Biomedical subjects

R D Abramson

Publications and source records attributed to R D Abramson.

16 recordsLinked to original sources

PubMed1993

Nucleic acid amplification technologies.

The polymerase chain reaction, Q beta replicase methodology, the ligase chain reaction, the self-sustained sequence replication system, and the new strand displacement assay have continued to progress with the development of improved reagents and new applications. These advances in enzymatic nucleic acid amplification strategies continue to provide research and medical communities with an ever-improving arsenal of ways to amplify RNA and DNA.

Gene Amplification↗

PubMed1993

High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.

Amino Acid Sequence↗

PubMed1991

Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.

Base Sequence↗

PubMed1991

Characterization of the human argininosuccinate lyase gene and analysis of exon skipping.

A cosmid clone containing the entire human argininosuccinate lyase gene was isolated and characterized. The gene is approximately 35 kb in length and is divided into 16 exons. The sequence surrounding all splice sites was determined, and one unusual 5' splice site sequence in which a GC dinucleotide occurred instead of the consensus GT dinucleotide was observed. The exon structure of the gene is identical to that of the rat and similar to that of the delta-crystallin genes in the chicken. The transcription initiation site was identified, and 252 bases 5' of that site were sequenced. Within that region, the GC content is 75%, no TATA element was observed, and multiple potential Spl and CACCC binding sites were noted. Also, a putative consensus sequence that may play a role in the regulation of urea cycle genes was identified within this region. Analysis of the structure of the mature mRNA derived from this gene revealed that the sequences encoded by exon seven were deleted in approximately 5-10% of the mature mRNA in all tissue sources examined.

Argininosuccinate Lyase↗

PubMed1989

Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay.

A new technique has been applied to the study of the RNA secondary structure unwinding activity of the eukaryotic initiation factors (eIFs) 4F, 4A, and 4B. Secondary structures were generated at the 5' ends of reovirus and globin mRNA molecules by hybridization with 32P-labeled cDNA molecules 15 nucleotide residues long. The dissociation of the labeled cDNAs from the mRNAs was assayed by a gel filtration chromatography procedure which separates the free cDNAs from mRNAs and mRNA/cDNA hybrids. When the three factors were tested alone, only eIF-4F stimulated dissociation of hybrids. The combination of eIF-4A plus eIF-4B also exhibited a strong hybrid dissociating activity, which was markedly temperature dependent. Under optimum conditions, up to 90% of the hybrid structures are disrupted in 60 min. These results demonstrate for the first time that stable double-stranded regions can be melted and dissociated by eIFs. They also characterize more precisely the first step in the structure unwinding reaction.

Chromatography, Gel↗

PubMed1988

Induction of eIF-4E phosphorylation by the addition of L-pyrroline-5-carboxylic acid to rabbit reticulocyte lysate.

Addition of L-pyrroline-5-carboxylic acid to reticulocyte lysates inhibits protein synthesis and induced phosphoproteins of 25 and 14 kDa. The 25 kDa phosphoprotein had the same Mr and pI as phosphorylated eIF-4E. Incubation of lysates with L-pyrroline-5-carboxylic acid did not alter the crosslinking of eIF-4E to reovirus mRNA caps. These results suggest that modifications of the translational apparatus other than eIF-4E phosphorylation may mediate the inhibitory effect seen with L-pyrroline-5-carboxylic acid and/or that phosphorylation of eIF-4E may effect functions subsequent to its interaction with the mRNA cap such as protein-protein interactions with other cap-specific translation factors.

Animals↗

PubMed1988

Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro.

Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.

Adenosine Triphosphate↗

PubMed1988

Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein.

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.

Animals↗

PubMed1988

Discriminatory interaction of purified eukaryotic initiation factors 4F plus 4A with the 5' ends of reovirus messenger RNAs.

The interaction of several reovirus mRNAs with cap-binding initiation factors has been investigated. Two quantitative experimental techniques have been applied to this question: (a) the rates of reaction of different mRNAs with tobacco acid pyrophosphatase and (b) the extent of cross-linking of different mRNAs to initiation factors in the presence and absence of ATP. The effects of ionic strength on these reactions have also been investigated. Our results demonstrate for the first time that the purified initiation factors interact differentially with purified reovirus mRNAs under competitive conditions and thus confirm earlier interpretations based on kinetic data. Comparison of the data from these studies with the translational behavior of the reovirus mRNAs, both in vitro and in vivo, has also led to specific predictions about features of these mRNAs that determine their competitive efficiencies. 1) Under ordinary ionic conditions, the steric accessibility of the m7G cap moiety of a reovirus mRNA appears to be a major determinant of its translation rate. 2) When the ionic strength is increased to supranormal levels, an additional feature, which may simply be the amount of secondary structure formed by sequences proximal to the cap, can become rate-limiting for several, but not all, of these mRNAs.

Animals↗

PubMed1988

Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA.

The role of eukaryotic initiation factor (eIF)4B in translation is somewhat uncertain, although it appears to stimulate a variety of activities of eIF-4A and eIF-4F. Using the model RNA-dependent ATP hydrolysis assay, the ability of eIF-4B to stimulate eIF-4A and eIF-4F was investigated. The most dramatic effect of eIF-4B is to increase the affinity of eIF-4A for RNA; no effect is seen on the affinity of eIF-4A for ATP. This is not the case for eIF-4F where stimulation occurs primarily through an increase in Vmax and not a change in the affinity for RNA. The finding that eIF-4A and eIF-4B can bind to an mRNA (lacking in secondary structure), with essentially the same degree of effectiveness and affinity as would occur for natural mRNAs in the presence of eIF-4A, eIF-4B, and eIF-4F, suggests a possible role for eIF-4A and eIF-4B in both cap-independent and internal initiation.

Adenosine Triphosphatases↗

PubMed1988

Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors.

Three mammalian eukaryotic initiation factors (eIF) are required for the ATP-dependent binding of mRNA to the 40 S ribosomal subunit. These three factors, eIF-4A, eIF-4B, and eIF-4F, have also been isolated from wheat germ. Three assays were used to measure the ability of the wheat germ factors to interact with and/or substitute for the mammalian factors. Two assay systems were used to measure partial reactions involving the interaction of the three factors, ATP, and mRNA: 1) RNA-dependent ATP hydrolysis and 2) cross-linking of the factors to the 5' cap of oxidized mRNA. A third assay system was used to measure the ability of the factors to support initiation of protein synthesis. The results of the ATP hydrolysis and cross-linking experiments indicate that the wheat germ factors can interact with or substitute for the mammalian factors. Wheat germ eIF-4A appears to be functionally equivalent to mammalian eIF-4A. Wheat germ eIF-4B and eIF-4F appear to be isozymes possessing functions similar to mammalian eIF-4F. Wheat germ eIF-4B does not appear to be a functional equivalent to the mammalian eIF-4B. In a complete translation system from wheat germ, mammalian factors partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system.

Adenosine Triphosphate↗

PubMed1987

The ATP-dependent interaction of eukaryotic initiation factors with mRNA.

The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to periodate-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single-stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m7GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independent activity in our assays. Evidence from the cross-linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5' cap region of the mRNA. From the data presented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF-4F is the first initiation factor to bind mRNA ind an ATP-independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F X eIF-4B X mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA.

Adenosine Triphosphatases↗

PubMed1986

Influence of 5' proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors.

The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure.

DNA↗

PubMed1986

Recycling of messenger RNA cap-binding proteins mediated by eukaryotic initiation factor 4B.

The ability of polypeptide components of eukaryotic initiation factor (eIF) 4F to bind to the m7G cap of an mRNA, to be released from that mRNA, and then to rebind to the cap of a second mRNA has been investigated. The release and rebinding steps have been termed "recycling." It was found that eIF-4B stimulates the recycling of the 24-26 kDa (p24) component of eIF-4F, and perhaps of other components as well. By contrast, eIF-4A seemed to have little or no effect on the recycling of eIF-4F components, either in the presence or absence of eIF-4B. The recycled p24 is capable of cross-linking to oxidized cap structures. The recycled factor is also able to stimulate the cross-linking of added eIF-4A, which cross-links poorly in the absence of eIF-4F. By these criteria it seems likely that the recycled eIF-4F components are active for a second round of translational initiation.

Animals↗

PubMed1985

ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors.

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.

Adenosine Triphosphate↗

PubMed1984

RNA-stimulated ATPase activity of eukaryotic initiation factors.

Previously, we have described an ATP-dependent recognition and binding of mRNA by eukaryotic initiation factors (eIF)-4A, eIF-4B, and eIF-4F (Grifo, J. A., Tahara, S. M., Leis, J. P., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1982) J. Biol. Chem. 257, 5246-5252; Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804-5810). This finding was consistent with other studies which implicated eIF-4A and eIF-4B in binding mRNA to the 40 S ribosomal subunit, an ATP-requiring process. As part of ongoing studies of this step, and, in particular its ATP requirement, we have examined ATPase activity of various initiation factors. In this communication we describe an RNA-dependent ATP hydrolysis catalyzed by eIF-4A and eIF-4F. Although eIF-4B has little or no ATPase activity it can stimulate the RNA-dependent ATPase activity of either eIF-4A or eIF-4F. Similar to the ATP-dependent mRNA binding assay, the RNA-dependent ATPase activity is inhibited by the cap analogue m7GDP when globin mRNA is used as the activator. In addition, a variety of polynucleotides stimulate the ATPase activity of these factors including rRNA, tRNA, poly(U), and poly(A) but not poly(dA). Finally, an attempt has been made to discern whether phosphorylation or ATP hydrolysis is responsible for the ATP-stimulated binding of mRNA by eIF-4A and eIF-4B. We present evidence which is consistent with the interpretation that ATP hydrolysis and not protein phosphorylation correlates with ATP-stimulated binding of mRNA.

Adenosine Triphosphatases↗