Structure and expression of the three MHC-linked HSP70 genes

A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3 untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a 2.4 kb mRNA in cells heat-shocked at 42°C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5 flanking sequence, is expressed as a 3 kb mRNA at low levels both constitutively and following heat shock.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M34267-9. Address correspondence and offprint requests to: R. D. Campbell.     

Idiotypic analysis of polyclonal and monoclonal anti-p-azophenylarsonate antibodies of BALB/c mice expressing the major cross-reactive idiotype of the A/J strain

The idiotypic cascade allows the induction of silent idiotypes, and as such, the immune system can be reprogrammed towards predetermined goals. To understand the genetic origin of silent idiotypes, we have used a system in which detailed structural and genetic information is available. The major cross-reactive idiotype (CRIA) of A/J mice (positive strain) immunized with arsonate coupled to a carrier can be regularly induced in BALB/c mice (negative strain) by anti-idiotypic treatment with or without subsequent antigen immunization. By using a panel of monoclonal anti-idiotypic antibodies, we have found that the germline-encoded CRIA displays a mosaic of at least five idiotopes. Polyclonal and monoclonal anti-arsonate antibodies prepared from idiotypically manipulated BALB/c mice have been studied. Four germline idiotopes are shared between the CRIA of the A/J strain and the CRIA-like idiotype induced in BALB/c mice. Furthermore, CRIA-like antibodies can appear "spontaneously" in some BALB/c mice immunized with antigen only. The data suggest that anti-idiotypic treatment in BALB/c mice selects a preexisting subset of antibodies. From the serological analysis, it is predicted that CRIA molecules from A/J and CRIA-like molecules from BALB/c employ different VH subgroups but share some components of the hypervariable regions. These predictions are tested in a forthcoming paper that describes the amino acid sequences of BALB/c monoclonal antibodies displaying the major cross-reactive idiotype of the A/J strain.     

Scale-dependent habitat selection by wintering geese: implications for landscape management

The management of Arctic migrant geese is complex, because they frequently use landscapes under intensive human use, and are conflict species in multiple respects. Some populations are of high conservation concern, but they also cause agricultural damage, are quarry for hunters, and may be particularly sensitive to infrastructure developments. In Bulgarian Dobrudzha, large wintering populations of greater white-fronted geese Anser albifrons and red-breasted geese Branta ruficollis feed in agricultural land, and cause management dilemmas. We developed linear models to investigate fine- and meso-scale foraging habitat selection of geese foraging on winter wheat in the area, and used these models to make suggestions for zoning landscape use in order to reduce conflict and conserve geese. Habitat selection was scale-dependent. Geese selected fields that were near to major roosts and had low proximity to roads and tree-lines, which may be a proxy for hunting disturbance. We found some evidence for selection of wheat fields with high nutritional quality. Within fields, geese strongly avoided features which cause landscape ‘clutter’: power-lines, tree-lines and wind-turbines, but primarily over distances of less than a few hundred metres. Optimal management might involve encouraging goose populations to feed in areas close to roosts, by means of agri-environmental measures and creation of hunting-free refuges. This would allow efficient use of agri-environment funds, might reduce conflict with farmers, and would mean that infrastructure development—notably wind farms—could be sited at greater distance from roosts with relatively minor impact on foraging habitat availability.     

Decline in Ecosystem <Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C and Mid-Successional Nitrogen Loss in a Two-Century Postglacial Chronosequence

Uncertainty about controls on long-term carbon (C) and nitrogen (N) balance, turnover, and isotopic composition currently limits our ability to predict ecosystem response to disturbance and landscape change. We used a two-century, postglacial chronosequence in Glacier Bay, Alaska, to explore the influence of C and N dynamics on soil and leaf stable isotopes. C dynamics were closely linked to soil hydrology, with increasing soil water retention during ecosystem development resulting in a linear decrease in foliar and soil δ¹³C, independent of shifts in vegetation cover and despite constant precipitation across sites. N dynamics responded to interactions among soil development, vegetation type, microbial activity, and topography. Contrary to the predictions of nutrient retention theory, potential nitrification and denitrification were high, relative to inorganic N stocks, from the beginning of the chronosequence, and gaseous and hydrological N losses were highest at mid-successional sites, 140–165 years since deglaciation. Though leaching of dissolved N is considered the predominant pathway of N loss at high latitudes, we found that gaseous N loss was more tightly correlated with δ¹⁵N enrichment. These results suggest that δ¹³C in leaves and soil can depend as much on soil development and associated water availability as on climate and that N availability and export depend on interactions between physical and biological state factors.     

A Novel Protein Expressed in Mammalian Cells Undergoing Apoptosis

Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 M_r protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca²⁺ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, nonmuscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the cytoskeleton, lead us to conclude that ASP forms part of, or at least strongly associates with, a modified cytoskeleton unique to cells undergoing apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis in vivo and in vitro.     

Phosphorylation of the carboxyl terminal region of dystrophin by mitogen-activated protein (MAP) kinase

Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy gene (DMD). The function of this protein remains to be elucidated. We have recently reported that dystrophin is phosphorylated,in vivo, in rat skeletal muscle primary cell culture (RE Milner, JL Busaan, CFB Holmes, JH Wang, M Michalak (1993) J Biol Chem 268: 21901–21905). This observation suggests that protein phosphorylation may have some role in modulating the function of dystrophin or its interaction with membrane associate dystroglycan. We report here that the carboxyl-terminal of dystrophin is phosphorylated by the MAP kinase p44^mpk (mitogen-activated protein kinase), from the sea star oocytes and by soluble extracts of rabbit skeletal muscle. Importantly we showed that native dystrophin in isolated sarcolemmal vesicles is phosphorylated by sea star p44^mpk. Partial purification and immunological analysis show that a mammalian kinase related to p44^mpk is present in the skeletal muscle extracts and that it contributes to phosphorylation of the carboxyl-terminal of dystrophin. This kinase phosphorylates dystrophin on a threonine residue(s). We conclude that phosphorylation of dystrophin may play an important role in the function of this cytoskeletal protein.Abbreviations MAP kinase mitogen-activated protein kinase - DMD Duchenne muscular dystrophy - GST Glutathione S-transferase - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MOPS 4-morpholinepropanesulfonic acid     

Prospects for biopesticides for aphid control

Diseases form an important component of the natural enemy complex of aphids. The most common and obvious of these diseases are entomophthoran fungi such asErynia neoaphidis Remaudiere & Herbert,Entomophthora planchoniana Cornu,Zoophthora radicans (Brefeld) Batko andConidiobolus obscurus (Hall & Dunn) Remaudiere & Keller. The pest status of some aphids such as the pea aphid,Acyrthosiphon pisum (Harris), is considerably reduced by natural epizootics of fungal disease. However, disease may contribute little to practical control as it is mainly effective in high density populations when weather conditions are suitable. Introduction of exotic diseases for classical biological control is only rarely possible since most diseases, like their aphid hosts, are distributed world-wide. One exception was the successful introduction into Australia of a strain ofZ. radicans for control of spotted alfalfa aphid,Therioaphis trifolii (Monell) f.maculata in 1979. Attempts to manipulate entomophthoran fungi have had limited success because of problems with mass production, the fragility of the conidia and the need for suitably moist conditions. Hyphomycete fungi such asVerticillium lecanii (Zimm.) Viegas,Metarhizium anisopliae (Metsch.) Sorokin,Beauveria bassiana (Bals.) Vuill. andPaecilomyces spp. are more suitable for development as mycoinsecticides as they are cheap to mass produce and form stable conidia. “Vertalec?”, a formulation ofV. lecanii, has been sold in small quantities commercially in Britain and parts of Europe for many years and used mainly in glasshouses. Recently promising results have been obtained with use ofM. anisopliae for control of lettuce root aphid,Pemphigus bursarius (L.) in the UK. Laboratory studies on selected isolates ofB. bassiana andPaecilomyces spp. show a promising level of activity. Problems may occur with these species as they can kill aphid predators such as coccinelids. In addition, more research is needed on developing improved formulations which enable control to be achieved under low humidity conditions.