A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.     

Isolation of microsatellite loci in the Capricorn silvereye,Zosterops lateralis chlorocephalus (Aves: Zosteropidae)

The Capricorn silvereye (Zosterops lateralis chlorocephalus) is ideally suited to investigating the genetic basis of body size evolution. We have isolated and characterized a set of microsatellite markers for this species. Seven out of 11 loci were polymorphic. The number of alleles detected ranged from two to five and observed heterozygosities between 0.12 and 0.67. One locus, ZL49, was found to be sex‐linked. This moderate level of diversity is consistent with that expected in an isolated, island population.     

Seasonal and annual variability in the phytoplankton community of the Raunefjord,west coast of Norway from 2001–2006

Changes in phytoplankton community composition potentially affect the entire marine food web. Because of seasonal cycles and inter-annual variations in species composition, long-term monitoring, covering many sequential years, is required to establish a baseline study and to reveal long-term trends. The current study describes the phytoplankton biomass variations and species composition in relation to hydrographic and meteorological conditions in the Raunefjord, western Norway, over a 6-year period from 2001 to 2006. The extent of inflow or upwelling in the fjord varied from year to year and resulted in pronounced differences in water column stability. The annual phytoplankton community succession showed some repeated seasonal patterns, but also high variability between years. Two to four diatom blooms were observed per year, and the spring blooms occurring before water column stratification in March were dominated by Skeletonema marinoi and Chaetoceros socialis, and other Chaetoceros and Thalassiosira spp. Blooms of the haptophytes Phaeocystis pouchetii and Emiliania huxleyi were irregular and in some years totally absent. Although E. huxleyi was present all year round it appeared in bloom concentrations only in 2003, when the summer was warm and the water column characterized by high surface temperatures and pronounced stratification. The annual average abundance of both diatoms and flagellates increased during the six years. Despite the high variation from year to year, our investigation provides valuable knowledge about annual phytoplankton community patterns in the region, and can be used as a reference to detect possible future changes.     

Lineage analysis of the olfactory epithelium using a replication-incompetent retrovirus

We have analysed the lineage of olfactory receptor neurons usinga replication-incompetent retrovirus injected beneath the olfactoryepithelium of young rats. There are two major types of clustersof infected cells seen at 5–40 days after infection: (i)horizontal basal cells (HBCs); (ii) variable numbers of globosebasal cells (GBCs), and immature and mature sensory neurons.Olfactory nerve lesion increased the frequency of the globose/sensoryneuron clusters, as well as the number of cells/cluster, butdid not change the number of HBC clusters or cells/cluster.No clusters contained sustentacular cells. These data indicatethat, at least in young rats: (i) HBCs are not precursors ofolfactory neurons; (ii) there is a lineage path from GBCs tomature neurons; and (iii) sustentacular cells arise from a separatelineage.     

Effect of change in particle number on pulmonary clearance of aerosolized 99mTc-DTPA

Pulmonary clearance (PCl) of inhaled aerosolized 99mTc-diethylenetriamine pentaacetic acid (DTPA) across the alveolocapillary membrane is diffusion limited. Therefore, if the mixing of the 99mTc-DTPA in the aqueous hypophase underlying surfactant is slow or incomplete or if there were no hypophase, an increase in the alveolar surface area occupied by 99mTc-DTPA particles would increase the absorption rate. The aim of this study was to examine whether there is an effect on PCl of changing the number of inhaled particles. The change in particle number was accomplished by a setup of four parallel jet nebulizers feeding a central delivery chamber of 400 cm3. We performed two kinds of experiments in eight healthy nonsmokers between 28 and 52 yr of age. In the first experiment, 99mTc-DTPA in saline was nebulized in one nebulizer, while saline was nebulized in the other three. In the second experiment the number of inhaled particles containing 99mTc-DTPA was increased by a factor of four by nebulizing 99mTc-DTPA in saline in all four nebulizers simultaneously. Increasing the number of inhaled 99mTc-DTPA particles caused an increase in PCl of 24.2% (P less than 0.01). We conclude that there is a slight but significant effect of changing the number of DTPA particles on PCl and that this is probably due to an uneven mixing of the 99mTc-DTPA in the aqueous hypophase underlying the surfactant lining and the alveoli.