Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.     

Interaction of the Stubble-Stubbloid Locus and the Broad-Complex of Drosophila Melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third instar. The region is coextensive with a complex genetic locus, the Broad-Complex (BR-C). The BR-C is a regulatory gene that contains two major functional domains, the br domain and the l(1)2Bc domain. BR-C mutants prevent metamorphosis, including morphogenesis of imaginal discs; br mutants prevent elongation and eversion of appendages and l(1)2Bc mutants prevent fusion of the discs. The Stubble-stubbloid (Sb-sbd) locus at 89B9-10 is best known for the effects of its mutants on bristle structure. Mutants of the BR-C and the Sb-sbd locus interact to produce severe malformation of appendages. Viable heteroallelic and homoallelic combinations of Sb-sbd mutants, including loss-of-function mutants, affect the elongation of imaginal disc appendages. Thus, the Sb-sbd(+) product is essential for normal appendage elongation. Sb-sbd mutants, however, do not affect eversion or fusion of discs. Correspondingly, only BR-C mutants deficient in br function interact with Sb-sbd mutants. The interaction occurs in deficiency heterozygotes using single, wild-type doses of the BR-C, of the Sb-sbd locus, or of both loci. These last results are formally consistent with the possibility that the BR-C acts as a positive regulator of the Sb-sbd locus. The data do not exclude other possible nonregulatory interactions between the two loci, e.g., interactions between the products of both genes.     

Inhibition of acetate and propionate assimilation by itaconate via propionyl-CoA carboxylase in isocitrate lyase-negative purple bacterium Rhodospirillum rubrum

The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741. The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned. The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified. Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively. The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes. Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E. coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate. At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E. coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate. Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case. This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD.     

The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. III. Hypomorphic and hypermorphic mutations affecting the expression of hairless

A lethal locus (l(2)br7;35B6-10), near Adh on chromosome arm 2L of D. melanogaster, is identified with Plunkett's dominant suppressor of Hairless (H). Of eight new alleles, seven act as dominant suppressors of H, the eighth is a dominant enhancer of H. One of the suppressor alleles is both a leaky lethal and a weak suppressor of H. Confirming Nash (1970), deletions of l(2)br7 are dominant suppressors, and duplications are dominant enhancers of H. A simple model is proposed to account for the interaction of l(2)br7 and H, assuming that amorphic (or hypomorphic) alleles of l(2)br7 suppress H and that hypermorphic alleles enhance H.     

Multifactorial genetics of exencephaly in SELH/Bc mice

BACKGROUND: The SELH/Bc mouse strain has 10-30% exencephaly and is an animal model for human neural tube closure defects. This study examined the number of causative genes, their dominance relationships, and linkage map positions. METHODS: The SELH/Bc strain (S) was crossed to the normal LM/Bc strain (L) and frequencies of exencephaly were observed in the F(1), BC(1), and F(2) generations. 102 F(2) males were individually testcrossed by SELH/Bc. The extremes, the 10 highest and 10 zero exencephaly-producing F(2) sires, were typed for 109 SSLP marker loci in a genome screen. Next, the resultant five provisional chromosomal regions were tested for linkage in 31 F(2) exencephalic embryos. Finally, 12 males, SS or LL for the Chr 13 region on an LM/Bc background, were testcrossed by SELH/Bc. RESULTS: The exencephaly frequencies in the F(1) (0.3%), BC(1) (4.4%), and F(2) (3.7%), and the distribution of F(2) males' testcross values (0-15.5%), indicated that the high risk of exencephaly in SELH/Bc is due to the cumulative effect of two or three loci. Linkage studies indicated the location of semidominant exencephaly-risk genes on Chr 13 near D13Mit13 (P < 0.001), Chr 5 near D5Mit168 (P < 0.025), and possibly Chr 11 near D11Mit10 (P < 0.07). The gene on Chr 13, Exen1, and the strong role of other loci were confirmed by the congenic males. CONCLUSIONS: The high risk of exencephaly in SELH/Bc mice is caused by the cumulative effect of two to three semidominant genes. Candidate genes include Msx2, Madh5, Ptch, and Irx1 (Chr 13) and Actb and Rac1 (Chr 5).     

Direct effect of PGF2α pulses on PRL pulses, based on inhibition of PRL or PGF2α secretion in heifers

The relationships between PRL and PGF_2α and their effect on luteolysis were studied. Heifers were treated with a dopamine-receptor agonist (bromocriptine; Bc) and a Cox-1 and -2 inhibitor (flunixin meglumine [FM]) to inhibit PRL and PGF_2α, respectively. The Bc was given (Hour 0) when ongoing luteolysis was indicated by a 12.5% reduction in CL area (cm²) from the area on Day 14 postovulation, and FM was given at Hours 0, 4, and 8. Blood samples were collected every 8-h beginning on Day 14 until Hour 48 and hourly for Hours 0 to 12. Three groups of heifers in ongoing luteolysis were used: control (n = 7), Bc (n = 7), and FM (n = 4). Treatment with Bc decreased (P < 0.003) the PRL concentrations averaged over Hours 1 to 12. During the greatest decrease in PRL (Hours 2-6), LH concentrations were increased. Progesterone concentrations averaged over hours were greater (P < 0.05) in the Bc group than in the controls. In the FM group, no PGFM pulses were detected, and PRL concentrations were reduced. Concentrations of PGFM were not reduced in the Bc group, despite the reduction in PRL. Results supported the hypothesis that a decrease (12.5%) in CL area (cm²) is more efficient in targeting ongoing luteolysis (63%) than using any day from Days 14 to ≥19 (efficiency/day, 10-24%). The hypothesis that PRL has a role in luteolysis was supported but was confounded by the known positive effect of LH on progesterone. The hypothesis was supported that the synchrony of PGFM and PRL pulses represents a positive effect of PGF_2α on PRL, rather than an effect of PRL on PGF_2α.     

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. VI. Induced Revertants of Scutoid

Twenty-six induced revertants of Scutoid (Sco), a dominant mutation of Drosophila melanogaster, have been characterized genetically. Sco is an unusual mutation, involving two small reciprocal transpositions within the region 35A4 to 35C5 of chromosome arm 2L. One of these transpositions juxtaposes the noc and l(2)br28 loci. We suggested previously that the Sco phenotype results from the "fusion" of noc and l(2)br28. In support of this idea we now show that 23 of 26 revertants of Sco are noc^-, indeed the majority are either chromosome aberrations broken between noc and l(2)br28 or deletions of these loci from the mutant chromosome. However, some revertants of Sco are rather more complex, and their properties suggest an interaction between the pu-noc and l(2)br28-l(2)br37 regions of chromosome arm 2L and also demonstrate the genetic complexity of the el-noc region.     

苏云金杆菌毒素调控基因plcR的特性与表达

根据蜡状芽胞杆菌plcR基因和papR基因序列设计特异引物,对6个Bt菌株（WB1、WB7、WB9、HD98、8010、8311）及5个Bc菌株（6A1、6A2、6A3、6A4、6S1）进行了PCR检测.结果显示,3个Bt菌株及4个Bc菌株含有plcR-papR基因.克隆了Bt8010、Bc6A2和6A3的plcR、papR基因,核苷酸序列分析表明,三个菌株的plcR、papR基因与NCBI数据库中的Bt、Bc及Ba相应序列都有很高的相似性.Bt8010的plcR基因编码框由846个核苷酸组成,可编码282个氨基酸;papR基因的编码框由144个核苷酸组成,可编码48个氨基酸.推导的氨基酸序列分析表明,Bt8010 的PapR有21个氨基酸的信号肽序列,PlcR没有信号肽序列.与Bc6A2、6A3和Bc 569相比,Bt8010 的PlcR和PapR在氨基酸序列上与Bc 相应序列存在相对较大的差异.将plcR-papR基因连接到表达载体pHT304中,并转化至大肠杆菌JM109中成功进行了表达,为研究Bt plcR基因的功能奠定了基础.     

Studies of the effect of retinoic acid on anterior neural tube closure in mice genetically liable to exencephaly

Previously we have shown that all SELH/Bc mouse embryos close their anterior neural tubes by an abnormal mechanism and that 10-20% of SELH/Bc embryos are exencephalic. The purposes of these studies were (1) to observe the effects of retinoic acid on the frequency of exencephaly in SELH/Bc embryos; (2) to compare the SELH/Bc response with those of normal strains and of other neural tube mutants; and (3) to compare, between SELH/Bc and a normal strain (SWV/Bc), the effects of retinoic acid on morphology of the closing anterior neural tube. SELH/Bc was more liable to retinoic acid-induced exencephaly than were normal strains. After maternal treatment with 5 mg/kg retinoic acid on day 8.5 of gestation, 53% of SELH/Bc embryos had exencephaly, compared with 22% in ICR/Bc and 14% in SWV/Bc. When these results were transformed according to the assumptions of the developmental threshold model, the effects of genotype and retinoic acid appeared to be additive. Similar treatment on day 9 or 10 of gestation had little or no effect on the frequency of exencephaly in SELH/Bc mice. These results are similar to the reported responses of the curly-tail and Splotch mutants, where frequencies of spina bifida but not exencephaly were decreased. This pattern suggests that studies of effects of periconceptional vitamin treatment on risk of human neural tube defects should consider anencephaly and spina bifida separately. The study comparing the morphology of anterior neural tube closure in SELH/Bc and normal SWV/Bc embryos showed that retinoic acid delays the elevation of the mesencephalic neural folds. This results in a "stalling" of many embryos in the first steps of neural tube closure, with their neural folds remaining convex and splayed wide apart. The delay in fold elevation was superimposed on the different closure patterns of the two strains. The overall conclusion is that there is no nonadditive interaction in the parameters studied between retinoic acid treatment and the SELH/Bc genotype.     

Developmental study of neural tube closure in a mouse stock with a high incidence of exencephaly

About 17% of embryos and fetuses in the SELH/Bc mouse stock have the anterior neural tube defect, exencephaly. No other malformations are seen. The genetic liability to exencephaly was shown to be probably genetically fixed in the SELH/Bc stock. This means that SELH/Bc embryos with successful neural tube closure are genetically the same as exencephalics. Females were significantly more likely to be affected than males (66% females). The pattern of morphological developmental events during anterior neural tube closure on days 8 and 9 of gestation was compared among 322 ICR/Bc (normal), 304 SWV/Bc (normal), and 265 SELH/Bc embryos. Anterior neural tube closure was found to follow a strikingly different pattern in almost all SELH/Bc embryos than in either of the normal strains or in previous published studies. SELH/Bc embryos lack the initial contact between the anterior folds in the posterior prosencephalon/anterior mesencephalon region (Closure 2). In spite of this, all but 17% manage to close the anterior neural tube by extending caudally the later occurring normal anterior zone of contact and fusion at the most rostral aspect of the prosencephalon (Closure 3) through the region of Closure 2 to meet the zone of closure of the rhombencephalon, Closure 4. Anterior neural tube closure was completed late, and in some SELH/Bc embryos, elevation and fusion in the mesencephalon did not occur at all. In histological sections of six- and eight-somite embryos, elevated numbers of pyknotic cells in the neuroepithelium and mesenchyme, and elevated numbers of unstained inclusions in the neuroepithelium were found; but their relationship, if any, to the abnormal pattern of neural tube closure is not clear.     

Role of LH in the progesterone increase during the bromocriptine-induced prolactin decrease in heifers

The luteotrophic effect of bromocriptine in heifers was studied to determine if the reported posttreatment increase in progesterone (P4) just before or at the beginning of luteolysis was attributable to loss of a luteolytic effect of prolactin (PRL) or to the stimulation of LH, a known luteotropin. Four treatment groups (n = 7) were used: control (Ct), bromocriptine (Bc; 16 mg/heifer), acyline (Ac; 3 μg/kg), and bromocriptine and acyline combined (BcAc). Bromocriptine (inhibitor of PRL) and acyline (antagonist of GnRH and therefore blocker of LH) were given at Hour 0 on Day 16 postovulation, and blood samples were taken hourly at Hours 0 to 8. Concentration of P4 was greater (P < 0.05) in the Bc group than in the Ct group at each of Hours 1 to 8. Concentration of LH increased (P < 0.05) between Hours 0 to 2 in the Bc group but not in the other three groups. The peak of the first posttreatment LH pulse occurred earlier in the Bc group than in the Ct group. Average concentration of PRL was lower (P < 0.05) and number of PRL pulses was less (P < 0.05) in the Bc group than in the Ct group. Acyline inhibited LH in the Ac and BcAc groups as indicated by a decrease (P < 0.05) in concentration between Hours 0 and 2 and a decrease (P < 0.001) in number of pulses/heifer during the 8 h. A decrease in PRL but not an increase in P4 and LH occurred in the BcAc group. Results supported the hypothesis that the P4 increase associated with PRL suppression by bromocriptine treatment is attributable to an increase in LH.     

The dengue virus NS2B–NS3 protease retains the closed conformation in the complex with BPTI

The C-terminal β-hairpin of NS2B (NS2Bc) in the dengue virus NS2B–NS3 protease is required for full enzymatic activity. In crystal structures without inhibitor and in the complex with bovine pancreatic trypsin inhibitor (BPTI), NS2Bc is displaced from the active site. In contrast, nuclear magnetic resonance (NMR) studies in solution only ever showed NS2Bc in the enzymatically active closed conformation. Here we demonstrate by pseudocontact shifts from a lanthanide tag that NS2Bc remains in the closed conformation also in the complex with BPTI. Therefore, the closed conformation is the best template for drug discovery.     

Interactions of 2-methyladenines and poly(m2A) with poly(br5U)

Mixing curve experiments and melting curve analyses have shown that poly(m2A) forms complexes with poly(br5U) with stoichiometries of either 1:1 or 1:2 in high ionic strengths. CD spectra of poly(m2A).poly(br5U) and poly(m2A).2 poly(br5U) both resemble quite well to those of poly(A). poly(br5U) and poly(A).2poly(br5U), respectively. This suggests that the corresponding complexes are closely related in the structural details. Significant similarities of the CD spectra were observed for poly(m2A).2poly(br5U) and complexes between 2,9-dimethyladenine or 2-methyladenosine and poly(br5U) in the presence of spermine, indicating also the 1:2 stoichiometry. Thus, a methyl group at the position 2 of adenine ring is not necessarily hindering a formation of the Watson-Crick type base pairings.     

Kinetics of Apoptosis and Expression of Apoptosis-Related Proteins in Rat CA3 Hippocampus Cells After Experimental Diffuse Brain Injury

The present study examined kinetics of apoptosis and expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 in the CA3 hippocampus cells after diffuse brain injury (DBI) induced experimentally in rats. Percentage of apoptotic cells and expressions of above proteins were examined by flow cytometry and immunohistochemistry. Substantial neuronal apoptosis was documented in the CA3 hippocampus cells after DBI (22.26 ± 2.97 % at 72 h after DBI vs. 2.92 ± 0.88 % in sham-operated animals). Expression of Bc1-2 decreased, while expression of Bax and caspase-3 increased after DBI, with caspase-3 expression peaking after that of Bax (72 vs. 48 h, respectively). Further, the Bc1-2/Bax expression ratio decreased prior to increase of caspase-3 expression. In conclusion, cell apoptosis and altered expressions of Bcl-2, Bax, and caspase-3 are present in the CA3 region of hippocampus after experimental DBI. Changes in the Bc1-2/Bax expression ratio may facilitate activation of caspase-3 and aggravate neuronal apoptosis after brain injury.     

A comparative study of the regioselectivity of the β-galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N-Acetyl-lactosamine in aqueous media

The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the β-galactosidases from Kluyveromyces lactis (KlβGal) and Bacillus circulans (BcβGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcβGal. BcβGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L(-1), corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlβGal, whereas the regioselectivity of BcβGal proved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcβGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.     

Ionization and self-association of unconjugated bilirubin, determined by rapid solvent partition from chloroform, with further studies of bilirubin solubility.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. 29: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At 21-25 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(2pH-B) + Bc.10(4pH-D)]; the bracketed terms reflect P for H2Bo (diacid), HB- (monoanion), B= (dianion), and (B=)2 dimer, respectively. Computer-fitted values for constants (+/- SD) were: Po = P for H2Bo = 5.79 x 10(-5); A = pK1 = 8.12 +/- 0.23; B = pK1 + pK2 = 16.56 +/- 0.10; pK2 = 8.44 +/- 0.33; D = pk22 + 2(pK1 + pK2) -log(2Po) = 37.64 +/- 0.07, and k22 = 0.26 microM-1 [formation constant of (B=)2 dimer]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H2Bo was 66 nM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of the central nervous system in Drosophila

The actions of steroid hormones on vertebrate and invertebrate nervous systems include alterations in neuronal architecture, regulation of neuronal differentiation, and programmed cell death. In particular, central nervous system (CNS) metamorphosis in insects requires a precise pattern of exposure to the steroid molting hormone 20-hydroxyecdysone (ecdysterone). To test whether the effects of steroid hormones on the insect nervous system are due to changes in patterns of gene expression, we examined Drosophila mutants of the ecdysterone-regulated locus, the Broad Complex (BR-C). This report documents aspects of CNS reorganization which are dependent on BR-C function. During wild-type metamorphosis, CNS components undergo dramatic morphogenetic movements relative to each other and to the body wall. These movements, in particular, the separation of the subesophageal ganglion from the thoracic ganglion, the positioning of the developing visual system, and the fusion of right and left brain hemispheres, are deranged in BR-C mutants. In addition, a subset of mutants shows disorganization of optic lobe neuropil, both within and among optic lobe ganglia. Optic lobe disorganization is found in mutants of the br and l(1)2Bc complementation groups, but not in those of the rbp complementation group. This suggests that the three complementation groups of this complex locus represent distinct but overlapping functions necessary for normal CNS reorganization. This study demonstrates that ecdysterone-regulated gene expression is essential for CNS metamorphosis, illustrating the utility of Drosophila as a model system for investigating the genetic basis of steroid hormone action on the nervous system.