The enzyme glutamine synthetase I of Drosophila melanogaster is associated with a modified RNA

Glutamine synthetase I (L-glutamate:ammonia ligase, ADP forming; EC 6.3.1.2) was purified from Drosophila melanogaster larvae. The complete enzyme has an apparent molecular weight of 380,000. The subunit of the active enzyme has an apparent molecular weight of 43,000 after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Routine preparations yield enzymes which have at least another polypeptide component of apparent molecular weight of 64,000. Several factors suggest that the 64,000-dalton polypeptide might be a transformation product of the 43,000-dalton subunit which occurs in association with enzyme inactivation. Distinct from its protein subunit, from pure glutamine synthetase I a material can be extracted which can be labeled with 32P-labeled gamma-ATP using polynucleotide kinase. After alkaline hydrolysis the majority of the radioactivity is recovered as 5'2' and 5'3' ribonucleotide diphosphates, and after venom phosphodiesterase digestion as 5' ribonucleotide. We therefore conclude that the native glutamine synthetase I enzyme contains, or at least is reproducibly associated with, an RNA component. Several characteristics of the labeled material indicate that the RNA is small in size and is bound to polymer molecules different from RNA.     

Genetic determinants of glutamine synthetase inDrosophila melanogaster: A gene for glutamine synthetase I resides in the 21B3-6 region

Recombinational and deletion mapping of electrophoretic variants of the glutamine synthetase I isozyme (GSI) in Drosophila melanogaster locates the gene in the 21B region on the second chromosome. We have conducted a genetic analysis of the region extending cytologically from 21A to 21B4-6. Recessive lethal mutations were generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU) mutagenesis and by hybrid dysgenesis (HD). These lethals fall into seven functional groups, which were partially ordered by complementation with cytologically defined deficiencies of this region generated by hybrid dysgenesis. Two of the EMS- and two of the ENU-induced lethals fulfill biochemical criteria expected for null alleles of the GSI gene.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Mutations of the withered (whd) gene in Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the carnitine palmitoyltransferase I (CPT I) gene.

Since some oxygen defense mutants of Drosophila melanogaster exhibit a crinkled wing phenotype, a screen was performed on strains bearing mutant alleles conferring a visible wing phenotype to determine whether any were hypersensitive to oxidative stress. One mutant, withered (whd), was found to be sensitive to both dietary paraquat and hyperoxia. New alleles of whd were induced on a defined genetic background and strains carrying these alleles were also found to be sensitive to oxidative stress. To identify the product of the whd gene we used a sequence-based positional candidate approach and by this method we determined that whd encodes carnitine palmitoyltransferase I (CPT I), an enzyme of the outer mitochondrial membrane that is required for the import of long-chain fatty acids into the mitochondria for beta-oxidation. Although this function is not vital under laboratory conditions, whd adults were found to be highly sensitive to starvation and to heavy metal toxicity relative to controls. This work uncovers a novel relationship between fatty acid metabolism and reactive oxygen metabolism. Further, these results in conjunction with past research on whd and on mammalian CPT I support the hypothesis that CPT I serves a vital function in the response to thymine supplementation.     

Purification of the glutamine synthetase II isozyme of Drosophila melanogaster and structural and functional comparison of glutamine synthetases I and II

Glutamine synthetase II was purified from Drosophila melanogaster adults. It was completely separable from the isozyme glutamine synthetase I by means of DEAE chromatography. The complete enzyme has an apparent molecular weight of 360,000. After two-dimensional electrophoresis it gave a single molecular species with an apparent molecular weight of 42,000. Structural analysis of the two isozymes showed that they are different both in subunit molecular weight and in isoelectric point. Peptide maps of the purified subunits showed considerable dissimilarity. Glutamine synthetase II is more active than glutamine synthetase I in the transferase assay, while the opposite is true in the biosynthetic assay. The kinetic parameters were determined, showing again noteworthy differences between the two isozymes. We therefore conclude that two forms of glutamine synthetase are present in Drosophila, with different primary structures, different kinetic behavior, and the possibility of different functional properties.     

A mutant dec-1 transgene induces dominant female sterility in Drosophila melanogaster

The Drosophila dec-1 gene produces three proproteins required for female fertility and eggshell assembly. The three proproteins are distinguished by their C termini. Fc106, the most abundant proprotein, is cleaved within the vitelline membrane to three mature derivatives in a developmentally regulated manner. To define sequences within fc106 that are critical for its function, we created wild-type and mutant versions of an fc106 cDNA transgene. The functional consequences of the mutations were assessed in dec-14, a female-sterile splicing mutant that does not produce the fc106 isoform. The fertility of dec-14 females was restored by the introduction of either a wild-type transgene or a transgene bearing a C-terminal deletion that included fc106-specific sequences. Surprisingly, the removal of internal coding sequences created an aberrant DEC-1 proprotein that induced female sterility when introduced into wild-type flies. Dominant female sterility was not associated with larger deletions that included the fc106 N terminus, suggesting that abnormal juxtaposition of N- and C-terminal sequences in the aberrant proprotein interfered with endogenous DEC-1 proteins. Changes in the fractionation behavior of the endogenous fc106 C-terminal derivative, s60, and morphological changes in the endochorion in response to expression of the aberrant proprotein support this interpretation.     

Cloning of the glutamine synthetase I gene from Rhizobium meliloti.

Glutamine synthetase is a major enzyme in the assimilation of ammonia by members of the genus Rhizobium. Two forms of glutamine synthetase are found in members of the genus Rhizobium, a heat-stable glutamine synthetase I (GSI) and a heat-labile GSII. As a step toward clarifying the role of these enzymes in symbiotic nitrogen fixation, we have cloned the structural gene for GSI from Rhizobium meliloti 104A14. A gene bank of R. meliloti was constructed by using the bacteriophage P4 cosmid pMK318. Cosmids that contain the structural gene for GSI were isolated by selecting for plasmids that permit ET8051, an Escherichia coli glutamine autotroph, to grow with ammonia as the sole nitrogen source. One of the cosmids, pJS36, contains an insert of 11.9 kilobases. ET8051(pJS36) grows slowly on minimal media. When a 3.7-kilobase HindIII fragment derived from this DNA is cloned into the HindIII site of pACYC177 and the plasmids are transformed into ET8051, rapid growth is observed when the insert is in one orientation (pJS44) but not the other (pJS45). Glutamine synthetase activity can be detected in ET8051(pJS44); most of this activity is heat stable. pJS36 hybridizes with the glnA structural gene from Escherichia coli. Insertion of a 2.7-kilobase Tetr determinant into a BglII site located within pJS44 abolishes all glutamine synthetase activity. This interrupted version of a glutamine synthetase gene was substituted for the normal R. meliloti sequence by homologous recombination in R. meliloti. Recombinants lose GSI activity, but retain GSII activity and grow well with ammonia as the sole nitrogen source. These mutants are unaffected in nodulation and nitrogen fixation.     

Mutations affecting glutamine synthetase activity in Salmonella typhimurium.

A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity.     

Non mendelian female sterility in Drosophila melanogaster,further data on chromosomal contamination

Molecular Genetics and Genomics - In relation to non Mendelian female sterility, Drosophila melanogaster strains can be divided into two main classes, inducer and reactive. The genetic element...     

Oogenesis in the female sterile (1)42 mutant of Drosophila melanogaster.

The sex-linked mutation fs(1)42 was induced by ethyl methane sulfonate. It has no effect on either the external morphology or longevity of adult hemizygotes or homozygotes. Heterozygotes and hemizygotes are fertile, but homozygotes are sterile. Egg chamber development proceeds through stages 8, and thereafter chambers degenerate. Dying follicle cells are seen in chambers at all positions in the ovarioles. Profollicle cells also die within germaria, and clusters of sister cystocytes take longer than normal to receive their coverings of follicle cells. Egg chambers in the vitellarium contain only about 60% the normal number of follicle cells, these generally have greater lateral dimensions, and their nuclei and nucleoli are also larger than normal. The follicular envelope of mutant chambers often contains gaps through which cystocytes send cytoplasmic projections. Abnormalities seen in development of the fs(1)42 oocyte are likely to be due to its envelope of defective follicle cells.     

Mutations that alter the covalent modification of glutamine synthetase in Salmonella typhimurium.

glnD and glnE mutant strains of Salmonella typhimurium lack three of the four activities required for reversible covalent modification of glutamine synthetase (GS; EC 6.3.1.2). The glnD strains, which are unable to deadenylylate GS and therefore accumulate the adenylylated or less active form of the enzyme, were isolated as glutamine bradytrophs. They lack the activity of PIIA uridylyl-transferase, one of the proteins required for deadenylylation of GS; in addition, they lack PIID uridylyl-removing activity. Mutations in glnD are suppressed by second-site mutations in glnE that eliminate the activity of GS adenylyltransferase (EC 2.7.7.42) and thus prevent adenylylation of GS. The glnD and glnE strains have one-third to one-half as much total GS as the wild-type strain when they are grown in a medium containing a high concentration of NH4+. The wild-type strain derepresses synthesis of GS fourfold in response to nitrogen limitation; glnD and glnE strains derepress synthesis of the enzyme fourfold and sevenfold, respectively. Thus, mutations that alter covalent modification of GS in Salmonella do not significantly affect derepression of its synthesis. The glnD gene lies at 7 min on the Salmonella chromosome and is 50% linked to pyrH by P22-mediated transduction.