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R J Milner

Publications and source records attributed to R J Milner.

At least 55 records · Page 3Linked to original sources
PubMed1986

Brain-specific gene expression.

The brain of an adult rat expresses approximately 30,000 different brain-specific mRNAs. To investigate their encoded proteins, we have selected cDNA clones corresponding to mRNAs expressed exclusively in rat brain, determined their nucleotide sequences and generated antisera against synthetic peptides mimicking short regions of the deduced protein sequences. The clone plB236 encodes a protein that defines a widely distributed neuronal system and may be the precursor for a family of novel neuropeptides. A second clone, plB208, encodes rat brain proteolipid protein, the major protein component of central nervous system myelin. These studies have also identified an 82 nucleotide genetic element called an ID (identifier) sequence that may be involved in the regulation of transcription of brain-specific genes.

Amino Acid Sequence↗
PubMed1986

The brain-specific gene 1B236 is expressed postnatally in the developing rat brain.

The rat brain-specific polypeptide 1B236 was previously characterized by molecular cloning and nucleotide sequence determination of its mRNA. It has been shown to exist in rat brain in discrete neuronal circuits, primarily as a 100,000 Da glycoprotein. We now have determined the time course of expression of 1B236 mRNA and protein in rat brain during fetal and postnatal development, detecting 1B236 mRNA by RNA blotting and assaying 1B236 protein by electroblotting and radioimmunoassay with antibodies against synthetic peptides. By both indices, expression of the 1B236 gene products is found to be a relatively late event in neuronal development. 1B236 mRNA is first detectable in extracts of whole rat brain at Postnatal Day 5 (PD 5) and increases to a maximum concentration at PD 25. In extracts of dissected brain regions, 1B236 mRNA is first detectable at PD 5 in hindbrain and cerebellum, at PD 9 in midbrain/diencephalon, but not until PD 13 in telencephalon. The appearance of 1B236 protein follows a very similar time course to that of its mRNA in both whole brain and dissected brain regions, suggesting that the expression of the protein during development is regulated largely by transcription of its mRNA. The pattern of 1B236 expression was confirmed by immunocytochemical localization of 1B236 protein: Immunoreactive material can be detected first in spinal cord at PD 3-PD 5 and then appears in progressively more rostral brain regions in increasingly older animals. Several brain regions, however, that do not contain 1B236 immunoreactivity in the adult, such as optic nerve and somatic efferent cranial nerve nuclei, show transient expression of 1B236 during postnatal development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein.

The 3200 and 1600 nucleotide mRNAs encoding rat brain proteolipid protein (PLP), the major protein component of central nervous system myelin, are heterogeneous at their 5' ends, differ in their 3' polyadenylation sites, and are transcribed from a single gene. The mRNAs, which first appear postnatally, encode identical 277 amino acid proteins that are 99% identical to the bovine protein sequence. Thus, PLP has been highly conserved during mammalian evolution. A single amino-terminal methionine is removed post-translationally, indicating that PLP does not require a signal peptide sequence for insertion into the myelin membrane. Mouse and monkey utilize the 3200 but not the 1600 nucleotide mRNA, suggesting that there is no functional necessity for two sizes of rat PLP mRNAs.

Amino Acid Sequence↗
PubMed1985

Brain-specific polypeptide 1B236 exists in multiple molecular forms.

The COOH-terminal amino acid sequence of the rat brain-specific polypeptide 1B236 was previously deduced from molecular cloning and nucleotide sequence determination of its mRNA and the 1B236 protein shown to exist in the rat brain. The amino acid sequence of 1B236 contained at least three peptide sequences demarcated by pairs of basic amino acids--a structure similar to known neuropeptide and hormone precursors--which suggested that the protein might be processed in vivo to generate peptides. We have developed radioimmunoassays specific for 1B236 with antibodies against three synthetic peptides corresponding to putative cleavage products of this protein and have used these assays to define the molecular forms of 1B236 in rat brain extracts. The most abundant form is of high molecular weight (ca. 100,000) and requires detergent for solubilization; hence, it is probably membrane-bound. However, a small fraction of the high molecular weight material is soluble in the absence of detergent. In addition, several low molecular weight species are detectable in brain extracts prepared under conditions preventing proteolysis. These molecules correspond in size to two of the possible products of proteolytic processing predicted from the amino acid sequence of 1B236. The multiplicity of 1B236 forms, together with other data, suggests that this protein undergoes extensive post-translational modification, including proteolytic processing to generate peptides that may be physiologically relevant.

Animals↗
PubMed1985

Immunocytochemical mapping of 1B236, a brain-specific neuronal polypeptide deduced from the sequence of a cloned mRNA.

The 318-amino acid, carboxy-terminal sequence of the putative brain-specific polypeptide 1B236 was deduced from the nucleotide sequence of its cloned brain-specific mRNA. Antisera raised against selected synthetic peptide fragments of this protein were used to map the cellular location of the presumptive gene product in the brains of normal or colchicine-pretreated adult rats. Antisera directed against any of three C-terminally located, but nonoverlapping, nonhomologous, synthetic peptide segments (P5, P6, or P7) produced virtually identical maps of intensely immunoreactive neuropil staining. The immunoreactivity was distributed heterogeneously and was most pronounced within olfactory, somatosensory, and limbic systems, and was more modest in certain motor and auditory structures. In colchicine-pretreated rats, large, multipolar perikarya were observed within the amygdala, caudate-putamen, cingulate, parietal, and piriform cortices, as well as in particular diencephalic and pontine nuclei. Smaller immunoreactive neurons with more limited dendritic extensions were observed in the olfactory bulb, the cerebellar cortex, and the dorsal horn and intermediolateral cell columns of the spinal cord. No immunoreactivity was observed in visceral structures innervated by the autonomic nervous system or in non-neural tissues. In addition to the virtually superimposable maps produced by antisera to all three synthetic fragments selected from the C-terminus of 1B236, some uniquely reactive sites were seen. Antisera to the most N-terminal of the three synthetic immunogens (P5) were reactive with neurons of the medial trapezoid nucleus and in nerve terminals surrounding the deep cerebellar nuclei. Antisera against the most C-terminal synthetic immunogen (P7) were reactive with neurons of the paraventricular and supraoptic hypothalamic nuclei. These data demonstrate that the 1B236 protein is located within selected neuronal elements within functionally related cellular circuits established more formally by other methods. Our data show that protein 1B236-immunoreactive cells share at least the expression of this protein and suggest that these cells may also be related epigenetically or evolutionarily. These data, together with other subcellular, ultrastructural, and electrophysiological properties of 1B236, suggest that this protein could be considered as a prohormone capable of yielding several final candidate transmitter products.

Animals↗
PubMed1984

Control of neuronal gene expression.

Some 30,000 genes are expressed exclusively in the rat brain, many of which contain a genetic element called an identifier sequence located in at least one of their introns. The identifier sequences are transcribed by RNA polymerase III exclusively in neurons to produce two RNA species, BC1 and BC2, of 160 and 100 to 110 nucleotides. This transcriptional event may define regions of chromatin that contain neuronal-specific genes and may poise these genes for transcription by polymerase II by rendering the gene promoters accessible to soluble trans-acting molecules.

Animals↗
PubMed1984

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization.

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.

Adrenal Glands↗
PubMed1984

Brain-specific genes have identifier sequences in their introns.

The 82-nucleotide identifier (ID) sequence is present in the rat genome in 1-1.5 X 10(5) copies and in cDNA clones of precursors of brain-specific mRNAs. One brain-specific gene contains more than one ID sequence in its introns. There is an excess of ID sequences to brain genes, and some ID sequences appear to have been inserted as mobile elements into other genetic locations. Therefore, brain genes contain ID sequences in their introns, but not all ID sequences are located in brain gene introns. A brain ID consensus sequence has been obtained by comparing 8 ID nucleotide sequences.

Amino Acid Sequence↗
PubMed1983

Gene expression in rat brain.

191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA. 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain. An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells. Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation. Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length. Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families. From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.

Animals↗
PubMed1983

Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos.

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.

Amino Acid Sequence↗
PubMed1983

Identifying the protein products of brain-specific genes with antibodies to chemically synthesized peptides.

From the nucleotide sequences of three cDNA clones of rat brain-specific mRNAs, we deduced the partial amino acid sequences of two previously unknown proteins. We raised antisera to synthetic peptides mimicking short regions of these putative brain-specific proteins, and used these sera in immunocytochemical studies to localize each protein in the brain. One protein is found in large neurons throughout the brain, asymmetrically distributed toward the dendritic pole of the cell cytoplasm, suggesting involvement in the synthesis and/or directional transport of dendritic substances. The sequence of the second protein contains pairs of basic residues similar to precursors for neurotransmitters. The protein is located in (and may be the precursor for the neurotransmitter of) a novel fiber network of major extent with ramifications in cerebellum, hippocampus, and cortex, and in cell bodies in the brain stem, hypothalamus, and caudate nucleus. Our approach provides a direct method for characterizing rare brain molecules which may not have been anticipated and for which there are no known functional assays.

Amino Acid Sequence↗
PubMed1983

Monoclonal antibodies against vasoactive intestinal polypeptide: studies of structure and related antigens.

Hybridomas secreting monoclonal anti-vasoactive intestinal polypeptide (VIP) antibodies were constructed from spleen cells sensitized to VIP in vitro. The secreted antibodies were characterized by binding to VIP in indirect radioimmunoassays and enzyme-linked immunosorbent assays. Two monoclonal antibodies, characterized for their binding activities with synthetic fragments of VIP, were found to bind different sites on the VIP molecule. These monoclonal antibodies may recognize tertiary structures of the VIP. A search was conducted for antigens recognized by the monoclonal antibodies in brain: brain proteins separated on polyacrylamide gels were electroblotted onto nitrocellulose filters and were reacted first with the mouse antibody and then with goat anti-mouse immunoglobulin coupled to horseradish peroxidase as a means of detection. The monoclonal antibodies were found to react with a protein of molecular weight 60,000, which was also recognized by polyclonal antibodies, although the latter reacted with a number of additional proteins. The relationship of the protein of molecular weight 60,000 to VIP is discussed.

Adrenal Gland Neoplasms↗
PubMed1982

beta-Endorphin enhances lymphocyte proliferative responses.

The opioid peptides alpha- and beta-endorphin and [D-Ala2, Met5]enkephalin were investigated for their effect on the proliferation of resting and activated rat splenic lymphocytes in vitro. beta-Endorphin enhanced the proliferative response of spleen cells to the T cell mitogens concanavalin A and phytohemagglutinin. The effect of beta-endorphin was dose dependent and occurred at peptide concentrations similar to those found in rat plasma. alpha-Endorphin and [D-Ala2, Met5]enkephalin did not affect the proliferative responses to any mitogen tested. Furthermore, the potentiating effect of beta-endorphin was not reversed by treatment with 10 microM naloxone. None of the peptides had any effect on resting, unstimulated spleen cells or on the response to a mixture of lipopolysaccharide and dextran sulfate, which is specifically mitogenic for B lymphocytes. The pharmacological properties of the beta-endorphin potentiation indicate that the effect may be mediated by a nonopiate but beta-endorphin-specific mechanism. These results suggest a possible role for peripheral beta-endorphin and may provide a link between stress and disease susceptibility.

Analgesics↗
PubMed1982

Common 82-nucleotide sequence unique to brain RNA.

Several randomly selected cDNA clones made from rat brain polyA+RNA have unusual properties. Although the cDNA inserts are 500-1,250 base pairs long, they hybridize to a 160-nucleotide RNA species that is present in brain but not in liver or kidney. Nucleotide sequence studies of two of the clones and hybridization studies show that a common 82-nucleotide sequence is responsible for the hybridization. The same sequence is located in the second intron of a rat growth hormone gene. These studies suggest that signals called "ID sequences" may be carried in the introns of genes and prescribe their tissue-specific expression.

Base Sequence↗
PubMed1979

An entomopoxvirus from Oncopera alboguttata (Lepidoptera: Hepialidae) in Australia.

A new entomopoxvirus has been isolated from field-collected larvae of Oncopera alboguttata at Ebor, New South Wales, Australia. The ovoid proteinaceous virus-containing bodies measured about 8 x 6 microns and contained up to about 17 centrally-embedded virus-free spindles and up to about 180 occluded virus particles. The virus particle had a beaded outer membrane and measured about 390 x 270 x 230 nm. The particle contained a single lateral body and a unilaterally concave core which in longitudinal section measured 320 x 80 nm. Dissolution of inclusion body and spindle protein to release virus particles occurred in carbonate/thioglycollate buffer at pH greater than 12.0. It is postulated that this virus has recently evolved from similar viruses in soil inhabiting coleopteran larvae.

Animals↗