamino lacto-N-tetraositol [Lc4Ose-PtdEtn(NAc)] and the radioactivly labeled analog [Lc4Ose-PtdEtn(N[14C]Ac)] were synthesized by coupling the corresponding oligosaccharide to phosphatidylethanolamine (dihexadecyl) via reductive amination and subsequent N-acetylation with unlabeled and [14C]acetic acid anhydride, respectively. Lc4Ose-PtdEtn(N[14C]Ac) was then incubated with homogenates of rat small intestine epithelial cells (IEC-6) at pH 4. The reaction products were shown to be the degradation products formed by glycosidases by fast atom bombardment mass spectrometry (FAB MS). On the other hand, incubation of Lc4Ose-PtdEtn(NAc) with IEC-6 cell homogenates in sialyltransferase assays yielded the corresponding sialylated product. When Lc4Ose-PtdEtn(N[14C]Ac) was fed to IEC-6 cells as BSA complex, up to 5% of the NeoGL administered were taken up by the cells. After extraction of the NeoGL and separation by thin layer chromatography (TLC) the catabolic products Lc3Ose-PtdEtn(N[14C]Ac), Lac-PtdEtn(N[14C]Ac), and Glc-PtdEtn(N[14C]Ac), as well as the main anabolic product NeuGc-Lc4Ose-PtdEtn(N[14C]Ac) could be identified by FAB MS. These results demonstrate that PtdEtn-derived NeoGL can be used as probes for studies on the metabolism of specific oligosaccharide structures in cell culture.     

Plasmids Responsible for Horizontal Transfer of Naphthalene Catabolism Genes between Bacteria at a Coal Tar-Contaminated Site Are Homologous to pDTG1 from Pseudomonas putida NCIB 9816-4

The presence of a highly conserved nahAc allele among phylogenetically diverse bacteria carrying naphthalene-catabolic plasmids provided evidence for in situ horizontal gene transfer at a coal tar-contaminated site (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330–2337, 1997). The objective of the present study was to identify and characterize the different-sized naphthalene-catabolic plasmids in order to determine the probable mechanism of horizontal transfer of the nahAc gene in situ. Filter matings between naphthalene-degrading bacterial isolates and their cured progeny revealed that the naphthalene-catabolic plasmids were self-transmissible. Limited interstrain transfer was also found. Analysis of the restriction fragment length polymorphism (RFLP) patterns indicated that catabolic plasmids from 12 site-derived isolates were closely related to each other and to the naphthalene-catabolic plasmid (pDTG1) of Pseudomonas putida NCIB 9816-4, which was isolated decades ago in Bangor, Wales. The similarity among all site-derived naphthalene-catabolic plasmids and pDTG1 was confirmed by using the entire pDTG1 plasmid as a probe in Southern hybridizations. Two distinct but similar naphthalene-catabolic plasmids were retrieved directly from the microbial community indigenous to the contaminated site in a filter mating by using a cured, rifampin-resistant site-derived isolate as the recipient. RFLP patterns and Southern hybridization showed that both of these newly retrieved plasmids, like the isolate-derived plasmids, were closely related to pDTG1. These data indicate that a pDTG1-like plasmid is the mobile genetic element responsible for transferring naphthalene-catabolic genes among bacteria in situ. The pervasiveness and persistence of this naphthalene-catabolic plasmid suggest that it may have played a role in the adaptation of this microbial community to the coal tar contamination at our study site.     

Genetic determination of osteoporosis: lessons learned from a large genome-wide linkage study

Osteoporosis is a common disease with strong genetic control. We performed an autosomal linkage scan in a large pedigree-based sample of 4,498 subjects for a composite osteoporosis phenotype that combines osteoporotic fracture (OF) and low bone mineral density (BMD). All of the subjects were U.S. Caucasians recruited in the Omaha area of Nebraska. Sex-specific linkage analyses and autosomal imprinting analyses were also conducted. For conventional linkage analyses in the total sample, we identified suggestive linkage on chromosomes 14q32 (LOD = 2.61), 7p14 (LOD = 2.42), and 11q25 (LOD = 2.09). In female subjects a significant linkage signal was detected on chromosome 14q22 (LOD = 3.53) and another two peaks were detected on chromosomes 7p14 (LOD = 3.07) and 9p21 (LOD = 2.29). Suggestive evidence of imprinted loci was found with paternally derived alleles on chromosomes 1q42 (LOD = 2.12) and 9q34 (LOD = 1.88). Some evidence of linkage to maternally derived alleles was found on chromosome 7q22 (LOD = 1.67). Our study provides new clues to osteoporosis genetic research and for the first time suggests that genomic imprinting effects may play a role in the etiology of osteoporosis.