Cytogenetic analysis of the cSOD microregion in Drosophila melanogaster

This report describes the genetic organization of a euchromatic region on the third chromosome of Drosophila melanogaster extending cytologically from 68A2 to C1, an interval comprising 10 or 11 polytene chromosome bands. The gene for cytoplasmic superoxide dismutase (cSOD) maps within this interval, as does low xanthine dehydrogenase (lxd).--Recessive lethal mutations were generated within the region by ethyl methanesulfonate mutagenesis and by hybrid dysgenesis. These lethals fall into 11 functional groups, which were partially ordered by complementation with deletions having breakpoints within the region. The distribution of dysgenesis-induced mutations in the region is highly nonrandom, the majority being within a single group. The mutability of this gene is comparable to that of singed (sn), a documented "hot-spot" for P-element insertion.--One of the EMS-induced lethals, l-108, fulfills biochemical criteria expected of a hypomorphic allele of cSOD. To our knowledge this is the first such allele recovered of this gene, and it should prove very useful in an analysis of the in vivo function of cytoplasmic SOD. Indeed, it has been demonstrated that cSOD is almost certainly a vital gene.     

I-R hybrid dysgenesis in Drosophila melanogaster. Use of in situ hybridization to show the association of I factor DNA with induced sex-linked recessive lethals.

The purpose of this paper is the genetic visualization by in situ hybridization of 130 sex-linked recessive lethals plus a non-lethal induced by I-R dysgenesis. This collection of lethals involves inducer strains which differ in the position of the I elements on the X chromosomes. The I-R interaction was strong. Our previous results have shown that about 30% of the induced recessive lethals are associated with cytologically visible chromosomal rearrangements. (1) The rearrangements induced by I-R-type hybrid dysgenesis often exhibit homology with the I factor at the level of one or both junction points, depending on the types of chromosome rearrangements. These results suggest that the chromosome rearrangements arise directly from the transposition of I elements. However, the breakpoints of some types of cytologically non-visible deficiencies and of 2 small cytologically visible deficiencies do not present detectable homology with the I factor. (2) The majority of rearrangements do not involve the I elements already present on the paternal X chromosome. (3) The hybridization signal distributions on the X chromosome are not uniform. They present peaks of various heights which may correspond to specific anchoring areas of copies of I in the course of integration. (4) The data presented here agree with the literature with respect to the mean number of copies of I per X chromosome and to the excess of copies of I at locus 1A. Two rearrangement formation mechanisms are envisaged: crossing-over and 'target' exchanges.     

The relationship between radiation-induced and transposon-induced genetic damage during Drosophila oogenesis

The combined effect of X-irradiation and transposon mobility on the frequencies of X-linked recessive lethals and dominant lethals was investigated in female hybrids in the P-M system of hybrid dysgenesis. X-linked lethals were measured in G2 hybrid dysgenic females whose X chromosome was derived from the M X P cross. To test for additivity or synergism, the mutation rate in irradiated dysgenic females was compared to that of unirradiated females as well as to irradiated nondysgenic hybrid females derived from M X M crosses. Eggs collected for 2 days after irradiation, were represented by the more radiation-sensitive A and B oocytes (about 75%) and the least sensitive C oocytes (about 25%). The production of X-linked lethal events in X-irradiated dysgenic females was 8.1%, as compared to 4.5% in dysgenic controls and 3.4% in irradiated, nondysgenic controls, demonstrating an additive effect of radiation and dysgenesis-induced genetic damage. The effect of irradiation on sterility of dysgenic hybrid females was a negative one, resulting in 20% less sterility than expected from an additive effect. The combined effect of radiation and dysgenesis on dominant lethality tested in A, B and C oocytes of the same hybrid females was synergistic. Egg broods collected for 3.5 days after irradiation showed that significantly more damage was induced in the presence of ionizing radiation in dysgenic females than in their nondysgenic counterparts. This effect was most obvious in B and C oocytes. The synergism observed may be related to the inability of cells to repair the increased number of chromosome breaks induced both by radiation and transposon mobility.     

Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.     

Streptomyces hygroscopicus has two glutamine synthetase genes.

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.     

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.     

I−R hybrid dysgenesis in Drosophila melanogaster: nature and site specificity of induced recessive lethals

This paper presents results of the genetic and cytological analysis of 144 sex-linked recessive lethals, plus 1 non-lethal. All of them were induced by I−R hybrid dysgenesis. This collection of mutants was pooled from experiments involving inducer chromosomes that differ in the chrosomal position of their I elements. Our results show that 30% of the recessive lethals are associated with chromosomal rearrangements which depend on the strength of the I−R interaction. These lethals are induced on both inducer- and reactive-origin chromosomes, and their frequency is dependent on the structure of the inducer chromosome used. The I−R-induced lethals occur along the entire length of the X chromosome. These sites probably correspond to specific loci which are more or less homologous with I. The complementation relationshups showed that some specific loci were more frequently involved in all the lethal mutations tested. The most sensitive loci are, in order of observation: l(1)J1, ct, f, ma¹ and m. Among induced recessive lethals considered to be point mutation, complementation tests showed that many of them are in fact multilocius deficiencies which can be detected only at the molecular level.

It seems that the production of I−R rearrangements (cytologically visible or not) may be the most important mechanism leading to lethal mutations. These mutations probably occur during the transposition of I elements, hence their importance from an evolutionary standpoint.     

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-a and GSI-. GSIa-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI--type genes occur in all other bacteria. GSI-- and GSI--type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision. The S. solfataricus GSI gene has a shorter GSI--type insertion, but like GSI-a-type genes, lacks conserved sequences about the adenylylation site. We suspect that the similarity of GSI- genes from Euryarchaeota and several bacterial species does not reflect a common phylogeny but rather lateral transmission between archaea and bacteria.Correspondence to: J.R. Brown 1073     

Characteristics of inserted mutations obtained in the P-M hybrid dysgenesis system in the 9F12-10A7 region of the Drosophila melanogaster X-chromosome

19 new mutations in the 9F12-10A7 region of Drosophila melanogaster X chromosome was obtained in the system of P-M hybrid dysgenesis. They appeared to be lethals, as judged from viability of homo- or hemizygous females. In situ hybridization of P DNA with polytene chromosomes revealed P-element insertion in the 10A1-2 band in the majority of the mutants. As a result of complementation analysis, all these mutations were localized at previously known loci: l(1)BP1, l(1)BP5, l(1)BP8, l(1)BP7. No insertion mutations were found at the vermilion locus. This can imply for non-random distribution of insertion mutations in the region studied. Further comparison of these mutations with previously EMS-induced ones revealed that insertion mutations are predominantly hypomorph lethals which do not influence the viability, morphology and fertility of homozygous males and females, but drastically reduce viability of hemizygous females.     

Characterization of a HindIII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium

The glnA gene, encoding glutamine synthetase in Salmonella typhimurium, has been cloned into the plasmid pBR322. One hybrid plasmid, pJB1, containing an 8.5 kb insert generated by a HindIII digest, was analyzed using eleven different restriction enzymes. Evidence that the region controlling glutamine synthetase expression remained on the insert was obtained by showing that the regulation is normal in cells carrying plasmids with the insert in the original and reversed orientation. Several new plasmids derived from pJB1 following SalI and EcoRI digestions were examined for their ability to complement a glnA202 mutation in order to locate the DNA segment needed for glutamine synthetase expression. The results show that cells containing plasmid pJB8, which has a 21 kb deletion, produce and regulate glutamine synthetase normally, whereas cells with a plasmid (pJB11) similar to pJB8, but lacking a 0.25 kb EcoRI fragment, do not exhibit glutamine synthetase activity. The analysis of proteins produced in minicells containing pJB8 and pJB11 show that they both produce a protein that migrates with the glutamine synthetase subunit. Because pJB11 makes an inactive protein of similar size to the glutamine synthetase subunit, the 0.25 kb deletion may encode only the C-terminus of this protein. Consistent with this finding is the presence of a strong RNA polymerase-binding site on pJB8 to the right of the 0.25 kb EcoRI that could correspond to a promoter near the N-terminus of the glnA gene.     

The occurrence of eukaryotic type III glutamine synthetase in the marine diatom Chaetoceros compressum

Glutamine synthetase (GS) has been described as one of the oldest functioning genes and thus a good molecular clock protein. GS is diverged into three distinct forms, type I (GSI), type II (GSII) and type III (GSIII), the last type of which is a member of the most recently discovered family among GSs and thus has been reported from a limited number of prokaryotes. In the present study, we determined the full-length sequence of GSIII from the marine diatom Chaetoceros compressum. The 3′ untranslated region of the diatom GSIII gene was composed of a polyadenylation signal followed by a poly (A)⁺ tail, clearly demonstrating that its mRNA is transcribed from the eukaryotic genome. We also screened available genome databases and identified full-length GSIII sequences from 5 eukaryotic species. These eukaryotic GSIIIs specifically contained regions A–D and a long additional sequence flanking region V toward the C-terminal site, both being specific to GSIII. Phylogenic analysis revealed that eukaryotic GSIIIs are not within a monophyletic relationship with the possible occurrence of lateral gene transfer in GSIII during evolution.     

Identification and characterization of three forms of glutamine synthetase unique to Rhizobia

Glutamine synthetase (GS) activities of Rhizobia were chromatographically resolved into three distinct forms, GSI, GSII, and GSIII on DEAE cellulose, being eluted with 0.3M, 0.5M and 0.8M KCl, respectively. GSIII was the major form inR. leguminosarum andR. phaseoli. InR. meliloti, however, GSI was the major form. The three forms of GS were also distinguished on the basis of (a) rapid heat inactivation of GSII, (b) insensitivity of GSI to inhibitors, (c) marked inhibition of GSII by thymidine, and (d) inability of Zn⁺⁺ to inhibit GSIII. The three forms of GS are also distinct molecular entities and are unique to Rhizobia.     

Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups

Abstract: The structure of glutamine synthetase (GS) enzymes from diverse bacterial groups fall into three distinct classes. GSI is the typical bacterial GS, GSII is similar to the eukaryotic GS and is found together with GSI in plant symbionts and Streptomyces , while GSIII has been found in two unrelated anaerobic rumen bacteria. In most cases, the structural gene for GS enzyme is regulated in response to nitrogen. However, different regulatory mechanisms, to ensure optimal utilization of nitrogen substrates, control the GS enzyme in each class.