A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

9-Hydroxydodecanoic acid,an acid from Blepharis sindica seed oil

A hitherto unknown hydroxy acid has been isolated from Blepharis sindica seed oil has been characterized as 9-hydroxydodecanoic acid by IR, NMR and mass spectral studies. The structure of this acid was further supported by its chemical transformations.     

Growth ofDiacrisia obliqua [Lep.: Arctiidae] with low doses ofBacillus thuringiensis var.Kurstaki

Early 3rd instarDiacrisia obliqua Walk. larvae were treated with concentrations ofBacillus thuringiensis var.kurstaki (Dipel^®) and the growth of treated larvae was assessed. All the doses reduced significantly the weight and survival of the insects (p<0.001).     

The effects of brain-stem section on the breathing and behavioural response to morphine in the fetal sheep

In the unanesthetized fetal sheep the administration of morphine causes initial apnoea followed by hyperpnoea. We thought that a section of the brain at midcollicular level might separate these two effects. Therefore we sectioned the brain stem of five fetuses at 132 +/- 1 (SEM) days of gestation and compared their responses to morphine (17 experiments) with that observed in seven intact fetuses at similar gestational ages (15 experiments). Brain stem sections were confirmed morphologically and histologically. Morphine, 1 mg/kg was injected in the fetal jugular vein during low-voltage electrocortical activity (ECoG). We measured ECoG, eye movements, diaphragmatic activity, blood pressure and amniotic pressure. Sectioned fetuses before the administration of morphine had a complete dissociation between ECoG and breathing activity. With the administration of morphine we found: (i) the length of the apnoea was 139.8 +/- 15.5 min in sectioned fetuses and 17.0 +/- 5.8 min in intact fetuses (P less than 0.01); and (ii) there was no hyperpneic response in the sectioned fetus whereas the length of hyperpnoea in the intact group was 99.1 +/- 11.8 min (P less than 0.001). The results support the idea of two central distinct areas of action of morphine in the fetal brain. The absence of hyperpnoea in the sectioned fetuses suggests that neurons inhibiting the 'respiratory neurons' are located rostrally to the mid-collicular line.     

In vitro flowering of Fortunella hindsii (Champ.)

Branch internodes of mature plants and stem internodes of seedlings of Fortunella hindsii flowered in vitro on half-strength MT (Murashige and Tucker 1969) basal medium supplemented with benzyladenine, adenine, 6---dimethylallylaminopurine and kinetin. The highest percentage of flowering was achieved with explants originating from branch internodes of flowering plants close to the apex on half-strength MT basal medium containing 5% sucrose and 0.01 mg 1^–1 BA in light. Exposure to darkness for more than 3 weeks followed by re-exposure to light reduced flowering. Flowering required a 4-day exposure to BA, but shoot formation could be initiated even without exposure to BA. First branch internode segments on MT basal medium containing 5% sucrose were prolific in flower (85%) production. The sucrose treatment affected the flower bud size distribution. There were about 13 flower buds per culture in the largest size category (>5 mm).     

Sterol and ochratoxin biosynthesis and miconazole action onAspergillus ochraceus activity

Miconazole (MC), an effective drug used in medicine as a topical broad-spectrum antifungal agents for treating tinea, aspergilloses and candidoses, was used for studying its effect on sterols, ochratoxin A and B biosynthesis byAspergillus ochraceus in vitro. Sterol biosynthesis and production of ochratoxin A and B were inhibited by MC when added into the culture medium together with the inoculum. MC, addition 2 d after culture inoculation, inhibited sterol biosynthesis after 5, 9 and 14 d, ochratoxin A production after 5 and 9 d and ochratoxin B production after 9 d of incubation. However, at 10, 50 and 100 ppm concentrations it induced a significant accumulation of both toxins after 14 d of incubation.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .