Species-Specific Oligonucleotides for Enumeration of Pseudomonas putida F1, Burkholderia sp. Strain JS150, and Bacillus subtilis ATCC 7003 in Biodegradation Experiments

Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism, Pseudomonas putida F1 and Burkholderia sp. strain JS150, and a third, Bacillus subtilis ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.     

Medium optimization for recombinant protein production by Bacillus subtilis

Summary Optimal concentrations of glucose, yeast extract and nutrient broth for the production of -lactamase by B. subtilis were predicted using a statistical experimental design and then tested. More yeast extract (13 vs. 1 g/L), less glucose (7.4 vs. 10 g/L), and less nutrient broth (12.6 vs. 15 g/L) were required to achieve high -lactamase activities (5700 U/L) instead of high cell growth rates (1.2 h^-1).     

Effect of seasonal placement ofCotesia melanoscela (Hym.: Braconidae) on its potential for effective augmentative release againstLymantria dispar (Lep.: Lymantriidae)

Cohorts ofCotesia melanoscela (Ratzeburg) (Hymenoptera: Braconidae) cocoons were exposed in the field at three Maryland locations to attack by natural enemies for two week periods, then were held in an outdoor insectary untilC. melanoscela adult or hyperparasitoid emergence. The timing of placement of theC. melanoscela cocoons in the field had a profound effect on the number ofC. melanoscela that survived and emerged as adults in synchrony with the field occurence of susceptible early-instarLymantria dispar (L.) larvae. The proportion of emerged adults available during susceptible host stages ranged from 1–92%, depending on dates of release. November or December placements ofC. melanoscela cocoons were most effective with 74–92 % emergence of adults during peak periods of susceptible host stages. Spring placements were least effective. The causes of ineffective placement, which varied with location and with date, were program (handling) loss, non-emergence, attack by hyperparasitoids, predation, andC. melanoscela adult emergence at times when appropriateL. dispar life stages would not be present. We concluded that November/December releases avoided natural enemies and promoted appropriate diapause and post-diapause development that enhanced survival and synchrony of adult emergence with host stage susceptibility.     

ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues.     

Development of Dryland Oilseed Production Systems in Northwestern Region of the USA

This report addresses the development of dryland oilseed crops to provide feedstock for production of biofuels in semi-arid portions of the northwestern USA. Bioenergy feedstocks derived from Brassica oilseed crops have been considered for production of hydrotreated renewable jet fuel, but crop growth and yields in the northwestern region are limited by a lack of plant available water. Based on a review of the scientific literature, several areas were identified where research could be directed to provide improvements. The current agronomic limitations for oilseed production are mainly due to seedling establishment under extreme heat, dry seedbeds at optimum planting times, survival under extreme cold, and interspecific competition with weeds. To improve emergence and stand establishment, future work should focus on developing soil management and seeding techniques that optimize plant available water, reduce heat stress, and provide a competitive advantage against weeds that are customized for specific crops, soil types, and soil and environmental conditions. Spring and winter cultivars are needed that offer increased seedling vigor, drought resistance, and cold tolerance.     

Molecular Analysis of Diepoxybutane-Induced Mutations at the rosy Locus of Drosophila melanogaster

We have analyzed at the molecular level diepoxybutane-induced mutants determined to have lesions affecting expression of the ry locus. Of the 21 mutants analyzed here, genetic analysis suggested that five were putative deficiencies involving ry and adjacent lethal loci. However, molecular analysis confirmed that only two of these five putative deficiencies were in fact deletions detectable by the methods used in the analysis. The remaining 16 mutants were viable as homozygotes, suggesting that their lesions were confined to the ry locus. Seven of these 16 intragenic mutants were determined to be deletions of genetic material as evidenced by altered restriction patterns relative to the wild type patterns. Thus, nine of 21 (43%) diepoxybutane-induced mutants are due to deletions ranging in size from approximately 50 base pairs to more than 8 kilobase pairs. Most of the deletions (seven of nine or 78%) are intragenic and less than 250 base pairs in size; it seems that most, if not all, affect coding rather than regulatory sequences.     

Amphotericin B renders stationary phase hepatoma cells sensitive to dipyridamole

This study provides evidence that the dipyridamole inhibitory effect on nucleoside incorporation changed with culture time. Lag and log phase hepatoma 3924A cells were highly sensitive to dipyridamole with IC50 values for thymidine incorporation of 0.20 and 0.31 microM, respectively. In contrast, stationary phase cells were comparatively insensitive to dipyridamole with an IC50 of 38.9 microM. Amphotericin B (10 microM) restored the sensitivity of stationary phase cells to dipyridamole, lowering the IC50 value for thymidine incorporation to 0.25 microM. Amphotericin B also enhanced the cytotoxicity of dipyridamole to hepatoma 3924A cells. The combination of amphotericin B and dipyridamole may be useful in cancer chemotherapy.     

Microdeletions in patients with gusher-associated, X-linked mixed deafness (DFN3)

Employing various probes from the proximal part of the Xq21 region, which is known to harbor the DFN3 gene, we have investigated 13 unrelated male probands with X-linked deafness, to detect possible deletions. For two of these patients, microdeletions could be detected by using probe pHU16 (DXS26). One of these deletions also encompasses locus DXS169, indicating that it extends farther toward the centromere. The presence of normal hybridization patterns in the DNA of 25 unrelated control males suggests that these deletions are the primary cause of progressive mixed deafness in these patients. If so, their molecular characterization may pave the way for the identification and isolation of the corresponding gene.     

Amino acid biosynthesis in plants: Approaching an understanding at the molecular level

Summary The genes encoding GS and EPSP synthase have already been cloned. Clones containing portions of the genes encoding aspartate amino transferase and asparagine synthetase tentatively have been identified by this and other laboratories. It is certain that many other genes encoding other important enzymes involved in amino acid biosynthesis will also be identified in the near future. It is evident that new techniques for the separation of proteins will greatly aid in identifying low abundance genes more rapidly and efficiently. Micro techniques need to be further developed and the necessary equipment made available to laboratories. Hopefully the cost of the required equipment will decrease and commercial enterprises will offer more contract services for work requiring the more expensive equipment. Graduate training in advanced techniques of separating proteins at high resolutions should be encour-aged expanded so that our future scientists are well versed in protein biochemistry as well as in techniques of molecular biology.