Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.     

Regulation of amidase formation in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity

The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation between amidase and urease formation was observed. The results suggest that amidase formation in strain PAO is subject to nitrogen control and that glutamine or some compound derived from it mediates the nitrogen repression of amidase.     

Maize mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of thylakoid membranes

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.     

Regulation of glutamine synthetase by regulatory protein PII in Klebsiella aerogenes mutants lacking adenylyltransferase.

A mutation of Klebsiella aerogenes causing production of an altered PII regulatory protein which stimulates overadenylylation of glutamine synthetase and also prevents its derepression was combined with mutations abolishing the activity of adenylyltransferase. The results support the idea that PII plays a role in the regulation of the level of glutamine synthetase which is independent of its interaction with adenylyltransferase.     

Use of nuclear mutants in the analysis of chloroplast development

Although a wide range of mutations in the nuclear genome also affect chloroplast biogenesis, their pleiotropic nature often limits their use in studying nuclear genes that regulate or facilitate chloroplast development. However, many mutations that cause a high-chlorophyll-fluorescent (hcf) phenotype exhibit limited pleiotrophy, causing the loss of functionally related sets of chloroplast polypeptides. Several hcf mutations are described that result in the loss of one specific protein complex from the thylakoid membrane. Chloroplast and cytosolic mRNAs coding for component polypeptides of the missing complex are unaffected in the mutants, suggesting that each mutation disrupts some process in the synthesis and assembly of the missing complex. Another hcf mutation causes both the loss of three protein complexes and grossly abnormal thylakoid membrane structures. The primary effect of this mutation might be in the assembly of thylakoid membranes or in the stable accumulation of the three protein complexes. Two other hcf mutations are more pleiotropic. Hcf*-38 causes a quantitative reduction of many chloroplast proteins and a reduction of some chloroplast RNAs, including several splicing intermediates. Hcf*-7 causes a major reduction of all chloroplast-encoded proteins examined. The range of pleiotropic effects of hcf mutations indicates that the mutations identify nuclear genes whose products are involved in a number of different steps in chloroplast development. Because some of the mutations described have been generated by transposon insertions, they can be cloned using the transposon to identify the mutant allele.     

Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas

Summary We have carried out a molecular and genetic analysis of the chloroplast ATPase in Chlamydomonas reinhardtii. Recombination and complementation studies on 16 independently isolated chloroplast mutations affecting this complex demonstrated that they represent alleles in five distinct chloroplast genes. One of these five, the ac-u-c locus, has been positioned on the physical map of the chloroplast DNA by deletion mutations. The use of cloned spinach chloroplast ATPase genes in heterologous hybridizations to Chlamydomonas chloroplast DNA has allowed us to localize three or possibly four of the ATPase genes on the physical map. The beta and probably the epsilon subunit genes of Chlamydomonas CF₁ lie within the same region of chloroplast DNA as the ac-u-c locus, while the alpha and proteolipid subunit genes appear to map adjacent to one another approximately 20 kbp away. Unlike the arrangement in higher plants, these two pairs of genes are separated from each other by an inverted repeat.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Phase variation: genetic analysis of switching mutants

Site-specific inversion of a controlling element is responsible for flagellar phase transition in Salmonella. When a 900 bp DNA sequence is in one configuration, it allows the expression of the H2 gene, a structural gene which codes for the flagellar antigen. When it is in the opposite configuration, the H2 gene is not expressed. A hybrid λ phage containing the invertible control region and the adjacent H2 gene was constructed, and expression of the H2 gene was shown to be regulated by the orientation of the inversion region. Transposon Tn5 insertion derivatives of this hybrid phage were isolated and λH2::Tn5 mutants defective for inversion (H2 switching) were selected and characterized. Two classes of switching phenotypes were observed among the mutants—those which had slightly reduced frequencies of transition from expression of the H2 gene (H2 on) to nonexpression (H2 off) (intermediate class) and those in which the frequency of transition was reduced at least three orders of magnitude (null class). Physical mapping of the Tn5 insertion sites revealed that in all mutants the insertion was located inside the inversion region. Tn5 insertion sites in the null class of mutants defined a region of DNA including approximately 500 bp which was necessary for inversion. Genetic complementation tests showed that these λH2::Tn5 mutants could invert the H2 gene control element if the 500 bp region was introduced in the trans configuration. It is concluded that a gene is located inside the inversion segment and codes for a protein which is required for the inversion event. Furthermore, the two sites at which the crossover event occurred functioned in a cis configuration and were required for inversion. The presence of a gene which is involved in controlling site-specific recombination events may be a general feature of transposon-like elements.     

Physiological and biochemical analysis of the glutamine synthetase-impaired mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Three types of glutamine synthetase (GS)-impaired mutants (gln) ofNostoc muscorum were isolated as ethylenediamine (EDA)-resistant phenotypes and characterized with respect to heterocyst development, nitrogen fixation, ammonium metabolism, photosynthetic characteristics, and glutamine synthetase activity. The criterion for categorizing the mutants was the extent of loss of GS activity (both in transferase and biosynthetic assays) compared with wild type; it was 70% in EDA-1, 30% in EDA-2, and more than 90% in EDA-3 strains. The level of nitrogenase activity in mutant strains was proportionate to heterocyst frequency and was found refractory to ammonium and EDA repression. In EDA-resistant strains, development of heterocysts and their spacing pattern remained unaffected and did not respond to treatment of amino acid analogues, drugs, and ammoniacal compounds which otherwise either stimulated or suppressed the number and altered the spacing pattern in wild type. A biphasic pattern of ammonium uptake indicating two transport systems was observed in all the strains except that the Km values for both high- and low-affinity systems were altered in mutant strains. In vivo treatment with MSX or EDA significantly inhibited the GS activity in wild type, whereas mutant strains did not respond to these treatments and were able to liberate NH ₄ ⁺ continuously into the medium without MSX treatment. During NH ₄ ⁺ uptake, percentage inhibition of O₂ evolution and changes in increase of fluorescence intensity were low in EDA strains compared with wild type. Assessment of GS protein with antibodies against GS and quantitative polyacrylamide gel electrophoresis (PAGE) suggested that loss in specific activity of GS per milligram of extractable protein in EDA mutants was owing to low production of GS-specific protein. SDS-PAGE of purified GS enzyme from all the strains revealed only one polypeptide band of molecular weight of about 51.28 kDa.     

Salmonella typhimurium mutants lacking protease II.

Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.     

Salmonella typhimurium mutants lacking NAD pyrophosphatase.

NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome.     

Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia.

Chloroplast DNA isolated from wild-type Nicotiana plumbaginifolia and 12 maternally inherited streptomycin-resistant mutants was digested with various restriction enzymes and the resultant patterns were compared. No gross structural alterations of the chloroplast genome were detected in any mutants; however, variant patterns owing to the gain or loss of a restriction site were found in three mutants, SR1007, SR1019, and SR1022. The variant patterns in SR1019 and SR1022 are identical and are the results of mutation in the psbG gene coding for a chloroplast membrane protein G, and that in SR1007 is due to mutation in the 16S rRNA gene. Inheritance of the variant patterns in mutants SR1007 and SR1019 was studied. The results showed that the variant patterns and streptomycin resistance were co-transmitted in reciprocal crosses.     

Isolation and genetic analysis of ethanol-sensitive mutants of thermotolerant Kluyveromyces marxianus

Six monogenic mutants of K. marxianus, affected in their ability to tolerate ethanol, were assigned to 6 loci etr1 through etr6, probably the hot spots for ethyl methane sulfonate as these loci were found mutated in each of the non-monogenic mutants as well. Differential ethanol tolerance of allelic hexagenic mutants suggests that even more than 6 genes may be involved in controlling ethanol tolerance in K. marxianus.     

Characterization and genetic analysis of antheridiogen-insensitive mutants in the fern Ceratopteris

WARNE, T. R., HICKOK, L. G. & SCOTT, R. S., 1988. Characterization and genetic analysis of antheridiogen-insensitive mutants in the fern Ceratopteris . The pheromone antheridiogen mediates the differentiation of male gametophytes in the fern Ceratopteris . Mutants insensitive to antheridiogen were isolated using an in vitro selection procedure. Antheridiogen-insensitive mutants exhibited partial or complete insensitivity to antheridiogen, but were normal in all other respects. Two mutants were completely insensitive to antheridiogen, whereas, another mutant was insensitive to supplemented antheridiogen, but produced male gametophytes in multispore cultures. Genetic analysis suggested a single gene basis for each mutant.     

The isolation and genetic analysis of sporulation-deficient mutants in Saccharomyces cerevisiae

Sporulation-deficient mutants were isolated from a homothallic strain of Saccharomyces cerevisiae. Sporulation was induced in these mutants by procedures to sporulate the products of protoplast fusion between mutants and wild-type strains. Spores formed in this way were crossed to wild-type strains in order to analyze them genetically. Twenty-three genes essential to sporulation were identified by tetrad analysis and complementation tests. Gene symbols spoT1 to spoT23 were tentatively assigned to them. These mutants fell into four classes by examination of premeiotic DNA synthesis and meiotic nuclear division: (i) Premeiotic DNA synthesis did not occur (spoT1 - spoT11); (ii) premeiotic DNA synthesis occurred but meiosis I did not occur (spoT12 - spoT15); (iii) meiosis II did not occur (spoT16 - spoT18); (iv) meiosis II occurred but mature spores were not formed (spoT19 - spoT23). Genes spoT4, spoT8, spoT20, and spoT23 were mapped on chromosomes IV, II, XVI and XI, respectively. SpoT18-1 was a UAG nonsense mutation.     

Parasexual genetic analysis of aggregation-deficient mutants of Dictyostelium discoideum.

One hundred and thirty-nine independent, nitrosoguanidine-induced mutants blocked early in development were isolated in two haploid strains of D. discoideum. Forty of these developmental mutants were completely aggregation-deficient on bacterial lawns (Class I mutants) and these mutants were selected for parasexual genetic analysis. By fusing the Class I mutants with developmentally-competent strains the developmental mutations in 39 of these mutants were shown to be recessive; the remaining mutation appeared to be partially dominant. Complementation analysis of the developmental mutations in the Class I strains identified 5 complementation groups. Statistical analysis of the complementation data suggests that there are approximately 40 genes in this organism which will completely block aggregation when mutated and perhaps as many as 150 genes involved in some aspect of the aggregation process. Linkage analysis of 18 Class I developmental mutations revealed that 10 of these mutations map in linkage group II at a minimum of 5 loci.