Cytological characterization and allelism testing of anther developmental mutants identified in a screen of maize male sterile lines.

Proper regulation of anther differentiation is crucial for producing functional pollen, and defects in or absence of any anther cell type result in male sterility. To deepen understanding of processes required to establish premeiotic cell fate and differentiation of somatic support cell layers a cytological screen of maize male-sterile mutants has been conducted which yielded 42 new mutants including 22 mutants with premeiotic cytological defects (increasing this class fivefold), 7 mutants with postmeiotic defects, and 13 mutants with irregular meiosis. Allelism tests with known and new mutants confirmed new alleles of four premeiotic developmental mutants, including two novel alleles of msca1 and single new alleles of ms32, ms8, and ocl4, and two alleles of the postmeiotic ms45. An allelic pair of newly described mutants was found. Premeiotic mutants are now classified into four categories: anther identity defects, abnormal anther structure, locular wall defects and premature degradation of cell layers, and/or microsporocyte collapse. The range of mutant phenotypic classes is discussed in comparison with developmental genetic investigation of anther development in rice and Arabidopsis to highlight similarities and differences between grasses and eudicots and within the grasses

Nuclear mutations affecting mitochondrial structure and function in Chlamydomonas

Wild type cells of the green alga Chlamydomonas reinhardtii can grow in the in the dark by taking up and respiring exogenously supplied acetate. Obligate photoautotrophic (dark dier, dk) mutants of this alga have been selected which grow at near wild type rates in the light, but rapidly die when transferred to darkness because of defects in mitochondrial structure and function. In crosses of the dk mutants to wild type, the majority of the mutants are inherited in a mendelian fashion, although two have been isolated which are inherited in a clearly nonmendelian fashion. Nine mendelian dk mutants have been analyzed in detail, and belong to eight different complementation groups representing eight gene loci. These mutants have been tentatively grouped into three classes on the basis of the pleiotropic nature of their phenotypic defects. Mutants in Class I have gross alterations in the ultrastructure of their mitochondrial inner membranes together with deficiencies in cytochrome oxidase and antimycin/rotenone-sensitive NADH-cytochrome c reductase activities. Mutants in Class II have a variety of less severe alterations in mitochondrial ultrastructure and deficiencies in cytochrome oxidase activity. Mutants in Class III have normal or near normal mitochondrial ultrastructure and reduced cytochrome oxidase activity. Eight of the nine mutants show corresponding reductions in cyanide-sensitive respiration

Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization.

We used polyclonal antisera and monoclonal antibodies (mAbs) to inhibit the growth of clonal populations of two strains of Borrelia burgdorferi, the Lyme disease agent, and thereby select for antibody-resistant mutants. mAbs were directed at the outer membrane proteins, OspA or OspB. Mutants resistant to the growth-inhibiting properties of the antibodies were present in the populations at frequencies ranging from 10(-5) to 10(-2). The several escape variants that were examined were of four classes. Class I mutants were resistant to all mAbs; they lacked OspA and OspB and the linear plasmid that encodes them. Two other proteins were expressed in larger amounts in class I mutants; mAbs to these proteins inhibited the mutant but not the wild-type cells. Class II mutants were resistant to some but not all mAbs; they had truncated OspA and/or OspB proteins. Class III mutants were resistant only to the selecting mAb; they had full-length Osp proteins that were not bound by the selecting antibody in Western blots. In two class III mutants resistant to different anti-OspA mAbs, missense mutations were demonstrated in the ospA genes. Class IV mutants were likewise resistant only to selecting antibody, but in this case the selecting antibody still bound in Western blots

Increased protein stability and decreased protein turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 mutant

In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical S-35 pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose

Fission yeast 26S proteasome mutants are multi-drug resistant due to stabilization of the pap1 transcription factor

Here we report the result of a genetic screen for mutants resistant to the microtubule poison methyl benzimidazol-2-yl carbamate (MBC) that were also temperature sensitive for growth. In total the isolated mutants were distributed in ten complementation groups. Cloning experiments revealed that most of the mutants were in essential genes encoding various 26S proteasome subunits. We found that the proteasome mutants are multi-drug resistant due to stabilization of the stress-activated transcription factor Pap1. We show that the ubiquitylation and ultimately the degradation of Pap1 depend on the Rhp6/Ubc2 E2 ubiquitin conjugating enzyme and the Ubr1 E3 ubiquitin-protein ligase. Accordingly, mutants lacking Rhp6 or Ubr1 display drug-resistant phenotypes

Global analysis of serine/threonine and tyrosine protein phosphatase catalytic subunit genes in Neurospora crassa reveals interplay between phosphatases and the p38 mitogen-activated protein kinase.

Protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. The genome of the filamentous fungus and model organism Neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. In this study, we have characterized 24 viable N. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. We found that 91% of the mutants had defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock

Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions

Calcium Homeostasis in Escherichia coli: Characterization of Mutants and Genome Expression of MG1655

While the role of calcium ions as secondary messengers has been well described in eukaryotic cells, little is known about calcium homeostasis in bacteria at the physiological and molecular levels. Genetic and genomic approaches were used to address calcium regulation and to identify genes (cal) involved in calcium homeostasis. Transposon mutagenesis of Escherichia coli generated several calcium-sensitive mutants that fell into three categories: (i) Ca2+-sensitive chemotaxis mutants, (ii) Ca2+-sensitive cell division mutants, and (iii) Ca2+-sensitive mutants that showed no defects in cell division or chemotaxis. The physiological properties of these Ca2+-sensitive mutants were determined. Besides calcium-sensitivity to 75 mM calcium, all of the mutants exhibited increased sensitivities to several divalent cations including Ni2+, Mg2+, Mn2+, Co2+, Zn2+, Cu2+, and Cd2+. To identify the cal gene sequence in the Ca2+-sensitive mutants, the region of the genes fused to the reporter gene (phoA) on the transposon TnphoA was amplified by PCR and sequenced. The sites of the gene fusion for three cal mutants were at the fdoG, gpt and pqi5 genes. The pleiotropic nature for the cal mutations suggested that many genes may be globally regulated by calcium. We then investigated global gene expression patterns of wild-type E. coli under calcium-depleted (addition of 10 mM EGTA) and calcium-elevated (addition of 75 mM Ca2+) conditions as compared to cultures grown under unstressed conditions. A comprehensive transcriptome analysis using macroarrys exhibited a global regulation of diverse genes within the E. coli genome during calcium homeostasis

Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase

The dnaQ gene of Escherichia coli encodes the Ɛ subunit of DNA polymerase III, which provides the 3\u27 - 5\u27 exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of Ɛ leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, Ɛ plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQgene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that Ɛ has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of e in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants