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.
Location of oligo(uridylic acid) sequences within messenger ribonucleic acid molecules of HeLa cells.
A significant fraction of the polyadenylated mRNAs of HeLa cells contain an oligo(uridylic acid) [oligo(U)] sequence of 15-30 nucleotides. Several different experimental approaches were used to determine if these oligo(U)'s occupied similar sites within all mRNAs. In one approach, poly(adenylic acid)-containing mRNAs [poly(A+) mRNAs] averaging 2800 nucleotides in length were reduced to an average size of 500 nucleotides by controlled alkaline hydrolysis. Over 20% of the oligo(U)-containing fragments isolated from the hydrolysate retained a poly(A) sequence, showing that oligo(U)'s were not exclusively located near 5' ends of mRNA although 20% were apparently close to 3' ends. To confirm these observations, oligo(U)-containing mRNA [oligo(U+) mRNA] was exposed to the 3'-exonucleolytic activity of polynucleotide phosphorylase to produce fragments containing the 5' regions of mRNA. Each of a set of fragments of decreasing length generated by increased times of exposure of the mRNAs to the enzyme was found to have about the same oligo(U) content, including the shortest that averaged 550 nucleotides. These data not only eliminated an exclusive location for oligo(U) in either 3' or 5' ends of mRNA but also suggested that oligo(U)'s might be close to the 5' ends of some mRNAs. To verify this last observation, periodate-oxidized poly(A+) mRNA was labeled at the 5' caps and at 3'-adenosine residues by sodium [3H]borohydride reduction before it was nicked 3-5 times with alkali to produce 5' and 3' end-labeled pieces that could be separated with oligo(thymidylic acid)-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)
Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells.
Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Polyadenylylated nuclear RNA contains branches.
A highly charged component can be isolated from a total RNase T2 digest of nuclear polyadenylylated RNA from HeLa cells that is separable from caps by (dihydroxyboryl)aminoethyl-cellulose chromatography. Chemical and enzymatic analyses show that the component contains a 2'-5' phosphodiester bond that creates a branch at the 2'-hydroxyl group of one nucleotide already linked to an adjoining nucleotide through the usual 3'-5' phosphodiester bond. [Formula: see text] This structure was confirmed by analysis of a similar component isolated from nuclease P1 digests of the same nuclear polyadenylylated RNA. Branches occur in roughly 10% of nuclear polyadenylylated RNAs, including those >10S in size, but are absent from cytoplasmic polyadenylylated RNA. Possible implications for branches as intermediates in mRNA processing are discussed.
A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells.
When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.
5'-Terminal cap structures of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells: comparison with other ribonucleic acid subpopulations.
The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.
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.