A recessive mutation leading to vertebral ankylosis in zebrafish is associated with amino acid alterations in the homologue of the human membrane-associated guanylate kinase DLG3.

We describe the characterization of the zebrafish homologue of the human gene DLG3. The zebrafish dlg3 gene encodes a membrane-associated guanylate kinase containing a single PDZ domain. This gene was cloned using a gene-trap construct inserted in the gene's first intron. The insertion co-segregates with a viable mutation called humpback (hmp), which leads to formation of ankylotic vertebrae in adult fishes. Insertion and mutation have both been mapped to chromosome 12, in a segment which is syntenic with region p12 to q12 of human chromosome 17. The hmp mutant phenotype, however, appears to be due to two point mutations in the guanylate kinase domain rather than to the transgene insertion itself. The results of this study are discussed in the light of the possible function of the guanylate kinase domain.     

Interfacial transfer kinetics of NO2 into pulmonary epithelial lining fluid.

Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.     

Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map

The ease of isolating mutations in zebrafish will contribute to an understanding of a variety of processes common to all vertebrates. To facilitate genetic analysis of such mutations, we have identified DNA polymorphisms closely linked to each of the 25 centromeres of zebrafish, placed centromeres on the linkage map, increased the number of mapped PCR-based markers to 652, and consolidated the number of linkage groups to the number of chromosomes. This work makes possible centromere-linkage analysis, a novel, rapid method to assign mutations to a specific linkage group using half-tetrads.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster

An ovary transplanted from a Drosophila melanogaster female into a male will mature and form morphologically normal yolk-filled oocytes. Since it has been supposed that the yolk polypeptides come only from the female fat body, it was hypothesized that the implanted ovary induces the fat body of the male host to synthesize and secrete yolk polypeptides (YPs). To test this hypothesis, fat body preparations from females, untreated males, and males containing transplanted ovaries were cultured in vitro with ³⁵S-methionine and the medium was examined for the presence of newly labeled YPs. Female fat body secreted newly labeled YPs, but no freshly synthesized YPs were secreted by fat bodies from untreated males or from males containing transplanted ovaries. In vitro cultured ovaries, however, both from females and from male hosts did secrete newly synthesized YPs. Therefore, the YPs in an ovary that matured in a male come mainly from endogenous synthesis by the implanted ovary. To find whether males were responsive to the hormones that stimulate YP production in isolated female abdomens, we treated males with the juvenile hormone analogue ZR-515 and with 20-hydroxyecdysone. The latter, but not the former, was able to cause synthesis and secretion of three bands migrating precisely as YPs in SDS gels. Partial peptide digests of the 20-hydroxyecdysone-stimulated polypeptides in males showed them to be identical with those stimulated by 20-hydroxyecdysone or ZR-515 in isolated female abdomens and with the three YPs found in normal female hemolymph. Finally, YP synthesis was assayed in mutants that affect the phenotypic sex of a fly. It was found that flies bearing two X chromosomes and the mutations dsx, dsx^D, ix or three sets of autosomes continued to make YPs, but tra-3-pseudomales did not. These results suggest that the process of sex determination involves steps leading to synthesis of an ecdysteroid in females, which then activates synthesis of the YPs by the fat body. A hypothesis is suggested to explain the fact that two different hormones can stimulate YP synthesis and two different organs can synthesize YPs.     

The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Nonvitellogenic female sterile mutants and the regulation of vitellogenesis in Drosophila melanogaster.

The genetic and endocrine regulation of vitellogenesis was investigated by studying 18 female sterile mutations that disrupt the development of normal vitellogenic follicles. Applications of exogenous juvenile hormone analog and reciprocal ovarian transplants between flies of different genotypes were employed to accomplish our first two objectives: to find (1) whether the mutation blocked development of the ovary directly, and (2) whether the mutation altered the hormonal milieu. In 15 of the mutants the developmental defect was localized to the ovary, but in the other 3 the ovary was competent to respond to a permissive environment. The internal milieu of these three mutants (ap⁴, fs(3)A1, fs(2)A18) was unable to provoke normal development in wild-type ovaries, suggesting that these mutations cause endocrine defects. Our third objective was to find whether an endocrine organ was itself defective in any of these mutants. The corpus allatum from two of the mutants was unable to provoke vitellogenesis in isolated wild-type abdomens, but corpora allata from wild-type females or from other mutants were able to promote maturation of ovarian follicles in isolated abdomens. Our fourth objective was to find whether any of the mutants were able to produce yolk proteins. Immunoelectrophoresis of fly hemolymph demonstrated that in all mutants tested vitellogenins were found in the blood. These experiments permit four main conclusions. First, they identify the first Drosophila mutants in which an endocrine gland is shown to be intrinsically defective during adulthood. Second, they show that the production of morphologically normal late previtellogenic follicles is not required for the induction of vitellogenin synthesis and secretion. Third, they show that juvenile hormone can cause ovarian follicles to sequester yolk in mutant flies. And finally, they show that mutants with defective corpora allata still synthesize and secrete vitellogenin. Taken together, these conclusions suggest that in Drosophila melanogaster the uptake of vitellogenin into follicles depends upon the availability of juvenile hormone, but that the synthesis and secretion of vitellogenin are independent of both normal ovaries and totally normal corpora allata.