Developmental changes of sepiapterin synthase activity associated with a variegated purple gene in Drosophila melanogaster

A variegated position effect on the autonomous gene, purple, has been studied enzymologically in Drosophila melanogaster. Sepiapterin synthase, the enzyme system associated with pr ⁺, was examined for activity in different developmental stages of the fly. The results indicate that T(Y:2) pr ^c5, cn/pr^c4 cn flies (flies in which pr ⁺ has been translocated and which exhibit variegation) have a reduced amount of enzyme activity as compared with both Oregon-R and pr ¹ flies. This reduction in activity was not found in larval stages, which suggests that the inactivation process probably occurs in late larval or early pupal stages. The phenotype of the variegated adult has white eyes with red-colored spots and patches where drosopterins occur. The phenotype of the fly carrying the translocation is modified by the presence of additional Y chromosomes. This extends the observation from other systems that extra heterochromatin acts to suppress the variegated position effect. The advantages of studying the variegation by measuring enzyme activity, as well as the phenotypic expression, are several; for example, the developmental time at which variegation occurs may be estimated even though drosopterin synthesis is not occurring.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the Department of Energy under Contract No. W-7405-eng-26.     

Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster

Kynurenine-H ³ transport and conversion to 3-hydroxykynurenine were studied in organ culture using the Malpighian tubules and developing eyes from wild type and the eye color mutants w, st, 1td, ca, and cn of Drosophila melanogaster. Malpighian tubules from wild type have the ability to concentrate kynurenine and convert it to 3-hydroxykynurenine. The tubules from w, st, 1td, and ca are deficient in the ability to transport kynurenine, as are the eyes of the mutants w, st, and 1td. This defect in kynurenine transport provides a physiological explanation for the phenotypic properties of the mutants. The relationship of these measurements to previous observations on these eye color mutants is discussed and the transport defect hypothesis is consistently supported. We have concluded that several of the eye color mutants in Drosophila are transport mutants.     

Mutations in the right operator of bacteriophage lambda: evidence for operator-promoter interpenetration

A set of virulent mutants of bacteriophage lambda have been selected from λv2 v3. The sites of mutation form two microclusters, both close to v3. Some of the mutants, selected for their ability to grow on a λ-lysogen, can also grow on a λdv carrier strain. They are called “supervirulent” and a mutation conferring super-virulence is called vs. The sites of mutation to vs lie between the presumed promoter mutants (x3, x7) and x13, implying that the operator and promoter interpenetrate each other. The relative affinities of λ repressor for binding, in vitro, to λv⁺, λv3, λvs326, and λv3 vs327 DNA were 1, $\text{1}\text{4}$, $\text{1}\text{20}$, and $\text{1}\text{4000}$, respectively. We suggest that two separate mutations in the right operator are needed to confer virulence because promoter sites lie within the operator.     

Mutations in the right operator of bacteriophage lambda: physiological effects

The right operator in bacteriophage lambda vs326 has one-twentieth the in vitro binding affinity for repressor as λv⁺; for comparison λv3 has one-quarter the affinity of λv⁺. In vivo, both mutants constitutively express genes in the right operon. Both λv3 and λvs326 express gene O constitutively because they complement λimm434Oam^? in a λ lysogen, vs, more efficiently than v3. The v3 allele in cis (but not in trans) to vs326 gives significantly greater phage yields in a λ lysogen than λvs326 alone, cro gene function, measured by arrest of exonuclease synthesis, suggested the following series of increasing degree of conatitutivity: v3, vs326, v3 vs326. λv2 vs326 forms plaques on lysogens that carry λcI857, but λv2 v3 does not. These results indicate that vs326, like v3, is an operator constitutive mutation but stronger in its effects. These mutants exemplify a uniform correlation between relative weakness of repressor binding and degree of constitutive gene expression.     

Reduction of drosopterin content caused by a 45-nt insertion in <Emphasis Type="Italic">Henna</Emphasis> pre-mRNA of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Phenylalanine hydroxylase is assumed to be a key enzyme in drosopterin metabolism, but direct in vivo evidence to support this hypothesis is still absent. In the present study, we found a new natural recessive purple eye mutant of Drosophila melanogaster, Hn ^bp , which was a 45-nt insertion mutant in the second exon of Henna. The insertion resulted in a predicted protein with 15 additional amino acids as compared to the wild-type protein. Further analysis of protein structure showed that the predicted mutant protein probably had two more β-sheets, which may cause instability of two α-helices near the catalytic centre of the enzyme in the Biopterin-Hydroxyl binding domain. Hn ^bp mutant showed eye color defect with decrease of mRNA level, as well as drosopterin content reduction. The drosopterin defect could be fully rescued by expression of wild type Henna in the Hn ^bp background by GMR-GAL4 UAS-Henna/UAS-Henna:Hn ^bp /Hn ^bp transgenic line. All taken together, it can be concluded that the mutation in Henna is responsible for drosopterin reduction in mutant Hn ^bp , which provides key in vivo evidence to support the hypothesis that Henna is involved in drosopterin synthesis.     

The ommochrome biosynthetic pathway in Drosophila melanogaster: The head particulate phenoxazinone synthase and the developmental onset of xanthommatin synthesis

Particulate fractions from the heads of Drosophila melanogaster catalyze the conversion of o-aminophenols to phenoxazinones. This particulate enzyme is stimulated by Mn²⁺. It has a number of features which distinguish it clearly from the Mn²⁺-dependent activity found in the soluble fraction. The particulate enzyme has a characteristic developmental pattern, showing a marked increase in activity at about the time of onset of xanthommatin synthesis. In addition, it is much reduced in activity in a number of xanthommatin-deficient mutants (v, cn, st, cd, and w). We believe that the head particulate enzyme is involved in xanthommatin biosynthesis and that the developmental onset of synthesis of this pigment is brought about by the synthesis or activation of this enzyme.     

Drosopterins in phototaxis-selected strains of Drosophila melanogaster

The age-dependent drosopterin concentration was investigated in phototaxis-selected strains (Röko, Röpo, Röne, Stab) of D. melanogaster using extracted head homogenates. The results are compared to the wild stock derived strain Plus. The drosopterin concentration increases with the age of the flies. Females have larger concentrations than males. There are no differences in the drosopterin concentrations among the phototaxis-selected strains. Plus shows a higher drosopterin content than all other strains. This difference might be based on the genetical background, which is different in these strains. The data indicate that the phototactic behaviour is not related to the drosopterin concentration of the eyes.     

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification

Summary The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd ₁) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c ₅₅₂ mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd ₁ lacking heme d ₁, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir^– mutants of another type that had low level synthesis of cytochrome cd ₁, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS ⁺ and nirD ⁺ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.     

Ultraviolet-irradiation induced and spontaneous mutation of Rhizobium trifolii 11B in relation to water-soluble and water-insoluble polysaccharide production ability

Rhizobium trifolii 11B was u.v. irradiated and nine u.v. mutants have been isolated. Among the mutants, only one, R. trifolii 21M11B, produced more (752 mg/100 ml) water-soluble polysaccharide than the parent (704 mg/100 ml). The composition of water-soluble polysaccharide from u.v. mutants differed from that of the parent, R. trifolii 11B, and none of its u.v. mutants produced water-insoluble polysaccharide as detected by the Aniline Blue method. Storage of u.v. mutants for 2 months at 5°C gave four spontaneous variants which acquired the ability to produce water-insoluble polysaccharide. The spontaneous mutants also retained their water-soluble polysaccharide producing ability. The water-soluble polysaccharide produced by these mutants was characterized as curdlan type. The chemistry of water-soluble and water-insoluble polysaccharides was also ascertained.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

A Study of Pneumococcal Merodiploids at the Molecular Level

The DNA of a sulfonamide-resistant Pneumococcal strain (heterozygous for sul^r-c) and that of three highly resistant and persistently heterozygous cd transformants, derived by introducing sul ^r-c marker into a stable sulfonamide resistant strain (sul ^r-d), were studied to analyze the genetic basis of their merodiploidy. The physical properties of the native and denatured DNA from the heterozygotes and the nonheterozygous strains were not distinguishable. The denaturability and the renaturability of biological activity for the heterozygous markers were essentially identical to those of the normal markers. The heterozygosity extends to the closely linked locus giving rise to four different configurations of cd and cd⁺ transformants, characterized by their frequencies of segregation and donor-marker activities. The marker-activity ratios and the frequency of co-transfer of heterozygous markers were found to remain the same in each when the donor DNA was native, denatured or reannealed without fractionation or reannealed after remixing of resolved strands. Possible models were weighed against these observations and these considerations led to the suggestion that tandem duplication of a gene region may be responsible for the heterozygosity and instability of this region. A more detailed examination of this model will be presented in an accompanying paper.     

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d₁ heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd₁ NiR in which Tyr¹⁰ has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369–377) that the topologically homologous Tyr²⁵ plays a crucial role in controlling the activity of the cd₁ NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr¹⁰ with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe³⁺ of the d₁ heme is provided by different side-chains in different species.     

Nutritional control of xanthine dehydrogenase. II. Effects on xanthine dehydrogenase and aldehyde oxidase of culturing wild-type and mutant Drosophila on different levels of molybdenum

Two new mutants, deficient in aldehyde oxidase and xanthine dehydrogenase, have been isolated from a wild-type stock of Drosophila melanogaster and have been provisionally termed lxd ^c and lxd ^d, respectively, as both mutants appear to be allelic with lxd (low xanthine dehydrogenase). An analysis has been made of the effects of dietary molybdenum on lxd, lxd ^c, lxd^d, lao (low aldehyde oxidase), mal (maroon-like eye color), and pac (Pacific) wild-type flies. On the lower dietary levels of 10 ^?3 M and 10 ^?2 M molybdenum, increases in specific activity of both enzymes were observed only in lxd. Furthermore, two- to three-fold increases in specific activity of both enzymes occurred in all strains, except mal, when cultured on 5×10 ^?2 M molybdenum. The lxd and lxd ^c strains failed to survive on this high concentration of the ion. Similar concentrations of molybdenum had no effect in vitro. An extra electrophoretic band of xanthine dehydrogenase was observed on polyacrylamide gel from extracts of wild-type flies cultured on certain levels of molybdenum, but its appearance was not always correlated with the increases in specific activity.     

Cytogenetics of Disomics, Monotelo- and Monoisodisomics and ml st Mutants of Chromosome 4 of Cotton

The linkage relationships and arm locations of the ml₁ and st₁ mutants of cotton were determined with the use of monotelodisomics, a monoisodisomic and disomics. The ml₁ st₁ mutants are more than 50 cM apart on chromosome 4. The ml₁ locus is in the short arm and 16 cM from the centromere, and st₁ is in the long arm and 48 or more cM from the centromere. Recombination in the monoisodisomic was twice the expected value and higher, but not significantly, than in its monotelodisomic counterpart. It was concluded that the increase indicates a greater frequency of proximal exchange in the monoisodisomic than in the monotelodisomic.     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20^th, 31^st, and 81^sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68^th, 79^th, and 95^th haplotypes), one was predominant in the South and Southwest China regions (the 18^th haplotype), one was relatively common in the Northeast and North China regions (the 94^th haplotype), and one was common in the Northeast, North and Northwest China (the 40^th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.