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.     

Ethological study of early language

Sign language studies of cross-fostered chimpanzees measure the effect of special rearing conditions on the development of very young chimpanzees. Cross-fostered chimpanzees, like human children, develop gradually in a process that takes many years. Here we discuss details of the procedure, the overlap between human and chimpanzee infants in the contents of the first 50-item vocabularies, and the ways in which the signs of the chimpanzees exhibit the fuzziness of natural language categories. We also compare the cross-fostering approach with more traditional modular approaches to the study of language-like behavior in nonhuman animals. Project Washoe was originally supported by grants MH-12154 from the National Institute of Mental Health and GB-7432 from the National Science Foundation. We gratefully acknowledge this support and the support that later sign language studies of chimpanzees have received since then from NIH, NSF, the National Geographic Society, the Grant Foundation, the Spencer Foundation, the University of Nevada, and the UNR Foundation.     

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.     

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.