Genetic and functional analysis of tryptophan transport in Malpighian tubules of Drosophila

Dissected Malpighian tubules from wild type and the eye color mutant white of Drosophila were compared with respect to their abilities to transport tryptophan and kynurenine into tubule cells. It was determined that mutation at white greatly impairs the ability of Malpighian tubule cells to take up tryptophan. Functional studies on the extracellular spaces and ultrastructural observations indicated no differences in these respects between wild type and white tubules. It is consistent with several observations that much of the tryptophan associated with white exists in the intercellular spaces. Furthermore, the uptake of tryptophan by the w ⁺ system of wild type tubules is inhibited by the analogue 5-methyl-tryptophan. However, the incorporation of radioactive tryptophan into protein in tubule cells from wild type and white occurs at the same rates and is not affected by 5-methyl-tryptophan. Therefore, it is apparent that Malpighian tubules have a transport system that enables entry of tryptophan into a cellular pool and that this cellular pool is initially independent of the tryptophan pool used for protein synthesis. The mutant white lacks this transport system. From these studies and others it appears that compartmentalization of cellular pools may be brought about via the utilization of specific membrane transport systems.     

Molecular characteristic of stable and unstable <Emphasis Type="Italic">white</Emphasis> gene alleles in highly mutable lines from natural <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> populations

Mutations in the white locus emerged in highly mutable isofemale Drosophila melanogaster lines from the populations of Novosibirsk 2013 (NS3 line), Nalchik 2014 (N119 line), and Sakhalin Island 2014 (S46 line). A single white-eyed male found in the NS3 line was sterile. Phenotypically mutant derivatives (white gene alleles) differing in eye color (pure white, different shades of yellow (honey), orange (apricot), cherry, and red (wild type)) emerged during the N119 and S46 line breeding in the laboratory. Molecular genetic study of the structure of wild type white locus in initial lines and white-mutant derivatives de novo emerging from them, as well as other white lines from the fund of the Laboratory of Population Genetics of the Institute of Cytology and Genetics (Siberian Branch, Russian Academy of Sciences), was conducted. The pairs of primers flanking different white gene regions were selected. Six such pairs overlapped the coding part of the gene. Molecular genetic analysis demonstrated that most DNA defects were limited to the region which includes the first exon (34 lines). Among them, four mutant events were accompanied by an insertion of DNA fragments of approximately 800 bp; one mutation event was accompanied by a deletion of approximately 200 bp; in 29 cases, no PCR product was obtained (this can indicate that as a minimum one of the primer binding sites is damaged). The inserted DNA fragments have no homology with known D. melanogaster sequences presented in the NCBI database. The complete white gene deletion with the manifestation of mutant “white eyes” phenotype was registered in four cases (and only in the N119 line derivatives). Normal PCR product was obtained in 22 cases for all six DNA fragments. Among them, there are both alleles phenotypically mutant by the eye color (white, cherry, or orange) and revertants to the wild type (red). The abundance of defects in the beginning of the gene can indicate a multiplicity of mobile genetic element insertion sites in this part of the white gene in D. melanogaster.     

Human Matrix Attachment Regions Insulate Transgene Expression from Chromosomal Position Effects in Drosophila melanogaster

Germ line transformation of white⁻ Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs′ elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and α1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.     

Genetic fine localization of the β-glucocerebrosidase (GBA) and prosaposin (PSAP) genes: implications for Gaucher disease

Mutations in the glucocerebrosidase (GBA) and prosaposin (PSAP) genes are responsible for Gaucher disease, the most prevalent sphingolipidosis. Somatic cell hybrid analysis and in situ hybridization experiments have localized the GBA gene to 1q21 and the PSAP gene to 10q21-q22. We performed pairwise and multi-point linkage analyses between the two genes and several highly polymorphic markers from the Généthon human linkage map. Our results show that six markers cosegregate with the GBA gene (Z_max = 8.73 at θ = 0.00 for marker D1S2714) and define a 3.2-cM interval between D1S305 and D1S2624 as the most probable location for the gene. Three of these markers (D1S2777, D1S303, and D1S2140), as well as the gene encoding pyruvate kinase (PKLR), are contained in a single YAC clone together with the GBA gene. A new polymorphism was identified within the PSAP gene (C16045T) and used for linkage studies. The multi-point analysis places the gene in a 9.8-cM interval between D10S1688 and D10S607. The fine localization of these genes provides a useful tool for cosegregation analysis, indirect molecular diagnosis, and population genetic studies. Received: 22 October 1996 / Accepted: 4 February 1997     

Mapping of the Gene for Machado-Joseph Disease within a 3.6-cM Interval Flanked by D14S291/D14S280 and D14S81, on the Basis of Studies of Linkage and Linkage Disequilibrium in 24 Japanese Families

The gene locus of Machado-Joseph disease (MJD) has recently been mapped within a 29-cM subregion of 14q chromosome. We did a linkage study of 24 multigenerational MJD Japanese pedigrees, in an attempt to narrow the candidate region of this gene. Pairwise and multipoint linkage analysis, together with haplotype segregation analysis, led to the conclusion that the MJD gene is located at the 6.8-cM interval between D14S256 and D14S81 (Z_max = 24.78, multipoint linkage analysis). D14S291 and D14S280, located at the center of this interval, showed no obligate recombination with the MJD gene (Z_max = 5.93 for D14S291 and 9.99 for D14S280). A weak, but significant, linkage disequilibrium of MJD gene was noted with D14S81 (P < .05) but not with D14S291 or D14S280. These results suggest that a 3.6-cM interval flanked by D14S291/D14S280 and D14S81 is the most likely location of the MJD gene and that it is closest to D14S81.     

Mutation p.Arg954Trp of KIF21A Causes Congenital Fibrosis of the Extraocular Muscles in a Chinese Family

Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is an autosomal dominant strabismus disorder associated with defects of the oculomotor nerve. In this study, we identified a Chinese family with CFEOM1 for four generations. Linkage analysis mapped the causative gene of the family to 12q with a Lod score 2.1 for polymorphic marker D12S85, where KIF21A is located. Direct DNA sequence analysis identified a 2860C→T change in exon 21, resulting in a tryptophan substitution for arginine in codon 954 of KIF21A. SSCP (single-stranded conformational polymorphism) analysis showed that mutation p.Arg954Trp of KIF21A co-segregated with the affected members, but was absent in the unaffected individuals in the family and 150 normal controls. Our results indicate that mutation p.Arg954Trp of the KIF21A is the genetic basis of the Chinese family with CFEOM1.     

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H₂O₂) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H₂O₂ stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.     

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.     

A new HaeIII polymorphism at the D21S13 locus

Summary DNA markers in the pericentromeric region of human chromosome 21 have shown linkage to a gene for Familial Alzheimer disease (FAD; St. George Hyslop et al. 1987). The limited informativeness of probes for the loci D21S13 and D21S16 have hindered precise mapping of the FAD locus and analysis of non-allelic heterogeneity in FAD (Schellenberg et al. 1988; St. George-Hyslop et al. 1987). We recently described a new EcoRII polymorphism at the D21S13 locus that was very informative in a large FAD pedigree (Pulst et al. 1990a, b). We now report another polymorphism for the D21S13 locus that further increases the informativeness of this locus.     

PTEN Redundancy: Overexpressing lpten,a Homolog of Dictyostelium discoideum ptenA,the Ortholog of Human PTEN,Rescues All Behavioral Defects of the Mutant ptenA?

Mutations in the tumor suppressor gene PTEN are associated with a significant proportion of human cancers. Because the human genome also contains several homologs of PTEN, we considered the hypothesis that if a homolog, functionally redundant with PTEN, can be overexpressed, it may rescue the defects of a PTEN mutant. We have performed an initial test of this hypothesis in the model system Dictyostelium discoideum, which contains an ortholog of human PTEN, ptenA. Deletion of ptenA results in defects in motility, chemotaxis, aggregation and multicellular morphogenesis. D. discoideum also contains lpten, a newly discovered homolog of ptenA. Overexpressing lpten completely rescues all developmental and behavioral defects of the D. discoideum mutant ptenA⁻. This hypothesis must now be tested in human cells.