Sequence of the Bacillus subtilis glutamine synthetase gene region

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.     

Genetic,molecular and developmental analysis of the glutamine synthetase isozymes ofDrosophila melanogaster

The glutamine synthetase isozymes ofDrosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. InDrosophila melanogaster two GS. isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the twoDrosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.     

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.     

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.     

Genetic basis for tissue isozymes of glutamine synthetase in elasmobranchs

Tissue-specific isozymes of glutamine synthetase are present in elasmobranchs. A larger isozyme occurs in tissues in which the enzyme is localized in mitochondria (liver, kidney) whereas a smaller form occurs in tissues in which it is cytosolic (brain, spleen, etc.). The nucleotide sequence of spiny dogfish shark (Squalus acanthias) liver glutamine synthetase mRNA, derived from its cDNA, shows there are two in-frame initiation codons (AUG) at the N-terminus which will account for the size differences between the two isozymes. Initiation at the up-stream and down-stream sites would yield peptides of 45,406 and 41,869 mol. wts. representing the precursor of the mitochondrial isozyme and the cytosolic isozyme, respectively. The additional N-terminal 29 amino acids present in the mitochondrial isozyme precursor contains two putative cleavage sites based on the Arg-X-(Phe,Ile,Leu) motif. The predicted two-step processing would remove 14 of the 29 N-terminal amino acids. These 14 amino acids can be predicted to form a very strong amphipathic mitochondrial targeting signal. Their removal would yield a mature peptide of 43,680 mol. wt. The calculated mol. wts. based on the derived amino acid sequence are therefore in good agreement with previous estimates of an approximately 1.5–2-kDa difference between the M_rs of the mitochondrial and cytosolic isozymes. A model for the evolution of the mitochondrial targeting of glutamine synthetase in vertebrates is proposed. Correspondence to: J.W. CampbellThe nucleotide sequence reported will appear in GenBank under accession number U04617     

A contribution to the genetic fine structure of the region adjacent toWhite inDrosophila melanogaster

A recombinational and cytogenetical analysis is reported of a new locus,sparse arista (sa), located immediately to the left ofwhite in salivary gland chromosome band 3Cl. The general problem of the cytogenetic localization of genes to giant chromosome bands is discussed. A bristle effect previously attributed to gene(s) located in band 3Cl is discussed in the light of the findings reported here. A portion of this study was taken from a dissertation submitted to the University of Illinois in partial fulfillment of the requirements of the Ph. D. degree. Supported by an N.I.H. postdoctoral traineeship under Public Health Training Grant GM 701-07. Supported by N.S.F. Grant GB 6127.     

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.     

Cytogenetic analysis of region 2B3-4-2B11 of the X-chromosome of Drosophila melanogaster

About 160 kb of DNA were cloned from the 2B region of the X chromosome, where the early ecdysone puff develops and the ecs locus is located. On the physical map of this sequence the positions of 13 chromosome rearrangement breakpoints interfering with both puff development and the ecs locus proximally and distally, were plotted by means of in situ hybridization. The maximal size of the ecs locus is about 100 kb (between the breakpoint of In(1)Hw ^49c and the proximal end of Df(1)St472) The DNA sequences essential for normal puffing are located within the ecs locus between the In(1)br ^lt103 and Df(1)St472 breakpoints and comprise about 65 kb. Thus the puff develops as a result of ecs activation. Since Df(1)P154, which reduces the puff size and removes the proximal part of the ecs locus, does not prevent puff induction by ecdysone, while removing the distal part of the locus by Df(1)St469 completely stops development of the puff, we conclude that the regulatory zone of the locus, which reacts to hormone is located in the distal parts of both the puff and the locus, proximal to the breakpoint of In(1)br ^lt103 .Since In(1)br ^lt103 , Df(1)pn7b and Df(1)br ^R1 damage ecs but do not prevent puffing it is proposed that there is a second regulatory zone for this locus with a minimal size of 15–20 kb (between the breakpoints of Df(1)br ^R1 and In(1)br ^lt103). After cytogenetic and electron microscopic analysis of 2B puff formation it seems very likely that the site of puff formation is situated in the proximal part of 2B3-4 and after enhancement of ecs expression by hormone it spreads proximally to the 2B6 band which does not puff. When the puff regresses at puff stages (PS)10-11 its material does not condense completely and a zone of residual puffing joins the condensed material located distal to it. This material can give the impression of a separate band, designated 2B5 in Bridges' map. For convenience we propose to call the site giving rise to the puff as 2B3-5.     

Formation and structure of 1-, 3- and 6+1-stranded helical cables of glutamine synthetase

Addition of millimolar concentrations of Co²⁺ to Escherichia coli glutamine synthetase induces aggregation along the 6-fold symmetry axes of the protein molecules, forming long strands. The strands subsequently aggregate laterally to form two types of helical cables, a large cable with six outer strands wrapped around a central strand (6+1-stranded cables) and a smaller cable in which three strands wrap around one another. Similar but less extensive aggregation is induced by other divalent metal cations: Cu²⁺, Ni²⁺ and Zn²⁺. The aggregates exhibit little enzymatic activity, and aggregation is completely reversible upon removal of Co²⁺ in the presence of millimolar Mn²⁺, including regeneration of nearly full enzyme activity.Each type of helical cable exists in a variety of related forms, which vary in their helical pitch: 6+1-stranded cables have 6-fold axial symmetry, and different specimens are observed with helical pitches from 320 to 540 nm; 3-stranded cables apparently do not have 3-fold axial symmetry and have pitches from 140 to 270 nm. The large variation in pitch for glutamine synthetase helical cables implies either a variation of the regions of intermolecular contacts of approximately 4–10 Å, or a movement of the bonding domains relative to the rest of the molecule by a similar amount.     

Identification of the structural gene for glutamine synthetase in Klebsiella aerogenes.

Mutations at two sites of the Klebsiella aerogenes chromosome, unlinked by transduction with phages PW52 and P1, result in the lack of enzymatically active glutamine synthetase. A mutation in the glnB site leads to a marked decrease in the formation of an apparently normal enzyme. Some of the mutations in the glnA site lead to the production of enzymatically inactive material capable of reacting with anti-glutamine synthetase serum. The revertant of a glnA mutant was found to produce a glutamine synthetase with less activity and less stability to heat than the enzyme of the wild type. These results locate the structural gene to the production of enzymatically inactive glutamine synthetase antigen, not subject to repression by exogenously added ammonia. This observation suggests that glutamine synthetase is itself involved in the regulation of the synthesis of glutamine synthetase.     

Intrachromosomal gene mapping in man: the gene for tryptophyl-tRNA synthetase maps in region q21 leads to qter of chromosome 14

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter leads to Xp22::14q21 leads to 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 leads to 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.