Biomedical subjects
M Edmonds
Publications and source records attributed to M Edmonds.
Antibodies specific for branched ribonucleic acids.
A chemically synthesized branched tetranucleotide, G3'p5'A [2'p5'G]3'p5'C corresponding to the consensus sequence at the branch point in introns undergoing RNA splicing, was used as a hapten to elicit antibranch antibodies. Binding assays with 32P-labeled hapten and unlabeled structurally related haptens indicated that the antibodies are highly specific for the branch structure and have some specificity for the A2'p5'G sequence at the branch point, but have essentially none for a variety of other 2'p5' or 3'p5' dinucleotides or for the linear trinucleotide G3'p5'A3'p5'C. Purification of these antibodies by binding to A2'p5'G covalently linked to Sepharose followed by covalent attachment of the purified antibodies to protein A-Sepharose has provided an adsorbent that immunospecifically retains branched oligonucleotides as well as branched introns released from RNAs during in vitro splicing.
Transcription vectors that facilitate the identification and mapping of RNA splice sites in genomic DNA.
Two transcription vectors were constructed that can identify the splice sites at exon-intron boundaries of inserted DNA fragments possessing the complementary splice site. One vector contains the 5' splice donor site and flanking exon-intron sequences from the 3' end of the adenovirus first late leader. The other vector contains the 3' splice acceptor site and the branch acceptor site, plus the flanking exon-intron sequences from the 5' end of the adenovirus second late leader. Both vectors contain a multiple cloning site for insertion of DNA fragments. DNA fragments supplying the complementary splice site, including the adjacent exon and intron sequences, were inserted into the vectors. The vectors were used as templates for the synthesis of chimeric RNA transcripts that were spliced in in vitro splicing extracts. Chimeric transcripts from the vectors containing complementary splice site boundary regions from the human growth hormone gene were accurately spliced in vitro. A splice site from a human growth hormone intron that is not normally spliced in vitro was spliced when paired with an adenovirus splice site. These vectors can be used to identify splice sites and to determine the lengths of exons and their attached introns within a DNA fragment of unknown coding content.
The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase.
Virtually all mRNAs in eucaryotes end in a poly(A) tail. This tail is added posttranscriptionally. In this report, we demonstrate that the enzyme that catalyzes this modification is identical with an activity first identified 30 years ago, the function of which was previously unknown. This enzyme, poly(A) polymerase, lacks any intrinsic specificity for its mRNA substrate but gains specificity by interacting with distinct molecules: a poly(A) polymerase from calf thymus, when combined with specificity factor(s) from cultured human cells, specifically and efficiently polyadenylates only appropriate mRNA substrates. Our results thus demonstrate that this polymerase is responsible for the addition of poly(A) to mRNAs and that its interaction with specificity factors is conserved.
Progression of diabetic autonomic neuropathy over a decade in insulin-dependent diabetics.
The prognosis for diabetics with autonomic neuropathy is little known. We therefore studied the progress of young insulin-dependent diabetics, first identified as having abnormal autonomic function 10-15 years ago. We have shown that the mortality of diabetics with symptomatic autonomic neuropathy is increased, but is less than previously reported. Mortality in asymptomatic diabetics with an isolated abnormality in autonomic function tests is not increased. The heart rate variability declines at 1.02 +/- 0.47 (SD) per annum in diabetics with an initially normal heart rate variability. While symptoms of autonomic neuropathy do not usually remit even over a decade, they do not commonly progress. Three groups of young insulin-dependent diabetics had heart rate variability tested between 1972 and 1977 and have been reviewed 10-15 years later. Group A (n = 49) had symptomatic autonomic neuropathy and an abnormal heart rate variability (less than 12), Group B (n = 24) were asymptomatic yet had an abnormal heart rate variability and Group C (n = 38) were asymptomatic and had a normal heart rate variability (16-26). The 10-year survival in Group A (73.4 per cent) was less (P less than 0.05) than in Groups B (91.7 per cent) or C (89.5 per cent) which did not differ from each other. The 18 Group A deaths were due predominantly to renal failure (n = 4), myocardial infarction in patients with nephropathy (n = 3) and sudden unexpected death (n = 3). The chief symptoms of autonomic neuropathy--diarrhoea, postural hypotension and gustatory sweating, were very persistent but did not necessarily deteriorate or become disabling in the majority of patients. The development of autonomic symptoms in asymptomatic patients with abnormal heart rate variability was uncommon over a decade.
Isolation and characterization of branched oligonucleotides from RNA.
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The quantitation and distribution of splicing intermediates in HeLa cells and adenovirus RNAs.
The steady state level of splicing intermediates in HeLa cells and in adenovirus RNA made late in the infectious cycle has been measured by a branch point analysis. About one in ten poly A(+) nuclear RNAs contained a branch point, but only 1/3 as many adenovirus RNAs were branched. Fewer branches were found in the poly A(-) RNAs of the nucleus and of late adenovirus transcripts suggesting that excised lariat introns do not accumulate in vivo. Branched RNAs were found in the poly A(+) RNAs from a nuclear matrix fraction, but several experiments failed to show an enrichment in these splicing intermediates in this matrix fraction. Branches were found in all size classes of poly A(+) nuclear RNA and were not exclusively associated with either the 3' or 5' regions, but were randomly distributed within RNA molecules. These results as well as the base and sequence data on branch points (1,18) are consistent with the conclusion that branched poly A(+) RNAs are splicing intermediates.
Chemical synthesis of branched RNA.
A branched tetranucleotide consisting of adenosine linked 2' and 5' to guanosine and 3' to cytidine was synthesized from appropriately protected nucleoside phosphoramidites as synthons. The product was characterized enzymatically.
Poly(a) sequences in Naegleria messenger RNA before and after initiation of differentiation. Absence of oligo(A).
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Properties of a small transcribed poly A sequence in heterogeneous nuclear RNA of HeLa cells.
A class of heterogeneous nuclear RNA (hnRNA) molecules contain an internal transcribed poly A sequence of close to 25 uninterrupted AMP residues. HnRNA molecules containing this sequence are separable from those containing the large 3' terminal poly A sequence on the basis of their differential affinity for oligo dT cellulose. The fact that the transcribed small poly A and the 3' terminal poly A are not found in the same hnRNA molecules even though both are present in similar size classes and that the small poly A is absent from cytoplasmic messenger RNA (mRNA) has led us to propose a scheme for mRNA processing in which the 3' end of the small poly A in hnRNA becomes a priming size for the post-transcriptional addiction of the large poly A.
Specificity of short-loop feedback of luteinizing hormone in the rabbit.
The specificity of the gonadotropin short-loop feedback mechanism was investigated in castrated rabbits by blood sampling from an indwelling jugular venous catheter for follicle-stimulating hormone (FSH) after the intravenous administration of purified human luteinizing hormone (hLH). Although we have previously shown that such hLH administration results in a decrease of rabbit LH, no decrease in rabbit FSH was seen in these experiments. This specificity of LH feedback is compatible with the site of feedback being at the (1) pituitary level or (2) at the hypothalamic level provided two separate LH and FSH releasing factors exist.
Characterization of the poly(adenylic acid) sequences in RNA synthesized in vitro by mouse myeloma nuclei.
The size and quantity of poly(A)sequences made by mouse myeloma nuclei in vitro are dependent on the concentration of KCI, ATP, other ribonucleoside triphosphates, as well as the nature of the divalent cation in the reaction medium. Reduction of the KC1 concentration from 120 mM to 5 mM, for example, stimulates poly(A) synthesis 10- to 20-fold. These poly (A) sequences are similar in size to cellular nuclear poly(A), but the RNA molecules to which they are attached are much shorter than poly(A) containing RNA molecules made at 120 mM KC1. Presence of Mn2+ in the medium led to a much more heterogenous population of poly(A) sequences. From such observations we have found reaction conditions in which nuclei synthesize molecules that resemble native nuclear poly(A) + RNA. Not only are the lengths and amounts of the poly(A) sequences similar, but they also undergo a terminal turnover like that of the poly(A) in hnRNA. An oligo(A) sequence that resembles the oligo(A) found in non-poly(A) containing hnRNA of mouse myeloma and HeLa cells is also synthesized in vitro. These observations suggest that some processing functions are retained during the in vitro incubation of these nuclei.
Poly(uridylic acid) sequences in messenger ribonucleic acid of HeLa cells.
The poly(uridylic acid [poly(U)] sequences of 30 to 40 nucleotides found in the heterogeneous nuclear ribonucleic acid (RNA) Of HeLa cells are also present in the poly(adenylic acid) [poly(A)] containing messenger RNA (mRNA) of these cells. Twenty-five percent of the total poly(U) sequences in the cell are found in the cytoplasm after HeLa cells have been labeled for 2.5 h with 32P. This value is close to 40% if only poly(A) containing RNA molecules of the cell are considered. The distribution of poly(U) sequences parallels that of poly(A) sequences in different size classes of cytoplasmic messenger RNA. From the concentrations and lengths of the two sequences it can be estimated that about 20% of the poly(A) containing mRNA molecules could contain one poly(U) sequence. Several lines of evidence have been developed to rule out poly(U) containing heterogeneous nuclear RNA (HnRNA) as the source of the poly(U) sequences in cytoplasm. Poly(U) sequences are also found in RNA molecules of the nucleus and cytoplasm which do not contain poly(A). Although poly(U) sequences are most abundant in nuclear RNA species lacking poly(A), they are well represented in a class of RNA molecules in cytoplasm which lack poly(A), but which otherwise resemble the polysomal mRNA of HeLa cells.
Transcribed oligonucleotide sequences in Hela cell hnRNA and mRNA.
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Preparation and properties of a poly A resin and its use in the isolation of naturally occurring poly U sequences.
A method for the covalent attachment of poly A, as well as other nucleic acids and nucleosides, to a methylene dianiline derivative of starch is described. The properties of this poly A resin and its use for the recovery of poly U sequences from both nuclear and cytoplasmic extracts of HeLa cells is described.