Isopycnography of intact cells — V: The kinetics of the initiation of DNA synthesis in E. coli undergoing nutrient shift-up

Manipulation of the ribosome content of E. coli by means of a nutrient shift-up leads to predictable changes in cellular specific gravity. Thus whole-cell pycnography can be used to monitor the proliferative status of the rRNA loci which cluster closely about the genetic origin of DNA synthesis. In this manner the rate of initiating new rounds of genome replication was followed during an upshift. The results indicate that after a short lag initiation of new rounds abruptly and completely shifts to the rate appropriate to the enriched conditions.     

Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function

Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F(+) and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F(+). The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.     

Microbial control of black cutworm (Lepidoptera: Noctuidae) in turfgrass using Agrotis ipsilon multiple nucleopolyhedrovirus

Agrotis ipsilon multiple nucleopolyhedrovirus (family Baculoviridae, genus Nucleopolyhedrovirus, AgipMNPV), a naturally occurring baculovirus, was found infecting black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), on central Kentucky golf courses. Laboratory, greenhouse, and field studies investigated the potential of AgipMNPV for managing black cutworms in turfgrass. The virus was highly active against first instars (LC50 = 73 occlusion bodies [OBs] per microl with 2-microl dose; 95% confidence intervals, 55-98). First instars that ingested a high lethal dose stopped feeding and died in 3-6 d as early second instars, whereas lethally infected fourth instars continued to feed and grow for 4-9 d until death. Sublethal doses consumed by third or fifth instars had little or no effect on subsequent developmental rate or pupal weight. Horizontal transmission of AgipMNPV in turfgrass plots was shown. Sprayed suspensions of AgipMNPV (5 x 10(8) - 6 x 10(9) OBs/m2) resulted in 75 to > 93% lethal infection of third or fourth instars in field plots of fairway-height creeping bentgrass, Agrostis stolonifera (Huds.), and on a golf course putting green collar. Virus spray residues (7 x 10(9) OBs/m2) allowed to weather on mowed and irrigated creeping bentgrass field plots significantly increased lethal infection of implanted larvae for at least 4 wk. This study, the first to evaluate a virus against a pest in turfgrass, suggests that AgipMNPV has potential as a preventive bioinsecticide targeting early instar black cutworms. Establishing a virus reservoir in the thatch and soil could suppress successive generations of that key pest on golf courses and sport fields.     

Functional insights from structural genomics

Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF_0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP_1951), and a 12-stranded β-barrel with a novel fold (V. parahaemolyticus VPA1032).     

A Clinical,Neuropathological and Genetic Study of Homozygous A467T POLG-Related Mitochondrial Disease

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.     

Effects of fire on abundance of Eragrostis intermedia in a semi-arid grassland in southeastern Arizona

Abstract. Eragrostis intermedia (Plains lovegrass) is a midheight perennial bunchgrass native to semi-arid grasslands of the southwestern USA, that becomes an abundant and dominant component of these grasslands in areas long protected from livestock grazing. Substantial mortality of plains lovegrass occurred on a large livestock exclosure in southeastern Arizona, after a period of declining precipitation, but only in areas that had not burned in the previous three years. Lovegrass abundance subsequently increased on both undisturbed and burned sites, but remained substantially higher on the burned area. Long-term abundance of plains lovegrass may depend on episodic fire, particularly during periods of reduced precipitation.     

Phosphorylation of Bruton's tyrosine kinase by c-Abl

Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.