The hook protein of Borrelia burgdorferi, encoded by the flgE gene, is serologically recognized in Lyme disease.

The periplasmic flagellum of Borrelia burgdorferi consists of a unipeptide flagellar filament, a hook, and a basal body. Here, we report the cloning and expression of the hook gene, flgE, of B. burgdorferi N40. The flgE gene is 1,119 nucleotides long and is located on the 950-kb linear chromosome of B. burgdorferi. The primary protein sequence of FlgE shows 73% similarity to the FlgE protein of Treponema phagedenis and approximately 50% similarity to the FlgG proteins of both gram-positive and gram-negative bacteria. The flgE gene was cloned into an Escherichia coli expression plasmid, pMX, to produce FlgE protein. Subsequently, FlgE murine antiserum was prepared by immunizing mice with the partially purified B. burgdorferi FlgE protein. By Western blot (immunoblot) analysis, the antiserum was found to react with a 40-kDa peptide in the whole-cell lysates, confirming the expression of the flgE gene in B. burgdorferi. Additionally, antibodies to FlgE were found in serum specimens from 19 of 42 patients with Lyme disease. Moreover, when other antigens, including 41G (the immunodominant domain of flagellin), OspE, OspF, and p22, were used to test for the development of corresponding antibodies in these patients, 67% of these patients (28 of 42) reacted to at least one of these five antigens, suggesting that a combination of FlgE with other available B. burgdorferi recombinant proteins is a good candidate for substrates in assays to aid in the diagnosis of Lyme disease.

Midkine is expressed early in rat fetal adrenal development.

Adrenal gland development is complex and poorly understood at the molecular level. Only a subset of patients with adrenal hypoplasia congenita (AHC) carry mutations in DAX1, a member of the nuclear hormone receptor superfamily. Therefore we set out to identify other candidate genes responsible for AHC by characterizing genes involved in fetal adrenal development. To identify these genes, we studied the differential expression of genes in fetal rat adrenals comparing tissues at 14 and 15 days postcoitum (dpc) since this period encompasses major morphological change in rat adrenal development. Fetal rat adrenals were dissected, cDNAs were prepared, and suppressive subtractive hybridization was performed. We isolated 126 clones of putatively differentially expressed clones and approximately 250 bp of each of the clones was sequenced. The most interesting putative developmental genes were examined. One member of the extracellular PTN/MDK (pleiotrophin/midkine) heparin-binding protein family involved in regulation of growth and differentiation was selected for initial study. We obtained full-length transcript by 3' rapid amplification of cDNA ends and performed Northern analysis on rat adrenal RNA from fetuses at 13, 14, 15, 17, and 19 dpc and newborns. Results from those analyses demonstrated the highest Mdk expression at days 13 and 14 followed by a moderate decrease of expression during the fetal stages thereafter. In the newborn, Mdk expression is nearly undetectable. Our results indicate that Mdk has a very specific pattern of fetal expression in the adrenals. We conclude that Mdk is involved early in fetal development of the rat adrenal. Therefore, MDK is a candidate gene for AHC not due to DAX1 mutations.

Up-regulation of WNT-4 signaling and dosage-sensitive sex reversal in humans.

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.