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R E Rhoads

Publications and source records attributed to R E Rhoads.

At least 91 records · Page 5Linked to original sources
PubMed1989

In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E.

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.

Animals↗
PubMed1989

Neuronal RNA in Pick's and Alzheimer's diseases. Comparison of disease-susceptible and disease-resistant cortical areas.

Comparative neuronal RNA analyses were conducted in disease-prone (frontal association, Brodmann's area 9) vs relatively disease-resistant (primary visual, occipital area 17) cortex of patients with autopsy-proved Pick's disease (PD) and Alzheimer's disease (AD). Azure B-RNA staining and scanning-integrating microdensitometry were used to determine total RNA contents of pyramidal neurons in layers 3 and 5. In both PD and AD (1) significant (15% to 47%) RNA loss was detected in neurons of both cortical areas and layers relative to those of aged, nondemented controls, and (2) RNA loss was not markedly enhanced in the damaged frontal cortex relative to that in the preserved occipital cortex. Neuronal RNA depletion was generally more marked in PD than AD. However, this impairment does not appear to be related to the formation of classic neuropathological abnormalities in either disease.

Aged↗
PubMed1989

Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus.

Full-length cDNA copies of tobacco vein mottling virus (TVMV) RNA were constructed downstream from bacteriophage T7 or T3 RNA polymerase promoters. The plasmids were designed to produce in vitro transcripts containing, respectively, one or two guanosine residues at the 5' terminus not derived from the TVMV sequence and a single cytidine residue at the 3' terminus following the poly(A) tail. Introduction of transcripts from either plasmid into tobacco mesophyll protoplasts resulted in the accumulation of TVMV coat protein and RNA. Neither coat protein nor viral RNA accumulated in protoplasts inoculated with linearized cDNA or with in vitro transcripts synthesized in the absence of 7-methylguanosine(5')triphospho(5')guanosine (m7GpppG). Tobacco seedlings inoculated with native TVMV RNA developed symptoms a few days before those inoculated with in vitro transcripts; however, 3 weeks after inoculation, the symptoms produced by the two inocula were indistinguishable.

Blotting, Western↗
PubMed1988

Juvenile hormone regulation of mRNA levels for a highly abundant hemolymph protein in larval Trichoplusia ni.

Several hemolymph proteins ranging in size from 73 to 76 kDa increase to very high levels just prior to metamorphosis in Trichoplusia ni (Lepidoptera). One of these proteins (pI = 5.8, Mr = 76,000) was selected for a study of hormonal regulation. The appearance of this protein could be suppressed in vivo by topical treatment with the juvenile hormone analog fenoxycarb. An antiserum for this protein was prepared and shown to react selectively with the 76-kDa protein in whole hemolymph. Translation of poly(A)-containing RNA from untreated larvae yielded the 76-kDa protein, whose identity was verified with the antibody, whereas mRNA from juvenile hormone analog-treated larvae did not. These data indicate that juvenile hormone acts to regulate the level of the mRNA of this hemolymph protein.

Animals↗
PubMed1988

Neuronal RNA in relation to Alz-50 immunoreactivity in Alzheimer's disease.

Both defective nucleic acid metabolism/protein synthesis and the expression of an abnormal antigen recognized by the Alz-50 antibody may be involved in the neuronal degeneration of Alzheimer's disease (AD). A multiparametric analysis involving Alz-50 immunocytochemistry and azure B--RNA microdensitometry was developed to compare nucleic acid alterations in Alz-50-positive neurons to those in Alz-50-negative neurons of the brain of AD patients. Alz-50-immunoreactive neurons of the hippocampal endplate (Rose's H3-H5 fields) and the subiculum exhibited significantly lower (by approximately 30%) total RNA contents than negative neurons of the corresponding region. The mean RNA content of Alz-50-positive neurons of the AD brain was also reduced in comparison to that of Alz-50-negative neurons of age-matched, nondemented controls, whereas there were no significant differences between negative neurons of AD patients and controls. The hippocampus of nondemented controls was also found to contain Alz-50-immunoreactive neurons, although 20- to 30-fold fewer than the hippocampus of AD patients. In the controls, there was also a tendency toward reduced RNA levels in Alz-50-positive versus -negative neurons. These data suggest a relationship between Alz-50 immunoreactivity and defective nucleic acid metabolism in the AD brain.

Alzheimer Disease↗
PubMed1988

Astrocyte RNA in relation to neuronal RNA depletion in Alzheimer's disease.

A new double-staining procedure, in which the techniques of immunocytochemistry of glial fibrillary acidic protein (GFAP) and quantitative microdensitometry of azure B-RNA were combined, was used to study nucleic acid alterations in fibrous astrocytes in Alzheimer's disease (AD). RNA contents of GFAP-positive cells of the hippocampal endplate (Rose's H3-H5 fields) and the dentate gyrus molecular layer were determined in ten autopsy-proven AD patients (ages 51-88) and ten age-matched, non-demented controls. In addition, RNA contents of pyramidal neurons of the endplate were examined. While there were no differences in RNA contents of astrocytes of either region between AD patients and controls, neuronal RNA was markedly depleted. These data suggest that astrocytes maintain protein synthetic capabilities in AD and that RNA loss is limited to the neuronal compartment.

Aged↗
PubMed1988

In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus-encoded protease.

Potential protease functions associated with the NIa nuclear inclusion protein of tobacco vein mottling virus (TVMV) were investigated. In the absence of treatments, in vitro translation of viral RNA produced various polypeptides representing each of the proposed TVMV cistrons--28K-HC-42K-CI-5.5K-NIa-NIb-CP. When viral RNA was first hybridized to DNA probes complementary to the NIa cistron, and then treated with RNase H prior to translation, a 48-kDa processing product, immunologically identified as the NIa protein, was abolished. In its place was observed a series of larger polypeptides, immunologically identified as fusion products of the cylindrical inclusion (CI) and NIa cistrons. The use of probes which permitted translation through as few as 15 nucleotide residues beyond the sequences encoding the proposed carboxyl terminus of NIa resulted in normal processing. None of the DNA probes affected an apparent cleavage between the helper component (HC) and 42K proteins. Cloned cDNA regions representing the NIa cistron and flanking sequences were inserted in transcription vectors. Translation of the in vitro transcript resulted in synthesis, not of a large fusion polyprotein, but, of a mature-sized NIa polypeptide. In vitro transcripts, lacking the 3'-most sequences that were expected to encode the protease active site of the NIa protein, were translated. These generated an apparent fusion polypeptide that reacted with antisera to both CI and NIa. The results indicate that the NIa gene product functions as a protease responsible for some but not all of the cleavage events which lead to the production of the mature forms of TVMV proteins.

Genes, Viral↗
PubMed1988

Mapping of the tobacco vein mottling virus VPg cistron.

The location of the cistron encoding the genome-linked protein (VPg) in the potyvirus tobacco vein mottling virus (TVMV) was investigated. Precipitation of 125I-labeled VPg with anti-tobacco etch virus 49K nuclear inclusion protein antiserum (which reacts with the NIa nuclear inclusion protein of TVMV) indicated that the TVMV VPg is immunologically related to NIa. Lysyl residues were found to be present at positions 2, 11, and 16 of the amino-terminal region of the VPg. A search of the TVMV polyprotein sequence for this distribution of lysyl residues revealed a unique location beginning at amino acid residue 1801, the proposed amino-terminus of the NIa protein.

Amino Acid Sequence↗
PubMed1988

A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase.

A cDNA that encodes what appears to be the inhibitory domain of the plasma membrane calcium-pumping ATPase (Ca2+-ATPase) has been isolated by screening a lambda gt11 bovine brain cDNA library with antibodies prepared against the human erythrocyte membrane Ca2+-ATPase. This screening resulted in isolation of a bacteriophage containing a 1.5-kilobase cDNA insert encoding a 71-residue polypeptide, the remainder being a large 3' terminal noncoding region. A portion of this deduced peptide sequence was identical to that of a peptide isolated from a V8 protease digest of the human erythrocyte Ca2+-ATPase except for 1 residue. Antibodies purified by immunoabsorption to the fusion protein containing this cDNA-encoded polypeptide reacted only with those fragments of a limited trypsin digest of the human erythrocyte Ca2+-ATPase that contain the inhibitory domain. Moreover, these antibodies were able to partially stimulate basal enzyme activity and block further activation by calmodulin. The encoded polypeptide bears homology to the glutamic acid-rich regions N-terminal to the Ca2+-binding loops of calmodulin and to a lesser extent with the loops themselves. This encoded polypeptide also represents the C terminus of the Ca2+-ATPase. Portions of the isolated cDNA were homologous to the 3' noncoding region of the sarcoplasmic reticulum Ca2+-ATPase cDNA, indicating a possible mechanism for the evolution of these distinct membrane Ca2+ pumps.

Amino Acid Sequence↗
PubMed1987

Phosphorylation site of eukaryotic initiation factor 4E.

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) was labeled in situ with [32P]orthophosphate in cultured HeLa cells and rabbit reticulocytes and purified by affinity chromatography. Tryptic digestion yielded one labeled peptide which contained predominantly serine and lysine. After treatment of the protein with citraconic anhydride to block epsilon-amino groups of lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure Trp-Ala-Leu-Trp-Phe-Phe-Lys-Asn-Asp-Lys-Ser(P)-Lys-Thr-Trp-Gln-Ala-Asn-L eu-Arg, one of the arginyl peptides predicted from the human eIF-4E cDNA sequence. The only serine in this peptide is located at position 53 of eIF-4E. Thus, it is concluded that eIF-4E contains a single site of phosphorylation for an endogenous protein kinase, which is Ser-53 in the human eIF-4E sequence.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of the mRNA cap-binding protein from human tissues.

The 25-kDa mRNA cap-binding protein (CBP) involved in translation was purified by affinity chromatography from human erythrocytes and rabbit reticulocytes. The sequences of eight human and seven rabbit tryptic and V8 proteolytic peptides were determined. Based on the peptide sequence data, oligodeoxynucleotide probes were synthesized and used to screen human fibroblast and lymphocyte lambda cDNA libraries. The DNA sequence obtained from recombinant lambda phage inserts was found to code for all but one peptide. A 23-base oligonucleotide was synthesized based on the DNA sequence and used to prime synthesis of cDNA from human placental mRNA to construct a third library in lambda gt10. Screening with a 22-base oligonucleotide, whose sequence was upstream from the 23-base primer, yielded numerous recombinant phages with approximately equal to 250-base inserts. The 1900-base-pair cDNA sequence compiled from all phage inserts appeared to represent the entire primary sequence of CBP (Mr 25,117). Blot analysis of human placental and HeLa mRNA revealed multiple CBP mRNA species ranging from 1925 to 2250 bases. The amino acid sequence of CBP showed homology to the cap-binding PB2 protein of influenza virus.

Amino Acid Sequence↗
PubMed1987

Neuronal RNA in relation to neuronal loss and neurofibrillary pathology in the hippocampus in Alzheimer's disease.

Topographic analyses were performed on the distribution of neuronal RNA loss in relation to local neuronal loss and neurofibrillary degeneration in the hippocampal region of brains of patients with Alzheimer's disease (AD). Compared to age-matched controls, pyramidal neuronal RNA was depressed (p less than 0.0001) in all areas of the hippocampus examined in AD, viz., the endplate (33%), Rose's H2 field (30%), Rose's H1 field (37%) and the subiculum (46%). Significant neuronal loss was observed in Rose's H1 field and the subiculum but not in the endplate or Rose's H2 field. The frequency of neurofibrillary tangle-bearing neurons was enhanced in all four regions of AD brains, the number of involved neurons being markedly greater in Rose's H1 field and the subiculum than in the endplate and Rose's H2 field. Overall, the data indicate the existence of a generalized disturbance in RNA metabolism within pyramidal neurons in the hippocampus in AD, occurring in regions of minimal (endplate, Rose's H2 field) as well as those of extensive (Rose's H1 field, subiculum) pathological alterations. Although there is focal accentuation of RNA depletion in the latter, the marked RNA depletion in regions of minimal pathologic change suggests that this effect is largely unrelated to local neuronal loss or neurofibrillary degeneration.

Aged↗
PubMed1987

In situ analysis of neostriatal RNA and chromatin in Alzheimer's disease.

Scanning-integrating microdensitometry of azure B-RNA- and Feulgen-Schiff-stained tissue sections was used to measure neostriatal neuronal RNA levels and susceptibility of neuronal and oligodendrocyte chromatin to acid hydrolysis in Alzheimer's disease (AD) patients and controls. AD was associated with neuronal RNA depletion (17 to 23%) in both caudate nucleus and putamen. While neuronal chromatin was found to be more acid-labile than that of the oligodendrocytes, there were no differences in either cell type between AD and controls. These data support the existence of a macromolecular disturbance (RNA loss) occurring within neostriatal neurons, perhaps related to the extrapyramidal dysfunction of AD, but fail to demonstrate that an alteration in chromatin is responsible for this effect.

Aged↗
PubMed1986

Terminal structure of hypovirulence-associated dsRNAs in the chestnut blight fungus Endothia parasitica.

The 3'- and 5'-terminal sequences of the five large double-stranded RNA species (L-dsRNA; 4.5-6.0 X 10(6) daltons) of EP713, a hypovirulent strain of Endothia parasitica, were determined by mobility-shift and enzymatic methods. All the L-dsRNAs appeared to have identical terminal sequences. A heteropolymer sequence was found at one 3'-terminus and a poly(A) sequence of variable length at the other. It was possible to label only one 5'-terminus using polynucleotide kinase and [gamma-32P]ATP, and this was shown to be a poly(U) sequence of variable length. We propose that the dsRNAs have the following structure, where X represents a blocking group: (Formula: see text). A recombinant plasmid containing dsRNA-related sequences was constructed. Hybridization analysis using the recombinant probe indicated that the sequence homology among the L-dsRNAs extended beyond these terminal regions and was also shared by small dsRNAs (0.3-0.45 X 10(6) daltons).

Ascomycota↗
PubMed1986

The nucleotide sequence of tobacco vein mottling virus RNA.

The nucleotide sequence of the RNA of tobacco vein mottling virus, a member of the potyvirus group, was determined. The RNA was found to be 9471 residues in length, excluding a 3'-terminal poly(A) tail. The first three AUG codons from the 5'-terminus were followed by in-frame termination codons. The fourth, at position 206, was the beginning of an open reading frame of 9015 residues which could encode a polyprotein of 340 kDa. No other long open reading frames were present in the sequence or its complement. This AUG was present in the sequence AGGCCAUG, which is similar to the consensus initiation sequence shared by most eukaryotic mRNAs. The chemically-determined amino acid compositions of the helper component and coat proteins were similar to those predicted from the nucleotide sequence. Amino acid sequencing of coat protein from which an amino-terminal peptide had been removed allowed exact location of the coat protein cistron. A consensus sequence of V-(R or K)-F-Q was found on the N-terminal sides of proposed cleavage sites for proteolytic processing of the polyprotein.

Base Sequence↗
PubMed1986

Structural analysis of the messenger RNA cap-binding protein. Presence of phosphate, sulfhydryl, and disulfide groups.

The messenger RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with [32P]orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with [1-14C]iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges. Species of CBP with faster or slower electrophoretic mobilities could be generated by treatment of the protein either with O2 in the presence of a catalyst or with dithiothreitol. The predominant form of the untreated protein migrated between these two forms.

Animals↗
PubMed1985

Immunological detection of the messenger RNA cap-binding protein.

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.

Animals↗