In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.     

Electron spin resonance evidence for the formation of free radicals in plants exposed to ozone

Electron spin resonance (ESR) spectroscopy was used to demonstrate that free radicals are formed in O₃-fumigated plant leaves prior to the formation of visible leaf injury. ESR signals with a g-value of 2.0037 to 2.0043, were observed in pea ( Pisum sativum L. cv. Feltham first) and bean ( Phaseolus vulgaris L. cv. Pinto) plants that had been fumigated for 4 h with 70–300 nl l⁻¹ of ozone after they had been treated with the spin-trap N- t -butyl-α-phenylnitrone (PBN). The size of the ESR signals increased with the concentration of ozone used but the nature of the trapped radicals could not be identified. However, further experiments using an inhibitor of ethylene biosynthesis, arninoethoxyvinyl glycine (AVG), showed that the reaction between ozone and ethylene is the cause for ozone toxicity.     

Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

Simultaneous fructose and ethanol production from sucrose,usingZymomonas mobilis 2864 co-immobilised with invertase

Summary Fructokinase negativeZymomonas mobilis UQM 2864, was co-immobilised with invertase in alginate and incubated on sucrose-based media in batch and fedbatch culture. The highest fructose concentration achieved was 138 g/l using fed-batch cultivation with sugar-cane syrup-simultaneously producing 79.9 g/l or 10.1% (v/v) ethanol in less than 24 hours. The ethanol and fructose yields were 95 and 84% respectively. Co-immobilisation resulted in faster fermentation times, particularly for the batch fermentations, and complete utilisation of substrate.     

TGGE and HIEF: a comparison of two methods in the detection of carriers of the Z mutation of the alpha-1-antitrypsin gene

Alpha-1-antitrypsin (-1-AT) deficiency can lead to juvenile liver cirrhosis and lung emphysema in adulthood. The deficiency Z allele is caused by a base transition. Temperature gradient gel electrophoresis (TGGE) and hybrid isoelectric focusing (HIEF) were used to detect carriers of the Z mutation of the -1-AT gene. The resulting data were compared. To verify carriers at the sequence level, a manual nonradioactive sequencing strategy was established. Among our sample of carriers of the Z mutation, two were not detected by HIEF that could be identified by TGGE. DNA of all TGGE identified individuals harboring the Z mutation of the -1-AT gene were sequenced nonradioactively. All carriers harbored a G to A transition at position 11.940. This mutation is described to cause the altered protein.     

A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters

Scrapie hamster brains contain at least 5–10 g of scrapie-associated fibrils (SAF) per brain as estimated by the amount of its major constituent, a protein of about 26 000 daltons (SAF-protein). It can be extracted efficiently by a 10% solution of sarkosyl and can be enriched by differentia] centrifugation and buffer extraction. Scrapie infectivity, SAF, and SAF-protein copurify.