HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Präadaptation, Wirtskreiserweiterung and Parallel-Cladogenese in der Evolution von phytophagen Insekten1, 2

Preadaptation, host shifts and parallel cladogenesis in the evolution of phytophagous insects In this contribution we investigate the possibilities to apply concepts developed for the evolution of animal parasites to insect-plant systems. We compare host parasite systems in animals with plant-herbivore systems and list similarities and differences. The terms preadaptation, predisposition, expansion and contraction of host ranges, and parallel cladogenesis are discussed. We enumerate general preadaptations for the evolution of herbivory in insects and preadaptations for shifts between herbivory and entomophagy. Examples are given for expansions of host ranges based on phytochemical or structural characters of host plants. Cases of parallel cladogenesis in herbivoreparasitoid systems and plant-herbivore systems are compiled from the literature. An analysis of the insect fauna of the “thistles” (Cynaroideae) in the Palearctic and Nearctic demonstrates the importance of the evolutionary history of the plant taxa and of the existence of preadapted pools of herbivores for the evolution of guilds of specialized herbivores. The members of the Curculionid taxon Cleoninae provide examples for multiple colonizations and radiations of herbivores on the Cynaroideae. The taxonomic and biological relationships of the weevil genera Rhinocyllus, Bangasternus and Larinus which exploit the flower heads of Cynaroideae, can be interpreted as result of a basic parallel cladogenesis between herbivore and host. A gelelectrophoretic analysis of Larinus spp. supports this hypothesis.     

Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative population ecology of Ephydra hians Say (Diptera: Ephydridae) at Mono Lake (California) and Abert Lake (Oregon)

The population dynamics of Ephydra hians Say final instar larvae and pupae were compared over a two year period in rocky littoral habitats of two alkaline saline lakes in the western Great Basin. Relative abundance increased from 1983 to 1984 at Mono Lake (California), during dilution from ca. 90 to 80 g 1^-1 TDS (total dissolved solids). In contrast, relative abundance decreased over the same period at Abert Lake (Oregon), accompanied by a dilution of salinity from ca. 30 to 20 g l^-1 and a marked increase in the number and abundance of other benthic macroinvertebrate species. These observations are consistent with a hypothesis that proposes biotic interactions limit E. hians abundance at low salinity, and physiological stress limits abundance at high salinity.Oviposition extends from early spring to early fall. Mixed instars present throughout this period indicates multivoltine population dynamics with overlapping generations. The standing stock biomass of final instars increases exponentially in late spring and peaks in late summer or early fall. Pupae increase in proportional representation and abundance from a spring minimum to a fall maximum. The body size of adults and pupae cycle seasonally from a spring maximum to a fall minimum, and may be related to either or both food limitation, or water temperature.     

A new isotype sequence (V kappa 27) of the variable region of kappa-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides.

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

A population model for the alkali fly at Mono Lake: depth distribution and changing habitat availability

The densities of alkali fly larvae and pupae were measured in relation to depth and substrate type at six locations around Mono Lake. Samples representing a mixture of different bottom features were taken to a depth of 10 m (33 ft) using SCUBA. This is at or near the depth limit of fly larvae and pupae. The biomass of larvae and pupae on hard substrate were maximum and approximately equal at depths of 0.5 m and 1 m, substantially lower at intermediate depths of 3 m and 5 m, and over an order of magnitude further reduced at 10 m. Densities of flies on hard or rocky substrates (mainly calcareous tufa deposits), were significantly greater than those found on soft substrates such as mud or sand, at all but the greatest depth surveyed.Bathymetric maps of the areas of hard and soft substrate occurring at different lake depths were used to estimate the fly population size over the whole lake, based on the density distribution of larvae and pupae with depth on different substrates. The mapped areas of soft and hard substrates were also calculated for different lake levels, and applying the same procedure, a population model comparing the abundance of flies at different lake levels was developed. This habitat-based population model predicts that the abundance of the alkali fly is maximized at 6380 ft (1945 m) lake surface elevation. Most of the tufa substrate submerged at this lake level will become exposed and unavailable as habitat as the lake declines to 6370 ft (1942 m). In late 1991, the lake level was just over 6374 ft (1943 + m).