Steven B. Ellis

The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos

The ability to determine the sex of embryos prior to transfer to recipients has commercial application in the embryo transfer industry. A number of methods of sex determination have been attempted; however, none of these has been commercially successful to date. The identification of repetitive, male-specific bovine chromosomal DNA fragments enables the use of DNA-probe technology in determining the sex of preimplantation embryos from a small embryonic sample. The identification and isolation of three repetitive, male-specific bovine chromosomal DNA fragments, and the application of these probes in a sensitive and highly accurate embryo sexing assay are reported.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum. Within the cerebellar cortex, these pore forming proteins were found to have differential localisations when examined in adjacent sections. The alpha 1A and alpha 1B subunits broadly colocalised and were both present, though at apparently different levels, in the molecular, Purkinje and granule cell layers whilst alpha 1E was predominantly expressed in Purkinje cells. In the dentate nucleus, an area which has received little attention in previous studies, alpha 1A was highly expressed in regions in which Purkinje cell nerve terminals form synapses with deep cerebellar neurones.

Comparative structure of human neuronal α2–α7 and β2–β4 nicotinic acetylcholine receptor subunits and functional expression of the α2, α3, α4, α7, β2, and β4 subunits

AbstractcDNA clones encoding human neuronal nicotinic acetylcholine receptor α2, α3, α4, α5, α6, α7, β2, β3, and β4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human α2 and α6 and full-length β3 and β4 clones have not been previously reported. Deduced amino acid sequences of the α2, α6, β3, and β4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (α2), 87 (α6), 89 (β3), and 84% (β4) identity to the corresponding rat sequences. The amino termini of the human α2 and β3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat α2 and β3. Recombinant receptors were expressed inXenopus laevis oocytes injected with in vitro transcripts encoding either α7 alone or α2, α3, or α4 in pairwise combination with β2 or β4. Inward currents were elicited by the application of acetylcholine (1–100 µM) and other agonists; these responses were blocked 65–97% by application of 10 µM d-tubocurare, confirming functional expression of human nicotinic receptors.

Distribution ofα1A, α1B andα1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

Abstract The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits,α 1A , α 1B andα 1E in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level,α 1A , α 1B andα 1E subunits were differentially localized. In general,α 1A -immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-a and pri-a cells. Theα 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. Theα 1E staining pattern shared features in common with bothα 1A andα 1B with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-α cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison ofα 1B andα 1E staining in the CA3 stratum lucidum with calbindin-immunoreactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.

The expression of voltage-dependent calcium channel beta subunits in human cerebellum

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.