Interactions of thePolycomb group of genes with homeotic loci ofDrosophila

Summary Members of thePolycomb (Pc) group of genes are required for the correct determination of segment identity, and are thought to be negative regulators of thebithorax andAntennapedia complexes. This hypothesis has been tested molecularly for only some members of thePc group. Here, we examine the distribution ofUltrabithorax (Ubx),Antennapedia (Antp), andSex combs reduced (Scr) proteins in the epidermis, central nervous system, and midgut of embryos homozygous for mutations in tenPc group genes. We show that zygotic loss of mostPc group genes causes ectopic expression ofUbx andAntp, but that there are differences in time and tissue-specificity. FivePc group mutations lack midgut constrictions and also exhibit ectopic or suppressedUbx expression and suppression ofAntp expression. Distribution ofAntp is upset earlier than distribution ofUbx in the central nervous system of everyPc group mutant affecting both genes. Loss of the zygotic products ofPolycomb, extra sex combs, andAdditional sex combs cause ectopic expression ofScr in epidermis, and allPc group genes exceptPsc have suppressedScr expression in the nervous system. These results are discussed with respect to the function of thePc group.     

Trithorax: A new homoeotic mutation of Drosophila melanogaster causing transformations of abdominal and thoracic imaginal segments

Summary A new homoeotic mutation, trithorax ¹ (trx ¹), of Drosophila melanogaster is described which causes intersegmental transformations in both the adult thorax and abdomen. Specifically, the metathorax and ventral prothorax are partially transformed to mesothroax, whilst abdominal segments are partially transformed towards the first abdominal segment. Three variables are shown to influence the expressivity of the mutant phenotype: (1) presence of wild type copies of the gene in the female parent; (2) culture temperature during the first four hours of embryogenesis; (3) exposure of embryos to ether vapour at the blastoderm stage. In addition it is shown that phenocopies of the mutant phenotype can be induced by ether treatment of heterozygous trx ¹ embryos with a frequency higher than that of bithorax phenocopies in wild type embryos.These findings are together taken to imply that the mutated gene functions early during embryogenests, and that a common mechanism underlies the determination of all the thoracic and abdominal segments.     

Positive and negative cis-regulatory elements in the bithoraxoid region of the Drosophila Ultrabithorax gene

Summary The Ultrabithorax (Ubx) gene is required during embryogenesis and larval development to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid (bxd) region, a 40 kb DNA stretch upstream of the Ubx promoter, affect cis-regulatory elements responsible for the ectodermal expression of the Ubx gene in the posterior compartment of the third thoracic segment and anterior compartment of the first abdominal segment. Our genetic data and the available molecular information are used to map the adult epidermal cis-regulatory elements within the bxd region. Genetic combinations involving mutations affecting the bxd region show that (1) redundant or cooperatively acting sequences are required for Ubx gene expression in the anterior compartment of the first abdominal segment, and (2) the expression of Ubx in the posterior compartment of the third thoracic segment is modulated by positive and negative cis-regulatory elements.The Wellcome Trust CRC Institute for Cancer Research and Developmental Biology, Tennis Court Road Cambridge, CB2 1QR, UKDivision de Genética, Departamento de Genética Molecular y Microbiología, Campus de San Juan, Apdo. 374, 03080 Alicante, Spain     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5 translated region. The 5 flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.     

Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity

Summary We evaluated the effects of vawrying aqueous Cl^– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl^– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl^– concentrations and/or PGO on the relation between holding voltageV _H -mV) and open-time probability (P _o).Reducingcis Cl^– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P _v) with a half-maximal reduction inP _o atcis Cl^–170mM. Channel activity was sustained by equimolar replacement ofcis Cl^– with F^–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl^– concentration andP ₀ at 10mm Cl^–. Reducingcis Cl^– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl^– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl^– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl^– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl^– channels.     

The use of the S haplotype-specific F-box protein gene,SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume)

Japanese apricot (Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene (SFB), a candidate gene for pollen-S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5- and 3-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB¹ and Pm-SFB⁷ of 'Nanko (S¹S⁷)'. As in the case of SFB of other Prunus species, Pm-SFB¹ and Pm-SFB⁷ showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S¹- to S⁸-haplotypes as well as a self-compatible S^f-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.Communicated by H.F. LinskensThe nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank/DDBJ database under accession numbers, AB101440 and AB101441, for SFB¹ and SFB⁷, respectively     

Steric Enhancement of Imidazole Basicity incis-Urocanic Acid Derivatives: Models for the Action of Chymotrypsin

To test the hypothesis that substrate-induced steric compression between His 57 and Asp 102 at the active site of chymotrypsin can increase the basicity of His 57, we have synthesized thecis- andtrans-isomers of 2-bromo-3-(N-tritylimidazole)-2-propenoic acid and 2-chloro-3-(N-tritylimidazole)-2-propenoic acid and compared selected properties with those ofcis-andtrans-urocanic acids. Thecis-isomers display low field¹H NMR signals at 17 ppm in dimethylsulfoxide, similar tocis-urocanic acid; whereas thetrans-isomers do not show strong hydrogen bonds. Increasing the size of the C2 substituent (H < Cl < Br) in thecis-isomers increases the pK_aof the imidazolium group from 6.78 for H to 7.81 and 9.10 for Cl and Br, respectively; whereas the pK_as of thetransisomers are all 6.0 ± 0.1. The results indicate that thecis-urocanic acid derivatives with large substituents at C2 act as proton sponges in water, and they support the concept that steric compression in the catalytic triad of chymotrypsin can increase the basicity of His 57.     

Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements

We dissected the regulatory region of the AVP1 gene encoding the vacuolar H⁺-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen, and imply the biological significance of the V-PPase in pollen maturation.     

TheUbx syndrome ofDrosophila: the prothoracic transformation (ppx) is independent ofbx,bxd andpbx

Summary If, early in development, theUbx ⁺ gene is removed by mitotic recombination from cells of the meso-and metathoracic leg primordia, theseUbx ^– cells develop as in the posterior prothoracic leg. We show that this transformation, termedpostprothorax, is a discrete genetic function that is independent of other homeotic transformations such asbx, pbx orbxd, which also result from the inactivation of theUbx gene.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Chromosomal location of a gene (idg) that specifies production of the blue pigment indigoidine inErwinia chrysanthemi

Production of a blue pigment, indigoidine, is a variable trait among wild-type strains ofErwinia chrysanthemi; it is also influenced by the composition of the growth medium. Starting with a nonpigmented wild-type strain (ICPB EC183) ofE. chrysanthemi, we obtained by nitrosoguanidine mutagenesis a pigmented (idg ⁺) mutant strain (AC6055), which simultaneously was Arg^–. Linkage betweenarg andidg was established in two-factor transductional and conjugational crosses. Coinheritance ofidg withhis ⁺,ilv ⁺,leu ⁺,ser ⁺,thr ⁺, orura ⁺ transductants was not observed. Spontaneous Arg⁺ revertants of AC6055 were invariably Idg⁺. The pigments produced by AC6055, Idg⁺ recombinants, and wild-type Idg⁺ strains were identical, judged by absorption spectra (_max = 615 nm) of the dimethylsulfoxide extracts of whole cells. We concluded that nitrosoguanidine caused comutation in thearg andidg loci that are linked on theE. chrysanthemi chromosome.     

Production of the taste/odor-causing compound,trans-2,cis-6-nonadienal,within the Synurophyceae

Although several species of the Synurophyceae have been associated with taste and odor problems in potable water supplies, electron microscopic-based field studies linked problematic blooms only toSynura petersenii Korshikov. Eventually, the organic compoundtrans-2,cis-6-nonadienal was implicated to cause the associated cucumberlike odors. The objective of this study was to survey unialgal cultures of various Synurophycean species for the occurrence oftrans-2,cis-6-nonadienal. The compound was detected throughout a 24-day growth assay with aS. petersenii isolate, but was not detected in an identical assay withSynura sphagnicola (Korshikov) Korshikov. In separate 24-day cultures,trans-2,cis-6-nonadienal was detected in two isolates from theS. petersenii species complex, but was not detected in isolates of twoMallomonas or fourSynura taxa not from theS. petersenii complex. These results support the hypothesis that production oftrans-2,cis-6-nonadienal is unique to taxa within theS. petersenii complex. When contrast-enhancing optics and specific specimen preparation techniques are employed, light microscopy can be used to distinguish taxa in theS. petersenii complex from all other Synurophycean taxa. These methods are suggested as an efficient way to monitortrans-2,cis-6-nonadienal-producing taxa in potable water supplies.Author for correspondence     

Interaction of non-allelic loci in expression of the extra-sexcomb phenotype inDrosophila melanogaster

Summary The expression of the extra-sexcomb phenotype is enhanced in males that are doubly heterozygous for mutants from two non-allelic loci,extra-sexcomb (Polycomb, Extra-sexcomb) andbithorax (bithorax, bithoraxoid, Ultrabithorax) as compared to the expression of the extra-sexcomb mutants when no bithorax mutants are present in the genotype. If the bithorax mutants are recessive, the expression is usually greater when the non-allelic mutants are cis-hererozygotes than when they are trans-heterozygotes, but there is no difference between cis-and trans-heterozygotes in extra-sexcomb andUltrabithorax compounds.This work, begun while the author was a member of the staff of the Zoology Dept. of the University of California at Berkeley, was completed, with the assistance of a National Institutes of Health research grant (RG-6754), in the Dept. of Genetics of the University of Turku.     

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens

Immune response (Ir) genes mapping in theI region of the mouseH-2 complex appear to regulate specifically the presentation of a number of antigens by macrophages to proliferating T cells. We have investigated the possibility that similarIr genes mapping in theH-2K andH-2D regions specifically regulate the presentation of target antigens to cytotoxic effector T cells. We report that the susceptibility of targets expressing specific non-H-2 H alloantigens to lysis by H-2-compatible, H-antigen-specific cytotoxic effector T cells is controlled by polymorphicH-2K/D genes. This control of susceptibility to lysis is accomplished through what we have defined operationally as antigen-specific regulation of non-H-2 H antigen immunogenicity. High immunogenicity of the H-4.2 alloantigen is determined by a gene mapping in theH-2K region ofH-2 ^b . However, high immunogenicity of H-7.1 is determined by a gene mapping in theH-2D region ofH-2 ^b . High immunogenicity of the H-3.1 alloantigen is determined by genes mapping in both theH-2K andH-2D regions ofH-2 ^b . Therefore, genes mapping in theH-2K andH-2D regions serve a function in presenting antigen to cytotoxic effector T cells. This function is analogous to that played byI-regionIr genes expressed in macrophages which present antigen to proliferating T cells. We present arguments for classification of theseH-2K/D genes as a second system ofIr genes and discuss the implications of twoH-2-linkedIr-gene systems, their possible functions, and their evolution.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.